QR1S6.S5 Am 1 blthr C.l G347532 The Balme Library Pill 3 0692 1078 5892 8 University of Ghana http://ugspace.ug.edu.gh PRODUCTION AND CHARACTERIZATION OF MONOCLONAL ANTIBODIES AGAINST SCHISTOSOMA HAEMATOBIUM SOLUBLE EGG AND URINE-BASED ANTIGENS JONES DARKWA AMANOR University of Ghana http://ugspace.ug.edu.gh PRODUCTION AND CHARACTERIZATION OF MONOCLONAL ANTIBODIES AGAINST SCHISTOSOMA HAEMA TOBIUM SOLUBLE EGG AND URINE-BASED ANTIGENS A Thesis Presented to The Board of Graduate Studies, University of Ghana, Legon Ghana. In Part fulfilment of the Requirements for the Degree of Master of Philosophy (M. Phil.) (Animal Science). By JONES DARKWA AMANOR BSc. (Hons.) Department of Animal Science, Faculty of Agriculture, University of Ghana, Legon, Accra, Ghana. January, 1995 University of Ghana http://ugspace.ug.edu.gh DECLARATION I do hereby declare that except for references to other poeple's work which I i » 4 have duly acknowledged, this exercise is a result of my own original research, and this thesis, either in whole, or in part, has not been presented for another degree elsewhere. o ___________ JONES DARKWA AMANOR (Student) PROF. R. K. G ASSOKU (Supervisor) (Co-supervisor) ■m Vv-vSA •___ DR. T. ARISHIMA (Co-supervisor) K G ASSOKU Department of Animal Science) University of Ghana, Legon Ghana University of Ghana http://ugspace.ug.edu.gh Ill D E D I C A T I O N To my teachers Prof. R.K.G. Assoku, Dr. K. M. Bosompem and Dr. T. Arishima University of Ghana http://ugspace.ug.edu.gh IV AKNOWLEDGMENTS I wish to express my sincere gratitude and deep appreciation to all individuals and institutions who, in diverse ways, helped me to successfully complete this work. My first thanks go to my supervisors, Professor R.K.G. Assoku, Head of the Animal Science Department, University of Ghana and Drs. K.M Bosompem and T. Arishima, both of the Noguchi Memmorial Institute for Medical Research (NMIMR), University of Ghana. This work could certainly not have been completed without the daily, patient and expert guidance of Dr. Bosompem and Dr. Arishima. Equally invaluable to the successful completion of the work was the experienced supervision of Professor Assoku. I am very much indebted to them. I am especially thankful to Professor F.K. Nkrumah, Director of NMIMR for granting me the permission to conduct research work at the Institute and indeed the entire staff of the institute for their warm reception, kindness and help. Special thanks, however, go to Dr. Mary Aiyeertey, Head of the Parasitology Unit of NMIMR, who accommodated me in the unit for all the period that my work lasted. Mrs. Irene Ayi of NMIMR helped in the running of some of the assays. I deeply appreciate her assistance and encouragement. I am indebted to Mr. George Mensah of the same Institute and my colleague Mr. Bomiface Kayang for introducing me to the use of the computer. I wish also to thank the lecturers and other staff of the Animal Science Department, University of Ghana, my friend Isaac Sackey as well as my collegues, for their encouragement, assistance, concern and companionship. University of Ghana http://ugspace.ug.edu.gh Finally, I wish to thank my employer, The Ministry of Agriculture which granted me study leave to persue this course. V University of Ghana http://ugspace.ug.edu.gh VI TABLE OF CONTENTS Page No. T ITLE P A G E ................................................................................................................................................................ i D ECLARAT ION ........................................................................................................................................................ » D E D I C A T I O N ............................................................................................................................................... iii AKNOW LEDGM ENTS ......................................................................................................................................... iv TABLE OF CONTENTS ....................................................................................................................................... vi L IST OF F IG U R E S ................................................................................................................................................... x L IST O F TABLES .................................................................................................................................................... xi A BBREV IA T ION S ................................................................................................................................................ xii S UM M ARY ............................................................................................................................................................. x iii CHAPTER 1 INTRODUCTION 1.2 Objectives o f the s tu d y ............................................................................................................................ 7 1.3 Justifica tion .................................................................................................................................................. 8 CHAPTER 2 L ITERATURE REV IEW 2.1 Schistosomes and sch is to som ias is .................................................................................................... 10 2.2 C lassification and m orphology o f sch isto som es........................................................................ 10 2.3 D istribution o f Schistosomes and th e ir in term edia te h o s ts ....................................................... 12 2.3.1 T he S. haem atob ium g ro u p ................................................................................................ 13 2.3 .2 T he 5. mansoni g ro up .......................................................................................................... 14 2.3.3 T he S. ja pon icum g roup ....................................................................................................... 15 2.4 Schistosom iasis in G hana ..................................................................................................................... 16 2.4.1 Population d istribu tion o f S. haematob ium in fection in G h an a .............................. 16 2.4 .2 Population d istribu tion o f S. mansoni in fection in G han a ................................ 19 2.4.3 D istribution o f the in term edia te sna il host o f sch istosom iasis in G h a n a 19 2.5 The life cycle o f sch istosom es............................................................................................................. 21 2.5.1 T he schistosom e egg ............................................................................................................. 22 University of Ghana http://ugspace.ug.edu.gh 2.5.2 The m irac id ium ........................................................................................................................ 23 2.5.3 T he m other and daugh ter sporocysts................................................................................ 24 2.5 .4 T he C ercariae ............................................................................................................................ 25 2.6 C ercarial penetra tion and developm ent o f schistosom es in th e defin itive h o s t................... 26 2 .7 Consequences o f schistosome infection - a general ov e rv iew .................................................. 27 2 .8 Schistsom e an tig en s ................................................................................................................................. 28 2.8.1 Schistosom ula polypeptide surface an tig en s .................................................................. 29 2.8.2 C arbohydrate surface an tig en s ............................................................................................ 31 2.8.3 Schistosome egg an tig en s ..................................................................................................... 32 2 .8 .4 Hepato toxic egg an tig en s...................................................................................................... 34 2.8.5 C ircu la ting schistosome an tig en s....................................................................................... 35 2.9 Immunity to sch isto som es..................................................................................................................... 36 2.10 D iagnosis o f sch isto som iasis ................................................................................................................ 39 2.10.1 The Parasito log ical d iagnostic m e th od s .......................................................................... 40 2.10.1.1 Feacal exam ination for eggs o f in testin a l sch isto som iasis .................... 40 2.10.1 .2 D irect faecal sm e a r .............................................................................................. 40 2.10.1.3 T he F orm a l-e ther m e th o d ................................................................................. 40 2.10.1 .4 T he Bell m e th od ................................................................................................... 41 2 .10.1.5 T he K ato m e th od .................................................................................................. 42 2 .10.1 .6 E xam ina tion for eggs o f S. ha em a tob ium ................................................... 42 2 .10.1 .7 D irec t exam ination o f u r in e ............................................................................. 42 2 .10.1 .8 U rine filtra tion m ethods..................................................................................... 43 2 .10.1 .9 P reserved u rin e sam ples..................................................................................... 43 2 .10.1 .10 D em onstra tion o f schistosome eggs in host t is s u e ................. 44 2 .10.2 Immunodiagnosis o f sc h isto som ias is ............................................................................... 44 2.11 P roduction o f m onoclonal an tibodies (M oA b s ) ........................................................................... 48 2.12 C haracteriza tion o f M onoclonal A n tib od ie s .................................................................................. 50 2.13 Usefulness o f MoAbs in immunolog ical stud ies........................................................................... 51 2.14 V accination against sch isto som iasis................................................................................................. 52 CHAPTER 3 GENERAL MATERIALS AND METHODS 3 .1 P repara tion o f a n tig e n s .......................................................................................................................... 56 3.1.1 F ixation o f m irac id ia w ith paraform aldehyde, acetone and e th a n o l ..................................................................................................................... 56 3.2 Myeloma Cell L ines and their M a in ten an ce .................................................................................. 57 3.3 Cell Fusion and Selection o f H ybridom as..................................................................................... 57 3.4 Screening, C lon ing and S tabilation o f H yb ridom as .................................................................... 59 vii University of Ghana http://ugspace.ug.edu.gh 3 .5 P ropagation and S torage o f H ybridom as............................................. ................ ......................... 60 3.6 Purifica tion o f M onoclonal an tibod ie s ............................................................................................... 62 3.6.1 Gel f i l t r a t io n ............................................................................................................................. 62 3.6.2 Ion exhcange ch rom atog raphy ............................................................................................ 63 3.7 P repara tion o f enzym e antibody con juga te s .................................................................................... 63 3.8 Sodium dodecyl su lphate-po lyacrilam ide gel electrophoresis (SD S -PA G E )...................... 65 3.8.1 A ssembly o f slab gel appara tus, and p repara tion o f reso lu tion and stack ing g e ls .......................................................................................................................65 3.8.2 P repara tion o f sam ples and electrophore tic ru n ........................................................... 68 CHAPTER 4 STUDIES OF ANTI-5. HAEM ATOB IUM M ONOCLONAL ANTIBODIES. I: PRODUCTION 4.1 In troduc tion ................................................................................................................................................. 70 4.2 M ateria ls and m e thod s ............................................................................................................................ 71 4.2.1 M ice ............................................................................................................................................. 71 4.2 .2 P repara tion o f p arasite an tig en s ........................................................................................ 71 4.2.2.1 E x trac tion o f crude schistosom e worm , egg and hookworm egg a n tig en s ............................................................................................................................ 71 P recip ita tion o f p ro teins from S. haem atob ium in fected hum an u r in e ............................... 72 4.2.3 Imm uniza tion o f M ice fo r M oAb P rodu c tio n .............................................................. 72 4.2.3.1 Imm uniza tion w ith S. haem atob ium Soluble E gg A ntigens (S h S E A )................................................................................................................................... 72 4.2.3 .2 Imm uniza tion w ith A ntigens Ex trac ted from the U rine o f S. haematob ium Infected In d iv id u a ls ........................................................................ 74 4.2 .4 Screening o f Immunized M ice fo r Antibody A ctiv ity ............................................... 75 4.2.5 Antibody-detection M icro-p late E L ISA ......................................................................... 75 4.2 .6 Dot-ELISA P ro c ed u re .......................................................................................................... 76 4.2 .8 Cell fusions, c lon ing and se lection o f hybridom as..................................................... 77 4.2 .9 D eterm ination o f Immunoglobulin C lass and S ub c la ss ........................................... 77 4.3 R e su lts .......................................................................................................................................................... 79 4.3.1 Cell fusion and selection o f hybridom as........................................................................ 82 4.3 .2 A nti-ShSEA MoAb Secreting H ybridom as ............................................................... 82 4.3.3 A n ti-U P 2-IP MoAb Secreting H y b r id om as ................................................................. 84 4.3 .4 C loning o f MoAb Secreting Hybridomas and th e Selection o f C lo n e s 84 4.4 D iscu ss io n .................................................................................................................................................. 87 4.5 S um m ary ........................................................................................................................................................ 89 CHAPTER 5 STUDIES OF ANTI-SCH ISTO SOMA HAEM ATOB IUM MONOCLONAL ANTIBODIES. II: CHARACTERISATION University of Ghana http://ugspace.ug.edu.gh IX 5.1 In troduc tion ................................................................................................................................................. 92 5.2 M ateria ls and m ethod s............................................................................................................................ 94 5.2.1 M onoclonal a n tib o d ie s ......................................................................................................... 94 5.2.2 W estern B lo ttin g ................................................................................................................. 94 5.2.3 D eterm ination o f th e B iochem ical N atu re o f A n tigen ic Ep itopes ....................... 94 5.2.3.1 D etection o f M oAbs specific for carbohydrate e p ito p e s ........................ 94 5.2 .3 .2 D etection o f M oAbs specific fo r p ro te in epitopes .................................. 95 5.2 .4 IF AT p rocedu re ........................................................................................................................ 96 5.2.5 Dot-ELISA Procedure ....................................................................................................... 97 5.3 R e su lts .......................................................................................................................................................... 98 5.3.1 Anti-5 ' haematob ium M oAbs selected for fu rth e r cha rac te riza tion ...................... 98 5.3.2 Immunoloca lization o f S. haematob ium an tigens bound by th e M oA bs 98 5.3.3 Reactivity o f th e selected M oAbs in the w estern imm unob lo t assay .................... 98 5.3.4 B iochem ical N ature o f th e Schistosom e A n tigen ic Ep itopes B ound by th e M oA bs.............................................................................................................................................. 101 5.3.5 Reactivity o f the M oAbs w ith A ntigens o f D ifferen t Schistosom e Species and H ookw orm ...................................................................................................................... 101 5.4 D isc u ss io n ................................................................................................................................................ 105 5.5 S um m a iy 5 .4 ................................................................................................................................................108 CHAPTER 6 GENERAL D ISCUSSION AND CONCLUSIONS ..................................................................... 110 REFERENCES ....................................................................................................................................................... 116 University of Ghana http://ugspace.ug.edu.gh XLIST OF FIGURES Page No. FIGURE 1 Extraction of UP0 P and UP2-IP from S. haematobium infected human urine............................................................................... 73 FIGURE 2a Serum antibody responses of mice immunized with ShSEA as determined by reactivity with ShSEA.................................. 80 FIGURE 2b Serum antibody responses of mice immunized with UP2-IP as determined with reactivity with UP2-IP...................... 81 University of Ghana http://ugspace.ug.edu.gh XI LIST OF TABLES Page No. TABLE 1 Classification of schistosomes.................................................................. 11 TABLE 2 Summary of Cell fusions and hybridoma development experiments from spleen cells of B ALB/c mice.................................... 83 TABLE 3 Selected MoAbs reactive with S. haematobium antigens as determined by micro-plate ELISA...................................... 86 TABLE 4 Immunolocalization of the S. haematobium antigens detected by the MoAbs by indirect fluorescent antibody test (IFAT)..............................................................................................99 TABLE 5 The molecular weights of the antigens detected by the S. haematobium reactive MoAbs as determined by Western Immunoblot analysis......................................................... 100 TABLE 6 The nature of antigenic epitopes detected by the MoAbs as determined by periodate oxidation and proteinase-K digestion..........................................................................102 TABLE 7 Reactivity of selected MoAbs with several antigens as determined by micro-plate ELISA and dot-ELISA..................................................................................... 103 University of Ghana http://ugspace.ug.edu.gh ABBREVIATIONS ABTS Azino-b is(3-e thy lbenzth iazo line-6-su lfon ic acid) APS Ammonium per su lphate DAB D iam inobenzid ine te trahydroch loride DE -52 D iethylam inoethyl cellulose DM SO Dymethylsulfoxide DNA Deoxyribonucleic acid EDTA E thy lened iam inete traceta te ELISA Enzym e-linked imm unosorben t assay FBS - Foetal bovine serum FITC F lourescein iso th iocynate gm G ramme(s) HAT -m edium - H ypoxanthine, am inop terin and thym id ine m edium HEPES N -2-hydrxyethy l-p iperazine-N -2-e thane sulfonic ac id h r Hour HRPO H orserad ish peroxidase HT -m edium - H ypoxanthine and thym id ine m edium IF AT Ind irect imm imoflourescenct antibody test Ig Immunoglobulin IgA Immunoglobulin A IgD - Immunoglobulin D IgE Immunoglobulin E IgG Immunoglobulin G IgM Immunoglobulin M 1 Litre 2-M E 2-m ercaptoethanol MoAb M onoclonal antibody m l M illilitre(s) m in M inutes MW M olecular weight |x m icrons Hg m icrogram s nm N anom etres NC N itrocellose NM IMR Noguchi M emmorial Institu te fo r M edical R esearch PBS Phosphate buffered sa line PEG Polyethylene glycol pH Negative logarithm base o f hydrogen ion concentration SDS Sodium dodecyl su lphate SDS-PAGE - Sodium dodecyl su lphate polyacrilam ide gel electrophoresis TBS T ris buffered saline TEM ED N ,N ,N '-,N '-te tram ethy lethy lened iam ine WHO W orld H ealth O rgan iza tion Xg - T im es gravitational force University of Ghana http://ugspace.ug.edu.gh SUMMARY Diagnosis of schistosome infections is principally based on the demonstration of parasite eggs in excreta, or by haematuria and /or proteinuria. These methods are, however limited by inadequate sensitivity or specificity. Furthermore, even though microscopy is very specific, it is also tedious and time consuming. The work reported in this thesis was conducted with the aim of producing monoclonal antibodies (MoAbs) against Schistosoma haematobium antigens, and to characterize them so as to determine if any of them would be useful in diagnosis, especially for detection of parasite antigens in the urine of infected persons. The availability of such MoAbs would pave the way for the development of more sensitive, specific and field-applicable immunological assays for diagnosis of urinary schistosomiasis caused by S. haematobium. The strategy employed in this study was to produce MoAbs, using S. haematobium soluble egg antigens (ShSEA) and infected human urine-based parasite antigens in protein extracts (UP2-IP) as immunogens. Both antigens induced substantial serum antibody responses in immunized mice, with titres as high as 1:5,000 for ShSEA and 1:50,000 for UP2-IP. The high immunogenicity of UP2-IP was attributed to human immunoglobulins associated with parasite antigens in immune complexes. All MoAbs produced, using this antigen, were found to react with UP2IP but not with ShSEA. Also, the MoAbs generated using ShSEA as immunogen did not react with UP2-IP. However, one MoAb Sh5/32.30 reacted with both ShSEA University of Ghana http://ugspace.ug.edu.gh X IV and UP2-IP. This MoAb was produced using both ShSEA and UP2-IP as immunogen, but the final booster, just before cell fusion was made with ShSEA. In all, six MoAbs were produced. These included two S. haematobium species-specific MoAbs (Sh2/15.F and Sh3/38.2), and four pan-schistosome MoAbs (Shl/71.7, Sh3/15.28, Sh4/14.3 and Sh5/32.30) that cross-reacted with antigens in S. haematobium, S. mansoni and S. japonicum egg or adult worms. Three of the MoAbs were also found to detect S. haematobium antigens in urine of infected humans. Characterization of the antigens detected, using the indirect immunofluorescent test (IFAT), Western immunoblot analysis, proteinase-K digestion and periodate oxidation, showed that the two S. haematobium species-specific MoAbs bound different antigens (one protein and one glycoprotein), and at least three different antigens. The glycoprotein species-specific antigens had molecular weights (MW) of approximately 37kDa and 46kDa. It was also found that all the glycoprotein antigenic determinants detected were located on the surface membrane, as well as on intracytoplasmic organelles in S. haematobium miracidia, whilst the protein epitopes were located only on the surface membrane. Of the six MoAbs that were produced and characterized, three of them bound protein epitopes, all of which were different from each other. One of the protein epitopes was detected by S. haematobium species-specific MoAb (Sh2/15.F), whilst the other two were detected by pan-schistosome MoAbs (Sh4/14.3 and Sh5/32.30). Interestingly, only the three protein antigens could be detected in the urine of S. University of Ghana http://ugspace.ug.edu.gh haematobium infected persons. One of these protein epitopes, bound by Sh4/14.3, was shown to occur on different peptides of MW, 73kDa and 78kDa. Cross-reactivity studies with soluble egg antigens of an Egyptian strain of S. haematobium revealed that only Sh2/15.F could not react with the north African parasite stain. This ability of most of the MoAbs to detect both Ghanaian and Egyptian strains of S. haematobium suggested that the MoAbs generated in this study might not only be useful for diagnosis of urinary schistosomiasis in Ghana, but also in other parts of Africa. X V University of Ghana http://ugspace.ug.edu.gh CHAPTER 1 INTRODUCTION University of Ghana http://ugspace.ug.edu.gh 1.1 General introduction 2 Schistosomiasis is an economically important disease of man and livestock caused by trematode parasites of the genus Schistosoma. The disease is wide-spread and endemic throughout many parts of the world infested by the intermediate fresh water snail hosts belonging to the family Planorbidae. The affected areas include; Africa, Madagascar, S. America, India, Sri Lanka, South East Asia, China, Japan, Philipines, Taiwan, Indonesia and the Caribbean (Rollinson and Southgate, 1987), 75 countries in all. It is estimated that 250 million people are infected with schistosomes whilst another 600 million people are exposed to the risk of infection. In endemic rural areas of many developing countries, schistosomiasis is an important occupational hazard (Doumenge, Mott, Cheng, Villenave, Chapuis, Perrin and Reaud-Thomas, 1987). Earliar reports indicated that, in Ghana, schistosomiasis was focal, occurring only in certain localized areas until the creation of the Volta dam in 1964 (Odei, 1964; Derban, 1983; Okoh, 1994). Today, schistosomiasis is known to occur in all regions of Ghana, especially among the riparian communities, some of which have registered prevalence rates as high as 100% for urinary schistosomiasis (Okoh, 1994). The creation of the Volta dams at Akosombo and Kpong, and the construction of dams and reservoires in various other parts of the country, have provided very siutable breeding sites for the Planorbid snail intermediate hosts, resulting in changes in the transmission patterns of the disease. The risk of transmission of bilharziasis continues to be University of Ghana http://ugspace.ug.edu.gh aggravated as fishermen attracted by the Volta lake move from place to place while fishing and thereby contribute to the spread of the disease to previously uncontaminated areas. Both Odei (1975) and Okoh (1994) emphasized that this situation has made several water bodies in Ghana public health liabilities, thus undermining their economic importance. The important schistosomes pathogenic to man are S. haematobium, S. mansoni, S. japonicum, S. mekongi, and S. intercalatum. The disease in domestic animals is caused by S. mattheei, S. curassoni, S. spindale, S. nasale, S. leiperi, S. indicum, S. bovis, S. incognitum and S. japonicum. The first four schistosome species infect cattle, sheep and goats, whilst S. leiperi and S. indicum infect horses and camels respectively, in addition. Schistosoma bovis and S. incognitum, also called S. suis, are restricted to cattle and pigs respectively. Schistosoma japonicum is the only important schistosome pathogen of man which also infect domestic animals, namely, cattle, sheep and goats. On rare occasions, man may also be parasitised by the animal schistosomes, S. bovis, S. curassoni, S. margrebowiei, S. mattheei and S. rodhainii (Rollinson and Southgate, 1987). In man, acute schistosomiasis may cause Katayama fever, anaemia, or fluid/electrolyte problems in severely toxaemic victims. A real threat of ectopic central nervous system involvement also exists during early infection (Lichtenberg, 1987). For these reasons, early anti-schistosomal treatment is important, as it helps to ameliorate 3 University of Ghana http://ugspace.ug.edu.gh the course of acute schistosomiasis. However, very few heavily infected patients have died from the acute disease. Chronic schistosomiasis caused by S. haematobium, is characterized by haematuria (bloody urine), which is caused by haemorrhages resulting from penetration of the bladder wall by schistosome eggs. It is known that a few egg patches at or near the ureterovesical junction can hinder urine flow. In general, pathological lesions may range from trivial to lethal (von Lichtenberg, 1987). On the other hand, chronic S. mansoni and S. japonica, are characterized by scattered granulomatous foci of the gut and liver, to full-blown bilharzial pipestem fibrosis of the liver. The risk of developing "bilharzial polyposis" and portal fibrosis remains benign until portal (venous) hypertension sets in. The standard method for identification of schistosome infections in man, is by microscopic demonstration of schistosome eggs in excreta or biopsy specimen. In the case of human urinary schistosomiasis, routine diagnosis is normally achieved by examination of terminal urine for the presence of blood or eggs of the parasite. Also, the determination of excess protein in urine (proteinuria) has been found to correlate well with S. haematobium infections, but it is less sensitive than some of the then known methods (Savioli and Mott, 1989). The presence of blood or schistosome eggs in human stool specimen may indicate infection due to intestinal schistosomiasis be caused by either S. mansoni, S. japonicum, S. intercalatum or S. mekongi. Differential diagnosis is made possible by a 4 University of Ghana http://ugspace.ug.edu.gh consideration of the geographical distribution of the different schistosome species, and the morphology of the parasite eggs. Diagnosis of schistosomiasis in man is, however, beset with several problems. Firstly, the identification of schistosome eggs in the urine or stool of infected persons is not sensitive enough, because of the great fluctuation of egg output and/or by the small numbers of eggs excreted. Secondly, the use of haematuria for the diagnosis of S. haematobium infections could be misleading since bloody urine could be the results of other conditions such as prostatic disease, genito-urinary tract infections and carcinoma of the genito-urinary tract (Goldsmith, 1985). Similarly, traces of blood in the stool of man, could be due to a variety of causes, including several gastro-intestinal tract infections, such as dysentery and hookworm infections (Savioli and Mott, 1989). Schistosome infections in man can also be identified by serological tests based on the detection of host antibodies directed against schistosome antigens. Some of these tests have proven sensitive and specific. However, the presence of specific antibodies does not always indicate an active infection, since antibody titres remain positive for a long time after spontaneous or chemotherapeutic cure. Furthermore, antibody levels seldom show a good correlation with worm burden. Consequently tests have been developed to detect schistosome circulating antigens in the blood of infected hosts. The most promising of these tests is a monoclonal antibody (MoAb) based enzyme-linked immunosorbent assay (ELISA) which utilises two schistosome specific antigens: the circulating anodic antigen (CAA) and the circulating cathodic 5 University of Ghana http://ugspace.ug.edu.gh antigen (CCA) (Deelder, DeJonge, Boerman, Fillie, Hilberath, Rotmans, Gerritse and Schutte, 1989; Deelder, Miller, DeJonge and Krijger, 1990). However, none of these tests have been successfully adopted for application in routine diagnosis of schistosomiasis in the field. The need for timely diagnosis and intervention of schistosomiasis in remote rural areas continues to call for the development of simple, accurate and field applicable assays. An attractive possibility in the case of human urinary schistosomiasis, would be to develop MoAb-based assays for detecting S. haematobium soluble egg antigens in patient urine. That this may be a feasible approach is evidenced by the works of Domingo and Warren (1968), Carter and Colley (1979) and Dunne and Doenhoeff (1983), in which the presence of immunogenic soluble egg antigens (SEAs) were demonstrated. Moreover, it has been shown by Inatomi (1962) that schistosome eggs possesses sub-microscopic egg-shell pores, and by De Jonge, Fillie, Hilberath, Krilger and Lengeleret (1989) that SEAs released into patient urine could be detected by MoAbs. Findings such as these led the WHO scientific working group (SWG) to recognise the advantage of supporting the incorporation of research on MoAbs into the overall strategy for schistosomiasis control (Bergquist, 1984). Based on these observations, the work described in this thesis was conducted to produce and characterize monoclonal antibodies reactive to S. haematobium soluble egg antigens, with the objective that some of the antibodies may be useful in developing more specific and sensitive assays for urinary schistosomiasis. 6 University of Ghana http://ugspace.ug.edu.gh 71.2 Objectives of the study 1.2.1 To produce monoclonal antibodies (MoAbs) against S. haematobium antigens. 1.2.2 To determine the specificity of the MoAbs through cross-reactivity studies with S. haematobium, S. mansoni, S. japonicum and Necator americanus (hookworm). 1.2.3 To investigate the ability of the MoAbs to detect urine-based S. haematobium antigens. 1.2.4 To characterize the antigens detected by the monoclonal antibodies using western immunoblot analysis and biochemical studies, and to localize them using immunocytochemical studies. University of Ghana http://ugspace.ug.edu.gh 81.3 Justification The production and characterization of MoAbs that detect S. haematobium urine-based antigens, would provide useful information on the schistosome antigens identified, and facilitate the development of alternative diagnostic assays that employ MoAbs in the detection of parasite antigens in patient urine. Furthermore, some of the identified antigens may prove useful in studies relating identification of protective molecules and development of a vaccine against schistosomiasis. University of Ghana http://ugspace.ug.edu.gh CHAPTER 2 LITERATURE REVIEW University of Ghana http://ugspace.ug.edu.gh 10 2.1 Schistosomes and schistosomiasis The first schistosome to be described was Schistosoma haematobium. Adult worms of this parasite were discovered in the veins of a man at autopsy in Cairo by the German surgeon Theodor Bilharz in 1851. The disease, bilharziasis was later named after him. Elucidation of the schistosome life cycle, however, was not made until 1913 when Miyairi and Suziki (cited by Rollinson and Southgate, 1987) showed that S. japonicum developed in the hydrobid, snail Oncomelania hupensis nosophora. 2.2 Classification and morphology of schistosomes Schistosomes are trematodes which belong to the family Schistomatidae (Webbe, 1982). Members of this family are dioecious Digenea, parasitic in the blood- vascular system of vertebrates. A general feature of the family is that the mature female is more slender than the male and is normally carried in a ventral groove called the gynaecophoric canal, which is formed by ventrally flexed lateral outgrowth of the male body (Rollinson and Southgate, 1987). Of the twelve genera within the family (Table 1), seven are confined to birds and five to mammals, but only the genus Schistosoma is associated with man. Epidemiological studies have shown that the Schistosoma has achieved the greatest geographic distribution and diversification in terms of members of recognized species and different hosts parasitized (Rollinson and Southgate, 1987). University of Ghana http://ugspace.ug.edu.gh University of Ghana http://ugspace.ug.edu.gh 12 The genus Schistosoma differs from most digenetic trematodes in being dioecious, a consequence of heteromorphic chromosomes in the ovum (Rollinson and Southgate, 1987). Thus a population of schistosomes in the final host may be unisexual (male or female) or mixed, comprising both male and female worms. In nature, however, the latter situation is the most frequent, resulting in the pairing of males and females and the production of eggs (Erasmus, 1987). The three principal schistosome species known to infect man are Schitosoma haematobium, S. mansoni and S. japonicum. Schitosoma haematobium causes urinary schistosomiasis which is also known as schistosomiasis haematobia or bilharziasis. S. mansoni, on the other hand, causes interstinal schistosomiasis also known as schistosomiasis mansoni, and likewise S. japonicum causes intestinal schistosomiasis referred to as schistosomiasis japonica or oriental schistosomiasis (Bergquist, 1987). The schistosomes are generally placed in 15 different groups which Kuntz (1955) referred to as species complexes. 2.3 Distribution of Schistosomes and their intermediate hosts Prevalence rates of schistosomiasis vary widely between different areas, and even, between localities within a single endemic area. The highest rates are found near large bodies of fresh water, where up to 100% of the population may be infected. According to Bergquist, (1987) the highest infection rates are found in Brazil, Egypt and Ghana. University of Ghana http://ugspace.ug.edu.gh 13 2.3.1 The S. haematobium group The distribution of S. haematobium is confined to Africa and some adjacent regions, extending through Arabia to the Khuzestan Province of Iran and to the Indian Ocean Islands of Madagascar and Mauritius (Rollinson and Southgate, 1987). There is evidence for geographical variation in many characters associated with this species. Thus for example, laboratory studies with hamsters have revealed differences between strains of the parasites that differ in many biological features including, intermediate host specificity, infectivity of cercariae, adult worm growth rates and maturation times, egg productivity and the distribution of eggs in infected host organs (Wright and Knowles, 1972). In general, S. haematobium in the tropical regions of Africa is transmitted by snails of the Bulimis africanus group. In the Mediterranean area and the South West of Asia, transmission is mainly by the tetraploid members of the B. tmncatus/tropicus complex and in Arabia and Mauritius by members of the forskalii group of snails. In West Africa, all three of these snail groups are known to act as intermediate hosts for S. haematobium, and in Arabia the B. truncatus group is also implicated. Of particular significance is the observation that S. haematobium from North Africa and the Middle East develops in B. truncatus and the parasite from tropical Africa develops in snails of the B. africanus group, but with few exceptions, neither of these forms can develop in the intermediate host of the other (Frandsen, 1979). University of Ghana http://ugspace.ug.edu.gh 14 Related to S. haematobium are other African schistosomes which differ in the shape of their eggs and in their definitive hosts. These include the widely distributed S. bovis of cattle (Majid, Bushara, Saad, Hussien, Taylor, Dargie and Marshall, 1980); S. matheei of cattle, sheep and goats in South Africa (Rollinson and Southgate, 1987); S. intercalatum of man in Zaire; S. curassoni of ruminants and S. margrebowiei which infect a wide range of hosts (Rollinson and Southgate, 1987). 2.3.2 The S. mansoni group Schistosoma mansoni is the most important parasite that causes intestinal schistosomiasis in man in both the New and Old Worlds. The parasite occurs in Oman, Saudi Arabia, Yemen, Poeple's Democratic Republic of Yemen, Libya and Madagascar, and it is distributed discontinuously over the greater part of Africa, South of the Sahara. In the Caribbean, it is endemic in Puerto Rico, St. Lucia, Guadeloupe, Martinique, Dominican Republic, Antigua and Monstreat. In South America, intestinal schistosomiasis due to S. mansoni is found in Brazil, Surinam, and Venezuela (Rollinson and Southgate, 1987). Of seventeen well-defined species of Biomphalaria in the Americas, only B. glabrata and B. straminae have been found naturally infected with S. mansoni. Other species such as B. amazonica (Correa and Paraense, 1971) and B. peregrina (Paraense and Correa, 1973) have been found to be susceptible to infection in the laboratory. Snails from different geographical areas tend to show variation in the levels of susceptibility to different strains of S. mansoni (Basch, 1976; Mchelson and Dubois, University of Ghana http://ugspace.ug.edu.gh 15 1978) and populations of snails from a given area may vary in their susceptibility to parasites isolated from anyone of the intermediate snail hosts. In Africa, twelve species of Biomphalaria are recognized (Brown, 1980) and all those that have been tested appear to show some compatibility with at least certain strains of S. mcmsoni. B. pfeifferi shows a broad compatibility and is, therefore, regarded as an important intermediate host, whereas species such as B. alexandria appears to be susceptible only to the local S. mcmsoni from Egypt (Frandsen, 1978). Other schistosome species found mainly in South East Asia are also placed in the same group as S. mcmsoni. They include S. rhodaini which infects rodents and dogs (Pitchford, 1977), S. edwardiense and S. hippopotami of hippopotamus (Pitchford and Viser, 1981), S. indicum of equines and a variety of other domestic animals on the Indian subcontinent (Montgomery, 1960), S. spindale of ruminants (Montgomery, 1960), S nasale of cattle , sheep, goats and buffaloes in India and surrounding regions (Dutt and Srivastava, 1968) and S. incognitum of pigs and humans in South East Asia (Ahluwala and Dutt, 1972). 2.3.3 The S. japonicum group The disease caused by S. japonicum is widespread in the Far East where it is endemic in parts of China. S. japonicum also occurs on the Philippine Islands of Leyete, Samar, Mindanao, Bohol, Mindoro and Luzon. In Japan, the parasite is limited to three main areas, namely Kufu, Katayama and the basin of University of Ghana http://ugspace.ug.edu.gh 16 the Chikugo River. It is also found in parts of Taiwan and Indonesia (Rollinson and Southgate, 1987). 2.4 Schistosomiasis in Ghana Urinary schistosomiasis was first reported in Ghana in the year 1895, whereas intestinal schistosomiasis was not identified until 1920 (Fura, 1987). However, until 1952, data on the distribution of schistosomiasis in Ghana was derived mainly from hospital records (Furu, 1987). Studies conducted after this period now form the basis for current available information. McCullough (1965), Papema (1969) and Wen and Chu (1984) studied and reported on the distribution of the vector snails, as well as urinary and intestinal schistosomiasis. Papema (1969) and Odei(1984) also considered the ecology of schistosomiasis transmission and the distribution of both aquatic weeds and the vector snails in the new man-made Volta Lake in Ghana. According to Furu (1987), both urinary and intestinal schistosomiasis were widely distributed in Ghana. Schistosomiasis haematobia was present in all the regions of the country whilst schistosomiasis mansoni was concentrated in the northern and southern quarters. 2.4.1 Population distribution o f S. haematobium infection in Ghana It was estimated in 1963 (Furu, 1987) that 15 to 20% of the inhabitants of Ghana were infected by S. haematobium at some time or other University of Ghana http://ugspace.ug.edu.gh 17 during their lives, often during childhood. The two most endemic areas were in the North-eastern and in the South-eastern parts of the country. High prevalence rates were reported in localities along the frontier with Cote d'Ivoire and the Central part of Ghana. The number of endemic areas identified was observed to increase steadily after 1965 (Furu, 1987). In most regions, prevalence rates in excess of 60% were recorded in the years 1970-1980. Lake Volta, with its 5000 km of shoreline, was completely filled in 1968 and surveyed for the first time in the 1970s following the observation that S. haematobium infection was spreading (Furu, 1987). The surveys revealed high urinary schistosomiasis prevalence among peoples living near the shores of the new lake even though earlier studies earned out by Medical Field Unit had indicated that schistosomiasis was absent in many populations living traditionally on or close to the Volta river. Similarly, in areas where transmission had been present for some time, the number of foci was found to have increased (Tagoe, 1965; Bergquist, 1987). An extensive epidemiological survey carried out between 1959 and 1961 in the Upper Regions of Ghana showed that the eastern part was markedly more affected than the western part. The prevalence of S. haematobium infection was over 30% in 19 out of 20 districts in the eastern part, and less than 30% in 16 out of the 18 districts in the western part (Hunter, 1981). The zone most affected by urinary schistosomiasis had prevalence above 50%. This area was bounded in the east by the White Volta and in the west by the Sisili river. Papema (1969) reported that the Northern region of Ghana had low schistosomiasis prevalence rates mostly below 5% before the filling of the Volta University of Ghana http://ugspace.ug.edu.gh 18 Lake. However, after the formation of the lake, very high prevalence rates greater than 50% have been recorded in most places in the north. A complete assessment of the prevalence among the shore dwelling populations of Lake Volta carried out in 1982 also indicated that the eastern part of central Ghana had become an important endemic area (Klumpp, 1982). The prevalence varied between 38% and 96.7% among the peoples settled in these areas. Scott, Senker and England (1982) recorded an average prevalence rate already in excess of 83.9% among the shore-dwelling peoples of the lower reaches of the Afram branch, and among those living along the Pawmpawm branch of the lake. In the area below the Akosombo dam and throughout the south east of Ghana, S. haematobium infection has become endemic (Bergquist, 1987 and Furu, 1987). In 1969, transmission of S. haematobium was significant around the marshes and ponds connected with the Volta river. Prevalence in excess of 75% was recorded in all localities surveyed (Papema, 1968). The prevalence was inversely proportional to the distance from transmission sites. This observation was confirmed in the Volta river delta, as well as, in the Eastern Region, where prevalence of S. haematobium was low (Papema, 1969). Papema (1969) also reported that, the prevalence in the Tafo forest sector was higher in the primary schools of New Tafo, located near the main site of transmission than in those of Old Tafo, which were farther away. Teenagers in secondary schools in Old Tafo, however, had a higher prevalence than their counterparts in primary schools because of their more extensive water contact. University of Ghana http://ugspace.ug.edu.gh 19 Around the city of Accra, epidemiological surveys conducted in the years 1966-1967 revealed extremely variable prevalence rates which were low at Madina or Ofankor (13%), but high at Pokuase and Mayera (70%), and Ashiaman (84%). In the Accra region, the prevalence was 30% in 1966 and 84% in 1967. 2.4.2 Population distribution o f S. mansoni infection in Ghana Intestinal schistosomiasis due to S. mcmsoni is far less prevalent in Ghana than urinary schistosomiasis. In 1955, only the north-east of the country was known to be an endemic zone and the overall prevalence was only 2.4%. Ten years later McCullough and Ali (1965) reported five foci in the Upper Region, four of which were to the east of the Sisili river and one in the vicinity of the Black Volta. They also reported two foci in the south-west (Western Region), two others in the south-east (Volta Region) and one in the Ashanti Region. Most of the prevalence rates recorded were low, between 5% and 7%. Until 1965, S. mcmsoni had not been reported in the vicinity of the new Lake Volta. However, Wen and Chu (1984) identified four new foci in the area between the Akosombo dam and the mouth of the river Volta. 2.4.3 Distribution o f the intermediate snail host o f schistosomiasis in Ghana The natural conditions for the development of the snail intermediate hosts of schistosomes are ideal over large parts of Ghana. Bulinus globosus, one of the principal snail hosts of urinary schistosomiasis, was present in three-quarters of University of Ghana http://ugspace.ug.edu.gh 20 the country before the filling of the Volta lake. This included both forest and savanna areas. Furu, (1987) observed that this snail was particularly common in the ponds and streams of the Volta plateau in the north-east, in the water collected on the Kwahu plateau in the south-west and in the Togo ranges as well as in the coastal plains from the Togolese frontier to Axim. On the other hand, B. globosus was practically absent from the region now occupied by Lake Volta. According to McCullough (1965), the porous nature of the mineral substrata accentuated the effects of the lack of water during the dry season and further limited the propagation of the snail. Since the creation of the lake, the snail has established itself only in the lower valley of Obosum in the Volta Region (McCullough, 1965). The other intermediate snail host of S. haematobium found in Ghana B. tnincatus rohlfsi was far more restricted in its distribution before the filling of the lake. This snail species was totally absent from the forest zone. It's presence was recorded in the Upper Region in the Wa area and between Bolgatanga and Bawku, in the valleys of the Black and White Volta; in the Northern region, from Tamale to Kete Krachi; in the Volta region, in the Dayi river valley, and lastly and most importantly, in the lower valley of the delta of the Volta river, from Akosombo to Agave, especially behind the Keta lagoon (Furu, 1987). Biomphalaria pfeifferi, the intermediate snail host of S. mansoni, had a distribution similar to that of B. globosus. The snail proliferated both in the savanna of the Upper region and the forest zone of the south-west. Furthermore, B. pfeifferi was identified in the Togolese Ridge. This snail was, however, not found in the Volta Lake (Furu, 1987). University of Ghana http://ugspace.ug.edu.gh 21 Generally, the snail intermediate hosts of schistosomiasis referred to above are found in small water courses, flood-water ponds and the fresh water mashes of the Volta delta and the coastal plain. These snails are characteristically absent from the salt water mashes and fast-flowing rivers. B. globosus is found mostly in permanent marshes and slow-flowing water courses particularly in the forest zone and the coastal plain. B. tnmcatus on the other hand is found mostly in the area between the confluence of the Black and White Volta and the Volta delta. The rapid growth of water plants, especially Pistia stratoites, Spirodela polyrhiza and Ceratophyllum demersum, in the Volta Lake has promoted the proliferation of B. tnmcatus rohlfsi (Papema, 1969) and hence the spread of S. haematobium. Ceratophyllum is plentiful in the Afram and Dayi branches of the Volta Lake and along the Oti and White Volta rivers. It is found up to a depth of 4.5m normally in association with tree trunks left in situ in the inundation of the original Volta valley (Odei, 1975). Pistia and Spirodela, on the other hand, often form floating islands that drift around the lake. In so doing, these plants contribute to the spread of schistosomiasis through dispersion of the snails and their eggs. 2.5 The life cycle of schistosomes Schistosomes undergo an alternation of generations with sexual reproduction taking place in the definitive host (man and other mammals) and asexual reproduction in the intermediate snail host. Schistosome eggs pass out of University of Ghana http://ugspace.ug.edu.gh 22 the body of the definitive host in the excreta (urine or faeces), and, on encountering fresh water, they hatch into free-living, ciliated embryos known as miracidia. The free- swimming miracidia seek and find a compatible snail species which they penetrate few hours after hatching from the egg. Within the tissues of the snail, these organisms develop over a month into mother sporocysts, which then develop into thousands of fork-tailed free-swimming larvae called cercariae. The cercariae are released in daily bursts over a fairly long period that may last several months if the snail survives the infection. It is the cercariae which is capable of infecting man. 2.5.1 The schistosome egg Schistosome eggs are generally oval in shape and non-operculate. They are characterized by a pointed expansion of the egg shell to form either a lateral or a terminal spine. This characteristic is one of the most important features considered in the identification of the different schistosome species using the morphology of the eggs. Kussel (1970) provided a detailed morphological description of S. mansoni eggs and showed that the miracidium within the egg is suspended by four vacuoles which together with the miracidium are surrounded by vitellin membrane. Earlier on Inatomi (1962) had shown that schistosome eggs possess sub-microscopic egg-shell pores through which various egg antigens including enzymes could be released. Indeed, it has been reported that numerous interacting factors, namely, the spine of the egg, blood pressure, peristalsis and proteolytic enzymes secreted by the miracidia inside the University of Ghana http://ugspace.ug.edu.gh 23 egg may be involved in the passage of eggs through the walls of vessels and parenchyma of the intestines or bladder (Smith, 1974; Bloch, 1980). The number of eggs excreted daily by schistosomes is estimated as the number of eggs per worm pair. This estimate differs greatly between different species of the parasite. For example Loker (1983) estimated 560-2200 for S. japonicum, 66-495 for .S', mansoni and 22-203 for S. haematobium. Once excreted, the eggs hatch to release the miracidia under suitable environmental conditions which normally increase the chances of contacting suitable snail hosts. Studies by Erasmus (1972), Morgan (1972), Bair and Etges (1973) have shown that the major factors involved are temperature, light and osmotic pressure. 2.5.2 The miracidium The miracidium represents the first free larval stage in the life-cycle of schistosomes. This larval stage ensures transmission between the vertebrate and the snail which it actively penetrates. Electron microscopy studies by Pan (1965), Schutte (1974a) and Eklu-Natey, Wuest, Swiderski, Striebel and Huggel (1985) have thrown more light on the morphology of the miracidium. A newly released miracidium is generally pyriform, and varies between 150 and 180 (J.m in length and 70 and 80 |im in width. The body of this larval stage is covered by 21 or 22 ciliated, enucleated cells responsible for its swift movement in water. The anterior extremity has a hemispherical structure called tetraborium, which is formed of anastomosed membrane folds with at University of Ghana http://ugspace.ug.edu.gh 24 least 12 ciliated sensory organelles. The secretary ducts of the apical and lateral glands emerge at this point. The miracidium has an excretory system which comprises an anterior and posterior pair of flame cells and a common duct which opens laterally in the posterior third of the body. It is likely that a large amount of soluble egg antigens found in the urine of infected persons are secreted by the miracidium via this route. The mechanism by which miracidia seek to penetrate susceptible snail hosts have been investigated by several workers. For example, Jourdane and Theron (1987) reported that physical stimuli in the aquatic medium appear to favour movement of miracidia to the part of the biotope where the host snails are most likely to be found. The parasite larvae then home in by responding to chemical stimuli released from the snail host (Chemin, 1970; Sponholtz and Short, 1975). Though it is generally accepted that penetration of miracidia into snail tissues occurs by a mechanical action, the role of histolytic secretions of the glandular cells of the miracidia could be implicated (Jourdane and Theron, 1987). In S. mansoni, S. haematobium and S. intercalatum, penetration occurs mostly via the foot of the snail (Jourdane and Xia, 1987). 2.5.3 The mother and daughter sporocysts Following penetration of the snail tissues, the miracidium, undergoes morphological transformations which culminate in the formation of mother sporocysts. Schutte (1974b) studied the morphogenesis of the mother sporocysts University of Ghana http://ugspace.ug.edu.gh 25 of S. mansoni and reported that some of the early changes that occur with the miracidium upon penetration are the loss of epidermal ciliated cells, loss of the musculature and loss of the papillae. By the fourth day, the sporocyst is amoeba-like in shape. It gradually takes on the shape of a long sac which becomes folded and rolled back on itself and which is closely bound to snail tissues. The germinal cells of the miracidium then undergo a series of multiplication and differentiation to form daughter sporocysts within the mother sporocysts. By the end of their differentiation the mother sporocysts look like vermiform larvae, measuring 150-250 |_im in length (Jourdane and Theron, 1987). Daughter sporocysts, leave the mother sporocyst by breaking through the tegumentary wall. Ten to seventeen days afterwards, the daughter sporocysts migrate towards the digestive gland of the snail host. This migration is either passive through the host's circulatory system (Becker, 1970; Meuleman, 1972; Schutte, 1974b) and/or active through the host's loose connective tissues (Pan, 1965). 2.5.4 The Cercariae Germinal cells contained in the cavity of daughter sporocysts only start to differentiate into cercariae after the sporocysts have reached their permanent location in the digestive gland of the snail (Cheng and Bier, 1972). With the exception of S. japonicum for which cercarial production can stop for a period and restart, the production of cercaria in other species of schistosomes occur daily and sometimes for periods exceeding eight months (Jourdane and Theron, 1987). University of Ghana http://ugspace.ug.edu.gh 26 2.6 Cercarial penetration and development of schistosomes in the definitive host Schistosome cercariae may remain infective for approximately 20 hours after emergence from the snail and can penetrate the unbroken skin of man and other mammals within minutes. During the penetration process, the cercaria loses its tail and external layer and changes from a fresh water organism into one which can survive only in salt water and is then called schistosomulum. The schistosomulum moves through the tissues into the lymph and blood vessels and ultimately reaches the lungs where they remain for several days. They then migrate to the liver via the bloodstream or directly through the tissues. In the liver, they grow into adult male and female schistosomes, depending on the sex of the cercariae. Male and female worms pair up and pass down the mesenteric or vesical venules depending on the schistosome species. Each worm pair produces 300-3000 eggs per day and may do so for many years. Approximately half of these eggs are excreted from the body, and the remainder remain trapped in the tissues (Warren, 1973). 2.7 Consequences of schistosome infection - a general overview After maturing in a permissive host, schistosome worm pairs take up residence in characteristic venous habitats depending on the parasite species, and engage in permanent copula and egg-laying for many years. Their life span in University of Ghana http://ugspace.ug.edu.gh 27 humans has been estimated to average 3.5-12 years with some worms surviving for 30 years or longer (Vermund, Bradley and Ruiz-Tiben, 1983). Consequently, schistosomiasis is a disease of long chronicity which normally begins with cercariae penetration upon contact with contaminated water during childhood and progresses into adult life. Nevertheless, it has been observed that schistosomiasis prevalence in humans peaks around age 15-17 and then decreases with increasing age (Mott, 1987). The schistosome parasites (adult worms) have an ability to evade the host's immune system, which probably, explains the chronic nature of the disease (Colley and Colley, 1989). The mechanisms involved in this immune evasion include rapid turnover of membrane components, enzymatic cleavage of attached antibodies, tegumental structural developments, the coating (or masquerade) of the parasite surface with host antigenic components [such as ABO blood group antigens, serum components and major histocompatibility (MHC) antigens], antigenic mimicry, direct immunosuppressive effects and the induction of humoral and cellular immunoregulatory mechanisms, including anti-idiotypic networks (Bloom, 1979; Colley and Colley, 1989). Even though a newly infected host is exposed to a wide variety of parasite antigens, only a few infected persons ever develop the acute febrile illness which begins one or two months after first cercarial exposure. This acute disease is referred to as "toxaemic schistosomiasis" in (Brazil) or as "katayama fever" in Japan. Most infected children have only minor early symptoms or none at all, and, they may continue in apparent good health during the subsequent chronic phase of the disease even though University of Ghana http://ugspace.ug.edu.gh 28 progressive pathological changes occur internally. Eventually, in five or more years, individuals with heavy parasite burdens begin to suffer advanced fibrovascular lesions to target organs such as the urinary bladder and genito-urinary tract in the case of S. haematobium infections, or the liver and gastrointestinal tract in the case of S. mansoni or S. japonicum infections. The prevalence of severe symptomatic schistosomiasis varies from one endemic setting to another. In heavily infected populations, it may reach 5% or more, with the bulk of infected persons continuing indefinitely in the subclinical state (von Lichtenberg, 1987). It is well established that the lesions that result from schistosome infections are largely caused by the parasite eggs, rather than the worms. Erasmus (1987) observed that much of the pathology in schistosomiasis is the result of host immunological responses to the accumulation of parasite eggs in host tissues. Indeed with S. haematobium most of the eggs laid by the female worm in the host fail to reach the lumen of the gut or urinary tract. Instead, they get trapped in the wall of the viscus or swept on into the portal radicles or lung arterioles (Cheever, 1969). 2.8 Schistsome antigens During a normal infection, the schistosome presents a complex array of antigens to its host, with major antigenic stimuli believed to come from the adult worm and the eggs (Kelly, 1987). Studies conducted by several workers including Kusel, Sher, Perez, Clegg and Smithers (1975), Haguya, Murrel, Taylor and Taylor (1979), University of Ghana http://ugspace.ug.edu.gh 29 Dissous, Dissous and Capron (1981), Arostein and Strand (1983) and Norden and Strand (1984) on antigens recognized by sera from patients infected with S. mcmsoni, S. haematobium, or S. japonicum revealed about 20-30 antigenic polypeptides of adult worms and about 20 glycoprotein egg antigens of each of the three schistosomes species. In the case of the glycoproteins, extensive cross-reactivity was observed. Thus, for example, sera from patients infected with S. haematobium precipitated all but three of the antigens recognized by S. mcmsoni infected sera. A slightly lower degree of cross-reaction was observed using sera from patients infected with S. japonicum. 2.8.1 Schistosomulapolypeptide surface antigens The existence of a set of polypeptide antigens on the surfaces of schistosomula has been demonstrated by radioiodination of intact live schistosomula, followed by immunoprecipitation with a variety of antisera (Dissous et al., 1981; Smithers, Simpson, Yi, Omer-Ali, Kelly and McLaren, 1987). However, Dissous, Gryzch and Capron (1982) confirmed earlier works by some including Dissous et al. (1981) and Smithers et al. (1987) that only a subset of the polypeptides available for surface labelling was recognized as antigenic by infected animals. In various studies, Ramasay (1979), Snary, Smith and Clegg (1980) and Smithers et al. (1987) identified polypeptides of Mr less than 100,000 by lactoperoxidase-catalased iodination labelling. Simpson and Smithers (1985) also studied schistosomula surface antigens. They used immunoprecipitation techniques utilizing either lactoperoxidase- or iodogen-catalyzed radioiodination and showed that University of Ghana http://ugspace.ug.edu.gh 30 sera from chronically infected mice recognized a slightly different profile of antigens compared with sera from mice immunized by exposure to radiation-attenuated cercariae. Collectively, however, both sera were found to precipitate antigens of M, > 200.000, 92,000, 38,000-32,000, 20,000, 17,000 and 15,000. Additionally, both sets of sera precipitated an antigen of Mr 22,000 following lactoperoxidase but not iodogen-catalyzed iodination. Using another labelling technique, the diazonium salt of 125I iodosulphanilic acid, Taylor, Haguya, and Vannier (1981) identified three major schistosomula surface antigens of Mr 15,000, 28,000 and 69,000. Several schistosomulum surface antigens have also been identified using monoclonal antibodies which bind to the surface of live schistosomula as determined by immunofluorescence. Using such a monoclonal antibody, Ham, Mitsuyama, Haguenel and David (1985) precipitated an antigen of M 22,000 from solubilized schistosomula surface membrane-enriched fractions labelled with the Bolton-Hunter reagent. Dissous et al. (1982) also produced a monoclonal antibody which precipitated an antigen of approximate M 38,000. This antigen was shown to be recognized during chronic infections of both rodents and man. Using iodogen-catalyzed labelling followed by immunoprecipitation with human immune sera, Simpson and Smithers (1985) identified the major surface antigens of S. haematobium schistosomulum to be of Mr 17,000 and a complex of M 24,000-30,000. These appeared species-specific since they were not recognized by human anti-5, mansoni serum. Sera from vaccinated mice or rabbits have also been successfully used to precipitate labelled S. haematobium antigens of Mr 94,000, 38.000, 26,000 and 10,500. University of Ghana http://ugspace.ug.edu.gh 31 2.8.2 Carbohydrate surface antigens In studies with schistosome antigens the emphasis has been laid on protein or glycoprotein antigens. This is because of the possibility of synthesizing them in large quantities in microorganisms using recombinant DNA technology to provide the basis of an anti-schistosome vaccine. Nevertheless the importance of existing glycolipid and polysaccharide antigens has not been ruled out (Simpson, James, and Sher, 1981). A number of studies have shown that carbohydrate components are significant contributors to the antigenicity of the schistosomula surface. Omer-Ali, Magee, Kelly and Simpson (1986) showed that under conditions of antibody excess, sera from chronically infected mice bound S. mansoni schistosomulum surface antigens at a 2-3 fold higher level than sera from vaccinated mice. Treatment of the schistosomula with reagents such as trifluoromethanesulfonic (TFMS) and sodium periodate which selectively remove or modify carbohydrates - indicated that most of the antibodies in sera from chronic infections reacted with specific carbohydrate epitopes not recognized by sera from vaccinated animals. These epitopes were shown to be in antigens of Mr > 200,000, 38,000, 32,000 and 17,000. It is, however likely that some of these epitopes could also be present on other membrane components such as glycolipids (Kelly, 1987). Indeed Weiss, Magnami and Strand (1986) have demonstrated the presence of antigenic glycolipids in eggs, cercariae and adult worms of S. mansoni. In studies with non-species-specific monoclonal antibodies that bound carbohydrate antigenic epitopes in glycoprotein antigens, Yi, Omer-Ali, Kelly, University of Ghana http://ugspace.ug.edu.gh 32 Simpson and Smithers (1986) suggested that carbohydrate epitopes may be responsible for inducing the non-species-specific concomitant immunity whereas polypeptide epitopes are more likely to be involved in species-specific immunity. However, the immunogenic stimuli for concomitant immunity could also come from either eggs or adult worms, since both share carbohydrate epitopes with the schistosomulum (Hamburger, Lustigman, Arap Siongok, Ouma and Mahmoud, 1982; Omer-Ali et al., 1986; Yi et al., 1986). Nevertheless, several workers have demonstrated the presence of IgM blocking antibodies in the sera of chronically infected rats and humans that are directed against carbohydrate epitopes (Gryzch, Capron, Lambert, Dissous, Torres and Dissous, 1985; Yi etal., 1986; Capron, Pearce, Balloul Gryzch, Dissous, Sondermeyer and Lecocq, 1987). The role of these blocking antibodies may explain in part the susceptibility to reinfection by S. mansoni in humans whilst the loss of the antibodies may ultimately lead to the acquisition of immunity (Capron et al., 1987). 2.8.3 Schistosome egg antigens The main pathologic feature of hepatosplenic schistosomiasis involves the formation of granulomatous inflammation around eggs entrapped in the tissues and portal vasculature. The immune response also leads to fibrosis which affects the organ's architecture and circulation (Warren, 1972 and Kelly, 1987). In S. mansoni infections, the granulomatous reaction around the eggs is essentially a cell-mediated immune response to antigens normally secreted by mature viable University of Ghana http://ugspace.ug.edu.gh 33 eggs (Boros and Warren, 1970; Hang, Warren, and Boros, 1974). Warren (1972) showed that the soluble egg antigens (SEA) which is the supernatant of ultracentrifuged egg homogenate can elicit granulomatous hypersensitivity and other immunologic reactions characteristic of intact eggs. This crude SEA preparation was shown by Carter and Colley (1978, 1979) and Pelley, Hamburger, Peters, and Warren (1976) to contain multiple glycoproteins and non-glycoconjugated proteins. Also, studies with SEA glycoproteins (purified from SEA by affinity chromatography on immobilized Concanavalin A) suggested their possible importance in the induction of granulomatous hypersensitivity as well as the elicitation of other delayed type responses (Pelley et al., 1976; Boros et al., 1977; Carter and Colley, 1979). Furthermore Cater and Colley (1979) showed that only the glycoprotein fraction of SEA was capable of eliciting T-cell responses. Investigations by Pelley et al. (1976) showed that sera from mice with light chronic S. mansoni infections identified three major serological antigens which formed the bulk of the glycoprotein fraction obtained with ion exchange chromatography on Concanavalin A. These antigens were designated MSAi, MSA2 and MSA3 and their molecular weights estimated by gel filtration to be 50, 450 and 80 kDa respectively. Hamburger et al. (1976) investigated the specificity of these antigens using antigen-binding radioimmunoassay and showed that antibodies that reacted with MSA2 and MSA3 showed cross-reaction with cercarial, but not adult worm antigens of S. mansoni. However they both cross-reacted with SEA from S. University of Ghana http://ugspace.ug.edu.gh 34 japonicum and S. haematobium. In contrast, MSAi appeared to be both egg stage- and species-specific. Further investigations with MSAi suggested that it may be a major immunopathological egg antigen (Kelly, 1987). Three glycoprotein antigens of S. japonicum were also identified by Tracy and Mahmoud (1982) who demonstrated the sensitizing activity of Concanavalin A binding fraction of S. japonicum SEA. The molecular weights of the antigens were determined by gel filtration to be 590, 245 and 46 kDa. The 46 kDa antigen was less sensitizing compared with the others. Boros and Warren (1970) and Warren, Boros, Hang, and Mahmoud (1975) studied S. japonicum and S. mansoni SEA and reported that SEA from S. japonicum elicited an immediate (antibody-mediated) inflammatory response when injected into the footpads of S. japonicum infected mice, whereas, S. mansoni SEA elicited a delayed type (cell-mediated) response. An enhanced granulomatous reaction was only observed when mice were pre-sensitized with eggs or SEA of S. japonicum injected subcutaneously as against intraperitoneal injection in the case of S. mansoni. Owashi and Ishi (1982) also purified a glycoprotein antigen of approximate Mr 900,000 from SEA of S. japonicum. The antigen showed eosinophilic chemotactic activity which appeared to be dependent on the integrity of the carbohydrate moiety. 2.8.4 Hepatotoxic egg antigens It has been observed that T-cell deprived mice infected with S. mansoni do not develop granulomatous reactions around eggs deposited in tissues within University of Ghana http://ugspace.ug.edu.gh 35 seven days of infection, however, they suffer an acute hepatotoxicity reaction (Byram, Doenhoeff, Musallam, Brink and von Lichtenberg, 1979). Injection of such mice with serum from chronically infected mice prevents the liver damage (Doenhoeff, Musallam, Bain and Mcgregor, 1979). Using chronic sera, Dunne, Lucas, Bickle, Peresan, Madgwick, Bain and Doenhoeff (1981) identified 12 of the S. mansoni egg antigens by immunoelectrophoresis. Also, work with a series of sera with partially overlapping specificities, showed that recognition of one particular antigen was required for sera to protect against liver damage. Subsequently, Dunne and Doenhoff (1983) purified and characterized this antigen coded wt a non-glycosylated polypeptide of approximate Mr 22-26 kDa. This antigen appeared to be stage-specific and has proved a valuable immunodiagnostic reagent (Kelly, 1987). 2.8.5 Circulating schistosome antigens Several studies have been conducted to detect circulating antigens in schistosome infected mammals. These studies are found necessary because existing assays utilizing crude or partially purified schistosome antigens to detect anti­ schistosome antibodies do not give information suitable for the estimation of worm burden (Nantulya, Musoke, Rurangirwa and Moloo, 1984), or for identification of active infection. However, similar to the egg detection method, detection of circulatory antigens in body fluids of the host would provide a basis for the identification of active infection and give a better correlation to infection intensity (Deelder, Komelis, Marck, Van Eveliegh and Egmond, 1980). University of Ghana http://ugspace.ug.edu.gh 36 Berggren and Weller (1967) described a circulating schistosome antigen in the serum of mice and hamsters heavily infected with S. mansoni. The antigen was later characterized by Gold, Rosen and Weller (1969) who also demonstrated its presence in the urine of infected hamsters. They found that the antigen is anodic (on the basis of its mobility in immunoelectrophoresis) and it is heat-stable and dialyzable. Nash (1974) reported that, the circulating antigen was a large molecular weight substance, most likely a polysaccharide. It was demonstrated in S. mansoni, S. haematobium and S. japonicum homogenates and in the serum of mice and hamsters heavily infected with S. mansoni and S. japonicum (Bawden and Weller, 1974). Nash (1974) and von Lichtenberg, Bawden and Shealy (1974) also showed that the same antigen was present in the epithelial cells of the schistosome gut. Deelder, Klappe, Van den Aardweg and Van Meerbeke (1976) confirmed the presence of circulating anodic antigens (CAA) in S. mansoni infected hamsters and also demonstrated the occurrence of a lower molecular weight circulatory cathodic antigen (CCA). Both the CAA and CCA were demonstrated in adult worm extracts as well as in the excretory and secretory products of the worms by Deelder et al. (1976) who also showed the presence of CAA in the urine of infected hamsters and humans. 2.9 Immunity to schistosomes Elucidation of the mechanisms involved in the immune response in schistosomiasis has been greatly facilitated by investigations with the animal model in the laboratory. This approach has been widely used because unlike University of Ghana http://ugspace.ug.edu.gh 37 other human parasites such as those that cause malaria and filariasis which have stricter host specificity, the principal human schistosomes are capable of infecting a range of laboratory animals (McLaren and Smithers, 1987). Some of the animals that have been extensively used are the rat, guinea pig, mouse and some primates. It is, however, necessary to mention that difficulties in maintaining the life cycle of S. haematobium under laboratory conditions has slowed down work with this parasite. Despite the obvious differences with pathology and immunity to schistosomes in man, the animal experiments have played a pivotal role in the accumulation of data contributing to an understanding of human schistosomiasis. For example, it is now established that immunocompetent mammals may become immune to reinfection following either chronic infection or exposure to radiation-attenuated cercariae (Kelly, 1987). Furthermore, several antigens that play important roles in the acquired immunity have been identified. Immunity induced following experimental infection and that obtained by the use of attenuated cercariae have been shown to differ in several ways. For example, resistance following exposure to normal cercariae develops about the time egg-laying commences and peak immunity is reached some 4-6 weeks later (Smithers and Terry, 1967), but with attenuated cercariae, immunity commences about two weeks following the death of larval schistosomula along their migratory pathway. It reaches a peak in about 5 weeks and remains indefinitely high. In the S. mansoni!mouse model, resistance has been shown to correlated with the presence of eggs in the tissues or with factors related to egg-associated pathology University of Ghana http://ugspace.ug.edu.gh 38 such as the degree of portal hypertension (Dean, Bukwoski,and Cheever, 1981). Nevertheless, intrahepatic transfer of adult worms into naive rhesus monkeys, baboons and mice has shown that immunity may be induced by the adult worm stage without a necessary prior exposure to cercariae or schistosomula (Smithers and Terry, 1967; Webbe, James, Nelson, Smithers and Terry, 1976; Peresan and Cioli, 1980). This immunity has been referred to as "concomitant immunity" because in this situation, schistosomula of challenge infection are destroyed whilst the adult worms of the primary infection remain unharmed (Smithers, Simpson, Yi, Omer-Ali and Kelly, 1987). In contrast, immunity following exposure to irradiated cercariae is called "vaccine immunity" It is clear that this type of immunity is not associated with the adult stage of the parasite nor with egg induced pathology, instead, similar to concomitant immunity, it is the migrating larvae or immature juveniles which constitute the target (McLaren, Pearce and Smithers, 1985). McLaren et al. (1985) reported that the crucial difference between concomitant and vaccine immunity appears to be their relative specificities. Concomitant immunity crosses the species barrier (Smithers and Doenhoff 1982), whereas vaccine immunity is species-specific ( Moloney and Webbe, 1987). Sher, Heiney, James and Asofsky (1982) demonstrated that vaccine immunity is based on both T-cell and B-cell responses. On the other hand, it has been suggested that concomitant immunity, is dependent on non-immunological factors, perhaps involving vascular changes in the liver which affect the circulatory patterns of challenge schistosomula (Dean et al., 1981; Harrison, Bickle and University of Ghana http://ugspace.ug.edu.gh 39 Doenhoeff, 1982; Wilson, Coulson, and Metting, 1983). However, James and Cheever (1985) observed from their experiments that a major part of the resistance associated with infected mice is immunologically based. They showed that "P" strain mice which have a genetic defect in macrophage function develop poor levels of both concomitant and vaccine immunity despite the fact that infected "P" strain mice show normal levels of egg induced pathology. Also studies with a monoclonal antibody NIMP/R.41 which binds and destroys mouse neutrophils have shown that depletion of these cells abrogates dermal inflammatory response to schistosomula leading to the suppression of both vaccine and concomitant immunity. 2.10 Diagnosis of schistosomiasis More simple, rapid, sensitive and reproducible diagnostic tests for schistosomiasis are needed not only for epidemiological studies and evaluation of drug efficacy, but also for the management of infected individuals (Hoffman, Lehman, Stott, Warren and Webbe, 1979; Peters and Kazura, 1978). The currently available schistosomiasis diagnostic tests may be divided into three categories. Firstly, there are the parasitological methods which are based on microscopic demonstration of parasite eggs in host excreta or tissues. Secondly, there are serological assays based on the detection of anti-parasite antibodies or circulatory parasite antigens, and thirdly there are imaging techniques for demonstration of pathological changes resulting from the University of Ghana http://ugspace.ug.edu.gh 40 disease (Peters and Kazura, 1987). 2.10.1 The Parasitological diagnostic methods 2.10.1.1 Feacal examination for eggs o f intestinal schistosomiasis Most of the eggs laid by adult worms of S. mansoni, S. japonicum and S. intercalation are eventually trapped in the gut wall or are excreted in the faeces. Diagnosis of these infections are, therefore, mostly based on direct examination of faeces or biopsy specimen of the rectal mucosa for parasite eggs. 2.10.1.2 Direct faecal smear Approximately 2mg of stool is emulsified following the addition of one or two drops of 0.9% saline solution on a glass slide and spread with a coverslip before the smear is scanned for parasite eggs by microscopic examination. This technique is not sensitive enough mostly because of the small amount of specimen examined. Also, the presence of large amounts of fibre may interfere with the visualization of the parasite eggs. For these reasons, Peters and Kazura (1987) concluded that although the method is simple, it is mainly for identification of heavily infected persons. 2.10.1.3 The Formol-ether method This method which leads to the preservation of specimen is also simple to perform (Garcia and Shimuzi, 1981). By this technique, approximately half a teaspoon of fresh stool is placed in 10ml of 10% formalin and allowed to "fix" for at least 30 University of Ghana http://ugspace.ug.edu.gh 41 minutes. The preserved specimen is then passed through two layers of gauze into a centrifuge tube. The filtered stool is sedimented and washed twice by centrifugation at 290 Xg in 0.9% NaCl and the final pellet resuspended in 7ml of 10% formalin. The mixture is then added with 3 ml of ether and shaken for 30 seconds in a capped tube before centrifugation again at 290 Xg for 2-3 min. This separates the mixture into four layers consisting of a small sediment containing schistosome eggs at the bottom with a layer of formalin above it followed by faecal debris and ether on top. The sediment containing parasite eggs is recovered and examined by microscopy. The method is semi-quantitative and, therefore, not suitable for accurate determination of intensity of infection. 2.10.1.4 The Bell method The Bell method for the detection of schistosome eggs in faeces was the first to be extensively used in field studies (Bell, 1963; Teesdale and Amin, 1976; Jordan, Batholomew and Petras, 1981). In this method, a sufficient volume of stool is added to 90ml of a formalin-glycerol solution to bring the final volume to 100ml. The mixture is then mixed at medium speed in a blender for 15-20 seconds to give a creamy suspension. An aliquot of 1ml (representing 0.1ml of stool) is placed on a 7 cm diameter piece of filter paper that is fixed on a suction apparatus and subjected to negative pressure to spread the specimen. The paper is then sprayed with ninhydrin, which stains the egg blue. Before counting the eggs by microscopic examination, distilled water is placed on the paper to improve the refractive index. University of Ghana http://ugspace.ug.edu.gh 42 2 . 1 0 . 1 .5 The Kato method This technique is generally useful for detecting the eggs of the intestinal schistosomes as well as of intestinal parasites in stool specimen. In the standard Kato method, a sample of stool is picked up with a wooden spatula and forced through a stainless steel screen to remove particulate and fibrous material. The specimen is then applied to fill a hole in a metal template that is set on a glass microscope slide. The template is carefully lifted off the slide to leave an intact plug of faeces. The stool specimen is then covered by a 25 x 35mm cellophane coverslip previously impregnated with 50%(v/v) glycerol in water containing 3% malachite green. The slide is subsequently turned face down on a flat surface, and pressed gently but firmly to spread the stool specimen evenly. The prepared slide is left for about 30min in the light to clear before microscopic examination for parasite eggs. This method is rapid and suitable for large epidemiological surveys in which sensitivity needed to detect light infections is not crucial. 2 . 1 0 . 1 . 6 Examination for eggs o f S. haematobium 2.10.1.7 Direct examination o f urine Urine may be examined directly for S. haematobium eggs by microscopy. The method is relatively simple and rapid but it is not sensitive enough. One way to increase the sensitivity of this method is by examination of the sediment from large volumes of urine (Peters and Kazura, 1987). Such urine sediment may be obtained by centrifugation or by gravity. University of Ghana http://ugspace.ug.edu.gh 43 2.10.1.8 Urine filtration methods With these techniques, urine is passed through a suitable membrane filter that traps schistosome eggs. The eggs are then observed microscopically for identification of schistosome species and counted for estimation of the intensity of infection. Polycarbonate and polyamide membrane filters have been shown to be very suitable for this purpose (Peters, 1976 and Mott, 1982) The procedure involves collection of urine between 1100 and 1400 hours, when egg excretion is known to be highest. A fixed volume of urine usually 10ml is drawn into a syringe and passed through a membrane filter secured in a swinnex support chamber. The filter is removed from the chamber with forceps and the side with trapped eggs laid face down on a microscope slide. When ready for microscopy, a drop of saline is placed on the filter to improve the refractive index (Peters, Warren, and Mahmoud, 1976). The sensitivity of this method is reported to be fairly good, especially when 10ml of urine is used. 2.10.1.9 Preserved urine samples In field situations where fresh urine samples cannot be processed immediately within a few hours, the excreted eggs may be preserved by adding an equal volume of 0.002% carbolfuchsine which consists of 100ml absolute ethanol, 50ml phenol, 850ml distilled water and 0.02 grammes of carbolfuchsine. When ready for examination, the treated urine may be subjected to centrifugation to obtain a sediment for direct inspection or it may be passed through no. 1 Whatman filter paper attached to a vacuum apparatus. Air-dried filters are University of Ghana http://ugspace.ug.edu.gh 44 placed egg side up under a microscope and examined. It has been reported that concentration of preserved urine by centrifugation is more sensitive than by filtration. 2.1 0 . 1 . 1 0 Demonstration o f schistosome eggs in host tissue The absence of schistosome eggs in feacal specimen do not necessarily exclude the possibility of infection (Peters and Kazura, 1987). This is because eggs may not be found in the faeces but they may be present in various host tissues such as the rectal mucosa, liver, lung, urethra and occasionally even in the central nervous system. Hence, the diagnosis of schistosomiasis by examination of biopsy specimen has proven very useful. By this approach, urethra biopsy may be taken where urinary schistosomiasis is suspected, while rectal biopsy is suitable for diagnosing intestinal schistosomiasis. The procedure involves the excision of l-2nm snips of tissue which is subsequently sandwitched between two glass slides and examined for parasite eggs by microscopy. 2.10.2 Immunodiagnosis of schistosomiasis Parasitological diagnosis by microscopic examination of stools or urine for parasite eggs has been the most widely accepted method of identifying individuals with schistosomiasis (WHO, 1992). However, the validity of the parasitological results obtained in any epidemiological study, depends on many factors, including the ability of a diagnostic laboratory to perform accurately the daily tasks of University of Ghana http://ugspace.ug.edu.gh 45 collecting samples, preparing and reading slides and recording the results, whilst avoiding the possibility of mislabelling specimens or contaminating apparatus (Jordan and Goddard, 1982). Moreover, the technique is labour-intensive, and relatively insensitive as it is influenced by daily fluctuations in the rate of egg excretion which tend to render especially negative results unreliable, particularly in areas characterized by low intensities of infection. Furthermore, schistosomes are less fecund than most helminths, and majority of their eggs are retained in tissues (Kloetzel, 1963). As a result, accurate determination of schistosomiasis requires more than one stool or urine examination (Ruiz-Tiben, FOllyer, Knight, Gomez de Rois and Woodall, 1979). It is, therefore, not surprising that the quest for simpler, more rapid, specific and sensitive quantitative field detection techniques for the diagnosis of schistosomiasis remains. An attractive possibility is the use of immunological diagnostic techniques (Sendo and Saito, 1991; WHO, 1992). Virtually all the well-established immunological assays including complement fixation (CF), indirect haemagglutination assay (IHA), thin layer immunoassay, gel precipitation, indirect immunofluorescent assay (IFA) and enzyme-linked immunosorbent assay (ELISA) have been applied to identification of schistosome species and the diagnosis of schistosomiasis (Kagan, 1968). however, very few of them have been advocated for large-scale use in diagnosis. Lack of sensitivity appears to be an important limitation in these applications. For example, complement fixation tests employing extracts of lyophilized worms were comparatively as specific as University of Ghana http://ugspace.ug.edu.gh 46 immunofluorescence utilizing adult worm sections, yet they could detect antibody in only 70% of infected cases investigated (Reviewed by Smithers and Doenhoff, 1982). Several tests have also been developed for purposes of diagnosing schistosomiasis. One of the most important of these assays is the Circumoval Precipitin Test (COPT) introduced by Oliver-Gonzales (1954). The COPT which was established with fresh S. mansoni eggs was, made simpler by the use of lyophilized eggs by Tanaka, Matsuda, Bias and Nonsenas (1975). This technique was made even more applicable under field conditions in the tropics following the introduction of the use of air-dried schistosome eggs (Kamiya, 1983). Both the ELISA and radioimmunoassy have improved the sensitivity of serodiagnostic procedures for schistosomiasis (Smithers and Doenhoff, 1982). However, ELISA has several advantages over radioimmunoassay. These advantages include the stability of antigen-antibody complexes and the need for no radioactive substances. In different studies with ELISA McLaren, Draper, Roberts, Minter- Goedbloed, Lighthart, Teesdale, Amin, Omer-Ali, Bartlet and Voller (1987) found that schistosome egg antigens were more reactive than worm antigens and was better for detecting antibodies in acute or earlier infections. On the other hand, using the MSAi antigen obtained from S. mansoni eggs, it has been shown that the radioimmunoassay was stage-specific and more sensitive in detecting parasite-specific antibodies in chronic infections (Hamburger, Pelley and Warren, 1976; Hillyer and Pelley, 1980). Using antibody detection ELISA McLaren, Long, Goodgame and Lilleywhite (1979) reported that 82-100% of intestinal schistosomiasis patients in St. Lucia could be diagnosed even University of Ghana http://ugspace.ug.edu.gh 47 though up to three different stool examination were required to detect some of those infections by parasitological means. It has, however, been pointed out that although antibody detection is the most sensitive serodiagnostic approach (Kelly, 1987), its field applicability is limited by the inability to differentiate active infections from previously cured ones (Nantulya, et al., 1984). Despite its high sensitivity, antibody detection ELISA using crude parasite antigens is also limited by extensive cross-reactivity between S. mansoni, S. japonicum and S. haematobium (McLaren et al, 1987). To overcome this lack of specificity, efforts have been made to use more purified parasite antigens (McLaren, Lilleywhite, Dunne and Doenhoeff, 1981). Also, the problems associated with antibody detection assays could be overcome by detecting parasite antigens instead (Gold, Rosen and Weller, 1969; Bawden and Weller, 1974). Nash (1974) and Deelder et al. (1980) studied and reported on circulatory schistosome antigens which could be exploited in diagnosis of schistosomiasis. Two important circulatory antigens, both of them proteoglycans associated with the gut of adult schistosomes have been identified, and named, circulatory anodic antigen (CAA) and circulatory cathodic antigen (CCA) on the basis of their electrophoretic mobilities (Deelder et al., 1976). Although both antigens are not strong immunogens, (Feldmeier, Nogueira-Queiros, Doehring, Dessaint, de Alencar, Daffalla and Capron, 1986) used polyclonal antisera in ELISA to detect CAA at very low concentrations in a two-site radioimmunoassay. More recently, DeJonge et al. (1989) used a MoAb produced against the CAA and showed that it could be used to detect the antigen in the urine of patients. University of Ghana http://ugspace.ug.edu.gh 48 Even though neither the CAA nor CCA are specific to any of the schistosome species, this new development has further demonstrated the applicability of MoAbs in diagnosis of schistosomiasis. 2.11 Production of monoclonal antibodies (MoAbs) According to the clonal selection theory which is generally accepted by immunologists, a single matured B-lymphocyte produces only one kind of antibody with respect to epitope specificity (Roit, 1991). B-lymphocytes are, however, incapable of continuous growth in vitro. It was, therefore, not possible to obtain large quantities of these mono-specific antibodies even though their potential applications were obvious. This limitation remained important until Kohler and Milstein (1975) discovered and introduced a technique for the production of mono-specific antibodies (monoclonal antibodies). The principle involves fusing a normal antibody secreting B- lymphocyte with a tumogenic (myeloma) non-secreting plasma cell to obtain a "hybridoma" which possesses both the ability to secret antibodies and to grow continuously in culture. The cell fusion procedure and rationale may be summarized as follows. A suitable mammal (such as a BALB/c mouse) is immunized with antigen (normally crude antigen preparations) containing epitopes to which it is desired to raise antibodies against. The spleen of the mammal is aseptically removed, minced and the splenocytes incubated with myeloma cells in the presence of a fusing agent such as polyethylene glycol. The myeloma cells are mutant cells selected for deficiency in the enzyme Hypoxanthine guanine phosphorybosyl transferase (HGPRT) or Thymidine Kinase University of Ghana http://ugspace.ug.edu.gh 49 (TK) based on their ability to grow in the presence of toxic drugs such as 8-azaguanine and 6-thioguanine, respectively. These drugs interfere with nucleic acid synthesis mediated by the respective enzymes (Goding, 1980). The lack of HGPRT, for example, provides the basis for selection of hybridomas after cell fusion. This selection is possible because cells synthesize nucleotides by two biosynthetic pathways. These are the de novo and salvage pathways. De novo synthesis is blocked by aminopterin, a folic acid antagonist. If the cell is, thus, grown in medium containing aminopterin, nucleic acid synthesis ceases. However, provided the enzyme HGPRT could be supplied, the cells can synthesis DNA by the salvage pathway which mainly involves the recycling of preformed nucleotides. Selection medium containing Aminopterin is, therefore, added with two nucleotides namely, Hypoxanthine and Thymidine, and abbreviated HAT. Fusing HGPRT+ spleen derived lymphocytes with HGPRT' myeloma cells results with three main cell types. These are fused HGPRT+ hybridoma cells unfused HGPRT myeloma cells and unfused HGPRT+ spleen cells. During HAT selection, the hybridoma cells survive because the presence of HGPRT enables salvage synthesis of DNA whilst the unfused myeloma cells are killed because of lack of the enzyme. The unfused splenocytes also die with time because they are incapable of continuous growth in culture. The selected hybridomas are then cloned to ensure that single hybridomas are separated. These proliferate into clones of antibody secreting cells secreting University of Ghana http://ugspace.ug.edu.gh 50 MoAbs (Goding, 1980; Lopes and Alves, 1984). The mouse is usually the animal of choice as the donor of splenocytes and for the production of myeloma cells. All of the currently available mouse myeloma cells which are suitable for cell fusion are of BALB\c origin (Levy and Diley, 1984). Successful fusion of cells between different species have, nonetheless, been achieved. Some of the myelomas available express their own heavy and light chains of immunoglobulins, example, P3-X63/AG8; while others express only one chain, example, NSl/lAg4.1; and a third type of myelomas are non-producers, example, X63/AG8.653. As a general rule, the non-producer line with a good fusion performance is the best choice. Two of such mouse myeloma cell lines are in widespread use. They are X63.Ag.653 (Kearney, Radbruch, Liesegang and Rajowsky, 1979) and SP2/O.Agl4 (Shulman, Wilde and Kohler, 1978). Another cell line, known to have a good overall performance, is NS1/1 Ag4.1, a kappa chain producer (Kohler, Howe and Milstein, 1976). 2.12 Characterization of Monoclonal Antibodies Being immunoglobulins, monoclonal antibodies (MoAbs) possess various characteristics (Ouchterlony, 1976). One of the first characteristic of MoAbs which one would want to determine is the immunoglobulin class or subclass. The range of immunoglobulin class or subclass depends on the species origin of the spleen and myeloma cells used for cell fusion. For mouse/mouse derived MoAbs, the likely immunoglobulin classes and subclasses are, IgM IgGi, IgG2a, IgG2b, IgG3, IgA and IgE (Lopes and Alves, 1984). University of Ghana http://ugspace.ug.edu.gh 51 Another characteristic usually determined is the biochemical nature of the antigenic epitope which a MoAb binds. That is, whether protein, carbohydrate, lipid, glycoprotein, lipoprotein or glycolipid. This is important because of the varied immunologic implications of the various epitope types in an immunocompetent host (Gryzch et al., 1984). Yet another important characteristic of MoAbs which gives them the advantages they have over polyclonal heterospecific antibodies, is their specificity with respect to antigenic epitopes. In most studies, this is the focus of attention (Goding, 1980; Lopes and Alves, 1984). 2.13 Usefulness of MoAbs in immunological studies Conventional animal antisera contain the products of many different antibody- secreting clones, even when the animal has been immunized with a purified antigen. Such a heterogenous reagent has obvious limitations whenever greater specificity is required (Lopes and Alves, 1984). Fortunately, the introduction of monoclonal antibodies (MoAbs) has greatly improved the performance of immunological assays (Sikoraand Smedley, 1984). The extraordinary specificity of epitope detection provided by MoAbs has made it possible to develop standardizable immunodiagnostic reagents of high specificity. As a result, MoAbs are now being used in immunoparasitology; (1) as probes for the detection and localization of antigen, and analysis of its organization and availability, (2) for studies of antigenic heterogeneity in parasite University of Ghana http://ugspace.ug.edu.gh 52 populations; (3) for detection of cloned DNA in various vectors; and (4) for parasite typing and parasite detection (Goding, 1980). Monoclonal antibodies are thus convenient tools, not only for immunodiagnosis, but also for characterization and isolation of parasite antigens. One area in which this application has been particularly useful is the identification of immunoregulatory, and protective antigens for use in immunizations. 2.14 Vaccination against schistosomiasis It is generally accepted that any breakthrough in the development of a vaccine for human schistosomiasis may require production of antigens by recombinant DNA technology or synthetic peptides constructed from knowledge of antigen gene sequences (Simpson, Chandler, Kelly, Walker, Knight and Smithers, 1987). The need therefore to employ recombinant DNA technology is necessitated by two requirements. Firstly, there is the need to ensure highly specific and controlled immunological intervention in which only antigens with a protective value are administered. Secondly, gene cloning and its associated technologies constitute the means for producing sufficient antigen for immunization. Both carbohydrate and polypeptide epitopes expressed on the surfaces of newly transformed schistosomula have been reported to induce protective immunity in schistosomiasis. However, the carbohydrate epitopes are reported to be unsuitable as candidate antigens for vaccine production because of their University of Ghana http://ugspace.ug.edu.gh 53 similarity with schistosome egg antigens in terms of their ability to induce pathological responses. The polypeptide epitopes, on the other hand, are present in much lower densities but they appear to be more suitable for use in vaccine production. This is because they appear not to cross-react with the egg antigens, and therefore, they are less likely to initiate or enhance pathology (Hacket, Simpson, Knight, Ali, Payeres and Smithers, 1986). So far, three major polypeptide antigens of Mr 32,000, 25,000 and 20,000 which are expressed on the surface of both schistosomula and adult worms have been identified. These antigens which are presented to the host immune system during natural infections have been the focus of antigen gene cloning experiments (Simpson, Hackett, Kelly, Knight, Payeres and Omer-Ali, 1986). Moreover, a variety of cloned and expressed polypeptide antigens are now becoming available from several laboratories. Some of these antigens are well defined while others are simply labelled as antigenic peptides. It is likely that this trend of investigation may lead to the identification of novel protective antigens (Simpson et al., 1987). Lanar, Pearce, James and Sher (1986) identified and cloned a 97 kDa antigen from S. mcmsoni parasites. This internally localized antigen known as paramyosin has been found to resemble the a-helical protein (paramyosin) present in invertebrate muscle. This molecule is important not only because of its immunological potential as a vaccine immunogen, but also because of its probable physiological function for the parasite. As a core structure for myosin filaments, paramyosin is suspected to be an important molecular component in a "catch" mechanism aiding adult schistosomes in continuously maintaining themselves University of Ghana http://ugspace.ug.edu.gh 54 against the venule wall and thus avoiding dislodgment by the blood flow. Several other antigens have been discovered and are being considered in vaccine trials. These include SRP and 53 from S. mansoni schistosomula; 155, p28 GST and gp68 from S. mansoni adult worm; Sj26, from S. japonicum; JM8-36, an anti-idiotype antibody, and Fh(Smin) from Fasciola hepatica adult worm (Colley and Colley, 1989). University of Ghana http://ugspace.ug.edu.gh CHAPTER 3 GENERAL MATERIALS AND METHODS University of Ghana http://ugspace.ug.edu.gh 56 3.1 Preparation of antigens 3.1.1 Fixation o f miracidia with paraformaldehyde, acetone and ethanol Miracidia were hatched from S. haematobium eggs obtained from the urine of infected persons. Large volumes of urine (more than 3 litres) were left standing undisturbed overnight at 4°C and the supernatant decanted away. The sediment was collected into 50ml conical tubes and centrifuged at 500 Xg for 5 min. The sediments resuspended in 1% saline and washed by centrifugation at 500 Xg for 5 min. The final sediment was resuspended in distilled water and transfered into a flat bottom flask covered with aluminium foil to leave only the upper quarter of the neck. The flask was filled with distilled water up to a level above the darkened portion and placed in front of a light source. The schistosme eggs hatched and the miracidia attracted to the light were concentrated in the small volume of exposed fluid. The organisms were harvested with a pasteur pipet into 50ml tubes and various fixatives diluted in PBS, pH 7.4 added to give final concentrations of 0.25% and 1% paraformaldehyde, 5% acetone, and 95% acetone. These preparations were kept at 4°C overnight to fix and stabilize membrane antigens after which the miracidia were washed thrice with PBS, pH 7.4 by centrifugation at 500 Xg for 15 min each. The final miracidia pellet was resuspended with a small volume of Dulbecco's PBS, pH 7.4 and the miracidia counted. Fixed miracidia were kept at 4°C until use. University of Ghana http://ugspace.ug.edu.gh 57 3.2 Myeloma Cell Lines and their Maintenance BALB/c-derived, 8-azaguanine-resistant parental myeloma cell lines were used. X63-AG8.653 were obtained from International Laboratory for Research on Animal Disease (ILRAD) while NS-1/1 Ag 4.1 were obtained from Tokyo University, Japan. These two myeloma cell lines were adapted for in vitro propagation in Minimum essential Medium (MEM, Gibco) or in Iscove's Modified Dulbecco's Medium (IMDM, Gibco), all supplemented with 10%(v/v) heat-inactivated (56°C for 30 mins) Foetal Bovine Serum (FBS). The myeloma cells were cultivated in 25cm2 or 75cm2 sterile tissue culture flasks (Sumilon, Sumitomo Bakellite Company Ltd., Japan) in a C 02 incubator set at 5% C02 and 37°C. The cells were allowed to grow to a concentration of about lx 107/ml and maintained in culture by subculturing a third of the cell suspension with two-thirds volume of fresh growth medium into new flasks. This was to ensure that the cells remain in the logarithmic growth phase for at least 4 days prior to cell fusion (Pearson, Pinder, Roelants, Kar, Lundin, Mayor-Whitney and Hewett, 1980). 3.3 Cell Fusion and Selection of Hybridomas Three to four days after the final booster with antigens, the highest responder mice were killed by terminal anaesthesia, with diethyl-ether and their spleens dissected out aseptically. Cell fusion was done according to the method described by Pearson et al. (1980) with some modifications. All the stages in this procedure were carried out under aseptic conditions in a Clean bench (Hitachi, Tokyo, Japan). University of Ghana http://ugspace.ug.edu.gh 58 The spleen from each mouse was separately minced with a curved pair of scissors and the splenocytes suspended in growth medium in a 15 ml conical tube. Clumps and membrane fragments were allowed to settle and the resulting cell suspension pipetted into another tube and washed once with serum-free medium by centrifugation at 290 Xg for 5 min. The cells were then counted using an improved Neubauer Counting Chamber (Hagayaki Works, Tokyo, Japan) and pipetted into a 50ml conical tube. The myeloma cells were similarly washed in serum-free medium and a volume of the cell suspension mixed with the spleen cells at a ratio of 1:5-10 myeloma to spleen cells. The mixed cell suspension was centrifuged at 290 Xg for 5 min and the supernatant completely removed. One millilitre of polyethylene glycol (PEG), warmed to 37°C, was added dropwise to the cells in a conical tube immersed in a beaker containing water at 37°C over a period of 1 min while gently mixing the cells by turning the tube in a circular manner. The cells were then gently resuspended by stirring with the same pipette for another 1 min. Soon after, 10ml of serum-free medium was added dropwise over a period of 3 min. The cell suspension was then centrifuged at 190 Xg for about 3 min and the supernatant removed. Care was taken to ensure that the total time from the beginning of the addition of the PEG to its removal did not exceed 8 min. The fused cell mixture was then resuspended in HAT medium consisting of O.lmM hypoxanthine, 4 x 10^mM aminopterin, and 0.016 mM thymidine dissolved in fusion medium [growth medium modified by addition of 38.57mM NaHCOs, lOmM N-2-hydroxyethyl-piperazine-N-2-ethane sulfonic acid University of Ghana http://ugspace.ug.edu.gh 59 (HEPES) and 0.198mM L-glutamine], The resuspended cells were distributed into two 24-well tissue culture plates, in 1ml of HAT medium per well. HAT-medium was replaced every 3 days until day 10 and changed to HT-medium consisting of 0.1 mM hypoxanthine and 0.016mM thymidine dissolved in fusion medium. Usually between day 8 amd 14, wells with large colonies of hybrid cells, as determined by observation under an Inverted microscope (Nikon, 46212, Japan) were marked and the media allowed to turn acidic (yellow) and then tested for antibody activity. Hybrid cells from wells showing positive antibody activity were immediately cloned, and some stabilated as soon as possible. More supernatants were tested for antibody activity as hybrids grew. When selected hybrids were growing well, the HT-medium was replaced with normal growth medium. 3.4 Screening, Cloning and Stabilation of Hybridomas Culture supernatants from wells containig hybridoma cell colonies were screened for antibody activity, using the antibody detection nitrocellulose membrane- based dot-ELISA or by micro-plate ELISA. Wells with detectable antibody activity as indicated by optical densities or intensity of staining on nitrocellulose membrane were selected for cloning. Some cells from these positive wells were transfered to 25cm2 tissue culture flasks and grown until 2xl07 cells could be frozen in liquid nitrogen. Cells were stabilated in growth medium containing 7.5%(v/v) Dimethylsulfoxide (DMSO). The remaining hybridomas were resuspended in 1% Trypan Blue in University of Ghana http://ugspace.ug.edu.gh 60 phosohate buffered saline, pH 7.4, which served as vital stain and live cells were counted in an improved Neubauer Counting Chamber. The hybridomas were then cloned by limiting dilution in growth medium. A cell suspension was made to give 1 cell750|il of medium and 2 cells/50|_il, and 50|il volumes of each cell concentration dispensed into the wells of different 96 well tissue culture plates previously incubated at 37°C overnight with 50|i 1/well of splenocytes and/or thymocytes from 2-4 week old BALB/c mice. The 96 well culture plates were incubated at 37°C in a humified atmosphere containing 5% C02 in air and left undisturbed for about 10 days. They were then examined for visible cell colonies by viewing the bottom of the wells against light or by observing under an inverted microscope. Wells with single visible cell colonies were marked and transferred into 0.5ml of growth medium in 48-well tissue culture plates. The cells were allowed to grow and increase in number and 1ml of fresh growth medium added to each well. The culture fluids were allowed to become acidic before testing for antibody. Antibody positive culture fluids were concentrated 20 times, and used for determination of immunoglobulin class by double immunodifussion. 3.5 Propagation and Storage of Hybridomas Cloned hybridoma cells secreting MoAbs of desired specificity were transferred from 100fj.l of growth medium in 96 well plates to 0.5ml of medium in 24 well plates, and the hybrids allowed to multiply before increasing the volume to lml. The hybridoma cells were grown in the 24 well plates with University of Ghana http://ugspace.ug.edu.gh 61 medium changes every three days until about 1 x 107 cells could be transferred into 25cm2 tissue culture flasks. The hybrid cells were then transferred to 75cm2 tissue culture flasks in 20-25ml of growth medium containing between 1 x 105 1 x 10s cells/ml and maintained by subculturing into other flasks when medium turned acidic. In order to produce more MoAbs, hybridoma cells were propagated in 1 to 2 litre glass flasks (Coming Glass Works, New York) in 500 to 2000ml medium. The cultures were then gassed with a mixture of 5% CO2 in air and incubated at 37°C in an incubator (Model IF-41, Yamato Scientific Co. Ltd., Tokyo Japan). Culture fluids, containing MoAbs were harvested after centrifugation at 290 Xg for 5 min to remove the cells, and the supernatants stored frozen at -20°C. Some of the hybridoma cell cultures were maintained in the logarithmic phase of growth for at least 4 days before preservation of the cells in liquid nitrogen. This was to ensure that healthy cells were preserved. Suspensions of cells in the logarithmic growth phase were centrifuged at 290 Xg for 5 min and the supernatant removed. The pelleted cells were resuspended in growth medium containing 7.5%(v/v) DMSO and 1ml aliquots pipetted into 2ml cryopreservation vials before freezing in liquid nitrogen using a Cryo-controller version 2.01 (Department of Biomedical Engineering, University Hospital, Copenhagen,Denmark). The cryo-controller was programmed to freeze cells over a period of lhr during which specimen were subjected to a temperature gradient from room temperature to -140°C. The vials were then transferred into liquid nitrogen at - 196°C. University of Ghana http://ugspace.ug.edu.gh 6 2 3.6 Purification of Monoclonal antibodies MoAb containing culture spematants stored at -20°C were retrieved and thawed at 37°C in a waterbath. The immunoglobulins were then precipitated by slow addition of an equal volume of saturated ammonium sulphate [(NHi^SCM] while mixing. The precipitates formed were pelleted by centrifugation at 1,200 Xg for 30 mins and dissolved in minimum amount of distilled water. The antibody solutions were then transferred into dialysis membrane (Spectro Medical Industries Incorporated, USA) of molecular weight cut off 1,000 and dialysed overnight against Phosphate buffered saline (9mM NaH2,P0 4 , 0.9mM Na2HPC>4, 15mM NaCl, pH 7.4) with one buffer change. Monoclonal antibody conentrated by this process was subjected to further purification. 3.6.1 Gelfiltration IgM monoclonal antibodies precipated and dialysed as described above were purified by gel filtration through a Sephadex G-200 (Pharmacia, Uppsala, Sweden) column using Phosphate buffered saline, pH 7.4 as eluting buffer. Sephadex G-200 was prepared according to the manufacturer's instructions. Ten ml of dialysed antibody was applied onto the Sephadex G-200 column and fractionated. The eluate was collected in 5ml fractions and assayed for antibody activity using the antibody detection microplate ELISA. Fractions containing antibodies were pooled and concentrated using the Amicon concentration chamber (Amicon, Corporation, Ireland) with University of Ghana http://ugspace.ug.edu.gh 63 ultrafiltration disk membranes of molecular weight cut-off 10,000 (Sigma Chemical Company, USA). 'l ‘ * \3.6.2 Ion exhcange chromatography t o \ ■ f \ * 'V ** Culture supernatants containing IgG MoAbs were also concentrated by the ammonium sulphate precipitation method and dialysed against PBS. IgG MoAbs were, however, purified by ion-exchange on diethyl aminoethyl cellulose (DE-52, Whatman, Kent, England). Sample was applied onto the column and washed with at least one column volume of dialysis buffer (PBS, pH 7.4). Bound MoAb was then eluted with a linear gradient of 15-300mM NaCl. 10ml fractions were collected in tubes and each assayed for antibody. Fractions with antibody activity were pooled and concentrated as described for IgM antibodies. 3.7 Preparation of enzyme antibody conjugates Schistosoma haematobium reactive MoAbs previously purified by gel chromatography, were conjugated to horseradish peroxidase (HRPO) using the periodate method described by Wilson and Nakane (1987). The protein concentration of antibody fractions was estimated using the Bio-Rad Protein Assay reagent (Bio-Rad Laboratories, USA). The desired amount of antibody IgG (MW 160 000) or IgM (MW 90 0000) in terms of protein to be conjugated was pipetted into a tube. HRPO enzyme (MW 40 000, Sigma Chemical Company, USA) was conjugated to antibody at a ratio of 1:1 (enzyme : antibody) by weight. Assuming an 80% coupling efficiency, University of Ghana http://ugspace.ug.edu.gh 64 the calculated value for enzyme was multiplied by 100 and divided by 80. For every X mg of IgG antibody and IgM antibody, therefore, X x 1/4 x 100/80 mg, or X x 1/22.5 x 100/80 mg of HRPO, respectively were required. For IgM antibody, this calculated value was multiplied by 5 to account for its pentameric structure. The required amount of HRPO was weighed and dissolved in 2ml of 50mM sodium acetate (CH3COONa) buffer, pH 4.0. HRPO was oxidized using sodium metaperiodate (NalO^ at a ratio of 1:1 by weight. The weighed NaI04 was dissolved in 1ml of 50mM sodium acetate buffer before adding it dropwise to the enzyme solution to mix in a 50ml centrifuge tube covered with aluminium foil. The mixed solution was then incubated with gentle rocking for 15 min at room temperature after which 300|al of ethyleneglycol (C2H6O2) were added and stirred. The mixture was then fractionated through a column packed with Sephadex G-25 equilibrated with 50mM sodium acetate buffer pH 4.0 and eluted with the same buffer. The pH of the eluted enzyme was adjusted to pH 9 .6 by adding lml of carbonate-bicarbonate buffer pH 9.6 and followed by dropwise addition of saturated sodium carbonate (Na2C0 3 ). The pH of the antibody solution was likewise adjusted to pH 9.6. The enzyme solution was then added to the antibody solution and allowed to mix for 1 hr at room temperature in a tube covered with aluminium foil. This was followed by the addition of 300mg of Glycine (NH2.CH2.COOH) and adjustment of the pH of the resulting solution to pH 8 .0 using 1M HC1. The conjugate was kept at 4°C overnight, after which it was precipitated with 33%(v/v) saturated ammonium sulphate solution for IgG or 50% for IgM and then centrifuged for 2 min at 9,900 Xg and the pellet dissolved with glycine/Na2EDTA University of Ghana http://ugspace.ug.edu.gh 65 buffer (0.4M glycine, 0.3M NaCl and 20mM Na2EDTA, pH 8.0). 20mg of ovalbumin per mg of conjugate was added and mixed to dissolve, and the solution again centrifuged to remove particulate matter. The conjugate solution was then filtered with 0.45um membrane filter (Millipore Products Division, Bedford, MA, USA) followed by a 0.22um filter to remove polymerized conjugate. An equal volume of glycerol was finally added, mixed and stored at -20°C. Conjugate potency was determined by antigen detection micro-plate ELISA. 3.8 Sodium dodecyl sulphate-polyacrilamide gel electrophoresis (SDS-PAGE) Electrophoresis of schistosome proteins was performed with the ATTO Corporation Slab Gel Apparatus (Bunkyo-Ku, Tokyo, Japan), following the SDS-Tris- glycine discontinuous buffer system (Laemni, 1970). 3.8.1 Assembly o f slab gel apparatus, and preparation o f resolution and stacking gels The gel casting apparatus was composed of a clamp apparatus, a pair of glass plates wiped clean with absolute methanol, and a rubber gasket. The gels were cast in a mold formed by a plain glass plate and a notched glass plate put together and sealed with a U-shaped gasket. The assembled plates were kept vertically upright by 4 spring clamps. University of Ghana http://ugspace.ug.edu.gh 66 Resolution acrylamide gradient gels (10-15%) were prepared as follows. Solution A, 10% resolution gel (one gel) 29.2% (w/v) acrylamide, 0.8%(w/v) N,N'-methylene bis-acrylamide 6ml 1.5M (Tris-aminomethane)-HCl pH 8 .8 , 0.4%(w/v) sodium dodecyl sulphate (SDS) 4.5ml 10% (w/v) Ammoniun persulphate (APS) 0.07ml N,N,N'-N'-tetramethylethylenediamine (TEMED) 0.01ml Distilled water 4.5ml Solution B, 15% resolution gel (one gel) 29.2% (W/V) Acrylamide, 0.8%(W/V) N,N'-methylene bis-acrylamide 9ml 1.5M (Tris-aminomethane)-HCl pH 8 .8 , 0.4% (w/v) sodium dodecyl sulphate (SDS) 4.5ml 10% (w/v) Ammonium persulphate (APS) 0.07ml N,N,N',N'-Tetramethylethylenediamine (TEMED) 0.01ml Distilled water 4.5ml Solution C, Stacking gel (one gel) 29.2% (w/v) acrylamide, 0.8%(w/v) N,N'-methylene bis-acrylamide lml University of Ghana http://ugspace.ug.edu.gh 67 1 5M (Tris-aminomethane)-HCl pH 6 .8 , 0.4% (w/v) sodium dodecyl sulphate (SDS) 1.5ml 10% (w/v) Ammonium persulphate (APS) 0.018ml N,N.N',N'-T etramethylethylenediamine 0.006ml Distilled water 3.6ml A 10-15% resolution acrylamide gradient gel was prepared by slowly mixing solution A and B using an improvised gradient former. Each solutiion was swirled to mix and poured into one of the two linked chambers of the gradient mixer. A tube joining the chambers at the bases was closed with a clip to avoid premature mixing of the solutions. Solution B with the higher acrylamide concentration was poured into the chamber closer to the outlet. The tube joining the two chambers was opened and a magnetic stirer placed in solution B started. An ATTO chromatographic peristaltic pump (ATTO, Kunkyo-ku, Tokyo, Japan) set at a flow rate of 3ml/min was used to deliver the gel mixture via rubber tubing into the space between the glass plates. The gel former was immediately rinsed with distilled water to prevent polymerization of gel in the connecting tubes. The gel in between the glass slabs was overlaid with about 800|j.l of distilled water using an adjustable pipette and left to polymerize. After it had polymerized, the overlaying water was drained away with tissue. A stacking gel solution (C) was then poured on top of the separating gel and a comb carefully inserted to cast the desired wells for sample application. University of Ghana http://ugspace.ug.edu.gh The stacking gel was allowed to polymerize overnight. The comb was removed and the cast gel units assembled in an ATTO Corporation Cell Electrophoresis apparatus. The upper electrophoretic chamber, at the cathode, was filled with 0.2 litres of running buffer (24.8mM Tris, 191.8mM Glycine and 3.47 mM SDS) and the lower chamber, at the anode, filled with 0 .6 litres of running buffer. 3.8.2 Preparation o f samples and electrophoretic run Crude antigens run on the gels were adjusted for protein and diluted with sample buffer [25mM Tris, 192mM Glycine, 0.1%(w/v) SDS and 20%(v/v) Methanol] in a 2:1 sample to buffer ratio, to give 15mg of protein per lane. The antigens included in this study were soluble egg antigens from Ghanaian strain(s) (ShSEAGh) S. haematobium and Egyptian strain(s) (ShSEAEgy), S. haematobium adult worm crude antigen extract (ShW), S. japonicum soluble egg antigens (SjE) and adult worm crude antigen extract (SjW), S. mansoni adult worm crude antigen extract (SmW), precipitated proteins from S. haematobium infected patient urine (UP2-IP) and (UP„- IP), and normal control urine precipitates (NUX) and (NUJ). Standard low molecular weight markers (Sigma, USA) were prepared as described by the manufacturer and used. The samples were boiled for 5 min and centrifuged for about 2 min at 9,900 Xg to remove particulate matter before they were loaded unto the gel. A constant current of 15mA was supplied by an electrophoresis power supply (ATTO Corporation, Japan) until the bromophenol blue tracer dye reached the interface between the stacking and separating gels. The current was then increased to 25mA and maintained until the bromophenol blue marker had barely run out of the separating gel. 68 University of Ghana http://ugspace.ug.edu.gh CHAPTER 4 STUDIES OF ANTI S. HAEMATOBIUM MONOCLONAL ANTIBODIES: I: PRODUCTION University of Ghana http://ugspace.ug.edu.gh 70 4.1 Introduction Schistosomiasis is an economically important disease which affects about 250 million people worldwide, with a further 500-600 million others exposed to the risk of infection. The principal methods for identifying people with the disease is by the demonstration of parasite eggs in excreta or by haematuria or proteinuria. These methods are, however, not sensitive enough. Furthermore, haematuria and proteinuria could be due to causes other than schistosomiasis. These limitations have made the search for more sensitive and specific alternative schistosomiasis diagnostic methods inevitable. One outcome of this search, has been the development of tests based on the detection of anti-schistosome antibodies or circulatory schistosome antigens in serum or urine (Sherif, 1962; Shoeb, Basma, Haseeb and Said El Din, 1968; Feldemeier, Stevens, Bridts, Dafalla and Buttner, 1983). None of these tests has, however, been able to replace the parasite egg detection method in routine field diagnosis of the disease. This is mainly because of the lack of adequate specificity and in some cases sensitivity. The introduction of the technology for producing MoAbs by Kohler and Milstein (1975), has offered great opportunities for the development of more sensitive and more specific immunodiagnostic techniques (Bergquist, 1984; Sikora and Smedley, 1984). An attractive possibility, in the case of urinary schistosomiasis, is the development of assays which utilize MoAbs in the detection of parasite-specific antigens released into patient urine. This study was, therefore, undertaken with the objective of producing University of Ghana http://ugspace.ug.edu.gh 71 MoAbs reactive with schistosome antigens, with the view that some of them may prove useful in the diagnosis of urinary schistosomiasis. 4.2 Materials and methods 4.2.1 Mice Inbred male and female BALB/c mice aged between 12 and 16 weeks and nursling BALB/c mice 7-14 days old, were used for the production of MoAbs. All the mice used were obtained from the NMIMR colonies. 4.2.2 Preparation o f Parasite Antigens 4.2.2.1 Extraction o f crude schistosome worm, egg and hookworm egg antigens Crude schistosome antigens were extracted using an extraction fluid consisting of phenylmethyl-sulfonylflouride (PMSF), N-ot-P-Tosyl-L-Lysine chloromethyl ketone (TLCK) and N-Tosyl-L-phenylalamine chloromethyl Ketone (TPCK) all from Sigma Chemical Company, USA. These were reconstituted in absolute ethanol and diluted in Dulbecco's PBS pH, 7.4 to a final concentration of ImM PMSF, 0.2mM TLCK and 0.05mM TPCK. Protease inhibitors, Leupeptine and trans-Epoxysuccinyl-leucylamido (4-guanidino) butane (E-64, Cambridge Research Biomedicals Incorporated, UK) were added to a final concentration of lOmg/ml. Antigen extracts were prepared from S. haemtobium, S. mansoni S. japonicum eggs and worms as well as Hookworm eggs. Parasite eggs frozen at -20°C were University of Ghana http://ugspace.ug.edu.gh 72 retrieved and added with extraction fluid before thawing. After resuspension, each specimen was pipetted into a glass homogenizer (Kudoguki, Keiki, Tokyo, Japan) and disrupted on ice at 1200rpm for 5 min. Homogenized eggs or worms were collected into different eppendorf tubes and centrifuged at 290 Xg for 5 min and the supernatants collected as primary extracts. Fresh extraction medium was added to each pellet and mixed, and the extraction process repeated to obtain secondary and then tertiary extracts. One ml aliquots of the extracts were kept at -20°C until used in experiments designed to investigate MoAb specificity. 4.2.2.2 Precipitation ofproteins from S. haematobium infected human urine The protocol for the extraction of urine protein as illustrated in figure 1 was provided by Dr. Kwabena M. Bosompem of NMIMR. 4.2.3 Immunization o f Mice fo r MoA b Production 4.2.3.1 Immunization with S. haematobium soluble egg antigens (ShSEA) Soluble egg antigens from the eggs of an Egyptian strain of S. haematobium (ShSEAEgy), were used for immunization of BALB/c mice. All immunogens were given by the intraperitoneal route. Initial immunizations were made with 30-50|_ig of schistosome proteins emulsified in Freund's complete adjuvant (Sigma, U.S.A.) supplemented with 1 mg/ml of Mycobacterium tuberculosis, while subsequent University of Ghana http://ugspace.ug.edu.gh 73 Figure 1: Extraction of UP0P and UP2-IP from S. haematobium infected human urine (Bosompem, unpublished data) Urine Centrifugation (2,900 xg, 15min) Pellet = UPoP UP0S (Supernatant) Added; Saturated (NH4)2S0 4) 50%(V/V) Centrifugation (2,900 xg, 30min) Supernatant = UPiS Pellet Added; H20 UPjP (Suspension) Centrifugation (2,900 xg, 15min) Pellet = UP [IP UPi-IP (Supernatant) Added; Saturated (NH4)2S0 4) 50%(V/V) Centrifugation (2,900 xg, 30min) Supernatant = UP2S Pellet Added; H20 UP2P (Suspension) C entrifugation (15,000 xg, 15min) Pellet = UP2IP UP2-IP (Supernatant) University of Ghana http://ugspace.ug.edu.gh 74 (booster) immunizations were made with Freund's incomplete adjuvant. Booster immunizations consisting of similar concentrations of schistosome antigens were given 3 and 6 weeks after the initial immunization. About 10 days after the second booster, the mice were bled from the tail veins and tested for antibody response to ShSEAEgy by dot-ELISA or micro-plate-ELISA. Mice with high antibody titre (1:5000 or more) were each given a final intraperitoneal booster of 50|ig of antigen. Pre-fusion sera were prepared from blood obtained by orbital bleeding 3-4 days after the final booster. Immediately after the bleeding, the mice were killed by terminal anaesthesia using diethyl-ether, and their spleens aseptically dissected out for cell fusion. 4.2.3.2 Immunization with antigens extracted from the urine o f S. haematobium infected individuals Proteins concentrated from S. haematobium infected human urine by ammonium sulphate precipitation were used to immunize male and female BALB/c mice. The mice were injected intraperitoneally with 100j_il total inoculum consisting of 50|j.g of the antigen emulsified in Freund's complete adjuvant. First and second intraperitoneal booster immunizations were given respectively, 2 and 6 weeks later, using similar concentrations of antigen in Freund's incomplete adjuvant. Ten days after the second booster, immunized mice were bled and screened for antibody response as described in (section 4.3.4). The best responder mice with an antibody titre of 1:5000 or more were each given a final intraperitoneal booster of 50(j.g of the antigen preparation in Freund's incomplete adjuvant. Three to four days later, the mice were University of Ghana http://ugspace.ug.edu.gh bled for pre-fusion serum, just before their spleens were aseptically removed for cell fusion. 4.2.4 Screening o f Immunized Mice fo r Antibody Activity The tip of the tail of each mouse was cut with a pair of scissors after cleaning with 70% ethanol and the tail gently squeezed from the base towards the tip. Drops of blood from the severed veins were aspirated using a 10|il Eppendorf pipette and then transferred each into an Eppendorf tube containing 490pil of Dulbecco's PBS pH 7.4 and mixed thoroughly. The tubes and their contents were then centrifuged at 9,900 Xg for 5 min and the supernatants pipetted into different tubes. The sedimented blood cells were discarded. Each supernatant was tested for antibody activity by micro-plate ELISA or dot-ELISA. 4.2.5 Antibody-detection Micro-plate ELISA This procedure was used for screening immunized mice sera and hybridoma culture supernatants for antibody activity. In this assay, polystyrene micro-plates (Sero-wel, Bibby Sterilin, UK) previously coated with 5 Ojj. 1/well of schistosome antigens were emptied and rinsed once before incubation with test samples for 15min at room temperature. Immunized mice sera were tested in two-fold dilutions beginning from 1 : 1 0 0 while 1 0 0 |ol of hybridoma culture supernatants were tested in duplicates without dilution. Normal mouse serum and pre-fusion serum were added as negative and positive controls, respectively. The plates were rinsed once to remove excess unbound antibody. Each 75 University of Ghana http://ugspace.ug.edu.gh 7 6 well was then incubated with 50(_il of goat anti-mouse HRPO conjugate diluted 1:500 in washing buffer and incubated for 15min at room temperature. Each plate was washed 3 times, each by lOmin incubation with washing buffer to remove excess unbound conjugate. The presence of bound conjugate was revealed by the addition of substrate solution consisting of 40mM 2,2'-azino-bis (3-ethylbenzethiazoline-6- sulphonic acid) (ABTS) and 0.01%(v/v) hydrogen peroxide in 50mM citric acid buffer, pH 4.0. The substrate was incubated for 30 min at room temperature. The colourless substrate solution changed to green in wells with bound enzyme conjugates. The optical densities were read at 410nm wavelength using a Dynatech MR 600 micro- ELISA plate reader (Dynatech Laboratories Incorporated, USA). 4.2.6 Dot-ELISA Procedure This assay was used to screen immunized mice supernatants for antibody. Schistosome egg or wonn extracts unto Nitrocellulose membrane filters, 0.4|im pore size (Sigma Chemical Company, USA). The antigen "dotted" membranes were cut into strips and incubated for 1 hour with "blocking solution" containing 5%(w/v) skimmed milk in Tris-buffered saline (TBS) (50mM Tris and 150mM NaCl, pH 8.0). The strips were removed from blocking solution and incubated for 2 hours with primary antibody, ie., immunized mice sera diluted 1:250 with blocking solution or hybridoma culture supernatants diluted 2:3 in blocking solution. The strips were then washed in TBS and incubated for lhr in University of Ghana http://ugspace.ug.edu.gh goat anti-mouse HRPO-conjugated immunoglobulin diluted 1. 500 in blocking solution, after which they were washed three times (10 min each). Finally the membrane strips were incubated for 3 min in a substrate solution containing 0.15%(v/v) Hydrogen peroxide and 0.05%(w/v) chromogen (3,3'-diaminobenzidine) in phosphate-Na^DTA buffer (lOmM NaH2P04, lOmM Na2HP04, lOmM Na2-EDTA). The strips were washed with distilled water and the substrate reaction stopped by the addition of a few drops of concentrated HC1. Positive reactions appeared as brown dots. 4.2.7 Cell Fusions, Cloning and Selection o f Hybridomas The procedures adopted for cell fusions, cloning and selection of monoclonal antibody secreting hybridomas have been described under sections 3.3 and 3.4 of Chapter 3. 4.2.8 Determination o f Immunoglobulin Class and Subclass The class and subclass specificity of the murine immunoglobulins (Igs) were determined using the double immunodifiision method described by Ouchterlony (1967). Commercially prepared antisera (Goat anti-mouse Ig against murine Ig isotypes: IgGi, IgG2a, IgG2b, IgG3, and IgM) were purchased from (Sigma, USA) and used as recommended. A l%(w/v) agarose (Bethesda Research Laboratories, USA) solution was prepared by melting solid agarose in Phosphate buffered saline (15mM NaCl, 1.1 mM Na2HP0 4 and 0.1 mM KH2PO4, pH 7.4) Five millilitres of the molten agarose was poured unto a microscope slide and allowed to solidify. Wells were then 77 University of Ghana http://ugspace.ug.edu.gh 78 cut into the solid gel in a circular arrangement surrounding a central well and each filled with approximately 15|ul of reagent. Antisera were placed in the central well and culture supernatants concentrated twenty-fold by ammonium sulphate precipitation were placed in the surrounding wells. A precipitin line formed in-between a sample well and the homologous antiserum in the central well. The precipitin reaction was allowed to develop in up to 48 hours at room temperature in a moist chamber and observed by viewing the gels against light. University of Ghana http://ugspace.ug.edu.gh 79 4.3 Results The BALB/c mice responded well to the Schistosoma haematobium antigen preparations that were used in the immunizations. In general, high antibody responses with titres far beyond 1:5,000 were obtained against the antigen preparations, as determined by the double-antibody sandwich micro-plate ELISA. Also, some of the sera could detect as low as 0.063|ig of S. haematobium soluble egg antigens (ShSEA). Differences were, however, found in the ability of the two antigen preparations, namely, ShSEA and crude protein extracts from the urine of S. haematobium infected persons (UP2-IP) to induce antibody responses. Figures 2a and 2b illustrate the mean serum antibody responses of the mice immunized respectively with ShSEA and UP2-IP as compared with serum reactivity of normal non-immunized mice. The individual curves in the figures show that antibody responses in mice immunized with UP2-IP remained high with optical density greater than 0.2 even at antibody titres up to 1;51,200. On the other hand, antibody responses in mice immunized with ShSEA remained high above optical density 0.2 at antibody titres of 1:6000. Antibody titres of the response in the non-immunized mice, on the other hand, were very low in each case, and remained below 0 . 1 optical density at all the dilutions tested from 1 : 1 0 0 to 1:51,200. 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