Measuring concentrations of insulin-like growth factor-l with an enzyme-linked immunosorbent antibody assay in plasma samples from Holstein cows

Abstract

This study describes the use of a commercial non extraction ELISA to quantify total concentrations of insulin-like growth factor-l (IGF-I) in plasma samples from Holstein cows fed pasture-based diets varying in dry matter and metabolizable energy density. The assays were conducted using the protocols provided by the manufacturer. The ELISA was verified for linearity, accuracy in measuring IGF-I from spiked plasma samples, and precision involving variation within and between assays. Validation also involved comparing results of the ELISA against an established RIA after defatted plasma samples were subjected to acid gel HPLC to dissociate and separate IGF and IGF binding protein complexes. Validation results had low coefficients of variation (CV; intraassay CV of less than 6% and interassay CV of less than 8%) and a high recovery percentage of IGF-I (79%) from samples spiked with unbound IGF-I. The coefficients of determination between the ELISA and the RIA reference assay were 0.90 in 2 separate assays. Associations between the RIA and the ELISA were higher, and the limits of agreement at 95% confidence intervals were narrower compared with those between the RIA and a modified ELISA procedure in which IGF binding protein complexes were extracted using HPLC. The samples were obtained from cows sampled over a period of 5 wk. These results demonstrated that the DSL-10-2800 nonextraction IGF-I ELISA was acceptably specific and sufficiently sensitive to be used to measure the extent and patterns of change in the plasma concentrations of IGF-I in samples from lactating Holstein cows