Comparison of radial immunodiffusion and enzyme-linked immunosorbant assay for quantification of bovine IgG in colostrum and plasma

Abstract Text: Radial immunodiffusion (RID) is the standard quantification method for bovine IgG in colostrum and plasma. Recent studies have published ELISA IgG values; however, RID and ELISA measurements have not been compared. Heating colostrum to 60^o C for 30 min does not decrease IgG concentration measured by RID; use of ELISA has not been tested. This study’s objective was to compare ELISA and RID values in plasma and in colostrum before and after heating. Colostrum (n = 58) and plasma (n = 99) were collected from individual cows and calves and frozen prior to IgG assessment. Colostrum was diluted 1:10 for RID and 1:1,000,000 for ELISA. Two dilution series were created for each sample and duplicated within each assay. Values were accepted when coefficients of variation (CV) were ≤ 10.5% among duplicates from a single dilution series and < 15% among 2 series from the same sample. Samples were retested until acceptable CV were acquired. The effect of heat-treatment was tested by heating 20 mL aliquots of each colostrum to 60^o C for 30 min, then cooling, freezing, and retesting using the same dilution factors. Plasma samples were tested by RID without dilution and by ELISA at 1:500,000. Requirements for CV were identical to those used for colostrum. Proc Corr and Proc Mixed in SAS were used to determine correlation coefficients and effect of heat on colostrum IgG quantification, respectively. Mean (± SD) IgG concentration of colostrum before heating was 39.7 mg/mL (± 22.7) and 81.2 mg/mL (± 29.7) by ELISA and RID, respectively; and 19.2 mg/mL (± 12.8) and 76.8 mg/mL (± 34.6) after heating. Heat treatment reduced colostral IgG concentration when measured by ELISA (P < 0.01) but not by RID (P = 0.73). Correlation coefficients (P - value) were 0.20 (0.14) and 0.27 (0.03) for unheated and heat-treated colostrum, respectively. Fat and non-IgG protein in colostrum may interfere with assays and may be a cause for low correlation. Mean (± SD) plasma IgG concentration was 11.4 mg/mL (± 7.9) by ELISA and 15.2 mg/mL (± 9.1) by RID. Relative to colostrum, plasma results were more strongly correlated (r = 0.55; P< 0.01); however, direct comparisons of ELISA and RID values merit caution. Results from ELISA were < RID for colostrum and plasma, and colostrum IgG decreased during heat-treatment when measured with ELISA. Further research is needed to determine effects of heat-treatment on colostrum IgG.

Keywords: IgG