A class capture enzyme immunoassay for immunoglobulin level determinations in bovine sera

An enzyme immunoassay for determination of individual isotype concentrations in bovine serum was developed. Polystyrene tubes were coated with affinity purified goat antibovine IgA, IgG1, IgG2 or IgM, washed and then incubated with purified isotypes to ascertain crossreactivity and sensitivity limits. Bound isotype was detected using the homologous affinity purified antibody conjugated to horseradish peroxidase and hydrogen peroxide-2,2-azino-di-(3-ethylbenzthiazoline sulfonic acid), as the substrate/chromogen. A standardized serum was diluted and used as a control for comparison. Several dilutions were used initially, however, determinations may be made with a single dilution, 1:200, for all isotypes. Results for 100 sera were compared to data obtained with the same samples using a radial immunodiffusion technique. A low correlation coefficient was noted between results from the two assays. Day to day variation and within test repeatability were determined for both assays using ten samples. For the enzyme immunoassay method, day to day variation for IgA, IgM, IgG1 and IgG2 determinations was 17.5, 19.3, 7.6 and 7.3% while variation in repeatability (within a test) was 6.2, 5.9, 3.3 and 4.5%, respectively. Day to day variation for the single radial immunodiffusion test for IgA, IgM, IgG1 and IgG2 was 15.4, 26.0, 11.5 and 18.3% and variation repeatability (within a test) was 11.6, 13.9, 5.9 and 8.3%, respectively. The procedures consistently detected 0.1 micrograms of immunoglobulin whereas the radial diffusion sensitivity limit was approximately 500 micrograms.