Measurement of IgG blocking antibody in human serum: comparison of ELISA with monoclonal antibody and fluorogenic substrate and Staphylococcus protein A solid-phase RIA.
We compared ELISA with mouse monoclonal antihuman gamma-chain antibody and a fluorogenic substrate with the Staphylococcus protein A solid-phase radioimmunoassay (SPRIA) in the measurement of specific IgG antibody to short ragweed pollen. Sera from 51 ragweed-allergic patients undergoing allergen immunotherapy were evaluated for ragweed-specific IgG antibodies with the same ragweed extract in the two assay systems. With optimal conditions, the ELISA and SPRIA displayed comparable positive thresholds (approximately 1 ng/ml of ragweed-specific IgG). Both assays also demonstrated consistently parallel dilution curves with 51 sera (mean interdilutional coefficient of variation [CV] less than 8.8% for ELISA and less than 8.6% for SPRIA). Reproducibility was determined by constructing precision profiles for intra- and interassay variations over the working ranges of each assay (ELISA, 0.8 to 100 ng/ml; SPRIA, 1 to 250 ng/ml). ELISA intra-assay CVs ranged from 13% near threshold to less than 5% at higher antibody concentrations; SPRIA intra-assay CVs ranged from 4.3% to 2.8%. Interassay reproducibility was somewhat better for SPRIA (4.6% to 9.6%) than for ELISA (10% to 18%). In direct comparison, 41 (80%) of the 51 sera were concordant in the two assays (r = 0.91; p less than 0.001). Although each assay result was reproducible, 10 (20%) of the sera elicited consistently discrepant results in the two assays. In eight of the 10 discordant sera, the SPRIA results were higher than ELISA, suggesting the possibility that some ragweed allergen may be better represented on the short ragweed-pollen extract agarose than on ELISA plate wells.(ABSTRACT TRUNCATED AT 250 WORDS)
Lee
H B
HB
Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Md.
Adkinson
N F
NF
D000906:Antibodies / Q000378:metabolism*; D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D001667:Binding, Competitive; D002863:Chromogenic Compounds; D004797:Enzyme-Linked Immunosorbent Assay; D005456:Fluorescent Dyes; D006801:Humans; D007074:Immunoglobulin G / Q000276:immunology*; D011863:Radioimmunoassay / Q000379:methods; D006255:Rhinitis, Allergic, Seasonal / Q000097:blood / Q000276:immunology; D013205:Staphylococcal Protein A
D000906:Antibodies; D000911:Antibodies, Monoclonal; D002863:Chromogenic Compounds; D005456:Fluorescent Dyes; D007074:Immunoglobulin G; D013205:Staphylococcal Protein A