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 γ-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 (~1 ng/ml of ragweed-specific IgG). Both assays also demonstrated consistently parallel dilution curves with 51 sera (mean interdilutional coefficient of variation [CV] <8.8% for ELISA and <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 <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 < 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. There is no apparent explanation for the two sera in which ELISA results were consistently higher than SPRIA. The effects of serum dilution on nonspecific binding was minimal (<2% in both assays). These two methods provide comparable results for ragweed IgG antibody measurement in most (>80%) sera. Occasionally, discrepant results would not be expected to be of clinical significance. Performance specifications for the ragweed IgC ELISA were acceptable, and the technical ease of ELISA is superior to the labor-intensive SPRIA. For routine clinical use, the ragweed ELISA is a suitable alternative to SPRIA.