A computerized micro-ELISA assay for allergen-specific IgE antibodies

Abstract

A computer-normalized micro-ELISA assay employing horseradish peroxidase and polystyrene microtiter plates (IP assay) for allergen-specific IgE antibodies is described. The specificity of the assay was confirmed by heat inactivation and multiple absorption experiments. Individual allergen blanks were used to account for the variability in the nonspecific binding among various allergens. The results obtained in milliunits of absorbance were normalized by a computer protocol using four reference sera. The coefficients of variation for the intraassay and interassay reproducibility ranged from 1.49 to 9.33% and 7.11 to 15.19%, respectively. Direct correlations between the results of the IP and the RAST assays (Absorbance vs. bound radioactivity) were excellent; the values for correlation coefficients for six pollens (June grass, Bermuda grass, Short ragweed, English plantain, Oak and Box elder), house dust mite, and two epidermal (cat and dog) allergens ranged from 0.88 to 0.99. Correlation between the two assays was lacking for the Alternaria and Penicillium mold allergens (r values: Alternaria 0.32, Penicillium 0.5). For sera from patients with a positive history and positive skin tests challenged with 11 allergens cited above, the IP assay detected low levels of specific IgE in many cases where the RAST assay was negative (from 10% for June grass to 48% for Alternaria and Penicillium mold antigens). The RAST positivity accompanied by the IP negativity, by contrast, was rarely seen. The superior sensitivity of the IP assay for specific IgE antibodies was achieved by lowering the background "noise" in the IP assay. This was attributed to the following factors: (1) use of individual allergen blanks; (2) 1:5 dilution of the serum sample that minimizes interference from high levels of total IgE antibodies and from allergen-specific IgG antibodies; (3) nonporous nature of the polystyrene immunosorbent surface; and (4) the use of a computer data reduction system to normalize the assay results.