Application of the dot immunobinding assay to allergy diagnosis

The recently developed dot immunobinding assay is operationally simple and facilitates performance of multiple simultaneous assays. Here, its use as the basis for determination of total and allergen-specific IgE is established. For total IgE, the same commercially available polyclonal anti-human IgE was used on the solid phase and as a peroxidase conjugate in the liquid phase. After incubation with a chromogenic substrate, IgE was determined from the color intensity of the resulting dots with a scanning reflectance densitometer. The limit of sensitivity was 50 pg/ml of IgE. Standardized conditions gave the dynamic range 50 to 2500 IU/ml in serum. The IgE measured was not subject to interference by serum components, was labile at 56 degrees C, was soluble at 30% saturation (NH4)2SO4, and was unaffected by anti-human immunoglobulins of other specificity. Coefficients of variation were 0.05 within run, and were 0.1 between run. Comparison with data on sera obtained with the PRIST method yielded a correlation coefficient of 0.96 and a linear regression of slope 1.15. Assays for allergen-specific IgE were established with bee venom and dust mite allergens in the solid phase. The same peroxidase-conjugated antibody was used as for total IgE. Comparisons with comparable RAST assays were performed.