A Sandwich Enzyme Immunoassay for the Detection of Human IgG Antibodies to Dog Allergens

Abstract

A double-sandwich enzyme immunoassay (DSEIA) was developed for the detection of human IgG antibodies to dog dander and hair (DH) allergens. Since DH allergens are immunogenic in rabbits, the gamma-globulin fraction of rabbit antiserum to DH (RGG-a-DH; 10 micrograms/ml) was used for coating of polystyrene microtiter plates. Dog allergens (1 microgram/ml of DH extract) were bound to RGG-a-DH. Binding of human IgG Ab to nonimmune RGG (10 micrograms/ml)--used as a background control--was substracted from the DH specific one and nonspecific binding was further eliminated by using 0.5% of normal rabbit serum in the dilution buffer. Sera were diluted 1:10 in this buffer. Specificity of the assay was shown by absorption experiments: protein A, anti-IgE discs (Phadebas PRIST) and dog or birch (e5, t3; Phadebas RAST) allergen discs were used to remove total and allergen specific IgG and IgE, respectively. The results were expressed as net absorbances or as a percentage of a reference serum. A significant correlation (r = 0.65, p less than 0.001) between IgG (DSEIA) and IgE (RAST) antibodies to dog allergens was observed in 40 untreated dog allergic subjects. The DSEIA was found to be more sensitive than conventional ELISA in detecting IgG Ab in 15 asthmatic children during hyposensitization: a significant rise was observed in 14 compared to 12 with ELISA, while no significant increases were observed in the placebo-treated group.