A nylon ball solid-phase radioimmunoassay for specific antibodies in human sera. Application to measurement of IgG antibodies to pollen allergens.
The principle of the radioallergosorbent test (RAST) has been used to measure IgG antibodies to timothy grass pollen allergens in sera from desensitized allergic subjects. 125I-labeled goat anti-human IgG was used as detector protein. Non-specific binding was eliminated by use of a non-porous nylon ball an antigen carrier and by use of a special buffer with high ionic strength and pH, containing 1% bovine gamma globulin and 5% normal rabbit serum as 'balance proteins'. At dilution 1:80 non-specific binding was only 0.28% and the binding ratio for a high-liter serum was about 10. By inhibition experiments the assay was demonstrated to be specific for IgG antibodies to timothy grass pollen. The results obtained with this assay correlated statistically significantly with those found th a double -antibody method (rs equal 0.68, n equal 20, t equal 3.93, P less than 0.001). Serum dilution curves were parallel, indicating that the assay is in allergen excess. The within-assay coefficient of variation ranged from 3.9 to 7.6%; the between-assay coefficient of variation from 8.4 to 19.5%. The assay is very simple to perform, requiring no centrifugation. The allergen-coated balls are stable for at least 3 months. The assay should be applicable to measurement of IgG antibodies and IgG subclass antibodies to any protein antigen of interest.
D000906:Antibodies / Q000032:analysis*; D000888:Antibodies, Anti-Idiotypic / Q000276:immunology; D001666:Binding Sites, Antibody; D002021:Buffers; D003429:Cross Reactions; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007074:Immunoglobulin G / Q000032:analysis*; D007700:Kinetics; D008863:Microspheres; D009994:Osmolar Concentration; D011058:Pollen / Q000276:immunology*; D011485:Protein Binding; D011863:Radioimmunoassay / Q000379:methods* / Q000592:standards
D000906:Antibodies; D000888:Antibodies, Anti-Idiotypic; D002021:Buffers; D007074:Immunoglobulin G