Investigation of IgG4 levels in atopic patients using a competitive inhibition assay employing biotinylated IgG4 myeloma and avidin peroxidase.
Modification of a 'sandwich' ELISA assay developed for the determination of serum IgE levels proved to be unsatisfactory for the measurement of IgG4. This was attributed to the limited capacity of the microtitre plate solid phase which required high serum dilutions in order to measure IgG4 levels. To overcome this problem a competitive inhibition assay was developed with monoclonal anti-IgG4 attached to the plate. In this system biotinylated IgG4 myeloma and sample IgG4 compete for the limited antibody binding sites present on the solid phase. The attached biotinylated myeloma is detected by addition of avidin conjugated with peroxidase and following development with substrate, IgG4 levels are calculated by reference to a calibrated inhibition curve. The inhibition ELISA assay has been used clinically to measure IgG4 levels in atopic and normal individuals and the values obtained correlated closely (r = 0.99) with the IgG4 levels determined by radial immunodiffusion. For 43 atopic dermatitis patients investigated the median IgG4 level was 1.1 g/l which was significantly elevated when compared to a median of 0.385 g/l for 60 blood donors (P less than 0.0001, Mann-Whitney U). Among the 47 hay fever patients investigated the median was 0.6 g/l which, although lower than in atopic dermatitis, was again significantly increased (P less than 0.025). Within this latter group, 25 patients were investigated for the effects of desensitization with commercial grass pollen injections. The total IgG4 showed a variable but significant rise between the start and finish of treatment (P less than 0.01 Wilcoxon signed ranks test).
D001667:Binding, Competitive; D001710:Biotin / Q000378:metabolism; D003876:Dermatitis, Atopic / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D006969:Hypersensitivity, Immediate / Q000276:immunology*; D007124:Immunoenzyme Techniques; D007074:Immunoglobulin G / Q000032:analysis*; D006255:Rhinitis, Allergic, Seasonal / Q000276:immunology
D007074:Immunoglobulin G; D001710:Biotin