The development of a rapid ELISA for IgE utilizing commercially available reagents

Abstract

An enzyme-linked immunosorbent assay (ELISA) for human immunoglobin E (IgE) has been developed. To coat the polystyrene tubes (the solid phase) an antibody concentration of 6 mg/1 in sodium carbonate buffer, pH 9.8, at 37°C for 24 h was found superior to other conditions. The maximum amount of globulin adsorbed to the polystyrene surface was estimated to be 2.7 mg × m −2. This is consistent with a monolayer being adsorbed. While some of the anti-IgE molecules are inactivated during adsorption, the remaining molecules, once adsorbed, appear to retain activity for extended periods, and the coated tubes were stable for several weeks. Kinetic studies of the association of analyte, of antibody-enzyme conjugate and enzymic catalysis were used for the optimisation of the assay and allowed us to reduce assay time to 6 h. The association of analyte to the coated tube and the association of anti-IgE-peroxidase conjugate to bound IgE, in the present design, had T 1 2 of approximately 0.5 h. Equilibrium is not obtained. The dissociation of analyte, surprisingly, was nearly undetectable. At least one of the two antibodies employed in the sandwich assay, preferably the anti-IgE-peroxidase conjugate, should be free of non-specific antibodies in order to eliminate non-specific reactions. The influence of serum matrix was eliminated by diluting serum samples and standards 1 : 21 in buffer. For the assay of very low concentrations (i.e. < 10 kIU/1) standards produced in IgE-free serum are required to compensate the influence of matrix. Comparison with a commercial kit (Hoechst-Behringwerke, Frankfurt/Main, FRG) showed good agreement. All reagents are commercially available and the assay is well suited for routine use.