THREE-LAYER SOLID PHASE BINDING ASSAY TECHNIQUES

摘要

SPECIFIC BINDING ASSAY TECHNIQUES The standardization of a labelled-constituent, such as anti-IgE, for use in a specific binding assay method is disclosed. The method involves standardizing a labelled ligand (eg., radiolabelled IgE) against a ligand reference substance (eg., WHO standard IgE) to determine the weight of IgE protein represented by the labelled ligand, expressed in terms of the reference substance (eg., WHO units). Antilight chain antibodies are attached to a solid phase and contacted with varying concentrations of the labelled ligand to bind the ligand to the anti-light chain antibodies. The ligand is then contacted with the labelled constituent and the amount of labelled constituent, in relation to the amount of ligand protein present, expressed in terms of the reference substancej is determined. The labelled constituent is thus standardized, since the actual weight of a ligand can be determined from the amount of labelled constituent bound thereto. Also disclosed is an improved specific binding assay test method for determining the potency of an allergen extract. The test method generally involves inhibition by the soluble extract, of the binding of a ligand (provided by serum from an allergic individual) to solid-phase allergen as assessed by the standardized-labelled constltutent (antiIgE). The improvement involves using a parallel model system of a second complex which consists of anti-light chain antibodies, labelled ligand and the standardized labelled constituent. Because the actual weight of a ligand bound to the labelled constituent is known, from the standardization procedure described above, the amount of standardized labelled constituent bound to the ligand in the first complex can be determined. The weight of ligand inhibited by addition of soluble allergen is then used as a measure of the potency of the allergen extract.