SANDWICH IMMUNOASSAY FOR THE HAPTEN ANGIOTENSIN-II

Immunoassays for haptens such as short peptides or drugs are usually b ased on the principle of competition for a limited number of binding s ites on antibody molecules. Owing to the small size of these antigens it has been thought that two specific antibodies cannot simultaneously bind a hapten. However, antisera containing so called anti-metatypic antibodies have been reported (Voss et al. (1988) Mol. Immunol. 25, 75 1-759) that bind to hapten-mAb complexes in a reaction where conformat ional changes on the primary antibody are important. Here, we report o n monoclonal antibody pairs able to form ternary complexes with the oc tapeptide angiotensin II. The first mAb (mAb1) is conventional and bin ds angiotensin II with high affinity (K-d 10(-11) M). The secondary (a nti-metatypic) mAbs (mAb2s) recognize the immune complex consisting of angiotensin II bound to mAb1, but only poorly recognize mAb1 alone. A n immunization technique involving tolerization with uncomplexed mAb1 was used to generate mAb2s. None of the mAb2s were able to bind angiot ensin II by themselves but all efficiently bound the complex of angiot ensin II and mAb1. All mAb2s stabilized the angiotensin II-mAb1 comple x and one mAb2 distinctly improved the specificity of the assay for an giotensin II. By either labelling mAb1 and immobilizing mAb2 (or vice versa) two-site immunometric assays with detection limits of 1 pg/ml a ngiotensin II have been established. The kinetics of the complex forma tion was investigated by fiber optic biospecific interaction analysis (FOBIA), a system allowing real time observation of binding events on the surface of a glass fiber. The association rate towards the ligande d conformation of mAb1 was higher than towards the free mAb1. By contr ast, the mAb2s dissociated at similar rates from complexed and uncompl exed mAb1.