Monoclonal antibodies generated against an affinity-labeled immune complexof an anti-bile acid metabolite antibody: an approach to noncompetitive hapten immunoassays based on anti-idiotype or anti-metatype antibodies

Conventional immunoassays for haptens such as steroids and synthetic drugs are dependent on the competitive reaction between an unlabeled antigen (ana lyte) and a labeled antigen against a limited amount of anti-hapten antibod y. Although noncompetitive immunoassay procedures such as two-site immunome tric assays offer a much higher sensitivity, direct application of this pri nciple to haptens has been difficult due to their small molecular mass prec luding simultaneous binding by two antibody molecules. Here, we have attemp ted to develop a noncompetitive immunoassay system based on anti-idiotype o r anti-metatype antibodies. Ursodeoxycholic acid 7-N-acetylglucosaminide (U DCA 7-NAG), which is a bile acid metabolite (molecular weight, 595.8), was selected as the model hapten. A/J mice were immunized with a monoclonal ant ibody against UDCA 7-NAG, which had been affinity-labeled with a relevant h apten derivative. The fusion between the immune spleen cells and P3/NS1/1-A g4-1 myeloma cells yielded four kinds of or-type and two kinds of beta -typ e monoclonal anti-idiotype antibodies, each recognizing the framework regio n and paratope of the anti-hapten antibody. The use of a selected combinati on between alpha -type and beta -type antibodies together with the anti-hap ten antibody provided a noncompetitive assay system with a subfemtomole ord er sensitivity (detection limit, 118 amol) and a practical specificity. (C) 2000 Elsevier Science B.V. All rights reserved.