Noncompetitive Hapten Immunoassays Using New-Generation Antibody Reagents

Noncompetitive Hapten Immunoassays Using New-Generation Antibody Reagents 

The invention of B cell hybridoma technology (K3) has allowed the generation of various kinds of useful antibodies, even very minor or rare antibody species elicited in serum by the conventional procedure, as a pure immunochemical reagent in almost unlimited amounts. Among such new-generation monoclonal antibodies, anti-idiotype antibodies and anti-immune complex (anti-metatype) antibodies have been successfully introduced as key reagents enabling noncompetitive hapten immunoassays (Fig. 11). 

Assay Systems Based on Anti-idiotype Antibodies 

Although these assay procedures for steroids were successfully applied to clinical samples, the sensitivity was not extensively improved, as shown by their femtomole range detection limits 2 fmoF/assay for E2, 0.32 nmol/liter (40 fmoF/assay) for progesterone, and 0.4 nmol/liter (8 fmol /assay) for Ei-3G, respectively. Difficulty in obtaining a higher sensitivity would come from the competitive substitution of the bound hapten with the /3 -type antibody and/or imperfect selectivity of the a-type antibody toward the hapten-antibody complex. 

We (K1) attempted to develop a noncompetitive assay based on the anti-idiotype antibodies for a conjugated bile acid metabolite, ursodeoxycholic acid 7-A-acetyl-glucosaminide (UDCA 7-NAG), which is expected to serve as a diagnostic index for an autoimmune disease, primary biliary cirrhosis. In our assay, the hapten UDCA 7-NAG, a /3-type antibody, and a biotin-labeled a-type antibody were simultaneously added to a microtiter plate coated with an F(ab )2 fragment of a specific anti-UDCA 7-NAG antibody, then incubated at room temperature for 8 h. Bound biotin was then detected with HRP-labeled streptavidin, whose enzyme activity was measured using o-phenylenediamine/H202 as a substrate. This noncompetitive assay system provided a subfemtomole-order sensitivity (detection limit 118 amol) that was 7 times lower than the competitive immunoassay using the same anti-hapten antibody (K2), even with a common colorimetric detection (Fig. 13). Somewhat improved specificity was also obtained namely, better 

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