Noncompetitive immunoenzymometric assays for a hapten molecule employing anti-idiotype antibodies

Noncompetitive immunoenzymometric assays for a hapten molecule employing anti idiotype antibodies (Ids) are introduced to enable sensitive determination of endogenous steroids in clinical specimens. Ids recognize an epitope in the variable region (idiotope) of anti-hapten antibody (primary antibody); alpha-type Id (Id-alpha) recognizes an idiotope in the framework region, while beta-type Id (Id-beta) recognizes an idiotope on the paratope. The target hapten was captured by an excess amount of primary antibody, and the unoccupied paratope was blocked with Id-beta. The hapten-primary antibody complex was selectively detected with biotin-labeled Id-alpha that does not bind to the antibody blocked by Id-beta because of steric hindrance arising by Id-beta. This method was applied to the determination of ursodeoxycholic acid 7-N-acetylglucosamindes, which are bile acid conjugates expected to be diagnostic markers for primary biliary cirrhosis. A similar assay procedure for 11-deoxycortisol (11-DC), a diagnostic index for pituitary-adrenal function, was also established where the hapten was captured with a biotin-labeled primary antibody and then the complex was captured by immobilized Id-alpha. These methods were approximately 10 times more sensitive than conventional competitive assays employing the same primary antibody. To achieve higher sensitivity, antibody-engineering techniques have been introduced to prepare fusion protein of the single-chain Fv fragment (scFv) specific for 11-DC and alkaline phosphatase (ALP). Employing fusion protein in combination with Id-alpha and Id-beta, recognizing the idiotope on scFv, a highly sensitive single-antibody immunoenzymometric assay for 11-DC was developed. The detection limit of this novel immunometric assay was approximately 500-times lower than a competitive radioimmunoassay based on the corresponding anti-11-DC antibody.