Competitive immunoassay metabolite detection

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... 

Two different types of immunoassays, a competitive-type and an inhibition-type, were developed and compared for the insecticide, endosulfan. The detection range of both assay types was similar, 3-500 ng/ml for the competitive-type and 5-500 ng/ml for the inhibition-type assay. Metabolites of endosulfan and other insecticidal chloro-hydrocarbons possessing a hexachlorocyclopentene structure exhibited considerable crossreaction in both assays, so these immunoassays could be used for detection of class-specific compounds. The inhibition-type immunoassay showed less susceptibility to interfering factors and, therefore, seemed to be more suitable for environmental analysis. 

Monoclonal antibodies were obtained against atrazine and its metabolite hydroxyatrazine by immunizing mice with atrazine or hydroxyatrazine protein conjugates. By competitive ELISA, we observed that the antibodies raised against hydroxyatrazine cross-reacted mainly with hydroxypropazine. The antibodies raised against atrazine cross-reacted with propa-zine, prometone, prometryne, and to a much lower extent with a few other s-triazines and hydroxy-s-triazines. Atrazine could be detected in water samples down to 50 ppt. Average recoveries measured by ELISA from soil samples fortified with atrazine or hydroxyatrazine were comparable to those measured by GLC or HPLC. Soil samples of unknown atrazine content were analyzed by GLC, GC-MS, and by ELISA. The results show that the ELISA immunoassay represents a valuable detection method for trace amounts of atrazine and hydroxyatrazine in soil. 

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