Enzyme-hapten conjugate immunoassay

Competitive homogeneous enzyme immunoassays using enzyme-hapten conjugates The prototype of these assays (Ruben-... 

In plant hormone enzyme-linked immunoassays (ELISA), the tracer is an enzyme-hapten conjugate, usually constructed in the same way as the immunogen. The separation of free from bound tracer is achieved by initially immobilising the antibody by adsorption... 

An alternative format for en me immunoassays makes use of an en me-hapten conjugate, and is illustrated in Figure 2B. In this format, me antibody is immobilized onto me solid phase, and the antibody partitions between this conjugate and me analyte in the sample. The antibotfy may eimer have been immobilized on a solid phase to begin wim, or may be trapp on a solid phase after me incubation period. The amount of enzyme-hapten conjugate retained by me antibody on me solid is then inversely proportional to the amount of free analyte in me sample, and may be quantified wim a suitable substrate for me enzyme. 

Kitagawa, T., Kawasaki, T., and Munechika, H. (1982) Enzyme immunoassay of blasticidin S with high sensitivity a new and convenient method for preparation of immunogenic (hapten-protein) conjugates. J. Biochem. (Tokyo) 92, 585-590. 

Figure 14.3. Distinction between homogeneous and heterogeneous immunoassay formats. Haptenic analytes are indicated as triangles, and conjugated fluorescent probes are indicated by the letter F. In this hypothetical depiction, the homogeneous immunoassay is quantitated in the original reaction mixture. The heterogeneous immunoassay requires removal of unreacted tracer, further addition of reagents such as an enzyme to release a fluorescent molecule F, followed by quantitation.

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... 

Schneider, P. and B.D. Hammock. 1992. Influence of the ELISA format and the hapten enzyme conjugate on the sensitivity of an immunoassay for S-triazine herbicides using monoclonal-antibodies. J. Agric. Food Chem. 40 525-530. 

Homogeneous enzyme immunoassays using avidin-ligand conjugates Interaction of avidin (or haptenated avidin) with... 

A homogeneous enzyme immunoassay based on antibody inhibition of enzyme conversion from the apo- to the holo- form has been developed for DNP-aminocaproic acid, DNP-ACA (Ngo et al., 1985). A competitive equilibrium is established between the analyte DNP-ACA and DNP-conjugate apoglucose oxidase, DNP-CAGO, which is the labeled hapten. Flavin adenine dinucleotide, FAD, added to the mixture binds to free DNP-CAGO to give DNP-CAGO FAD, which is enzymatically active and... 

Nonisotopic immunoassays Use of nonisotopic immunoassays has, in the last few years, become increasingly common in both research and clinical laboratories. Although these immunoassays do not employ radioactive labels, they may have sensitivities similar to those of classic RIA. The immunochemical mechanism and principles are the same as for RIA only the label and the method of measurement are different. The most common labels are enzymes and fluorescent conjugates, which may be covalently bound to antibodies or haptens. 

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