Enzyme-hapten conjugate

Rubenstein et al. (1972), subsequently, developed a homogeneous system in which the enzyme is labeled, generally with a hapten, without seriously affecting its activity. This method is applicable if the antibody reacting with the hapten either reduces or increases enzyme activity. The presence of free hapten in the added sample lowers the fraction of antibody able to react with enzyme-hapten conjugate and thus lowers the effect of the antibody on the enzyme. 

One of the 6 lysyl residues (residue 97) of lysozyme is near the active site. Conjugation of a hapten to this group does not affect the activity of the enzyme, but incubation of the enzyme-hapten conjugate with the specific anti-hapten antibody prior to the addition of the bulky substrate, produces serious inhibition. The smaller Fab is not as effective for inhibition as complete IgG. 

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. 

Remove small molecules by dialysis or passage through Sephadex G-25. About 75% of the enzyme is conjugated to hapten (using nortryptyline instead of desmethylnortryptyline, only about 15% is conjugated). 

In this approach double-stranded oligonucleotide PCR amplicons are labeled with a hapten on one end and another different hapten on the other end during PC R. The transducer surface (gold or carbon screen-printed chips) is treated with protein based anti-haptens that capture one of the hapten label on the PCR amplicons. The second hapten label on the PCR amplicons will bind to a specific anti-hapten-conjugated HRP or AP reporter enzyme to labeled PCR amplicons. Addition of the enzyme-specific redox substrate and application of a fixed potential between working and reference... 

A reporter enzyme may be directly coupled to the DNA probe such that no secondary binding step is required. Alternately, the enzyme is conjugated to avidin or a monoclonal antibody that binds to a hapten label on the DNA probe. Assays that use direct enzyme-labeled probes are capable of extremely low backgrounds and can be operationally simpler and hence less expensive, faster, and more amenable to high-throughput screening. 

SMCC frequently is used to prepare hapten-carrier or antibody-enzyme conjugates. In both applications, one of the molecules is activated (usually the carrier or the enzyme) with the... 

The following is a generalized protocol for the activation of a protein with sulfo-SMCC with subsequent conjugation to a sulfhydryl-containing second molecule or protein. Specific examples of the use of this crosslinker to make antibody-enzyme or hapten-carrier conjugates may be found in Chapter 20, Section 1.1 and Chapter 19, Section 5, respectively. 

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. 

---