Labeling antibodies with enzymes

Labeling antibody with an enzyme can be accomplished rapidly and efficiently by the gluteraldehyde linkage method. Other widely used methods for labeling antibodies with enzymes include those of Nakane and Kawaoi and Engvall and Perlmann. Voller has described in techni-... 

In addition to labeling immunoglobulins with enzymes to provide detectability through their catalytic action on a substrate, antibody molecules also can be labeled or tagged with small... 

Enzyme immunoassay (El A) is one of such methods that label antigen or antibody with enzyme. The most representative form of El A is the enzyme-linked immunosorbent assay (ELISA) in which bound antigen or antibody is detected by a linked enzyme that converts a colorless substrate into a colored product (Figure 6.11). 

The use of biotinylated antibodies provides perhaps the greatest versatility and sensitivity of all methods The affinity of biotin for avidin or the more usually used streptavidm is very high, and the latter can be conjugated to radioisotope, fluorescent moiety, or enzyme Again, the basic procedures are the same as outlined for l25I-labeled antibodies with the additional steps required for streptavidin binding and subsequent incubation with enzyme substrate (see also Chapters 17 and 18)... 

The first scientists who started to label proteins with enzymes were histochemists. They labeled different antibodies and antigens for easier detection by light and electron microscopy of certain proteins in histochemical preparations Further... 

ELISA Using Enzyme-Labeled Antibody. Another type of competitive ELISA employs enzyme-labeled antibody with the antigen attached to a solid phase. In this technique, the binding of enzyme-labeled antibody to... 

Labeling of Antigens and Antibodies with Enzyme. Many methods exist for coupling haptens, proteins, and carbohydrates to proteins. Some of the most commonly used are summarized in the table together with some selected references. An extensive review on protein-protein coupling reactions has recently appeared and could be used as a source of additional chemical details on the coupling methods. 

The adsorption process, unlike antigen-antibody interactions, is nonspecific. Thus, during the incubation of the immobilized antigen or antibody with enzyme-labeled antigen or antibody, the latter binds specifically to the immobilized immune reactant, but may also be adsorbed directly onto the solid phase. This nonspecific adsorption of enzyme activity can be minimized by inclusion of a nonionic detergent such as Triton X-lOO or Tween 20. These do not interfere with the antigen-antibody reaction but prevent formation of new hydrophobic interactions between added proteins and the solid phase without disrupting to any appreciable extent the hydrophobic bonds already formed between the previously adsorbed protein and the plastic surface. 

Enzymes are currently the most widely used and investigated labels for immunoassays, because a single enzyme label can provide multiple copies of detectable species. This catalytic amplification results in immunoassay detection limits that rival those of radioimmunoassay without the storage and disposal problems associated with radioisotopes. Enzyme immunoassays label either ligands or antibodies with enzyme, and enzyme activity in bound or free fractions is measured. Heterogeneous immunoassays employing enzymatic labels have been named enzyme-linked immunosorbent assays (ELISAs). ELISA methods usually employ antibody immobilized onto the wells of polystyrene microtiter plates, and may be... 

Incubate 50 /i of enzyme-labeled antibody with various amounts of antigen (50 /il) for 30 min at 37 C. 

Thus, a worker with relatively few reagents and the ability to label antibodies with an enzyme may have enough materials to develop assays. This brief description of system possibilities has concentrated on antibody detection. Note that most of these comments are relevant to antigen detection. 

This chapter provides protocols for the most convenient methods of labelling mouse IgG monoclonal antibodies with enzymes (alkaline phosphatase and peroxidase), a fluorescent molecule (fluorescein), biotin, and DIG. A general protocol for the evaluation of the labelled monoclonal antibody is also given. 

As an alternative, extremely sensitive detection can be achieved with reporter antibody probes tagged with intensely SERS-active compounds or with enzymes that react with substrates to yield SERS-active products. These methods often involve sandwich immunoassay techniques, which increase the number of required steps but offer the advantages of excellent sensitivity and the potential for label multiplexing. For example, Nie and coworkers recently reported the simultaneous detection of two types of antigens in a... 

In a direct immunoassay the immobilized antibody binds to the corresponding antigen. The competitive immunoassay relies upon the competition of the analyte with a labelled analyte for antibody binding. These formats are widely used for high throughput affinity arrays. A sandwich immunoassay is based on the trapping or capture of the analyte by another antibody. In ELISA (enzyme linked immunosorbent assays) the second antibody is conjugated with an enzyme. The bound enzyme labelled antibody is detected by its ability to break down its substrate to a colored product. 

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