Enzyme amplification methods

An ingenious method of decreasing detection limits in biosensors that is frequently employed is enzyme amplification. An example is enzyme multiplied immunoassay technology (emit). 

Self, C. H. (1985) Enzyme amplification—a general method applied to provide an immunoassisted assay for placental alkaline phosphatase. J. Immunol. Methods 76, 389-393. 

If the major aim of the ELISA is to obtain quantification of substances present in extremely low concentrations there are a number of adaptations to the technique that can be used. Such techniques often use alkaline phosphatase enzyme systems, which can be used, for example, to lock into a circular redox cycle producing an end product such as red formazan which, is hugely amplified in comparison to standard amplification methods (4). Chemiluminescent amplified ELISA principles have also been shown to give very high... 

Fig. 7.8. Amplification generated by the production of a catalytically active NAD. The main problem with this technique is that even trace amounts of NAD in the enzyme or NADP preparations, will lead to high backgrounds. Alternative amplification methods include enzyme cascades (Blake et al., 1984) and catalyzed reporter deposition (Bobrow et al., 1991).

One advantage of indirect avidin-biotin method is an amplification of the detection system. For example, after biotin conjugated 2° antibody, avidin is incubated, binding to the available biotins. After the remaining free avidin is rinsed off, a biotin conjugated to 488 fluorophore is added and it binds to the remaining sites on the avidin. With this method, amplification occurs because any avidin can be attached to multiple biotins conjugated with either fluorescent or enzyme. This method requires two additional incubation steps of the indirect immunocytochemistry with fluorescent-labeled 2°. 

Further enhancement in detection sensitivity of reporter enzymes is achievable by enzyme amplification or cascade reactions often termed enzyme cycling assays. One approach is to use alkaline phosphatase as the reporter enzyme. Phosphatase cleavage of NADP forms NAD, which enters cyclic reactions catalyzed by alcohol dehydrogenase and diaphorase. Each turnover of phosphatase substrate initiates a cascade resulting in numerous detectable product molecules. Such approaches, perhaps incorporating chemiluminophores as the terminal product, hold promise for further extension of the sensitivity of immunoenzymatic methods. 

Work with algae described above shows that the roost sirople to the roost coroplex (antibody/antigen with enzymic amplification) biological systems provide ready-made microstructures and molecular systems for coupling to electrodes for highly sensitive and selective electroanalytical methods. 

A diagnostic method using fluorescence labeled DNA probes to detect and quantify the number complementary chromosomal sequences on a cellular resolution. A related technique that also allows assessment of gene amplifications, but without precise quantification of copy numbers is the chromogenic in situ hybridization (CISH). Here, instead of a fluorescent dye an enzyme that can generate a colored precipitate in the tissue samples is coupled to the DNA probe. 

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