Enzyme-labeled antibody technique

Noncompetitive ELISA. The usual principle here is the sandwich technique, which requires the antigen to have at least two antibody binding sites (epitopes). Unlabelled antibody is first fixed to microtitre plates a food sample containing antigen (analyte) is then added and allowed to react with the fixed unlabelled antibody (Figure 8.3). Unadsorbed material is washed out and enzyme-labelled antibody then added which reacts with a second site on the bound antigen. Unadsorbed Ab-E is washed off and enzyme activity assayed activity is directly related to the concentration of antigen. 

The antigen then is mixed with serial dilutions of the enzyme-labeled antibody. A chromogenic substrate mixed with the conjugated enzyme yields a water-soluble product, the absorbency of which can be measured by a spectrophotometer. Recent technology has led to the development of rapid tests that do not require intact cells, live organisms, or cell cultures. ELISAs using monoclonal antibody techniques for the rapid detection of HSVs, adenoviruses, and C. trachomatis are highly sensitive and specific. 

Nakane, P. R. and Pierce, G. B., Jr. (1966) Enzyme-labelled antibodies. Preparation and applicauon for the localization of antigens./. Hislochem. Cytochem. 14, 929-931. StembCTger, L. A. (1979) Immunocytochemistry, 2nd Ed., John Wley, New York. Mason, D. Y. (1985) Immunocytochemical labelling of monoclonal anubodies by the APAAP immunoalkaline phosphatase technique, in Techntquestnimmunocylochermstry, Vol. 3 (Bullock, G. R. and Petrusz, P., eds.), Academic, London, pp. 25-42. 

There is increasing interest in the use of nonisotopic labels in immunoassay. The main advantages of such labels in labeled antibody techniques are the expected longer shelf life of the reagent and the ability to use a detection apparatus that may for one reason or another prove to be more convenient—e.g., for automation or cheapness. Some alternatives that are being explored are bacteriophages, spin labels, enzymes, fluorescent compounds, and luminescent compounds. ... 

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... 

Enzyme-Linked Immunosorbent Assay. LlSAis a heterogeneous EIA technique that is widely used in clinical analyses. In this type of assay, one of the reaction components is nonspecifically adsorbed or covalently bound to the surface of a solid phase, such as that of a microtiter well, a magnetic particle, or a plastic bead. This attachment facilitates separation of bound- and free-labeled reactants. In the most common approach to using the ELISA technique, an aliquot of sample or calibrator containing the antigen to be quantitated is added to and allowed to bind with a solid phase antibody. After washing, enzyme-labeled antibody is... 

Enzyme labels are usually associated with solid-phase antibodies in the technique known as enzyme-linked immunosorbent assay (ELISA). There are several variants of this technique employing both competitive and non-competitive systems. However it is best used in combination with two monoclonal antibodies in the two-site format in which an excess of antibody is bound to a solid phase such as a test-tube or microtitre plate the test antigen is then added and is largely sequestered by the antibody (Figure 7.12). After washing... 

The most common of these systems is the enzyme-multiplied immunoassay technique or EMIT, which is particularly suited to the measurement of small molecules (haptens) such as drugs. EMIT is a trade mark of the Syva Corporation of Palo Alto, California. Although it does not involve the separation of bound fraction from free it is nevertheless a competitive assay system. The antigen is labelled with an enzyme in such a way that the enzyme retains its catalytic activity. When the antigen binds to the antibody the enzyme becomes inhibited, probably by an induced conformational change or by steric hindrance of the enzyme active site (Figure 7.15). 

Amplification can be attained with this type of method by employing two indirect techniques simultaneously. After the specimen is thoroughly rinsed, following the labeled secondary antibody incubation, a third antibody may be used, which is enzyme-labeled and reactive against the species immunoglobulin responsible for the secondary antibody. 

The technique described here is for use with monoclonal primary antibodies of mouse origin, but can easily be adapted for use with polyclonal antibodies from other species (i.e., rabbit). This method uses a secondary biotin-labeled antibody and a detection system that employs a biotin-avidin horseradish peroxidase complex linker step, the so-called ABC (avidin-biotin complex) detection system (5) (see Chapter 25). In this detection system, avidin acts as a bridge between the biotinylated secondary antibody and a biotin-labeled peroxidase enzyme. The anchored enzyme, in the presence of H2O2 can then convert the substrate, diaminobenzidine, to a brown or black reaction product that is easily identifiable in the tissue section. 

Enzyme Multiplied Immunoassay Technique (EMIT). This technique employs enzyme-labelled antibiotics which react analogously to the fluroimmunoassay in that a reduction of enzjrme activity is attributed to antibody binding. Higher concentrations of unlabelled drug in the sample result in less enzyme-labelled drug bound to the antibody. 

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