Enzyme-labeled antigen methods

Falini et al. (1980) compared the indirect enzyme-labeled antibody method, the SpA-POase method, the PAP method and the indirect enzyme-labeled antigen method. The big difference of the SpA-POase method with the other methods was the almost complete absence of background staining, though the detectability was lower than... 

Figure 7.15 Enzyme-multiplied immunoassay (EMIT). The three reactants, test (or standard) antigen, enzyme-labelled antigen and a limited amount of antibody are allowed to react and reach an equilibrium position. The unbound labelled antigen which remains is the only source of enzyme activity, the bound enzyme being inactivated. This free enzyme can be quantitated using a direct kinetic assay method and is proportional to the amount of unlabelled antigen originally present.

Rubenstein, Schneider, and Ullman described a HOIA method for morphine using lysozyme as the enzyme label. The covalent enzyme labeled antigen (Ag ) competes with sample antigen (Ag) for a limited concentration of antibody (Ab) to form a complex. The resultant complex exhibits very little enzyme activity because of either steric hindrance or... 

Enzyme-Labeled Antigen. In this method, the sample is first incubated with a moderate excess of solid-phase immobilized antibody (Ab ). After washing, excess enzyme-labeled antigen (Ag ) is allowed to bind to unreacted Ab . 

These four methods have been combined to develop a number of EIAs for quantifying antigens, antibodies, and haptens. For example, the enzyme multiplied immunoassay technique (EMIT ) is a commercially available homogeneous and competitive EIA for the determination of a variety of drugs. In addition, double antibody methods that utilize a heterogeneous and competitive EIA system are available. In this case, enzyme-labeled antigen and sample antigen are mixed... 

For indirect immunoassay methods, the antigen (analyte) is bound to support materials and excess binding sites are blocked. Analyte and primary antibody are then added simultaneously, followed by the addition of enzyme-labeled secondary antibody and color reagent. The bound analyte (coating antigen) and free analyte (in... 

Ishikawa, E., Hashida, S., Kohno, T. and Tanaka, K. Methods for enzyme-labeling of antigens, antibodies and their fragments , in Ngo, T. T. (ed.), Nonisotopic Immunoassay. Plenum Press, New York, 1988, p. 37. 

The principle behind the test method(s) is that antibodies are made of proteins that recognize and bind with foreign substances (antigens) that invade host animals. Synthetic antibodies have been developed to complex with petroleum constituents. The antibodies are immobilized on the walls of a special ceU or filter membrane. Water samples are added directly to the cell, while soils must be extracted before analysis. A known amount of labeled analyte (typically, an enzyme with an affinity for the antibody) is added after the sample. The sample analytes compete with the enzyme-labeled analytes for sites on the antibodies. After equilibrium is established, the cell is washed to remove any um-eacted sample or labeled enzyme. Color development reagents that react with the labeled enzyme are added. A solution that stops color development is added at a specified time, and the optical density (color intensity) is measured. Because the coloring agent reacts with the labeled enzyme, samples with high optical density contain low concentrations of analytes. Concentration is inversely proportional to optical density. 

Noncompetitive ELISA methods are based on sandwich assays in which an excess supply of immobilized primary antibody, the capture antibody, quantitatively binds the antigen of interest and an enzyme-labeled secondary antibody is then allowed to react with the bound antigen forming a sandwich. A color reaction product produced by the enzyme is then used to measure the enzyme activity that is bound to the surface of the microtiter plate. Sandwich ELISA (noncompetitive) methods yield calibration curves in which enzyme activity increases with increasing free antigen concentration. 

Enzyme-linked immunosorbent assay (ELISA) is a new method in alkaloid studies. The application of ELISA to alkaloid study is based on antibody incubations. It differs from classical precipitation-based methods in that specific antigen-antibody interactions are recognized by assaying an enzyme label conjugated to one reactant, usually an antibody. Because of the sensitivity with which enzyme... 

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). 

Most electrochemical immunosensors use antibodies or antigens labelled with an enzyme that generates an electroactive product which can be detected at the electrochemical transducer surface. The combination of high enzyme activity and selectivity with the sensitive methods of electrochemical detection provides a basis for the development of immunosensors. Horse radish peroxidase (HRP) and alkaline phosphatase (AP) are popular enzyme labels and can be used with a variety of substrates. 

When the deoxynucleotide is associated with a fluorochrome, the cells can be observed under a fluorescence microscope, whereby apoptotic cells present an intense fluorescence and, in advanced stages, nuclear fragmentation can be visualized. For a quantitative analysis, either a hemocytometer in an optical microscope or a flow cytometer can be used (Tinto et al., 2002). Peroxidase-marked deoxynucleotides can be quantified by chromo-genic tests that use the enzyme substrate. Indirect methods use antigens linked to the nucleotide and recognized by labeled antibodies. However,... 

Immunoassays, electrochemical — A quantitative or qualitative assay based on the highly selective antibody-antigen binding and electrochemical detection. Poten-tiometric, capacitive, and voltammetric methods are used to detect the immunoreaction, either directly without a label or indirectly with a label compound. The majority of electrochemical immunoassays are based on -> voltammetry (-> amperometry) and detection of redox-active or enzyme labels of one of the immunochemical reaction partners. The assay formats are competitive and noncompetitive (see also -> ELISA). 

Bovine serum albumin (BSA) and cyclic AMP (cAMP) are determined by a competitive binding enzyme immunoassay (315). With urease as label, an ammonia gas-sensing electrode is used to measure the amount of urease-labeled antigen bound to a double-antibody solid phase by continuously measuring the rate of ammonia produced from urea as substrate. The method yields accurate and sensitive assays for proteins (BSA less than 10 ng/mL) and antigens (cAMP less than 10 nM), with fairly good selectivity over cGMP, AMP, and GMP. 

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