Antigens binding immunoassay

Homogeneous immunoassays rely on a change in the intensity of the label signal that occurs when labeled antigen binds with antibody. When the label is an antibody, a reduction in the rate of enzyme catalysis forms the basis for the assay. This technique... 

Competitive immunoassays may also be used to determine small chemical substances [10, 11]. An electrochemical immunosensor based on a competitive immunoassay for the small molecule estradiol has recently been reported [11]. A schematic diagram of this immunoassay is depicted in Fig. 5.3. In this system, anti-mouse IgG was physisorbed onto the surface of an SPCE. This was used to bind monoclonal mouse anti-estradiol antibody. The antibody coated SPCE was then exposed to a standard solution of estradiol (E2), followed by a solution of AP-labeled estradiol (AP-E2). The E2 and AP-E2 competed for a limited number of antigen binding sites of the immobilized anti-estradiol antibody. Quantitative analysis was based on differential pulse voltammetry of 1-naphthol, which is produced from the enzymatic hydrolysis of the enzyme substrate 1-naphthyl phosphate by AP-E2. The analytical range of this sensor was between 25 and 500pg ml. 1 of E2. 

Antibody molecules have no inherent characteristic that facilitates their direct detection in immunoassays. A second important step in developing a successful immunoassay, therefore, involves the incorporation of a suitable marker . The marker serves to facilitate the rapid detection and quantification of antibody-antigen binding. Earlier immunoassay systems used radioactive labels as a marker (radioimmunoassay RIA) although immunoassay systems using enzymes (enzyme immunoassays EIA) subsequently have come to the fore. Yet additional immunoassay systems use alternative markers including fluorescent or chemiluminescent tags. 

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). 

Competitive binding immunoassays require a pure, labelled sample of the antigen... 

The presence of a cross-reacting antigen in a sample will result in falsely increased test values when using competitive binding immunoassays... 

Other immunoassays are based on the same antibody-antigen binding reaction but use a different labeling system for detection. Instead of an enzyme label, there are radioactive isotopes, and fluorescent and luminescent labels. Some important immunoassays are defined below ... 

Surface-plasmon resonance (SPR) has been used to detect surface-bound chemical species. SPR is achieved to detect the binding events of antibody and antigens in immunoassays. A gold-coated PMMA chip that was sealed by a PDMS channel plate was used. The antibodies were first immobilized on the gold layer. Upon binding with benzo[a]pyrene (BaP) 2-hydroxybiphenyl (HBP), the SPR signal was recorded [740]. 

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). 

Immunoassays derive their imique characteristics from two important properties of antibodies the strength of the antibody-antigen binding force and the narrow selectivity within a class of compoimds, sometimes even with absolute specificity for the analyte they were designed for. These two properties allow selective measurement of very low concentrations of analyte even in very complex matrices (e.g., biological fluids, residual waters, etc.). For clarity of presentation, the main terms and concepts used in immunoassays are listed in Glossary . 

Because radioisotopes were the first labels used in immunoassay methods, there exists a vast array of radioimmunoassays. Radioisotopes are easily detected at low levels, and labeling procedures are simple. The label has virtually no effect on antibody-antigen binding. However, radioisotopes are costly, hazardous, and require inconvenient monitoring and disposal procedures. In addition, isotopic decay necessitates the regular replacement of the labeled component. 

Polymer (2) was used as a test substance. It contains the dinitrophenyl-moiety, a classical hapten (DNP). Production of DNP-specific antibodies was measured by a radio-immunoassay (antigen-binding test). 

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