Antibodies binding sites

Novotny, J., et al. Molecular anatomy of the antibody binding site. /. Biol. Chem. 258 14433-14437, 1983. 

Figure 3 Immobilized antibody ELISA. Primary antibody (Y) is passively adsorbed to the surface of a polystyrene microtiter plate. Analyte (free H) and an enzyme-labeled hapten (H-E) compete for the fixed number of primary antibody binding sites. Following a wash step (dotted line), the substrate for the enzyme is added O) and a colored product formed ( ). The amount of product is inversely proportional to the amount of analyte present...

Direct and indirect competition formats, illustrated in Figure 1, are widely used for both qualitative and quantitative immunoassays. Direct competition immunoassays employ wells, tubes, beads, or membranes (supports) on to which antibodies have been coated and in which proteins such as bovine semm albumin, fish gelatin, or powdered milk have blocked nonspecific binding sites. Solutions containing analyte (test solution) and an analyte-enzyme conjugate are added, and the analyte and antibody are allowed to compete for the antibody binding sites. The system is washed, and enzyme substrates that are converted to a chromophore or fluorophore by the enzyme-tracer complex are added. Subsequent color or fluorescence development is inversely proportionate to the analyte concentration in the test solution. For this assay format, the proper orientation of the coated antibody is important, and anti-host IgG or protein A or protein G has been utilized to orient the antibody. Immunoassays developed for commercial purposes generally employ direct competition formats because of their simplicity and short assay times. The price for simplicity and short assay time is more complex development needed for a satisfactory incorporation of the label into the antibody or analyte without loss of sensitivity. 

Antisen, any substance that stimulates the immune system. Antigens are often foreign substances such as bacteria or viruses that invade the body. An antigen is a ligand that contains a region or epitope which is specifically recognized by an antibody binding site. 

In a so-called competitive immunoassay format the antigen competes with a labeled antigen for a limited number of antibody-binding sites. It can be shown that in this case the ultimate sensitivity of the assay (when the [Ab] approaches zero) is dependent on the equilibrium constant K and the reliability of the signal measurement of the bound fraction at zero dose [15],... 

Immunoradiometric assays (IRMAs) are like RIAs in that a radiolabeled substance is used in an antibody-antigen reaction, except that the radioactive label is attached to the antibody instead of the hormone. Furthermore, excess of antibody, rather than limited quantity, is present in the assay. All the unknown antigen becomes bound in an IRMA rather than just a portion, as in a RIA IRMAs are more sensitive. In the one-site assay, the excess antibody that is not bound to the sample is removed by addition of a precipitating binder. In a two-site assay, a molecule with at least two antibody-binding sites is adsorbed onto a solid phase, to which one of the antibodies is attached. After binding to this antibody is completed, a second antibody labeled with 125I is added to the assay. This antibody reacts with the second antibody-binding site to form a sandwich, composed of antibody-hormone-labeled antibody. The amount of hormone present is proportional to the amount of radioactivity measured in the assay. 

Monodisperse microspheres imprinted with theophylline or 17 (3-estradiol were used in competitive radioimmunoassays showing the MIP s high selectivity for the template molecule. In this case the assay is based on the competition of the target molecule with its radioactively labeled analogue for a limited number of antibody binding sites [77,118]. Figure 15 demonstrates that displacing the radioactively marked theophylline from the imprinted polymer was only possible with theophylline as competitor. Structurally related molecules showed effects solely at elevated concentrations [77]. 

Specificity of Antibody binding of Chlordiazepoxide A good number of benzodiazepines are tested for their ability to complete with labelled chlordiazepoxide for the respective antibody binding site. The various competitors are adequately tested at a concentration of 200 ng i.e.., 10-times the concentration of chlordiazepoxide required to produce a 50% inhibition of binding as shown in Table 32.2. 

Despite many novel developments in immunoassay design the principles are confined to two broad approaches those that rely on the competition between antigens labelled with a molecule which may be readily observed (for example, a radioisotope) and unlabelled antigens for a limited number of antibody binding sites and those in which the antibody is available in excess and for which there is no competition for binding sites. 

Fig. 9 Three haptens, [15]—[17], containing a 1,2-aminoalcohol functionality were investigated as alternatives for esterase and amidase induction. Of antibodies raised against hapten [15], 50% were shown to catalyse the hydrolysis of ester [18], thereby establishing the necessity for a compact haptenic structure. Hapten [19] along with [16] was employed in a heterologous immunization programme to elicit both a general and acid/base function in the antibody binding site.

Figure 14.17. Liposome fluoroimmunoassay depiction, in which antigen-liposome conjugates containing fluorescent dye (concentration quenched) compete with analyte antigen for antibody binding sites, followed by wash and detergent lysis, to release the fluorophore for fluorescence measurement.

An example of an evanescent wave fiber optic immunoassay and the associated optics has been described in detail for measurement of anti-rabbit IgG.(130) Rabbit antibody is immobilized on the distal face of an optical fiber. Unlabeled anti-rabbit IgG competes with fluorescein-labeled anti-rabbit IgG for rabbit antibody binding sites... 

ELISA assays may be competitive or noncompetitive. As the name imphes, in a competitive ELISA, enzyme-labeled antigen competes with free antigen (the analyte of interest) for a fixed and limited quantity of immobihzed antibody binding sites. After incubation, the microtiter plate (sohd support) is rinsed to remove all unbound species and the enzyme substrate is added in saturating concentration. The conversion of substrate to produce can be measured continuously (kinetic assay) or, more commonly. 

The substance to be assayed—e.g., the hormone thyroxine in a serum sample—is pipetted into a microtiter plate (1), the walls of which are coated with antibodies that specifically bind the hormone. At the same time, a small amount of thyroxine is added to the incubation to which an enzyme known as the "tracer" (1) has been chemically coupled. The tracer and the hormone being assayed compete for the small number of antibody binding sites available. After binding has taken place (2), all of the unbound molecules are rinsed out. The addition of a substrate solution for the enzyme (a chromogenic solution) then triggers an indicator reaction (3), the products of which can be assessed using photometry (4). 

RIA Development. The essential components of an RIA are (36) 1) competition between a radiolabeled hapten and the analyte for antibody binding sites and 2) separation of the label which is bound to the antibody from label which is free in solution. The assay is based on the expected result that at higher concentrations of competing analyte more label will remain free in solution. 

Immunometric Assays Based on Masking Unoccupied Antibody Binding Sites. . 155... 

The modified single-antibody immunometric assays discussed below are based on principle B2 in Fig. 4. In these assays, unoccupied antibody-binding sites are masked by reaction with a multiply hapten-labeled macromolecule to permit subsequent selective determination of hapten-occupied antibodies. 

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