Antigen-binding site formation

Figure 7.4 Basic structure of an IgG molecule. Two heavy chains (440 residues) and two light chains (214 residues) are joined by disulphide bonds and each shows a relatively constant amino acid sequence in one section (C-terminal end) and a variable sequence section (N-terminal end). The variable regions of both heavy and light chains are involved in the formation of the antigen-binding site.

Enzyme-multiplied immunoassay technique Perhaps the best known homogeneous assay format is the enzyme-multiplied immunoassay technique (EMIT), in which the analyte is covalently attached to an enzyme, and the formation of an analyte-antibody complex blocks the active site and inhibits enzyme activity. When this blocked enzyme is mixed with the experimental sample, there is competition between the enzyme-linked analyte and the sample analyte for occupation of the antibody s antigen-binding site. The more of the analyte present in the sample, the more of the enzyme is released from inhibition, and the level of enzyme activity can thus be used to determine the quantity of the analyte. 

Figure 2 Immobilized antigen ELISA format. Antigen is immobilized to a solid phase by passive adsorption. Following removal of unbound antigen, analyte (free H) and antigen (H-protein) compete for a fixed number of primary antibody (Y) binding sites. Unbound materials are removed (dotted line). Secondary antibody-enzyme conjugate (Y-E) is added to bind to primary antibody followed by another wash step. Substrate (A) for the enzyme is added to detect the bound enzyme. The amount of colored product ( ) detected is inversely proportional to the amount of analyte present...

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],... 

The basis for this technique lies in the competition between the test antigen and a labelled antigen for the available binding sites on a fixed amount of antibody. While the binding sites are traditionally associated with an antibody, any source of specific reversible binding sites may be used to create an assay in this format. Examples of such are specific transport proteins such as thyroxine-binding globulin and certain cellular receptors such as opiate or benzodiazepine receptors. Under these circumstances the equilibrium mixture may be represented thus ... 

Anthrax toxin is a bacterial toxin from Bacillus anthracis consisting of three parts protective antigen (PA), lethal factor (LF) and edema factor (EF). Both LF and EF compete for binding sites on the PA protein. The PA protein binds with high affinity to an as yet unknown receptor on macrophages and related cell types. When PA is internalized by the target cells, it functions as a shuttle protein for either EF or LF. Intracellularly, in the acidic environment of the endosome, EF and LF are capable of entering the cytosol by pH-dependent pore formation [139]. 

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