Antibodies antigen-binding sites

The active site of an enzyme differs from an antibody-antigen binding site in that the enzyme active site... 

Differences in Electrostatic Properties at Antibody-antigen Binding Sites Implications for Specificity and Cross-reactivity. 

A major consideration in choosing an appropriate ratio of TRITC molecules per protein molecule was based on the possibility of the TRITC binding at a site near to the antibody-antigen binding site. Therefore, in order to preserve binding specificity of the... 

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...

Another method to confirm that the peptide is specific is to test for specific inhibition of the binding of the antibody to its native protein target (i.e., in a tissue section) using soluble peptide. This tests whether the peptide was binding at or very close to the antigen binding site on an antibody. For each of the peptides described in this chapter, there was specific inhibition by soluble peptide but not by other, antigenically irrelevant peptides.6,7... 

In addition to the covalent binding, some methods derived from bioaffinity chromatography can be used for non covalent attachment of antibodies to a surface by the inactive Fc portion. The advantage is that antigen binding sites stay undamaged and accessible for the analytes due to the orientation of antibody with the active Fab portions towards the tested medium. 

One of the best ways to ensure retention of activity in protein molecules is to avoid doing chemistry at the active center. The active center is that portion of the protein where ligand, antigen, or substrate binding occurs. In simpler terms, the active center (or active site) is that part that has specific interaction with another substance (Means and Feeney, 1971). For the preparation of enzyme derivatives, it is important to protect the site of catalysis where conversion of substrate to product happens. For instance, when working with antibody molecules, it is crucial to stay away from the two antigen binding sites. 

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