The specificity of antigen:antibody binding interactions can be exploited for the purification of a specific antigen from a complex mixture, or alternatively, for the purification of specific antibodies from antiserum containing a mixture of different antibodies. The technique employed is called affinity chromatography (Fig. A.4). For purification of an antigen, antigen-specific antibodies are bound, often covalently, to small, chemically reactive beads, which are loaded into a column. The antigen mixture is allowed to pass over the beads. The specific antigen binds; all the other components in the mixture can then be washed away. The specific antigen is then eluted, typically by lowering the pH to 2.5 or raising it to greater than 11. Antibodies bind stably under physiological conditions of salt concentration, temperature, and pH, but the binding is reversible because the bonds are noncovalent. Affinity chromatography can also be used to purify antibodies from complex antisera by using beads coated with specific antigen. The technique is known as affinity chromatography because it separates molecules on the basis of their affinity for one another.
A-4 Radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), and competitive inhibition assay.
Radioimmunoassay (RIA) and enzymelinked immunosorbent assay (ELISA) are direct binding assays for antibody (or antigen); both work on the same principle, but the means of detecting specific binding is different. Radioimmunoassays are commonly used to measure the levels of hormones in blood and tissue fluids, while ELISA assays are frequently used in viral diagnostics, for example, in detecting cases of infection with the human immunodeficiency virus (HIV), which is the cause of AIDS. For both of these methods, one needs a pure preparation of a known antigen or antibody, or both, in order to standardize the assay. We will describe the assay that is used to determine the amount of a specific antigen in a sample, for instance, the amount of HIV p24 protein in a patient’s serum. For this, a preparation of pure antibody specific for the antigen is required. One can also use RIA or ELISA to determine the amount of specific antibody in a mixture, such as serum; in this case, a preparation of pure antigen is needed as a starting point.
For the determination of antigen concentration using RIA, pure antibody against the antigen is radioactively labeled, usually with 125I; for ELISA, an enzyme is linked chemically to the antibody. The unlabeled component, which in this case would be the solution containing an unknown amount of antigen, is attached to a solid support, such as the wells of a plastic multiwell plate, which will adsorb a certain amount of any protein. Following this, the labeled antibody
Fig. A.4 Affinity chromatography uses antigen–antibody binding to purify antigens or antibodies. To purify a specific antigen from a complex mixture of molecules, a monoclonal antibody is attached to an insoluble matrix, such as chromatography beads, and the mixture of molecules is passed over the matrix. The specific antibody binds the antigen of interest; other molecules are washed away. Specific antigen is then eluted by altering the pH, which can usually disrupt antibody– antigen bonds. Antibodies can be purified in the same way on beads coupled to antigen (not shown).
Add anti-A antibody covalently linked to enzyme Wash away unbound antibody sample 1 (antigen A) sample 2 (antigen B) Enzyme makes colored product from added colorless substrate Measure absorbance of light by colored product
Fig. A.5 The principle of the enzyme-linked immunosorbent assay (ELISA).