Specificity total binding antibody assays

Another technique, the enzyme-linked immunosorbent assay (ELISA), combines the specificity of antibodies with the sensitivity of an enzyme assay. The ELISA can be performed in a variety of combinations that involve either a specific antibody or the total cellular protein immobilized on a solid support, such as the wells of a plastic microplate. In one version of the method, the sandwich ELISA, the primary antibody is bound to the wells. When a mixture of proteins is added, the protein of interest binds to the antibody, and other proteins are washed away. A second labeled antibody, specific to a different epitope on the protein, is added, and the amoruit of signal is proportional to the amoimt of the particular protein in the sample. The method can be modified to detect specific antibodies in a mixture by using their antigen as the immobilized bait. ELlSAs also have the advantage of being able to be performed in 96-weU plates so many samples can be analyzed in one experiment. 

About 10% of serum proteins are immunoglobulins (Igs). After immunization, the specific antibodies produced are about 1"C5% of this fraction, so the required Ig (in ELISA) may be from 0.1 to 2.5% of the total protein in a serum. Some assays are favored by the relatively crude fractionation of serum to obtain Igs, e.g., for use in binding to plates in trapping (sandwich assays) to avoid competition for plastic binding sites by other serum proteins. Several methods for separation of Igs are available for use in ELISA. These procedures are suitable for polyclonal antibodies but not necessarily for mAbs. The isolation of total Igs as compared to the purification of specific Igs, is relatively simple. 

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