These assays illustrate two crucial aspects of all serological assays. First, at least one of the reagents must be available in a pure, detectable form in order to obtain quantitative information. Second, there must be a means of separating the bound fraction of the labeled reagent from the unbound, free fraction so that the percentage of specific binding can be determined. Normally, this separation is achieved by having the unlabeled partner trapped on a solid support. Labeled molecules that do not bind can then be washed away, leaving just the labeled partner that has bound. In Fig. A.5, the unlabeled antigen is attached to the well and the labeled antibody is trapped by binding to it. The separation of bound reagent from the free fraction is an essential step in every assay that uses antibodies.
RIA and ELISA do not allow one to measure directly the amount of antigen or antibody in a sample of unknown composition, because both depend on the binding of a pure labeled antigen or antibody. There are various ways around
Add labeled antigen A Add labeled antigen A + unlabeled antigen 100 50 1 2 0 standard curve Anti-A antibody bound Wash away unbound antigen Count bound label Known concentration of antigen A Mixture containing low concentration of antigen A Amount of competitor added Percent control binding Compete with standard curve curve 1 less inhibition curve 2
Fig. A.6 Competitive inhibition assay for antigen in unknown samples.
A fixed amount of unlabeled antibody is attached to a set of wells, and a standard reference preparation of a labeled antigen is bound to it. Unlabeled standard or test samples are then added in various amounts and the displacement of labeled antigen is measured, generating characteristic inhibition curves. A standard curve is obtained by using known amounts of unlabeled antigen identical to that used as the labeled species, and comparison with this curve allows the amount of antigen in unknown samples to be calculated. The green line on the graph represents a sample lacking any substance that reacts with anti-A antibodies.
this problem, one of which is to use a competitive inhibition assay, as shown in Fig. A.6. In this type of assay, the presence and amount of a particular antigen in an unknown sample are determined by the antigen’s ability to compete with a labeled reference antigen for binding to an antibody attached to a plastic well. A standard curve is first constructed by adding various amounts of a known, unlabeled standard preparation; the assay can then measure the amount of antigen in unknown samples by comparison with the standard. The competitive binding assay can also be used for measuring antibody in a sample of unknown composition by attaching the appropriate antigen to the plate and measuring the ability of the test sample to inhibit the binding of a labeled specific antibody.
A-5 Hemagglutination and blood typing.
The direct measurement of antibody binding to antigen is used in most quantitative serological assays. However, some important assays are based on the ability of antibody binding to alter the physical state of the antigen to which the antibody binds. These secondary interactions can be detected in a variety of ways. For instance, when the antigen is displayed on the surface of a large particle such as a bacterium, antibodies can cause the bacteria to clump, or agglutinate. The same principle applies to the reactions used in blood typing, only here the target antigens are on the surface of red blood cells and the clumping reaction caused by antibodies against them is called hemagglutination (from the Greek haima, blood).