The Antigen

Thus, if theantibody has a high affinity for the antigen, it has a high association constant. Typical association constants range from lO to 10 ° L/mol, and as high as 10 L/mol for some monoclonal antibodies. 

Definition of the association (or avidity) constant for such multivalent antibody—antigen reactions must consider not only the heterogeneity of the antibodies and the antigen determinant site(s), but also an apparent additive effect of binding two antigen molecules to a single antibody. Such effects lead... 

There are many possible means for quantification of the antigen—antibody reaction. Immunoassays may be classified according to the technology used for detection and quantification of the analyte being detected. 

Turbidimetric Agglutination Immunoassays. Agglutination—precipitation immunoassays were among the first practical appHcations of the antigen—antibody reaction in diagnostic tests. These assays are not as widely used in the 1990s as EIA and FIA because they are either not quantitative enough or lack the sensitivity limits of RIA, EIA, and EIA. 

Most modem RJAs utilize a competitive assay format (Fig. 2) in which radiolabled antigen, Ag, competes with unlabeled antigen, Ag, in a sample for binding to the antibody. Ah. The free antigens are then separated from the antigen—antibody complexes, and the amount of radioactivity in the... 

The final vacciae coataHis the two toxoids, as weU as pertussis (whole ceU or aceUular), a buffer, and an adjuvant, ie, a substance that Hicreases the response to an antigen when combHied with the antigen, eg, aluminum. As noted above, the final vacciae can also contain a component that protects against Haemophilus disease. 

Standardization and Testing. RequHements for DTP have been described (17). Standardization of potency for the toxoids reHes on antigenic and flocculation tests. In principle, the antigenic tests are conducted to measure the abUity of the vacciae to Hiduce specific antibodies Hi guHiea pigs. The flocculation test provides a quantitative estimate of the amount of toxoid Hi the vacciae. 

Common Procedures. The general analytical scheme for immunochemical methods is rather simple. The analyte of interest, the antigen (Ag), reacts with the analytical reagent, the corresponding antibody (Ah), forming an immunochemical antigen—antibody complex ... 

The immunochemical interaction between the antigen and antibody is very specific. By labeling either the antigen or antibody, the method s sensitivity is increased. The most frequently used labels to increase sensitivity are radionucHdes (see Radioisotopes) where the assay process is called radioimmunoassay (RIA), or en2ymes where the assay is named en2yme immunoassay (ElA) (see Enzyme applications). 

Va.ria.tions in Methods. The various immunochemical methods can differ in a number of ways. For example, the analytical reagent may be cmde antisemm, monoclonal antibodies, isolated immunoglobulin fractions, etc. The conditions under which the method is mn, detection of the antigen—antibody complex, and the techniques used to increase sensitivity or specificity of the reaction all maybe varied. 

Fig. 5. Scheme of the last steps for the sandwich ELISA. Where Ah 1 represents the the surface-bound antibody, Ag, the antigen, and Ah 2, the... 

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