Immunoassay antigens/antibodies

Immunoassays can also be classified as homogeneous or heterogeneous. In a homogeneous immunoassay, antigen, antibody, and sample are mixed in the solution phase, while in a heterogeneous assay, one constituent (typically the antibody) is immobilized on a solid surface while the other constituents are delivered via the solution phase. 

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. 

The microplate ELISA testis conducted in standard 96-well microplates. A microplate consists of a 12 X 8 grid of wells for test solutions. The three most widely used ELISA formats are immobilized antigen competitive immunoassay, immobilized antibody competitive immunoassay and sandwich immunoassay. " ... 

While antibodies display similar structures - in fact, various types of IgGs are commonly used in immunoassays, antigens and proteins show tremendous variations in size, structure and charge strongly effecting the array performance. 

Liposome conjugates may be used in various immunoassay procedures. The lipid vesicle can provide a multivalent surface to accommodate numerous antigen-antibody interactions and thus increase the sensitivity of an assay. At the same time, it can function as a vessel to carry encapsulated detection components needed for the assay system. This type of enzyme-linked immunosorbent assay (ELISA) is called a liposome immunosorbent assay or LISA. One method of using liposomes in an immunoassay is to modify the surface so that it can interact to form biotin-avidin or biotin-streptavidin complexes. The avidin-biotin interaction can be used to increase detectability or sensitivity in immunoassay tests (Chapter 23) (Savage et al., 1992). 

Immunoassays employ monoclonal or polyclonal antibody preparations (Chapter 13) to detect and quantify the product (Box 7.1). The specificity of antibody-antigen interaction ensures good assay precision. The use of conjugated radiolabels (RIA) or enzymes (EIA) to allow detection of antigen-antibody binding renders such assays very sensitive. Furthermore, when compared with... 

Ishikawa, E., Hashida, S., Kohno, T. and Tanaka, K. Methods for enzyme-labeling of antigens, antibodies and their fragments , in Ngo, T. T. (ed.), Nonisotopic Immunoassay. Plenum Press, New York, 1988, p. 37. 

A partially purified HIV viral lysate is laid onto a sodium dodecyl sulfate (SDS)-polyacrylamide gel slab and then electrophoresed, which distributes the HIV peptides through the gel by their relative molecular mass. The higher-molecular-mass proteins form bands near the top of the gel. The proteins on the gel are then transferred electrophoretically onto nitrocellulose paper. The paper is sliced into thin strips, each having the full distribution of HIV antigen bands. The strip is used as a solid support of an indirect immunoassay, and antigen-antibody reactions form insoluble colored bands on the strip. 

The basis for this procedure is the antigen-antibody "reaction"—i e., specific binding of an antibody to the molecule being assayed. Among the many different immunoassay techniques that have been developed—e.g., radioimmunoassay (RIA), and chemoluminescence immunoassay (CIA)—a version of the enzyme-linked immunoassay (ElA) is shown here. 

Methods for the Detection of Antigens/Antibodies Equilibrium and kinetic inhibition assays based upon fluorescence polarization, 70, 3 fluorescence excitation transfer immunoassay (FETI), 70, 28 indirect quenching fluoroimmunoassay, 70, 60 the homogeneous substrate-labeled fluorescent immunoassay, 70, 79 fluorescence immunoassays using plane surface solid phases (FIAPS), 70, 87. 

Using an antibody specifically recognizing the antigen-antibody complex, more direct noncompetitive hapten immunoassays, which can be regarded as semi two-site immunometric assay, could be established (S3). Figure 14 depicts two typical procedures of noncompetitive assays using anti-metatype antibodies, which are based on principle C in Fig. 4. 

Chuang further cautions that In solid phase immunoassay, it is generally assumed that antigens adsorbed to a surface, such as polystyrene microtiter dishes, will react with specific antibody in a manner similar to that antigen-antibody reaction in solutions such as occur in immune precipitation. However, our evidence and others89) seem to point out that data obtained from solid phase immunoassays should be interpreted with caution since adsorption of a nonantigen to a polymer surface could render it immunoreactive to previously unreactive antibodies. ... 

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