ELISA interactions

It has already been noted that the phenotype of an acquired immune response is considered to reflect the early cytokine environment in which naive CD4+ T cells interact with antigen. Again, it has been suggested, for example, that early exposure to IL-4 can push an immune response in a Th-2 direction (Swain et al., 1990). We therefore investigated (by ELISA) whether ES-62 was able spontaneously to induce IL-4 secretion in naive murine spleen cells (48 h exposure). Ironically, given that the molecule induces a Th-2 antibody response and seems to be able to induce the release of a number of other cytokines, IL-4 was not detected (Harnett et al., 1999a). It was noted, however, that IL-4 was produced by spleen cells from mice that had been pre-exposed to ES-62. This established Th-2 phenotype is consistent with the antibody data. 

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). 

The model immunoassay is the enzyme-linked immunosorbent assay (ELISA) in which a non-specific capture antibody is bound to a surface, such as a multi-well plate or small tube [13]. In the basic form of ELISA, a second antibody tagged with an enzyme interacts specifically with the analyte. The enzyme assay produces a colored product that is read with a spectrophotometer. There are many variations on the basic immunoassay format that serve to increase sensitivity, specificity, linear range, and speed. Many commercial instruments have been developed to take advantage of various technologies for reporter molecules. The immunoassay may be coupled to an electronic sensor and transducer, such as a surface acoustical wave (SAW) sensor. Electrochemiluminescence (ECL) is a method in which the detector antibody is tagged with a ruthenium-containing chelate [13-15]. When the tag is... 

Product variants can also be generated by in-process procedures, such as those used for viral inactivation, for example. These procedures could alter the protein structure, forming new epitopes. These types of changes could potentially be detected by ELISA because of the specificity of the antigen-antibody interaction. In the case of vaccine production, an ELISA could be used to monitor viral inactivation. For this, a panel of MAbs, if available, could be used. 

Braun and Alsenz6 used an ELISA to detect aggregates in interferon-alpha (IFN-a) formulations. They analyzed IFN-a formulations for possible aggregate formation because all marketed interferons are reported to induce antibodies to some extent. Because of its stabilizing effects, human serum albumin (HSA) is used in the formulation of marketed IFN-a at a great excess over IFN-a itself. HSA can also interact with other proteins. Braun and Alsenz developed an ELISA for the detection of both IFN-a-IFN-a and HSA-IFN-a aggregates. A MAb was used for the capture and detection of the IFN-a and a polyclonal for the detection of HSA. The assay is shown schematically in Figure 11.4. 

Host Cell Impurities Various organisms have been used to produce recombinant proteins yeast, bacteria (e.g., E. coli), insect cells, and mammalian cells such as Chinese hamster ovary (CHO) cells. During the purification process, some HCPs can copurify with the protein product. Because of the specificity of the antigen-antibody interaction, an ELISA can be used to detect and quantitate the contaminating HCPs. Detecting host impurities is important for quality process control as well as for product safety issues. The intent is to avoid unsafe levels of residual HCPs which might lead to adverse reactions.11... 

Enzyme-linked immunosorbent assay (ELISA) is a new method in alkaloid studies. The application of ELISA to alkaloid study is based on antibody incubations. It differs from classical precipitation-based methods in that specific antigen-antibody interactions are recognized by assaying an enzyme label conjugated to one reactant, usually an antibody. Because of the sensitivity with which enzyme... 

To date, complex proteins with biological activity (6), for use in X-ray crystal structure analysis (7) and ELISA systems (8), and for the development of animal drugs (9) have successfully been produced by using this system. Therefore, the Kaiko-baculovirus protein production system has broad applicability across the field of reverse chemical genetics for the analysis of protein function on the basis of interactions with chemical compounds. 

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