Enzyme-linked-immunosorbent optimizing ELISA

An enzyme-linked immunosorbent assay (ELISA) has two major components. The first is the immunological reaction that occurs between an antigen and antibody. This reaction is crucial and needs careful optimization. The second compo-... 

There are several important advantages RPMAs have over antibody arrays and other proteomic techniques such as immunohis-tochemistry or tissue arrays. Antibody arrays usually require a second specific antibody, made in a different species, for each captured protein to be visualized in a manner analogous to enzyme-linked immunosorbent assays (ELISA). Therefore, it becomes difficult to simultaneously optimize the antibody-antigen hybridization conditions for so many antibodies at once present on antibody arrays while minimizing nonspecific cross-reactivity and ensuring that proteins over a wide range of concentrations can be quantitated in a linear fashion (14). Antibody arrays also consume or require much higher inputs of protein than reverse phase arrays. With antibody arrays. 

Enzyme-linked immunosorbent assays (ELISA) are by far the most common immunological assay used in biochemical and clinico-chemical laboratories (Crowther 1995). The method is just as specific as RIA and it can achieve comparable sensitivities under optimal conditions. It can be carried out in various ways, but the common feature is that detection relies on turnover of a chromogenic substrate by an... 

Assay optimization. An optimization step not always taken, but nonetheless important to the success of any immunochemical method of analysis is the selection of materials, such as test tube or plastic plates, which maximize assay performance. An example of this is the selection of 96-well microtiter plates for enzyme-linked immunosorbent assay (ELISA) which give maximum protein binding capacity and minimum interwell variability (28>32 also Harrison, R.O. Nelson, J.O. J. Immunoassay. submitted). The type of microtiter plate may be the most important single determinant of ELISA performance and this selection should not be made carelessly. Significant error may also occur in the reading of assays performed in 96-well microtiter plates, due to alignment errors of automatic plate readers undetected in normal use (Harrison, R.O. Nelson, J.O. J. Immunoassay. submitted Harrison, R.O. Hammock, B.D. J. Assoc. Off. Anal. Chem.. submitted), and a plate reader test should allow further reduction of error. These steps should be taken before a major investment in time, effort, or money is made in an assay system which may later be found to be less than acceptable. 

FIGURE 16.5 Metabolites of testosterone in Japanese quail show the resolution of LC-AMS used in defining the optimal metabolite for developing a field enzyme-linked immunosorbent assay (ELISA) test. Fractional resolution is shown for 8 Gaussian-fitted peaks. Two unresolved peaks have dashed Gaussian fits that predict a 2.6-min separation for a resolution of 0.93. 

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