ELISA maximization

Ten blood serum samples from sick cows were investigated, for which the extinction coefficient in the ELISA assays was 2.0-2.5 at 1 500 dilntion. PAH, dodecanethiol and dextran snlfate were used for the intermediate layer formation. The maximal sensitivity was observed when the transducer surface was pre-treated with PAH and this method was used in further experiments. 

Column fractions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and ELISA. Tubes containing maximal anti-albumin activity were pooled and concentrated. A portion of this concentrate was analyzed by SDS-PAGE. 

Supernatants from positive clones can be analyzed directly by ELISA, or individual clones can be isolated and expanded for further analysis and purification. Thej558L cells should be grown to adensity of 2 X lOVmL or greater for maximal production of hybrid andbody. Conditioned medium is stored at -20 C after addidon of protease inhibitors NEM and PMSF (20 mL/L of each) and NagEDTA (20 mL/L). 

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. 

This chapter defines the terms and examines the configurations used for most applications of ELISA. Such a chapter is important because the possibilities inherent in the systems of ELISA must be understood in order to maximize their versatility in assay design. All heterogeneous systems have three basic parameters ... 

H36.12J clonally derived, C57BL/6N-mouse hybrid precursor macrophages were found useful to evaluate the mechanisms by which Be stimulates macrophage cytokine production, and by which T cell derived IFN-y amplifies TNF-a production in granulomatous disease (Sawyer et d. 2000). The response was maximal at 100 jiM BeS04 and did not occur when 12j cells were stimulated with either aluminium sulphate of cobalt sulphate. Beryllium stimulated the production of 725125 pg/ml TNF-a protein by 12j cells as measured by ELISA of culture supernatants after 24 h. As measured by RT-PCR, Be-stimulated 12j cell TNF-a protein production was accompanied by an increased intracellular TNF-a mRNA at 3 and 24 h. The addition of 10 U or 1(X) U of recombinant-Mu-IFN-y to Be-stimulated 12j cells further increased TNF-a production 1.5-4... 

The basic data given by the competitive ELISA can be analysed to determine the concentration of each sample of DNA that is necessary to cause 50% reduction in assay signal. In order to calculate from this the absolute level of adducts in the samples, it is necessary to include in the assay a sample of DNA carrying a known level of modified bases. This should be treated to maximize immuno-reactivity in the same manner as DNA samples being analysed. Preparation of this standard and the method of determining its level of base modification will depend on the modification being studied... 

Maximization of immunoreactivity of heat stable adducts by thermal denaturation of DNA 421 Maximization of immunoreactivity of heat labile adducts by digestion of DNA 421 Competitive ELISA 423 Analysis of data 425... 

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