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The use of enzyme linked immunosorbent assay, or ELISA, provides an economical, rapid and highly sensitive method for screening a large number of samples. ELISA can be used to detect and quantitate peptides, proteins or antibodies. The assay is based upon an antigen-antibody interaction and subsequent enzymatic action on a substrate yielding a soluble colored product. Variations of the basic method exist for specialized applications. A basic method is outlined below.
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It may be required to optimize the amount of a reagent in the assay by performing a checkerboard titration. This is accomplished by serial dilution of one reagent across the plate and serial dilution of the other reagent down the plate. This design permits you to analyze different concentrations of the two reagents in each well and to obtain the optimal combination of both reagents. It is important that the coating solution is absolutely free of detergents because competition for binding may cause low and/or uneven binding. Excessive concentrations of coating protein may actually lead to less coating. Always use high quality antibody conjugates. For alkaline phosphatase conjugates replace PBS with TBS. The intensity of the resultant color produced when the substrate is added should correlate to the concentration of the primary antibody and the respective antigen.
1. Phelps, D.C., Nemec, S., Baker, R. and Mansell, R. (1990) Immunoassay for Napthazarin Phytotoxins Produced by Fusarium solani. Phytopathology 80; 298- 302.
2. Harlow, E. and Lane, D., Antibodies - A Laboratory Manual. Cold Spring Harbor Laboratory (1988).
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