Abstract
A sensitive, specific, competitive enzyme-linked immunosorbent assay (ELISA) was developed for quantitative analysis of tyrosinase. Binding sites of anti-tyrosinase antibodies were competed for by purified tyrosinase adsorbed onto microtiter plates and a known (standard) or unknown (sample) amount of tyrosinase in solution. Adsorbed antibodies were detected by goat anti-rabbit IgG F(ab')2 labeled with peroxidase. A sensitivity range of 2.1 to 14 ng (30-200 fmol)/well was obtained. SDS was found to be the most suitable detergent for solubilizing the enzyme. Tyrosinase was extracted from B16 mouse melanoma and assayed by the ELISA. The tyrosinase content per mg melanoma protein was 505 +/- 106 (S.D.) ng. This assay is not only useful for measuring the content of normal tyrosinase in crude extracts but also is possibly applicable to detecting the unprocessed tyrosinases.
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