Abstract
A micro-fluorometric ELISA which required only 50 μl of reagent in each step is reported here for rapid detection of anti-estrogen receptor (ER) antibody production by hybridoma cultures. In this study, several factors which influence the sesitivity of ELISA, e.g., incubation time, substrates, microtiter plates, concentration of coating antigens, choice and concentration of specific antibody and enzyme were evaluated. We observed that micro-fluorimetric ELISA using 4-methylumbelliferyl phosphate-phosphatase system and MicroFluor W plates was the most sensitive; it detected readily 1 ng coating antigens or 1 ng antibody during a 15 min incubation. This system was used successfully for the detection of anti-ER antibodies in mouse immune sera, and in spent media from in vitro immunized spleen cells or hybridoma cells. This method is simple, sensitive, rapid and reliable for ER or anti-ER antibody determination.
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