Abstract
An investigation was made on optimal condition for detection of complement 3 (C3) in rat serm by enzyme linked immunosorbent assay. By box titration, dilution ranges of anti-rat C3 antibody, goat serum, and enzyme-linked anti-rat C3 antibody was decided. Anti-rat C3 antibody consentrations were 0.1μg/ml, 1.0μg/ml, 10μg/ml and 100μg/ml. Goat serum was diluted at 40, 200, 1000 and 5000 fold. Enzyme-linked anti-rat C3 antibody was diluted at 200, 400, 800 and 1600 fold. Using these diluted solutions optimal conditions were determind. Each well was coated with 100μl of diluted antibody solution (10μg/ml). The well plates were incubated at 37℃ for 15 h. Antibody coated wells were washed with PBS contained Tween 20 three times. Wells were coated with 100μl goat serum (1 : 200). The well plates were incubated at 37℃ for 2 h. Wells were washed with PBS-Tween 20 three times. Each of them was added with 100μl of rat serum that diluted form 1 : 800 to 1 : 200000. The well plates were incubated at 25℃ for 2 h. After that, each well was washed with PBS-Tween 20 three times Each well was added with 100μl of enzyme-linked anti-rat C3 antibody and incubated at 25℃ for 2 h. Wells were washed with PBS-Tween 20 three times, added with 100μl of the substrate solution, incubated for sixty minutes at room temprature and absorbance at 405 nm was measured using EIA-READER (BIO-RAD).
