The Pregnenolone BioAssay ELISA Kit （Universal） is a quantitative competitive assay for the detection of Pregnenolone in serum，plasma，tissue homogenates and other biological fluids.Detection Range：15.6-1,000pg/mlSensitivity：<9.375pg/mlPrecision：Intra-Assay CV： <8%Inter-Assay CV： <10%Assay Principle：The microtiter plate provided in this kit has been pre-coated with Pregnenolone. Pregnenolone in the standard or sample competes with a fixed amount of plate-coated Pregnenolone for antibody binding sites on the Antibody-Biotin reagent. Excess conjugate and unbound sample or standard are washed from the plate，after which Streptavidin-HRP （SABC） is added to each well and incubated. TMB substrate is then added to each well. The enzyme-substrate reaction is terminated by the addition of a sulfuric acid solution and the resulting color is measured spectrophotometrically at 450nm. The concentration of Pregnenolone in the samples is determined by comparing the absorbance of the samples to the standard curve.Kit Components：*358304A： Microtiter Strips，8x12 wells.*358304B： Standard，2 vials358304C： Sample/Standard Dilution Buffer，1x20ml358304D： Antibody-Biotin Concentrate，1x160ul358304E： Antibody Dilution Buffer，1x10ml358304F： Streptavidin-HRP Conjugate （SABC），1x120ul358304G： SABC Dilution Buffer，1x10ml358304H： TMB Substrate，1x10ml358304J： Stop Solution，1x10ml358304K： Wash Buffer （25X），1x30mlStorage and Stability：Store unopened *358304A at 4ºC; store at -20ºC once opened. Store unopened *358304B at 4°C; once reconstituted store at 4°C for up to 12 hours or at -20°C for up to 48 hours. Store other components at 4°C. Kit is stable for 6 months after receipt. For maximum recovery of product，centrifuge the original vials after thawing and prior to removing the cap.Assay Summary：1. Wash plate 2 times before adding standards，samples and control （zero） to wells!2. Add 50ul standard or sample to each well.3. Immediately add 50ul Antibody-Biotin working solution to each well and incubate for 45 minutes at 37°C4. Aspirate and wash 3 times.5. Add 100ul SABC working solution to each well. Incubate for 30 minutes at 37°C6. Aspirate and wash 5 times.7. Add 90ul TMB substrate. Incubate 15-20 minutes at 37°C8. Add 50ul Stop Solution. Read at 450nm immediately.9. Calculate results.
