The Complement Component 3a (C3a) BioAssay(TM) ELISA Kit is a quantitative sandwich assay for the detection of C3a in rat serum, plasma, tissue homogenates and other biological fluids. Detection Range: 0.625-40ng/ml Sensitivity: 0.375ng/ml Precision: Intra-Assay CV: <8% Inter-Assay CV: <10% Assay Principle: The microtiter plate provided in this kit has been pre-coated with an antibody specific to C3a. Standards and samples are added to the appropriate microtiter plate wells followed by a biotin-conjugated antibody specific to C3a. Streptavidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. After TMB substrate solution is added, only those wells that contain C3a, biotin-conjugated antibody and enzyme-conjugated streptavidin complex will exhibit a change in color. The enzyme-substrate reaction is terminated by the addition of sulfuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450nm 10nm. The concentration of C3a in the sample is then determined by comparing the O.D. of the sample to the standard curve. Kit Components: *354385A: Microtiter Strips, 1x48 wells or 1x96 wells *354385B: Standard, 1x1vial or 2x1vials 354385C: Sample/Standard Dilution Buffer, 1x10ml or 1x20ml 354385D: Antibody-Biotin Concentrate, 1x60ul or 1x120ul 354385E: Antibody Dilution Buffer, 1x5ml or 1x10ml 354385F: Streptavidin-HRP Conjugate (SABC), 1x60ul or 1x120ul 354385G: SABC Dilution Buffer, 1x5ml or 1x10ml 354385H: TMB Substrate, 1x5ml or 1x10ml 354385J: Stop Solution, 1x5ml or 1x10ml 354385K: Wash Buffer, 25X, 1x15ml or 1x30ml Storage and Stability: Store unopened *354385A at 4C, store at -20C once opened. Store unopened *354385B at 4C, once reconstituted store at 4C for up to 12 hours or at -20C for up to 48 hours. Store other components at 4C. 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 100ul standard or sample to each well and incubate for 90 minutes at 37C. Aspirate and wash 2 times. 3. Add 100ul Antibody-Biotin working solution to each well and incubate for 60 minutes at 37C 4. Aspirate and wash 3 times. 5. Add 100ul SABC working solution to each well. Incubate for 30 minutes at 37C 6. Aspirate and wash 5 times. 7. Add 90ul TMB substrate. Incubate 10-20 minutes at 37C 8. Add 50ul Stop Solution. Read at 450nm immediately. 9. Calculate results
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