The Complement Component 4a （C4a） BioAssay ELISA Kit （Human） is a sandwich enzyme immunoassay for the in vitro quantitative measurement of C4a in human serum，plasma and other biological fluids.Detection Range：0.312 - 20ng/mlSensitivity：<0.112ng/mlPrecision：Intra-Assay： CV<10%Inter-Assay： CV<12%Test Principle：The microtiter plate provided in this kit has been pre-coated with an antibody specific to C4a. Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated antibody specific to C4a. Next，Avidin conjugated to Horseradish Peroxidase （HRP） is added to each microplate well and incubated. After TMB substrate solution is added，only those wells that contain C4a，biotin-conjugated antibody and enzyme-conjugated Avidin 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 C4a in the sample is then determined by comparing the O.D. of the sample to the standard curve.Kit Components：*365883A： Microtiter Plate，1x96 wells. Pre-coated; ready to use.*365883B： Standard，2x1vial 365883C： Standard Diluent，1x20ml*365883D： Detection Reagent A，1x120ul *365883E： Detection Reagent B，1x120ul 365883F： Assay Diluent A，1x12ml365883G： Assay Diluent B，1x12ml365883H： TMB Substrate，1x9ml365883K： Stop Solution，1x6ml365883L： Wash Buffer，30X，1x20ml Storage and Stability：Store *365883A，*365883B，*365883D and *365883E at -20°C. Store all the other components at 4°C. Unused kit is stable for 6 months. Once kit components are opened，it is highly recommended to use remaining reagents within 1 month provided this is within the expiration date of the kit. For maximum recovery of product，centrifuge the original vials after thawing and prior to removing the cap.Assay Procedure Summary：1. Prepare all reagents，samples and standards.2. Add 100ul standard or sample to each well. Incubate 1 hour at 37°C.3. Aspirate and add 100ul prepared Detection Reagent A. Incubate 1 hour at 37°C.4. Aspirate and wash 3 times.5. Add 100ul prepared Detection Reagent B. Incubate 30 minutes at 37°C.6. Aspirate and wash 5 times.7. Add 90ul TMB Substrate. Incubate 10-20 minutes at 37°C.8. Add 50ul Stop Solution. Read at 450nm immediately.
