The RNASE3/ECP (Ribonuclease A3/Eosinophil Cationic Protein) BioAssay(TM) ELISA Kit (Human) is a quantitative sandwich assay for the detection of RNASE3/ECP in human serum, plasma and other biological fluids. Cell lysates and tissue homogenates, though not tested, may potentially be used as samples. Detection Range: 39.063-2500pg/ml Sensitivity: <23.438pg/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 RNASE3/ECP. Standards and samples are added to the appropriate microtiter plate wells followed by a biotin-conjugated antibody specific to RNASE3/ECP. 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 RNASE3/ECP, 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 RNASE3/ECP in the sample is then determined by comparing the O.D. of the sample to the standard curve. Kit Components: *358576A: Microtiter Strips, 8x12 wells. *358576B: Standard, 2 vials 358576C: Sample/Standard Dilution Buffer, 1x20ml 358576D: Antibody-Biotin (Concentrated), 1x120ul 358576E: Antibody Dilution Buffer, 1x10ml *358576F: Streptavidin-HRP Conjugate (SABC), 1x120ul 358576G: SABC Dilution Buffer, 1x10ml *358576H: TMB Substrate, 1x10ml 358576J: Stop Solution, 1x10ml 358576K: Wash Buffer (25X), 1x30ml Storage and Stability: Store unopened *358576A at 4C, store at -20C once opened. Store unopened *358576B at 4C, once reconstituted store at 4C for up to 12 hours or at -20C for up to 48 hours. Store *358576F and *358576H in the dark at 4C. Store all 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 3. Aspirate the liquid and wash plate 3 times. 3. Add 100ul Antibody-Biotin working solution to each well and incubate for 60 minutes at 37C 4. Aspirate the liquid and wash plate 3 times. 5. Add 100ul SABC working solution to each well. Incubate for 30 minutes at 37C 6. Aspirate the liquid and wash plate 5 times. 7. Add 90ul TMB substrate. Incubate 15-30 minutes at 37C 8. Add 50ul Stop Solution. Read at 450nm immediately. 9. Calculate results.
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