The Interferon gamma (IFN-gamma) BioAssay(TM) ELISA Kit (Human) is a quantitative sandwich ELISA for the quantitative determination of human interferon gamma (IFN-gamma) concentrations in serum, cell culture supernates, urine, tissue homogenates and cerebrospinal fluid (CSF). Sensitivity: 1.56pg/ml Detection Range: 6.25-400pg/ml. Specificity: This assay has high sensitivity and excellent specificity for detection of human IFN-gamma. No significant cross-reactivity or interference between human IFN-gamma and analogues was observed. Precision: Intra-assay: CV%<8% Inter-assay: CV%<10% Test Principle: An antibody specific for IFN-gamma has been pre-coated onto a microplate. Standards and samples are pipetted into the wells and any IFN-gamma present is bound by the immobilized antibody. After removing any unbound substances, a biotin-conjugated antibody specific for IFN-gamma is added to the wells. After washing, avidin conjugated Horseradish Peroxidase (HRP) is added to the wells. Following a wash to remove any unbound avidin-enzyme reagent, a substrate solution is added to the wells and color develops in proportion to the amount of IFN-gamma bound in the initial step. The color development is stopped and theintensity of the color is measured. Kit Components: Assay plate, 1x96wells Standard (Freeze dried), 2 Biotin-antibody (100X), 1x120ul HRP-avidin (100X), 1x120ul Biotin-antibody Diluent, 1x15ml HRP-avidin Diluent, 1x15ml Sample Diluent, 1x50ml Wash Buffer (25X), 1x20ml TMB Substrate, 1x10ml Stop Solution, 1x10ml Storage and Stability: Store powder at 4C liquid at -20C. Store other components at 4C. Stable for 6 months after receipt. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap. Assay Summary: 1. Prepare reagents, standards and samples as instructed. 2. Add 100ul standard or sample to each well. Incubate for 1 hour at 37C 3. Aspirate and wash 3 times. 4. Add 100ul MAU/ALB (Biotin), 1X to each well. Incubate for 1 hour at 37C 5. Aspirate and wash 3 times. 6. Add 100ul Avidin (HRP), 1X to each well. Incubate for 1 hour at 37C 7. Aspirate and wash 5 times. 8. Add 90ul TMB substrate to each well Incubate for 20 minutes at 37C. Protect from light 9. Add 50ul Stop Solution to each well. Read at 450nm within 5 minutes.
