The Human Prostaglandin D2 Synthase, Hematopoietic (PTGDS) ELISA Kit is a sandwich enzyme immunoassay for the in vitro quantitative measurement of PTGDS in human serum, plasma and other biological fluids Detection Range: 78-5,000pg/ml Sensitivity: <35pg/ml Precision: 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 PTGDS. Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated antibody specific to PTGDS. 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 PTGDS, 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 PTGDS in the sample is then determined by comparing the O.D. of the sample to the standard curve. Kit Components: *027668A: Microtiter Plate, 1x96 wells, Pre-coated, ready to use *027668B: Standard, 2x1vial 027668C: Standard Diluent, 1x20ml *027668D: Detection Reagent A, 1x120ul *027668E: Detection Reagent B, 1x120ul 027668F: Assay Diluent A, 1x12ml 027668G: Assay Diluent B, 1x12ml 027668H: TMB Substrate, 1x9ml 027668K: Stop Solution, 1x6ml 027668L: Wash Buffer, 30x, 1x20ml Storage and Stability: Store *027668A, *027668B, *027668D and *027668E at -20C. Store all the other components at 4C. 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. Materials Required But Not Supplied: 1. Microplate reader with 450 10nm filter. 2. Precision single or multi-channel pipettes and disposable tips. 3. Eppendorf Tubes for diluting samples. 4. Deionized or distilled water. 5. Absorbent paper for blotting the microtiter plate. 6. Container for Wash Solution Sample Preparation and Storage: Serum: Use a serum separator and allow samples to clot for two hours at room temperature or overnight at 4C before centrifugation for 20 minutes at approximately 1000xg. Assay freshly prepared serum immediately or store samples in aliquots at -20C or -80C for later use. Avoid repeated freeze/thaw cycles. Plasma: Collect plasma using EDTA or heparin as anticoagulant. Centrifuge samples for 15 minutes at 1000xg at 2-8C within 30 minutes of collection. Remove plasma and assay immediately or store samples in aliquots at -20C or -80C for later use. Avoid repeated freeze/thaw cycles. Note: Serum/plasma samples require about a 500-fold dilution. To prepare a 1:500 dilution of sample: Transfer 20ul sample to180ul PBS, this yields a 1:10 dilution. Dilute further by transferring 10ul of the 1:10 diluted sample to 490ul PBS, this yields a 1:500 diluted sample. Mix thoroughly at each stage of the dilution process. Samples should be diluted using 0.01M PBS (pH 7.0-7.2). Other Biological Fluids: Centrifuge samples for 20 minutes at 1000*g. Remove particulates and assay immediately or store samples in aliquots at -20C or -80C. Avoid repeated freeze/thaw cycles. Assay Procedure Summary: 1. Prepare all reagents, samples and standards. 2. Add 100ul standard or sample to each well. Incubate 2 hours at 37C. 3. Aspirate and add 100ul prepared Detection Reagent A. Incubate 1 hour at 37C. 4. Aspirate and wash 3 times. 5. Add 100ul prepared Detection Reagent B. Incubate 30 minutes at 37C. 6. Aspirate and wash 5 times. 7. Add 90ul TMB Substrate. Incubate 15-25 minutes at 37C. 8. Add 50ul Stop Solution. Read at 450nm immediately.
