Angiotensin 1-9 (Ang1-9) BioAssay(TM) ELISA Kit (Human)

The Human Angiotensin 1-9 (Ang1-9) ELISA Kit is a competitive inhibition immunoassay for the in vitro quantitative measurement of Ang1-9 in human serum, plasma, tissue homogenates, cell lysates, cell culture supernatants and other biological fluids. Detection Range: 2.47-200pg/ml, Sensitivity: <0.97pg/ml, Precision: Intra-Assay CV: <10%, Inter-Assay CV: <12%, Test Principle: This assay employs the competitive inhibition enzyme immunoassay technique. A monoclonal antibody specific for Ang1-9 has been pre-coated onto a microplate. A competitive inhibition reaction occurs between biotin-labeled Ang1-9 and unlabeled Ang1-9 (standards or samples) for limited binding sites on the pre-coated antibody. After incubation the unbound conjugate is washed off. Next, avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. The amount of bound HRP conjugate is inversely proportional to the concentration of Ang1-9 in the sample. After addition of the substrate solution, the intensity of color developed is inversely proportional to the concentration of Ang1-9 in the sample. Kit Components: *152267A: Microtiter Plate, 96 wells, Pre-coated, ready to use, *152267B: Standard, 2x1vial , 152267C: Standard Diluent, 1x20ml, *152267D: Detection Reagent A, 1x120ul , *152267E: Detection Reagent B, 1x120ul , 152267F: Assay Diluent A, 1x12ml, 152267G: Assay Diluent B, 1x12ml, 152267H: TMB Substrate, 1x9ml, 152267K: Stop Solution, 1x6ml, 152267L: Wash Buffer, 30X, 1x20ml, Storage and Stability: Store *152267A, *152267B, *152267D and *152267E 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. 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:, Allow samples to clot for two hours at room temperature or overnight at 4°C before centrifugation for 20 minutes at approximately 1000xg. Assay freshly prepared serum immediately or store samples in aliquots at -20°C or -80°C 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 2-8° within 30 minutes of collection. Remove plasma and assay immediately or store samples in aliquots at -20°C or -80°C for later use. Avoid repeated freeze/thaw cycles. Note: Serum/plasma samples require about 5-fold dilution (example: 40ul sample +160ul PBS). Samples should be diluted using 0.1M PBS, pH 7.0-7.2. , Tissue Homogenates:, The preparation of tissue homogenates will vary depending upon tissue type. For this assay, tissues were rinsed thoroughly in ice-cold PBS (0.02M pH 7.0-7.2) to remove excess blood and weighed before homogenization. The tissues were minced to small pieces and homogenized in 5-10ml of PBS with a glass homogenizer on ice (Micro Tissue Grinders works too). The resulting suspension was sonicated with an ultrasonic cell disrupter or subjected to two freeze-thaw cycles to further break the cell membranes. After that, the homogenates were centrifuged for 5 minutes at 5000×g. The supernatant was removed and assayed immediately or aliquoted and stored at -20°C or lower. Cell Lysates: Cells must be lysed before assaying according to the following directions. , 1. Adherent cells should be detached with trypsin and then collected by centrifugation (suspension cells can be collected by centrifugation directly). , 2. Wash cells three times in cold PBS. , 3. Resuspend cells in PBS (1×) and subject the cells to ultrasonication 4X (or freeze cells at -20°C. Thaw cells with gentle mixing. Repeat the freeze/thaw cycle 3X.) , 4. Centrifuge at 1500×g for 10 minutes at 2-8°C to remove cellular debris. Cell Culture Superntants and Other Biological Fluids: Centrifuge samples for 20 minutes at 1000×g. Remove particulates and assay immediately or store samples in aliquots at -20°C or -80°C. Avoid repeated freeze/thaw cycles. Note: 1. Samples to be used within 5 days may be stored at 4°C, otherwise samples must be stored at -20°C (<1 month) or -80°C (<2 months) to avoid loss of bioactivity and contamination., 2. Sample hemolysis will influence the results so hemolyzed specimens should not be used., 3. When performing the assay, bring samples to room temperature. Assay Procedure Summary: 1. Prepare all reagents, samples and standards., 2. Add 50ul standard or sample to each well, then add 50ul prepared Detection Reagent A immediately. Shake and mix. Incubate 1 hour at 37°C., 3. Aspirate and wash 3 times., 4. Add 100ul prepared Detection Reagent B. Incubate 30 minutes at 37°C., 5. Aspirate and wash 5 times., 6. Add 90ul Substrate Solution. Incubate 15-25 minutes at 37°C., 7. Add 50ul Stop Solution. Read absorbance at 450nm immediately., ,