Hemoglobin (HB) BioAssayª ELISA Kit (Human)

The Human Hemoglobin (HB) ELISA Kit is a competitive inhibition immunoassay for the in vitro quantitative measurement of HB in human serum, plasma, tissue homogenates, erythrocyte lysates, cell culture supernatants and other biological fluids. Detection Range: 1.23-100ug/ml, Sensitivity: <0.51ug/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 HB has been pre-coated onto a microplate. A competitive inhibition reaction occurs between biotin-labeled HB and unlabeled HB (standards or samples) for limited binding sites on the pre-coated antibody specific for HB. 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 HB in the sample. After addition of the substrate solution, the intensity of color developed is inversely proportional to the concentration of HB in the sample. Kit Components: *025597A: Microtiter Plate, 96 wells, Pre-coated, ready to use, *025597B: Standard, 2x1vial , 025597C: Standard Diluent, 1x20ml, *025597D: Detection Reagent A, 1x120ul , *025597E: Detection Reagent B, 1x120ul , 025597F: Assay Diluent A, 1x12ml, 025597G: Assay Diluent B, 1x12ml, 025597H: TMB Substrate, 1x9ml, 025597K: Stop Solution, 1x6ml, 025597L: Wash Buffer, 30X, 1x20ml, Precaution: The Stop Solution (025597K) suggested for use with this kit is an acid solution. Wear eye, hand, face, and clothing protection when using this material. Storage and Stability: Store *025597A, *025597B, *025597D and *025597E 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:, Centrifuge whole blood for 20 minutes at approximately 1000xg. Remove serum and assay immediately or store samples in aliquots at -20°C or -80°C. Avoid repeated freeze/thaw cycles. Plasma: Collect plasma using EDTA as anticoagulant. Centrifuge samples for 15 minutes at 1000xg at 2-8°C 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. 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. Note: If free HB is detected in non-hemolyzed serum and plasma, samples require about 10-fold dilution. A suggested 10-fold dilution is 20ul sample + 180ul PBS. Samples should be diluted using 0.01M PBS, pH 7.0-7.2. Erythrocyte Lysates: 1. Centrifuge whole blood for 20 minutes at approximately 1000xg, remove the supernatant and collect the cells., 2. Wash cells three times with PBS (0.01M, pH 7.0 - 7.2)., 3. Resuspend cells in ice-cold PBS and freeze cells at -20°C or lower. Thaw cells with gentle mixing. Repeat the freeze/thaw cycle 3 times., 4. Centrifuge at 5000xg for 10 minutes at 2-8°C to remove cellular debris., 5. Assay the supernatant immediately or store samples in aliquots at -20°C or -80°C. Avoid repeated freeze-thaw cycles. 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.