The Glycated Hemoglobin (HbA1C) BioAssay™ ELISA Kit (Human) is a competitive inhibition immunoassay for the in vitro quantitative measurement of HbA1c in human plasma, serum and erythrocyte lysates.
Test Principle:This assay employs the competitive inhibition enzyme immunoassay technique. A monoclonal antibody specific for HbA1c has been pre-coated onto a microplate. A competitive inhibition reaction occurs between biotin-labeled HbA1c and unlabeled HbA1c (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 HbA1c in the sample. After addition of the substrate solution, the intensity of color developed is inversely proportional to the concentration of HbA1c in the sample.
Kit Components:*351956A: Microtiter Plate, 96 wells, Pre-coated, ready to use
Storage and Stability:Store *351956A, *351956B, *351956D and *351956E 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:Use a serum separator tube and 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 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.
Note: Serum/plasma samples require about 10-fold dilution (e.g., 20ul sample + 180ul PBS). Samples should be diluted using 0.01M PBS, pH 7.0-7.2.
Erythrocyte Lysates:1. Centrifuge whole blood for approximately 20 minutes at 1000xg. Remove the supernatant and collect the cells
2. Wash cells three times in cold PBS (0.02M, pH 7.0-7.2)
3. Resuspend the cells in ice-cold PBS. Perform a freeze-thaw cycle by freezing the cells at -20°C or lower and then thawing the cells with gentle mixing. Perform the freeze-thaw cycle 3 times
4. Centrifuge for 10 minutes at 5000xg at 2-8°C to remove cellular debris
5. Assay the supernatant immediately or store samples in aliquots at -20°C or -80°C for later use. Avoid repeated freeze/thaw cycles.
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 10-20 minutes at 37°C
7. Add 50ul Stop Solution. Read absorbance at 450nm immediately.
