Collagen Type IV (COL4) BioAssay™ ECL Kit (Human)

The Human Collagen Type IV (COL4) ELISA, ECL Kit is a sensitive chemiluminescent sandwich enzyme immunoassay for the in vitro quantitative measurement of COL4 in human serum, plasma, tissue homogenates and other biological fluids. ||Detection Range:|0.14-100ng/ml||Sensitivity:|<0.07ng/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 COL4. Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated antibody specific to COL4. Next, Avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. The addition of substrates A and B mixture generates glow light emission kinetics. Upon plate development, the intensity of the emitted light is proportional to the COL4 level in the sample or standard.||Kit Components:|*351750A: Microtiter Plate, 1x96 wells, Pre-coated with antibody; ready to use.|*351750B: Standard, 2x1vial |351750C: Standard Diluent, 1x20ml|*351750D: Detection Reagent A (green), 1x120ul |*351750E: Detection Reagent B (red), 1x120ul |351750F: Assay Diluent A (2x), 1x12ml|351750G: Assay Diluent B (2x), 1x12ml|351750H: Substrate A, 1x10ml|351750K: Substrate B, 1x2ml|351750L: Wash Buffer (30x), 1x20ml ||Storage and Stability:|Store *351750A, *351750B, *351750D and *351750E 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. Luminometer capable of reading 96-well microplates with the following parameters: lag time 30.0secs; read time 1.0 sec/well . |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 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°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 (example: 20ul sample + 180ul PBS). Samples should be diluted using 0.01M 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 2500×g. The supernatant was removed and assayed immediately or aliquoted and stored at -20°C or lower.||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.||Assay Procedure Summary:|1. Prepare all reagents, samples and standards.|2. Add 100ul standard or sample to each well. Incubate 2 hours at 37°C.|3. Aspirate and add 100ul prepared Detection Reagent A. Incubate 1 hour at 37°C.|4. Aspirate and wash 3 times.|5. Add 100ul prepared Detection Reagent B. Incubate 30 minutes at 37°C.|6. Aspirate and wash 5 times.|7. Add 100ul Substrate Solution. Incubate 10 minutes at 37°C.|8. Read RLU immediately.|