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.
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), 1x12ml351750G: Assay Diluent B (2x), 1x12ml351750H: Substrate A, 1x10ml351750K: Substrate B, 1x2ml351750L: 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
