The mouse type IV collagen (Col IV) ELISA kit operates on the principle of a double-antibody one-step sandwich ELISA. The microplates are pre-coated with a specific capture antibody for Col IV. After adding the sample, standard, and HRP-conjugated detection antibody in sequence, the plate is washed thoroughly to remove unbound components. A TMB substrate is then added, which turns blue under peroxidase activity and changes to yellow when an acidic stop solution is introduced. The intensity of the color is directly proportional to the concentration of Col IV in the sample. The optical density (OD) is measured at 450 nm using a microplate reader, and the sample concentration is calculated based on a standard curve.
Sample collection and preparation are critical for accurate results. For serum, collect blood in endotoxin-free tubes, centrifuge at 3000 rpm for 10 minutes, and carefully separate the serum. Plasma should be collected using anticoagulants such as EDTA, citrate, or heparin, followed by centrifugation at 3000 rpm for 30 minutes. Cell supernatants require centrifugation at 3000 rpm for 10 minutes to remove debris. Tissue homogenates should be prepared by chopping the tissue in saline and centrifuging at 3000 rpm for 10 minutes. Store samples in single-use aliquots at -20°C, avoiding repeated freeze-thaw cycles. Thaw samples at room temperature before use.
For optimal performance, ensure all reagents are well mixed before use. The kit should be stored at 2–8°C and equilibrated at room temperature for 20 minutes before starting. If the washing buffer crystallizes after refrigeration, gently warm it in a water bath before use. Unused microplates should be returned to the sealed bag immediately. Avoid diluting the samples unless specified; typically, 10 μL of sample is sufficient. Follow the incubation times and steps precisely to ensure consistency.
The kit includes 96-well or 48-well configurations, along with standard solutions, detection antibodies, wash buffers, substrates, and stop solutions. The standards are diluted to concentrations ranging from 1600 pg/mL down to 0 pg/mL. Reagents like the 20× wash buffer should be diluted with distilled water at a 1:20 ratio. Washing can be done manually or with an automated washer, ensuring five complete washes.
During the procedure, first remove the required wells from the foil pouch and seal the rest. Set up standard, sample, and blank wells. Add 50 μL of standard or sample, followed by 10 μL of sample and 40 μL of diluent for the test samples. Then add 50 μL of HRP-labeled detection antibody to each well, seal, and incubate at 37°C for 60 minutes. After washing, add TMB substrate A and B, incubate in the dark for 15 minutes, and finally add the stop solution. Measure OD values within 15 minutes at 450 nm.
To analyze results, plot the standard curve in Excel, with concentration on the x-axis and OD on the y-axis. Use linear regression to determine the equation and calculate sample concentrations accordingly. The kit demonstrates high accuracy (R ≥ 0.99), sensitivity (less than 1.0 pg/mL), and specificity, with low inter- and intra-assay variability (<15%). It has a shelf life of 6 months when stored properly.
This ELISA kit is intended for research purposes only. It should not be used in clinical trials or animal experiments without proper authorization. Users must strictly follow the instructions, and different batch numbers should not be mixed. Any deviation from the protocol may lead to inaccurate results, and the company will not be responsible for any consequences.
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