Human high molecular weight cytokeratin (CK-HMW) ELISA kit

The Human High Molecular Weight Cytokeratin (CK-HMW) ELISA Kit operates on the principle of a double-antibody one-step sandwich ELISA. The process begins by coating microwells with specific capture antibodies against CK-HMW. Following this, samples, standards, and HRP-labeled detection antibodies are added sequentially, followed by incubation and thorough washing steps. A TMB substrate is then introduced, which turns blue under the action of peroxidase and changes to yellow when an acid is added. The intensity of the color produced directly correlates with the concentration of CK-HMW in the sample. The absorbance is measured at 450 nm using a microplate reader, allowing for precise quantification of CK-HMW levels. 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, then centrifuged 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 tissue in saline and centrifuging at 3000 rpm for 10 minutes. Store samples in aliquots at -20°C, avoiding repeated freeze-thaw cycles. Thaw samples at room temperature before use. To ensure accuracy, all reagents must be properly handled. The kit should be stored at 2–8°C and allowed to equilibrate at room temperature for 20 minutes before use. Crystallization of the concentrated wash buffer is normal; it can be dissolved by warming in a water bath. Unused strips should be returned to the ziplock bag immediately. Pre-treated samples do not require dilution, and 10 μL of sample can be directly added. Strict adherence to incubation times, volumes, and sequence is essential. All liquid components should be shaken well before use. The kit includes a 96-well or 48-well plate format, along with standard solutions, sample diluent, detection antibody-HRP, 20× wash buffer, TMB substrates A and B, stop solution, sealing films, and detailed instructions. Reagents should be prepared according to the provided guidelines. The 20× wash buffer is diluted 1:20 with distilled water. Washing can be done manually or with an automated system, with five wash cycles recommended. The procedure involves removing the required microplate strips, setting up standard, sample, and blank wells, adding 50 μL of standards and 10 μL of sample (with 40 μL diluent), followed by the addition of HRP-labeled detection antibody. After incubation at 37°C for 60 minutes, the plate is washed thoroughly. TMB substrate A and B are added, and the reaction is stopped after 15 minutes. The OD value is measured at 450 nm within 15 minutes, and a standard curve is plotted using Excel. The sample concentration is calculated based on the regression equation. The kit demonstrates high performance, including a correlation coefficient (R) of ≥0.99, sensitivity below 1.0 ng/mL, and specificity that avoids cross-reactivity with other analogs. The inter-assay and intra-assay variation is less than 15%. It should be stored at 2–8°C, away from light and moisture, and has a shelf life of six months. The detection range spans from 6.25 ng/mL to 200 ng/mL. Important disclaimers apply: this kit is intended for research purposes only and must not be used in clinical trials or human experiments. The manufacturer is not responsible for any misuse or deviations from the instructions. Users must strictly follow the protocol, and different batch numbers should not be mixed. Any deviation may lead to unreliable results, and the user assumes full responsibility.

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