The mouse H5N1 ELISA kit is designed to detect the presence of H5N1 influenza A virus antigens using a double-antibody one-step sandwich ELISA method. The microplates are pre-coated with specific antibodies that capture the target antigen from the sample. After adding the specimen, 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 produced is directly proportional to the concentration of H5N1 in the sample. The absorbance is measured at 450 nm using a microplate reader, allowing for the quantification of the analyte.
Sample collection and preparation are crucial for accurate results. For serum, blood is collected into pyrogen-free tubes, centrifuged at 3000 rpm for 10 minutes, and the supernatant is carefully separated. Plasma samples should be collected with anticoagulants like EDTA, citrate, or heparin and centrifuged at 3000 rpm for 30 minutes. Cell culture supernatants are obtained by centrifuging at 3000 rpm for 10 minutes to remove debris. Tissue homogenates are prepared by mixing tissue with physiological saline, followed by centrifugation to collect the supernatant. All samples should be aliquoted and stored at -20°C to avoid repeated freeze-thaw cycles. When thawing, allow samples to reach room temperature and ensure even mixing before testing.
For optimal performance, the following items are required: a microplate reader set at 450 nm, precision pipettes (0.5–10 µL, 2–20 µL, 20–200 µL, 200–1000 µL), and a 37°C incubator. Before use, the kit should be allowed to equilibrate at room temperature for 20 minutes. If the washing buffer crystallizes after removal from the fridge, it can be dissolved by warming it in a water bath. Unused wells should be returned to the sealed bag and stored at 2–8°C. No dilution is needed for pre-treated samples; simply add 10 µL of the sample directly. Strict adherence to incubation times, reagent volumes, and procedural steps is essential for reliable results. Always mix all liquid reagents well before use.
The kit includes microwells, standards, diluents, detection antibodies, wash buffer, substrates, stop solution, and sealing films. Standards are diluted to concentrations ranging from 1000 pg/mL down to 0 pg/mL. The 20× wash buffer is diluted 1:20 with distilled water. Manual or automated washing methods are both acceptable, with five washes recommended. The procedure involves setting up standard, sample, and blank wells, adding reagents in the correct order, incubating at 37°C for 60 minutes, washing, developing the color, stopping the reaction, and measuring OD values. A standard curve is plotted in Excel using concentration vs. OD, and sample concentrations are calculated accordingly.
Performance characteristics include high accuracy (R ≥ 0.99), sensitivity (<1.0 pg/mL), specificity (no cross-reactivity), and repeatability (CV <15%). The kit should be stored at 2–8°C, protected from light and moisture, and has a shelf life of six months. The detection range spans from 31.2 pg/mL to 1000 pg/mL.
**Disclaimer:** This kit is intended for research purposes only and should not be used in clinical trials or animal experiments. The user assumes all risks and responsibilities associated with its use. Following the manufacturer’s instructions is mandatory. Different batch numbers should not be mixed, and deviations from the protocol may lead to inaccurate results. The company is not liable for any consequences arising from improper use.
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