The FIV ELISA kit is based on a double-antibody one-step sandwich ELISA principle. The microplate wells are pre-coated with feline immunodeficiency virus (FIV) capture antibodies. After adding the samples, standards, and HRP-labeled detection antibodies sequentially, the plate is incubated, washed thoroughly, and then developed with TMB substrate. Under the action of peroxidase, TMB turns blue and then yellow when an acid is added. The intensity of the color correlates directly with the amount of FIV in the sample. The optical density (OD) at 450 nm is measured using a microplate reader to determine the concentration of FIV in the sample.
Sample collection and preparation should be done carefully. For serum, use endotoxin-free tubes, centrifuge at 3000 rpm for 10 minutes after blood collection. Plasma samples require anticoagulants like EDTA or heparin, followed by centrifugation at 3000 rpm for 30 minutes. Cell supernatants should be centrifuged at 3000 rpm for 10 minutes to remove debris. Tissue homogenates need to be prepared in saline and centrifuged similarly. Store samples at -20°C if not tested immediately, avoiding repeated freeze-thaw cycles.
For the experiment, you will need a microplate reader (set to 450 nm), precision pipettes, and a 37°C incubator. Before use, store the kit at 2–8°C and allow it to equilibrate at room temperature for 20 minutes. If the washing buffer crystallizes after being taken from the fridge, warm it gently before use. Unused strips should be returned to the ziplock bag and stored properly. No dilution is needed for the samples; simply add 10 μL directly.
Follow the instructions strictly regarding incubation time, volume, and sequence. Shake all reagents well before use. The kit includes microwells, standards, dilutions, detection antibody-HRP, wash buffer, substrates, stop solution, and sealing films. Prepare the 20× wash buffer by diluting it 1:20 with distilled water. Manual or automated washing methods can be used, with 5 washes required.
The procedure involves setting up standard, sample, and blank wells. Add 50 μL of standard solutions, 10 μL of sample plus 40 μL diluent, and 50 μL of HRP-labeled antibody. Incubate at 37°C for 60 minutes, then wash five times. Add 50 μL of TMB A and B, incubate in the dark for 15 minutes, and stop the reaction with the stop solution. Measure OD values within 15 minutes. Use Excel to plot a standard curve and calculate sample concentrations.
Performance characteristics include high accuracy (R ≥ 0.99), sensitivity (<1.0 ng/mL), specificity, and good repeatability (CV <15%). Store the kit at 2–8°C away from light and moisture. It has a shelf life of 6 months and detects FIV in the range of 15.6–500 ng/mL.
This kit is for research purposes only. Not suitable for clinical or in vivo testing. The company is not responsible for misuse or deviations from the instructions. Always follow the protocol carefully and do not mix reagents from different batches. Any errors due to improper handling are the user’s responsibility.
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