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The Brucellosis-Ag ELISA kit operates on the principle of a double-antibody one-step sandwich ELISA. The microtiter plate is pre-coated with specific antibodies against Brucellosis-Ag. During the assay, the sample, standard, and HRP-labeled detection antibody are sequentially added to the wells. After incubation and thorough washing, the substrate TMB is introduced. Under peroxidase catalysis, TMB turns blue and then yellow when an acid is added. The intensity of the color correlates directly with the amount of Brucellosis-Ag in the sample. The optical density (OD) at 450 nm is measured using a microplate reader, and the concentration of Brucellosis-Ag is calculated based on a standard curve.
Sample collection and handling are critical for accurate results. For serum, blood should be collected in endotoxin-free tubes, centrifuged at 3000 rpm for 10 minutes, and the supernatant carefully separated. Plasma samples require anticoagulants such as EDTA, citrate, or heparin, followed by centrifugation at 3000 rpm for 30 minutes. Cell culture supernatants should be centrifuged at 3000 rpm for 10 minutes to remove debris. Tissue homogenates are prepared by adding physiological saline, chopping the tissue, and centrifuging at 3000 rpm for 10 minutes. All samples should be aliquoted and stored at -20°C, avoiding repeated freeze-thaw cycles. Thaw samples at room temperature before use.
To ensure accurate results, proper equipment is essential. A microplate reader set to 450 nm, along with high-precision pipettes (ranging from 0.5–10 µL to 200–1000 µL), is required. The kit should be stored at 2–8°C, and all reagents should be allowed to equilibrate at room temperature for 20 minutes before use. If the washing buffer crystallizes after removal from the fridge, it can be dissolved by warming in a water bath.
During the experiment, unused plates should be returned to the sealed bag immediately. No dilution is needed for pre-treated samples, and 10 µL of the sample can be directly added. Incubation times and volumes must strictly follow the protocol. All liquid components should be thoroughly mixed before use.
The kit includes a 96-well or 48-well configuration, with standard solutions, diluted standards, sample diluent, detection antibody-HRP, wash buffer, substrates A and B, stop solution, and sealing films. The standard dilutions range from 500 ng/mL down to 0 ng/mL. The 20× wash buffer should be diluted 1:20 with distilled water. Washing can be done manually or automatically, with five wash cycles recommended.
The procedure involves setting up standard, sample, and blank wells, adding 50 µL of standard solutions and 50 µL of HRP-labeled antibody, followed by incubation at 37°C for 60 minutes. After washing, TMB is added, and the reaction is stopped with the stop solution. OD values are read within 15 minutes, and the standard curve is plotted in Excel to determine sample concentrations.
Performance characteristics include high accuracy (R ≥ 0.99), sensitivity (less than 1.0 ng/mL), and specificity without cross-reactivity. Repeatability is below 15% between plates. The kit should be stored at 2–8°C, away from light and moisture, and has a shelf life of six months. The detection range is 15.6–500 ng/mL.
This kit is for research purposes only. It should not be used in clinical trials or animal experiments, as the user assumes full responsibility for any consequences. Always follow the instructions carefully, and do not mix different batch numbers. Any deviation from the protocol may affect the reliability of results.
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