The rat adenosine ELISA kit is based on a double-antibody one-step sandwich ELISA technique. The microtiter plates are pre-coated with adenosine-specific capture antibodies. After adding the sample, standard, and HRP-conjugated detection antibody in sequence, the plate is incubated, washed thoroughly, and then developed using TMB substrate. Under the catalytic action of horseradish peroxidase (HRP), TMB turns blue and then changes to yellow when an acidic stop solution is added. The intensity of the color is directly proportional to the amount of adenosine present in the sample. The optical density (OD) is measured at 450 nm using a microplate reader, allowing for accurate quantification of adenosine levels.
Sample collection and preparation are crucial for reliable results. For serum, blood is collected in pyrogen-free tubes, centrifuged at 3000 rpm for 10 minutes, and the supernatant is carefully separated. Plasma samples should be collected using anticoagulants such as EDTA, citrate, or heparin, followed by centrifugation at 3000 rpm for 30 minutes. Cell culture supernatants require centrifugation at 3000 rpm for 10 minutes to remove debris. Tissue homogenates are prepared by grinding tissue in physiological saline and centrifuging at 3000 rpm for 10 minutes. All samples should be aliquoted, stored at -20°C, and avoid repeated freeze-thaw cycles. Thaw samples at room temperature before use to ensure even thawing.
For optimal performance, the following equipment is recommended: a microplate reader set to 450 nm, precision pipettes with various volume ranges (0.5–10 μL, 2–20 μL, 20–200 μL, 200–1000 μL), and a 37°C incubator. Before starting, 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, gently warm it in a water bath before use. Unused strips should be returned to the ziplock bag immediately and stored under low-temperature dry conditions.
All reagents must be properly diluted according to the instructions. The standard curve is prepared by diluting the stock standard (300 ng/mL) into serial concentrations (300, 150, 75, 37.5, 18.7, and 0 ng/mL). Samples should not be diluted unless specified; simply add 10 μL of the sample directly. Follow the protocol strictly, including incubation times, volumes, and sequence of steps. Shake all solutions well before use to ensure uniformity.
The kit includes 96-well or 48-well configurations, with components such as microwells, standards, detection antibody-HRP, wash buffer, substrates A and B, stop solution, seal films, and storage bags. The 20× wash buffer should be diluted 1:20 with distilled water. Washing can be done manually or with an automated washer, with five washes required for each well.
Following the procedure, after incubation, the plate is washed, and TMB is added. After 15 minutes of incubation in the dark, the reaction is stopped, and OD values are read within 15 minutes. A standard curve is plotted in Excel, with concentration on the x-axis and OD on the y-axis. The sample concentration is determined using the regression equation.
The kit offers high accuracy (R ≥ 0.99), sensitivity (<1.0 ng/mL), and specificity, with minimal cross-reactivity. Repeatability is ensured with a coefficient of variation <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 is 9.3–300 ng/mL.
This kit is intended for research purposes only and is not suitable for clinical trials or animal testing. Users are responsible for following the instructions precisely. Mixing different batch numbers is not recommended. Any deviation from the protocol may lead to inaccurate results, and the company assumes no liability.
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