Human Nicotinamide Adenine Dinucleotide Phosphate (NADPH) ELISA Kit Instructions

l This kit is used for quantitative determination of nicotinamide adenine dinucleotide phosphate (NADPH) in serum, plasma, tissue, cell supernatant and related liquid samples in vitro.

l Validity period: 6 months

l Storage conditions: 2 -8 ° C

Experimental principle of human nicotinamide adenine dinucleotide phosphate (NADPH) ELISA kit

The kit uses a double antibody one-step sandwich enzyme-linked immunosorbent assay (ELISA). To the coated microwells pre-coated with human nicotinamide adenine dinucleotide phosphate (NADPH) capture antibody, specimens, standards, HRP-labeled detection antibodies were sequentially added, incubated and thoroughly washed. Using the substrate TMB to develop color, TMB is converted to blue under the catalysis of peroxidase and converted to the final yellow color by the action of an acid. The color depth is positively correlated with human nicotinamide adenine dinucleotide phosphate (NADPH) in the sample. The absorbance (OD value) was measured at 450 nm using a microplate reader to calculate the sample concentration.

Nicotinamide adenine dinucleotide phosphate (NADPH) ELISA kit sample processing and requirements

1. Serum : Whole blood samples should be placed at room temperature for 2 hours or 4 °C overnight, centrifuged at 1000g for 20 minutes, the supernatant can be detected, or the specimens can be stored at -20 ° C or -80 ° C, but should be avoided repeatedly melt.
2. Plasma : EDTA or heparin can be used as anticoagulant. The specimen should be centrifuged at 2-8 °C for 1000 minutes within 20 minutes after collection, or the specimen should be stored at -20 °C or -80 °C, but repeated freezing and thawing should be avoided. .
3. Cell culture supernatant or other biological specimens : centrifuge at 1000g for 20 minutes, take the supernatant to detect, or store the specimen at -20 °C or -80 °C, but avoid repeated freezing and thawing.
Note: Specimen hemolysis will affect the final test results, so hemolysis specimens should not be tested.

Reagents and equipment that are needed but not provided

• Microplate reader (450nm)
• High-precision sampler and tip: 0.5-10uL, 2-20uL, 20-200uL, 200-1000uL
• 37 ° C incubator
• Distilled or deionized water

Precautions

• Incubation is carried out strictly in accordance with the specified time and temperature to ensure accurate results. All reagents must reach room temperature 20-25 °C before use. Store the reagents refrigerated immediately after use.
• Incorrect cleaning can result in inaccurate results. Make sure to drain the liquid in the well as much as possible before adding the substrate. Do not let the micropores dry during the incubation process.
• Eliminate residual liquid and fingerprints at the bottom of the board, otherwise it will affect the OD value.
• The substrate coloring solution should be colorless or very light, and the substrate liquid that has turned blue cannot be used.
• Avoid cross-contamination of reagents and specimens to avoid erroneous results.
• Avoid direct exposure to strong light during storage and incubation.
• After balancing to room temperature, the sealed bag is opened to allow water-repellent drops to condense on the cold slats.
• Any reagents should not be exposed to strong gases emitted by bleaching solvents or bleaching solvents. Any bleaching component will destroy the biological activity of the reagents in the kit.
• Cannot use expired products.
• If the disease is likely to spread, all samples should be managed and the sample and test device processed in accordance with the prescribed procedures.

Reagent preparation

The kit should be taken out of the refrigerated environment and should be balanced at room temperature before use.

Dilution of 20× Wash Buffer: Distilled water was diluted 1:20, ie 1 part of 20× Wash Buffer plus 19 parts of distilled water.

Steps

1. The required slats were taken out from the aluminum foil pouch after equilibrating for 20 min at room temperature, and the remaining slats were sealed back to 4 ° C with a ziplock bag.
2. Set standard and sample wells, standard wells with different concentrations of standard 50μL;
3. Add 50 μL of the sample to be tested to the sample well; blank holes are not added.
4. In addition to the blank wells, 100 μL of horseradish peroxidase (HRP)-labeled detection antibody was added to each well of the standard well and the sample well, and the reaction well was sealed with a sealing plate, and incubated at 37 ° C in a water bath or incubator for 60 min.
5. Discard the liquid, pat dry on the absorbent paper, fill each well with the washing solution (350 μL), let stand for 1 min, remove the washing solution, pat dry on the absorbent paper, and repeat the washing 5 times (can also be washed with a washing machine) .
6. 50 μL of each of the substrates A and B was added to each well, and incubated at 37 ° C for 15 min in the dark.
7. 50 μL of the stop solution was added to each well, and the OD value of each well was measured at a wavelength of 450 nm within 15 min.

Experimental result calculation

The OD value of the tested standard is the abscissa, the concentration value of the standard is the ordinate, the standard curve is drawn on the coordinate paper or with the relevant software, and the linear regression equation is obtained, and the OD value of the sample is substituted into the equation to calculate the sample. concentration.

Kit performance

• Detection range: 3.75 ng/mL – 120 ng/mL.
• Sensitivity: The minimum detection concentration is less than 0.1 ng/mL.
• Specificity: Does not cross-react with other soluble structural analogs.
• Repeatability: The intra-plate variation coefficient is less than 10%, and the inter-plate variation coefficient is less than 15%.

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