Rat Atrial Natriuretic Peptide (ANP) ELISA Kit
principle
This experiment uses the double antibody sandwich ABC-ELISA method. The anti-rat ANP monoclonal antibody was coated on the enzyme plate. The ANP in the standard and the sample was combined with the monoclonal antibody. Biotinylated anti-rat ANP was added to form an immune complex and connected to the plate. The enzyme-labeled Streptavidin is combined with biotin, the substrate working solution is added, and the final solution is added with sulfuric acid. The OD value is measured at 450nm. The ANP concentration is proportional to the OD value. concentration.
Kit composition (stored at 2-8 ° C)
Coated Wells
96 wells
Enzyme Conjugate
12ml
10 × Sample Buffer
12ml
20 × concentrated washing solution (Wash Buffer)
50ml
Standards (Standards): 40ng / bottle
2 bottles
Substrate working solution (TMB Solution)
12ml
The first antibody working solution (Biotinylated Antibody)
12ml
Stop Solution (Stop Solution)
12ml
Prepare reagents and collect blood samples
1. Collect specimens: serum, plasma (EDTA), cell culture supernatant, tissue homogenate, urine, etc. as soon as possible, stored at 2-8 ℃ for 48 hours; longer time must be frozen (-20 ℃ or -70 ℃) Store to avoid repeated freezing and thawing. Serum and plasma should be diluted at least 1: 2 (take 50ul, add 50ul of sample dilution, and dilute twice). The cell supernatant is at least 10-fold diluted.
2. Standard solution preparation: add 1ml of distilled water to mix well before use to make a solution of 40ng / ml. Set 8 standard tubes, add 900ul of sample dilution in the first tube, and 500ul of sample dilution in the second to eighth tubes. Add 100 ul of 40 ng / ml standard solution to the first tube and mix well, then aspirate 500 ul with the sampler and move to the second tube. Repeat the dilution in this manner repeatedly, draw 500ul from the seventh tube and discard it. The eighth tube is a blank control.
3. The 10 × specimen dilution is diluted 1:10 with distilled water (example: 1ml concentrated dilution + 9ml distilled water).
4. Washing solution: 1:20 dilution with redistilled water (example: 1ml concentrated washing solution is added with 19ml of redistilled water)
Testing procedures
1. Add sample: add 100ul of standard or sample to be tested to each well, mix the reaction plate thoroughly and place at 37 ℃ for 120 minutes.
2. Wash the plate: wash the reaction plate 4-6 times with the washing solution, and print it on the filter paper.
3. Add 100ul of the first antibody working solution to each well. The reaction plate was mixed thoroughly and then placed at 37 ° C for 60 minutes.
4. Wash board: same as above.
5. Add 100ul of enzyme-labeled antibody working solution to each well. Place the reaction plate at 37 ° C for 30 minutes.
6. Wash board: same as above.
7. Add 100ul of substrate working solution to each well, and place in a dark place at 37 ℃ for 15 minutes.
8. Add 100ul of stop solution to each well and mix well.
9. Measure absorbance at 450nm with a microplate reader within 30 minutes.
Result calculation and judgment
1. All OD values ​​should be calculated after subtracting the blank value.
2. With the standard products 4000, 2000, 1000, 500, 250, 125, 62.5, 0 pg / ml as the abscissa and the OD value as the ordinate, draw a graph on the graph paper and draw a standard curve.
3. Find the corresponding ANP content on the graph according to the OD value of the sample, and then multiply it by the dilution factor.
Kit performance
1. Sensitivity: The minimum detection concentration of ANP is less than 30pg / ml.
2. Specificity: simultaneous detection of recombinant or natural rat ANP. Does not cross-react with other cytokines in rats.
3. Repeatability: The coefficient of variation within the board and the board is less than 8.9%.
Precautions
1. The above standard holes and samples to be tested are recommended to be re-holes, and the standard curve should be made at the same time for each measurement.
2. The washing process is critical. Insufficient washing will lead to an increase in accuracy error and OD value.
3. After opening the slats, the remaining slats should be sealed again to keep the slats dry.
4. This kit should be stored in a 4oC refrigerator.
5. This kit is for scientific research only, not for clinical diagnosis!
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