Rat PINP (PINP) ELISA kit instruction manual

Rat PINP (PINP) ELISA kit

  ( used in serum, plasma, cell culture supernatants and other biological fluids )

principle

This experiment used double antibody sandwich ABC-ELISA. The anti-rat PINP monoclonal antibody was coated on the microtiter plate, the standard and the PINP in the sample were combined with the monoclonal antibody, and biotinylated anti-rat PINP was added to form an immune complex attached to the plate, horseradish peroxidation. The enzyme-labeled Streptavidin is combined with biotin, and the substrate working solution is blue. Finally, the stop solution sulfuric acid is added, and the OD value is measured at 450 nm. The PINP concentration is proportional to the OD value, and the PINP in the specimen can be obtained by drawing a standard curve. concentration.

Kit composition ( 2-8 ° C preservation)

Prepare reagents and collect blood samples

1. Collection of specimens: serum, plasma (EDTA, citrate, heparin anticoagulation), cell culture supernatant, tissue homogenate, etc., as early as possible, stored at 2-8 ° C for 48 hours; longer time must be frozen (-20 °C or -70 °C) Save to avoid repeated freezing and thawing.

2. Standard solution preparation: Add 0.5ml of distilled water before use and mix well to form a 10000ng/ml solution. Set 8 tubes of standard tube, add 800ul of standard dilution to the first tube, and add 500ul of specimen dilution to the second to eighth tubes. Add 10,000 ng/ml of the standard solution 200 ul to the first tube, mix and aspirate 500 ul with a pipette, and transfer to the second tube. Repeat the dilution as described above, and remove 500 ul from the seventh tube and discard it. The eighth tube is a blank control.

3. The 10× specimen dilution was diluted 1:10 with distilled water (example: 1 ml concentrated dilution + 9 ml distilled water).

4. Washing solution: diluted 1:20 with distilled water (example: 1 ml concentrated washing solution added to 19 ml of distilled water)

Test procedure

1. Loading: Add 100 ul of standard or sample to be tested in each well. Mix the reaction plate thoroughly and let it stand at 37 °C for 120 minutes.

2. Wash the plate: Wash the plate thoroughly with washing solution 4-6 times, and dry it on the filter paper.

3. Add 100 ul of the first antibody working solution to each well. The reaction plate was thoroughly mixed and placed at 37 ° C for 60 minutes.

4. Wash the board: the same as before.

5. Add 100 ul of enzyme-labeled antibody working solution per well. The reaction plate was placed at 37 ° C for 30 minutes.

6. Wash the board: same as before.

7. Add 100 ul of substrate working solution per well, set 37 The reaction was carried out in the dark at °C for 15 minutes.

8. Add 100 ul of stop solution to each well and mix.

9. Measure the absorbance at 450 nm using a microplate reader within 30 minutes.

Result calculation and judgment

1. All OD values ​​should be subtracted from the blank value before calculation.

2. Using standard products 2000, 1000, 500, 250, 125, 62.5, 31.2, 0 ng/ml as the abscissa and OD as the ordinate, plot on the coordinate paper and draw the standard curve.

3. Find the corresponding PINP content on the graph based on the sample OD value.

Kit performance

1. Sensitivity: The minimum PINP detection concentration is less than 16 ng/ml.

2. Specificity: Recombinant or natural rat PINP can be detected simultaneously. Does not cross-react with other cytokines in rats.

3. Repeatability: The coefficient of variation in the plate and plate is less than 9.6%.

Precautions

1. It is recommended to make double holes for the above standard holes and samples to be tested. The standard curve should be made at the same time for each measurement.

2. The washing process is critical. Insufficient washing will result in an accuracy error and an erroneous rise in the OD value.

3. After the slats are opened, the remaining slats should be sealed again to keep the slats dry .

4. This kit should be stored in a 4 ^o C refrigerator.

5. This kit is for scientific research only and cannot be used for clinical diagnosis!