Instruction manual of rat pregnancy-associated plasma protein A (PAPP-A) ELISA kit

This kit is for research use only
Intended application
Quantitative determination of PAPP-A in rat serum, plasma or other related fluids by ELISA.
Experimental principle This kit uses double antibody sandwich enzyme-labeled immunoassay to determine the level of PAPP-A in the specimen. The microtiter plate is coated with purified antibody to make a solid-phase antibody. PAPP-A antigen, biotinylated anti-rat PAPP-A antibody, and HRP-labeled avidin are added to the monoclonal antibody-coated microwells in sequence. After thorough washing, the color is developed with the substrate TMB. TMB is converted into blue under the catalysis of peroxidase, and into the final yellow under the action of acid. The color depth is positively correlated with PAPP-A in the sample. The absorbance (OD value) was measured with a microplate reader at a wavelength of 450 nm, and the sample concentration was calculated.
Kit composition
1. ELISA plate: one piece (96 wells)
2. Standard product (lyophilized product): 2 bottles, each bottle is diluted with the sample diluent to 1ml before use, and then left to stand for more than 10 minutes after being capped, and then repeatedly inverted / rubbed to help dissolve. / L, after serial dilutions, dilute to 10 mg / L, 5 mg / L, 2.5 mg / L, 1.25 mg / L, 0.62 mg / L, 0.31 mg / L, 0.15 mg / L, and their stock solutions It is directly used as the highest standard concentration, and the sample dilution is directly used as the standard concentration of 0 mg / L, prepared within 15 minutes before use.
3. Sample diluent: 1 × 20ml / bottle.
4. Test the diluent A: 1 × 10ml / bottle.
5. Test diluent B: 1 × 10ml / bottle.
6. Test solution A: 1 × 120ul / bottle (1: 100) is diluted with test diluent A1: 100 before use. Before dilution, according to the pre-calculated total amount required for each experiment (100ul per well), 0.1-0.2ml should be prepared in actual preparation. For example, 1ul detection solution A plus 99ul detection dilution A is prepared in proportion, mix gently and prepare within one hour before use.
7. Detection solution B: 1 × 120ul / bottle (1: 100) is diluted with detection diluent B1: 100 before use. The dilution method is the same as that of Test Solution A.
8. Substrate solution: 1 × 10ml / bottle.
9. Concentrated washing solution: 1 × 30ml / bottle, each bottle is diluted 25 times with distilled water.
10. Stop solution: 1 × 10ml / bottle (2N H2SO4).

Collection and preservation of specimens
1. Serum: Please leave the specimen at room temperature for 2 hours or overnight at 4 ° C and centrifuge at 1000 xg for 20 minutes. Take the supernatant for testing, or store the specimen at -20 ° C, but avoid repeated freezing and thawing.
2. Plasma: EDTA or heparin can be used as an anticoagulant. Centrifuge at 1000 xg at 2-8 ° C for 15 minutes within 30 minutes after the specimen is collected, or store the specimen at -20 ° C, but avoid repeated freezing and thawing.
Note: Hemolysis of specimens will affect the final test results, so hemolysis specimens should not be tested.

Operation steps Each reagent is equilibrated to room temperature before use.
1. Add sample: set blank hole, standard hole and sample hole to be tested respectively. In addition to the blank wells, add 100ul of the standard solution or the sample to be tested to the remaining wells and mix gently, at 37 ℃ for 120 minutes.
2. Discard the liquid and spin dry without washing.
3. Add 100ul of detection solution A working solution to each well at 37 ℃ for 60 minutes. Wash the plate 3 times, 350ul / per well, spin dry.
4. Add 100ul of detection solution B working solution to each well at 37 ° C for 60 minutes, wash the plate 5 times and spin dry.
5. Add 90ul of substrate solution to each well in sequence, and develop color at 37 ° C in the dark for 30 minutes.
6. Add 50ul of stop solution to each well in sequence to stop the reaction. The optical density (OD value) of each well was measured sequentially with an enzyme-linked instrument at a wavelength of 450 nm.
Note:
1. One hole is left for each experiment as a blank zero-adjusting hole. No reagents are added to this hole, only the substrate solution and 2NH2SO4 are added at the end. Use this hole to adjust the OD value to zero when measuring.
2. To prevent the sample from evaporating, place the reaction plate in a closed box covered with a damp cloth during the test.
Plate washing method Manual plate washing method: suck (do not touch the wall) or shake off the liquid in the microplate; place a few layers of absorbent paper on the experimental table, and force the microplate down several times; pat the recommended wash buffer Inject at least 0.4ml of solution into the hole and soak for 1-2 minutes. Repeat this process several times as needed.
Automatic plate washing: If there is an automatic plate washing machine, it should be used in the formal experiment process after being used skillfully.
Specificity This kit can detect recombinant or natural rat PAPP-A at the same time without cross-reactivity with other related proteins.
Calculate the standard concentration as the abscissa (logarithmic coordinate), the OD value is the ordinate (ordinary coordinate), draw a standard curve on semi-logarithmic coordinate paper, and find the corresponding concentration from the standard curve according to the OD value of the sample ; Multiply by the dilution factor; or use the standard concentration and OD value to calculate the linear regression equation of the standard curve, substitute the OD value of the sample into the equation, calculate the sample concentration, and multiply by the dilution factor, which is the actual concentration of the sample .
Precautions
1. The washing process is very important, inadequate washing is easy to cause false positives.
2. It is best to control the sampling time within 5 minutes. If there are many specimens, it is recommended to use a volley gun to add samples.
3. Please make a standard curve at the same time of each measurement, it is best to make a double hole.
4. If the content of the substance to be tested in the specimen is too high, please dilute it and then determine it. When calculating, please multiply by the dilution factor.
5. When preparing the standard and the working solution of the test solution, please prepare it with the corresponding diluent, not to be confused.
6. Please keep the substrate away from light.
examination range:
0.15 mg / L -10 mg / L
Explanation
1. Avoid exposing the reagent to strong light during storage and incubation. All reagent bottle caps must be tightly closed to prevent evaporation and contamination. Reagents should be protected from microbial contamination, because the interference of proteolytic enzymes will lead to erroneous results.
2. Aspirate the reagents carefully and strictly observe the given incubation time and temperature. Please note that when drawing samples / standards, enzyme conjugates or substrates, if the time interval between the first well and the last well is too large, it will result in different "pre-incubation" time, which obviously Affect the accuracy and repeatability of the measured value. Moreover, insufficient washing will affect the test results.
3. Storage of the kit: some reagents are stored at -20 ° C, and some reagents are stored at 2-8 ° C, depending on the label.
4. Salt will precipitate out in the concentrated washing liquid, and it can be heated and dissolved in the water bath when diluted.
5. There may be some water-like substances in the well of the enzyme-linked plate just opened. This is a normal phenomenon and will not have any impact on the experimental results.
6. There may be inconsistencies between the Chinese and English instructions. Please refer to the English instructions.
7. All samples should be managed, and the samples and testing devices should be processed according to the prescribed procedures.
8. Validity: 6 months

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