ELISA kit instruction manual-human full parathyroid hormone (I-PTH)
This kit is for research use only
Intended application
Quantitative determination of total parathyroid hormone (I-PTH) content in human serum, plasma or other related fluids by ELISA.
Experimental principle
PTH is a straight-chain peptide of 84 amino acids secreted by parathyroid gland cells, with a molecular weight of 9000, and its biological activity is determined by amino acid residues 1-27 of the N-terminus. Synthesize a pro-parathyroid hormone (pre pro-PTH) containing 115 amino acids in the parathyroid main cells, and then take off the N-terminal twenty-five peptide to generate ninety peptide proparathyroid hormone -PTH), after removing 6 amino acids, it becomes PTH. After PTH is released into the blood, it is called whole PTH (intact PTH, I-PTH).
This kit uses double antibody sandwich enzyme-labeled immunoassay to determine the level of I-PTH in the specimen. The microtiter plate was coated with purified antibody to make a solid phase antibody. I-PTH antigen, biotinylated anti-human I-PTH antibody, and HRP-labeled avidin were added to the monoclonal antibody-coated microwells in turn After thorough washing, color was developed with 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 I-PTH 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 and reagent preparation
1. ELISA plate: one piece (96 wells)
Standard product (lyophilized product): 2 bottles, each bottle is diluted with sample diluent to 1ml before use, and left to stand for more than 10 minutes after being capped, and then repeatedly inverted / rubbed to help dissolve, its concentration is 1000 pg / ml , After serial dilution, diluted to 1000 pg / ml, 500 pg / ml, 250 pg / ml, 125 pg / ml, 62.5 pg / ml, 31 pg / ml, 15.6 pg / ml, the original solution is directly used as The highest standard concentration, the sample dilution is directly used as the standard concentration of 0 pg / ml, and prepared within 15 minutes before use.
For example, to prepare a 500 pg / ml standard: take 0.5 ml 1000 pg / ml of the above standard and add it to an Eppendorf tube containing 0.5 ml of sample diluent, mix well, and so on for the remaining concentrations.
2. Sample diluent: 1 × 20ml / bottle.
3. Test the diluent A: 1 × 10ml / bottle.
4. Test dilution B: 1 × 10ml / bottle.
5. Detection solution A: 1 × 120ul / bottle (1: 100), diluted with detection diluent A 1: 100 before use, prepared according to the pre-calculated total amount required for each experiment before dilution (100ul per well) , The actual preparation should be more 0.1-0.2ml. For example, 1ul detection solution A plus 99ul detection dilution A is prepared in proportion, mix gently and prepare within one hour before use.
6. 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.
7. Substrate solution: 1 × 10ml / bottle.
8. Concentrated washing solution: 1 × 30ml / bottle, each bottle is diluted 25 times with distilled water.
9. 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 x g 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 the sample at 2-8 ° C 1000 x g for 15 minutes within 30 minutes after collection, 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 100 ul of standard solution or sample to be tested to the remaining wells, taking care not to have air bubbles, mix gently, cover the enzyme plate, and react at 37 ° C 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 ℃ for 60 minutes, wash the plate 5 times and spin dry.
5. Add 90ul of substrate solution to each well in sequence, and develop the color for 30 minutes at 37 ° C in the dark (at this time, the first 3-4 wells of the standard product have a clear blue gradient, and the latter 3-4 wells are not obvious).
6. Add 50ul of stop solution to each well in sequence to stop the reaction (in this case, the blue color turns to yellow). 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 human I-PTH at the same time, and does not cross-react 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:
15.6 pg / ml -1000 pg / ml
Explanation
1. Kit storage: -20 ℃ (when not in use for a long time); 2-8 ℃ (when used frequently).
2. Validity: 6 months
3. The concentrated washing liquid will have salt precipitation, and it can be heated and dissolved in the water bath when diluted.
4. The well of the ELISA plate just opened may contain a little water-like substance. This is normal and will not have any impact on the experimental results.
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