Instruction Manual of Human Heat Shock Protein 60 (Hsp-60) ELISA Kit
This kit is for research use only
Intended application
Quantitative determination of human heat shock protein 60 (Hsp60) in human serum, plasma, cell culture supernatant or other related fluids by ELISA.
Experimental principle
Heat shock protein 60 (HSP60) is a family of highly conserved proteins. In the physiological state, it assists the correct transposition, folding and assembly of peptides or proteins, and acts as a "molecular chaperone"; under stress, HSP60 is overexpressed or ectopically expressed as a self-antigen recognized by the immune system and induced The body's protective immune response can also function as a signaling molecule in signal transduction. Recent studies have confirmed that HSP60 plays an important role in the pathogenesis of autoimmune diseases, infectious diseases, atherosclerosis and chronic infections The role.
This kit uses double antibody sandwich enzyme-labeled immunoassay to determine the level of HSP60 in the specimen. The microtiter plate is coated with purified antibody to make a solid phase antibody. HSP60 antigen, biotinylated anti-human HSP60 antibody, and HRP-labeled avidin are added to the monoclonal antibody-coated microwells in turn. After thorough washing, use Substrate TMB color development. 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 HSP60 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)
2. Standard product (lyophilized product): 2 bottles, each of which is diluted with sample diluent to 1ml before use. After being capped, it is allowed to stand for more than 10 minutes, and then repeatedly inverted / rubbed to help dissolve. Its concentration is 10000 pg / mL, after serial dilution, dilute to 10000 pg / mL, 5000 pg / mL, 2500 pg / mL, 1250 pg / mL, 625 pg / mL, 312 pg / mL, 156 pg / mL As the highest standard concentration directly, the sample dilution is directly used as the standard concentration of 0 pg / mL, prepared within 15 minutes before use.
For example, to prepare a 5000 pg / mL standard: Take 0.5 ml of the above-mentioned standard 10000 pg / mL and add it to an Eppendorf tube containing 0.5 ml of sample diluent, mix well. The rest of the concentration can be deduced by analogy.
3. Sample diluent: 1 × 20ml / bottle.
4. Test the diluent A: 1 × 10ml / bottle.
5. Test diluent B: 1 × 10ml / bottle.
6. 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.
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. Supernatant of cell culture: collect the supernatant after centrifugation, and store the specimen at -20 ℃, and avoid repeated freezing and thawing.
2. 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.
3. 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.
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 ° 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 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 have no obvious gradient).
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
The kit can detect recombinant or natural human HSP60 at the same time, and has no cross reaction with other related proteins.
Calculation
Taking the concentration of the standard as the abscissa (logarithmic coordinate) and the OD value as 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 to obtain 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:
156 pg / mL -10000 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.
5. There may be inconsistencies between the Chinese and English instructions. Please refer to the English instructions.
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