This reagent is for research use only. Specimen: serum or plasma
Test principle:
The HSP-70 kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA). Standards of known HSP-70 concentration and samples of unknown concentration are added to the microplates for detection. HSP-70 and biotinylated antibodies were first incubated simultaneously. After washing with the human heat shock protein-70 ELISA test kit, avidin-labeled HRP was added. After incubation and washing, the unbound enzyme conjugate is removed, then the substrates A, B, and the enzyme conjugate are added simultaneously. Produce color. The depth of the color is proportional to the concentration of HSP-70 in the sample.
Self-contained materials
Distilled water.
Sampler: 5ul, 10ul, 50ul, 100ul, 200, 500ul, 1000ul.
Oscillators and magnetic stirrers, etc.
safety
Avoid direct contact with the stop solution and substrates A, B. Once exposed to these liquids, rinse with water as soon as possible.
Do not eat, drink, smoke or use cosmetics during the experiment.
Do not use your mouth to take any ingredients from the kit.
Operational precautions
Reagents should be stored according to the label instructions and returned to room temperature before use. Standards after dilution should be discarded and cannot be stored.
The slats not used in the experiment should be immediately put back into the bag and sealed to prevent deterioration.
Other reagents not used should be packaged or covered. Do not mix reagents of different batches. Use before warranty.
Use a disposable tip to avoid cross-contamination. Avoid pipettes with metal parts when drawing stop solution and substrate A and B.
Use a clean plastic container to configure the wash solution. Mix all the ingredients and samples in the kit thoroughly before use.
Wash the enzyme plate should be fully patted dry, do not put the absorbent paper directly into the enzyme standard reaction well to absorb water.
Substrate A should be volatilized to avoid opening the lid for extended periods of time. The human heat shock protein-70 ELISA assay kit substrate B is sensitive to light and avoids prolonged exposure to light. Avoid contact with hands and be toxic. The OD value should be read immediately after the experiment is completed.
The order of addition of reagents should be consistent to ensure that all wells are incubated for the same time.
The incubation was carried out according to the time indicated in the instructions, the amount of addition and the order.
Sample collection, processing and storage methods
Serum ---- Avoid any cell irritation during the procedure. Use tubes without pyrogens and endotoxins. After collecting the blood, the serum and red blood cells were quickly and carefully separated by centrifugation at 1000 x g for 10 minutes.
Plasma-----EDTA, citrate, heparin plasma can be used for detection. The pellet was removed by centrifugation at 1000 x g for 30 minutes.
The cell supernatant - 1000 x g was centrifuged for 10 minutes to remove particles and polymer.
Storage ------If the sample is not used immediately, it should be stored in small portions at -70 °C to avoid repeated freezing. Do not use hemolysis or hyperlipemia as much as possible. If there are large amounts of particles in the serum, centrifuge or filter before testing. Do not heat thaw at 37 ° C or higher. It should be thawed at room temperature and ensure that the sample is fully thawed evenly.
Reagent preparation
Standard: The serial dilution of the standard should be prepared during the experiment and cannot be stored. Mix the standard shakes before dilution.
Dilution of wash buffer (50 x): 50-fold dilution of distilled water.
Steps
Mix all reagents thoroughly before use. Do not allow the liquid to generate a large amount of foam, so as to avoid adding a large amount of air bubbles during the loading, resulting in errors in the loading.
The number of slats required is determined by the number of samples to be tested plus the number of standards. Multiple holes are recommended for each standard and blank hole. Each sample is determined according to its own quantity, and it is possible to use the double hole as much as possible to make a double hole.
50 ul of the diluted standard was added to the reaction well, and 50 ul of the sample to be tested was added to the reaction well. Immediately add 50 ul of biotinylated antibody. Cover the membrane, mix gently by shaking, and incubate for 1 hour at 37 °C.
Remove the liquid from the well, fill each well with the washing solution, shake for 30 seconds, remove the washing solution, and pat dry with absorbent paper. Repeat this operation 3 times. If washing with a washer, the number of washings is increased once.
80 ul of affinity streptavidin-HRP was added to each well, and the mixture was gently shaken and incubated at 37 ° C for 30 minutes.
Remove the liquid from the well, fill each well with the washing solution, shake for 30 seconds, remove the washing solution, and pat dry with absorbent paper. Repeat this operation 3 times. If washing with a washer, the number of washings is increased once.
Add 50 μl of each of the substrates A and B to each well, mix gently by shaking, and incubate at 37 ° C for 10 minutes. Avoid lighting.
Remove the microplate and quickly add 50 ul of stop solution. Immediately after adding the stop solution, the results should be determined.
The OD value of each well was measured at a wavelength of 450 nm.
Limit
The results above the No. 6 standard are non-linear and no accurate results can be obtained from this standard curve.
Kit performance
1. Sensitivity: The minimum detection concentration is less than the No. 1 standard. The linearity of the dilution. The linear regression coefficient of the sample and the expected concentration correlation coefficient R value was 0.990.
2. Specificity: Does not react with other human cytokines.
3. Repeatability: The coefficient of variation between the plate and the plate is less than 10%.
Result judgment and analysis
1. Instrument value: read the OD value of each hole on a microplate reader with a wavelength of 450 nm.
2. The absorbance OD value is the ordinate (Y), and the corresponding HSP-70 standard concentration is the abscissa (X). The corresponding curve is obtained. The HSP-70 content of the sample can be converted from the standard curve according to the OD value. The corresponding concentration.
3. Detection range: 0-80ng/ml
4. Sensitivity: 0.1 ng/ml
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