Instruction manual of human corticotropin releasing hormone (CRH) ELISA kit

This kit is for research use only

Detection range: 1 ng / ml-40 ng / ml
Minimum detection limit: 1 ng / ml
Specificity: This kit can detect natural or recombinant human CRH at the same time without cross-reactivity with other related proteins.
Validity: 6 months Expected application: ELISA method for quantitative determination of CRH content in human serum or other related biological fluids.
Explanation
1. Kit storage: -20 ℃ (when not in use for a long time); 2-8 ℃ (when used frequently).
2. The concentrated washing liquid will be salted out at low temperature, and it can be heated and dissolved in the water bath when diluted.
3. There may be inconsistencies between the Chinese and English instructions. Please refer to the English instructions.
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.
Experimental principle The purified sheep anti-rabbit is coated into a microwell plate to make a solid phase carrier, and then the specimen or standard, HRP-labeled CRH and anti-CRH antibody are added to the microwell in sequence, and washed with a substrate solution after thorough washing. color. The substrate 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 CRH 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. Assay plate: one piece (96 wells).
2. Standard product (Standard): 5 × 0.5 ml / bottle.
Standard product S1 S2 S3 S4 S5
Concentration (ng / ml) 0.8 2.4 8 20 40

3. Enzyme conjugate (HRP-Conjugate): 1 × 6 ml / bottle.
4. Antibody: 1 × 6 ml / bottle
5. Substrate A: 1 × 7ml / bottle.
6. Substrate B: 1 × 7ml / bottle.
7. Wash Buffer: 1 × 15ml / bottle, each bottle is diluted 20 times with distilled water.
8. Stop solution (Stop Solution): 1 × 10ml / bottle (2N H2SO4).

Reagents and equipment needed but not provided
1. Standard Specification Microplate Reader
2. High-speed centrifuge
3. Electric heating thermostat incubator
4. Clean test tubes and Eppendof tubes
5. Series adjustable pipettes and tips. When testing more samples at one time, it is best to use multi-channel pipettes
6. Distilled water, volumetric flasks and other specimen collection and preservation of serum: Whole blood specimens should be placed at room temperature for 2 hours or 4 ℃ overnight after centrifugation at 1000 xg for 20 minutes, take the supernatant for testing, or put the specimen at -20 ℃ Or store at -80 ℃, but avoid repeated freezing and thawing.
Note: Hemolysis of specimens will affect the final test results, so hemolysis specimens should not be tested.
Dilution principle of specimens:
First of all, we should know the approximate content of the sample to be tested through literature search, and determine the appropriate dilution factor. Only when it is diluted to the range of the standard curve, the test result is accurate. Detailed records should be made during the dilution process. When calculating the concentration at the end, it was diluted "N" times, and the concentration of the specimen should be multiplied by "N".
Before starting the experiment, please configure all reagents in advance. When diluting the reagents or samples, they should be mixed evenly. Try to avoid foaming when mixing. A standard curve should be made for each test. If the sample concentration is too high, dilute it first to make the sample meet the detection range of the kit.
1. Add sample: set a blank control well without adding any solution. Add 50μl of the standard or the sample to be tested to the remaining wells. Be careful not to have air bubbles. Add the sample to the bottom of the well of the microplate and try not to touch the wall of the well.
2. Add 50μl of enzyme conjugate and 50μl of antibody to each well, and no blank well. Mix well and incubate at 37 ° C for 2 hours.
3. After incubation, discard the liquid in the wells, spin dry, wash the plate 3 times, soak for 1-2 minutes each time, 200μl / well, spin dry.
4. Add 50μl of substrate solution A and B to each well in sequence, and develop at 37 ℃ in the dark for 15 minutes.
5. Add 50μl of stop solution to each well in sequence to stop the reaction (at this time, the blue color turns to yellow). The order of adding the stop solution should be the same as that of the substrate solution. In order to ensure the accuracy of the experimental results, the termination solution should be added as soon as possible after the substrate reaction time expires.
6. Measure the optical density (OD value) of each well in sequence using an enzyme-linked instrument at a wavelength of 450 nm. Test within 15 minutes after adding stop solution.
Note:
1. When using the reagent kit for the first time, the user should centrifuge various reagent tubes for several minutes so that the reagents are concentrated to the bottom of the tube.
2. Leave one well for each experiment as a blank zero-adjusting well. No reagents are added to this well, only the substrate solution and 2N H2SO4 are added at the end. Use this hole to adjust the OD value to zero when measuring.
3. To prevent the sample from evaporating, place the reaction plate in a closed box covered with a damp cloth during the test, and add a cover or film to the enzyme label plate.
4. It is recommended to set a double-hole test when testing samples to ensure the accuracy of the test results.
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 0.3ml 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.
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. When mixing protein solutions, try to be as gentle as possible to avoid foaming.
2. The washing process is very important. Insufficient washing can easily cause false positives.
3. 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.
4. Please make the standard curve at the same time of each measurement, it is better to make the complex hole.
5. 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.
6. Please keep the substrate away from light.
7. Do not replace the reagents in the kit with reagents from other manufacturers.

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