Rat PTH ELISA Kit

1. Sample handling and requirements

1. Serum:
Whole blood samples collected in serum separation tubes were placed at room temperature 2 Hour or 4℃ Overnight, then 1000×g Centrifugation 20 Minutes, take the supernatant, or place the supernatant in -20℃ Or -80℃ Store, but repeated freezing and thawing should be avoided.

2. Plasma:
use EDTA Or heparin as an anticoagulant to collect specimens, and collect the specimens after collection 30 Within minutes 2-8℃ 1000×g Centrifugation 15 Minutes, take the supernatant to detect, or place the supernatant in -20℃ Or -80℃ Store, but repeated freezing and thawing should be avoided.

3. Tissue homogenate:
With pre-cooled PBS (0.01M, pH=7.4) The tissue is flushed to remove residual blood (lysed red blood cells in the homogenate can affect the measurement), and the tissue is weighed and cut into pieces. Combining the shredded tissue with the corresponding volume PBS (Generally according to 1:9 Weight to volume ratio, such as 1g The tissue samples correspond to 9mL Of PBS The specific volume can be appropriately adjusted according to the experimental needs and recorded. Recommended in PBS Add protease inhibitor) into a glass homogenizer and ground thoroughly on ice. For further lysis of tissue cells, the homogenate can be sonicated, or freeze-thawed repeatedly. Finally, the homogenate was mixed in 5000×g Centrifugation 5~10 Minutes,
Take the supernatant for detection.

4. Cell culture supernatant or other biological specimen:
Please 1000×g Centrifugation 20 Minutes, take the supernatant to detect, or place the supernatant in -20℃ Or -80℃ Store, but repeated freezing and thawing should be avoided.

Note: Hemolysis of the specimen will affect the final test result, so hemolyzed specimens should not be tested.

Two, Reagent Preparation

The kit should be removed from the refrigerated environment and equilibrated at room temperature before use.

20× Dilution of wash buffer: distilled water according to 1 ： 20 Dilution, i.e. 1 Share 20× Wash buffer plus 19 Distilled water.

Three, Operation steps

1. Equilibration from room temperature 20min
Take out the required slats from the aluminum foil bag after that, and seal the remaining slats with ziplock bag and put them back 4℃ 。

2. Set up standard wells and sample wells, add different concentrations of standards to each standard well 50μL ；

3. The sample to be tested is added to the sample well 50μL ；
Blank holes are not added.

4. In addition to the blank wells, horseradish peroxidase ( HRP ) Labeled detection antibody
100μL Sealing the reaction hole with a sealing plate membrane, 37℃ Incubation in water bath or incubator 60min 。

5. Discard the liquid, pat dry on absorbent paper, and fill each well with wash liquid ( 350μL ), let stand 1min , throw off the washing liquid, pat dry on absorbent paper, and repeat washing the plate 5 Times (the plate can also be washed with a plate washing machine).

6. Substrate added per well A 、 B  Each 50μL ， 37℃ Incubate in the dark 15min 。

7. Add stop solution to each well 50μL ， 15min  Inside, in 450nm Wavelength measurement of each well OD  Value.

Calculation of experimental results

Based on the tested standard OD The value is the abscissa, and the concentration value of the standard product is the ordinate. Draw the standard curve on coordinate paper or with relevant software, and obtain the linear regression equation. The sample OD Values are substituted into Eq., and the concentration of the sample is calculated.


Standard curve

remark ：

1. The standard concentrations are as follows: 2000 、 1000 、 500 、 250 、 125 、 62.5 pg/mL

1. Incubate in strict accordance with the specified time and temperature to ensure accurate results. All reagents must reach room temperature 20-25 °C prior to use. Store reagents in refrigeration immediately after use.

2. Incorrect plate washing can lead to inaccurate results. Make sure to drain the liquid from the wells as much as possible before adding the substrate. Do not allow the wells to dry out during incubation.

3. Eliminate the residual liquid and fingerprints at the bottom of the plate, otherwise it will affect the OD value.

4. The substrate color development solution should be colorless or very light in color, and the substrate solution that has turned blue cannot be used.

5. Avoid cross-contamination of reagents and specimens to avoid wrong results.

6. Avoid direct exposure to strong light during storage and incubation.

7. After equilibrating to room temperature, open the sealed bag to prevent water droplets from condensing on the cold slats.

8. Any reaction reagent cannot come into contact with the bleaching solvent or the strong gas emitted by the bleaching solvent. Any bleaching component will destroy the biological activity of the reaction reagents in the kit.

9. Expired products cannot be used.

10. If it is possible to spread diseases, all samples should be managed, and the samples and testing devices should be handled according to the prescribed procedures.