Human 1,25 dihydroxyvitamin D (1,25- (OH) 2D) ELISA Kit

Sample handling and requirements:

1 、 The detection range of the kit is not equivalent to the concentration range of the test substance in the sample , it is recommended to pass the phase before the experiment Relevant literature estimates the concentration of the test substance in the sample and determines the actual concentration of the sample through pre-experiments. Such as If the concentration of the test substance in the sample is too high or too low, please dilute or concentrate the sample appropriately.

2 If the sample tested is not among the sample types listed in the instructions, it is recommended to conduct pre-experiments to verify its detection effectiveness.

3 、 Serum: Whole blood samples collected in serum separation tubes were placed at room temperature 2 Hour or 2-8℃ Overnight, then 1000×g Centrifugation 20 Minutes, take the supernatant, or place the supernatant in -20℃ Or -80℃ Save, However, repeated freezing and thawing should be avoided.

4 、 Plasma: use EDTA Or heparin as an anticoagulant to collect a sample, and collect the sample 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.

5 、 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 test results), 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 grind thoroughly on ice or grind in a homogenizer. 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.

6 、 Cell culture supernatant: 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.

7 、 Cell lysate: Precooling for adherent cells PBS Gently washed, followed by trypsinization, 1000×g Centrifugation 5 Cells were collected after minutes; The suspended cells can be collected directly by centrifugation. Collected Cells Pre-cooled with PBS Washing 3 Times, every 1×10^6 Added to cells 150-200μL PBS Resuspension (recommended at PBS Adding a protease inhibitor; If the content is very low, it can be appropriately reduced PBS Volume) and the cells were disrupted by repeated freeze-thaw or sonication. The extracts were mixed in 2-8℃ ， 1500×g Centrifugation 10 Minutes, Take the supernatant for detection.

8 、 Other biological samples: 1000×g Centrifugation 20 Minutes, take the supernatant to detect.

9 、 Sample Appearance: The sample should be clear and transparent, and the suspended solids should be centrifuged to remove.

10 、 Sample Preservation: After sample collection, if 1 Those tested within weeks can be stored in 4℃ , if not in time For testing, please pack it according to the amount used once, and store it frozen in -20℃ （ 1 Within months), or -80℃ （ 6 Test within months) to avoid repeated freezing and thawing. Hemolysis of the sample will affect the final test results because This hemolyzed sample is not suitable for this test.

Sample dilution protocol:
Please estimate the concentration range of the sample in advance. If your test sample needs to be diluted, refer to the dilution plan as follows:
Dilution 100 Times : One-step dilution. Take 5μL Sample to 495μL Within universal diluent, do 100 Double dilution;

Dilution 1000 Times: Two-step dilution. Take 5μL Sample to 95μL Within universal diluent, do 20 Double dilution, Take again 5μL 20 Double dilute sample to 245μL Within universal diluent, do 50 Double dilution, total dilution 1000 Times;

Dilution 100000 Times : Three-step dilution. Take 5μL Sample to 195μL Within universal diluent, do 40 Dilute, and then take 5μL 40 Double dilute sample to 245μL Within universal diluent, do 50 Time dilution, and finally take 5μL 2000 Double dilute sample to 245μL Within universal diluent, do 50 Double dilution, total dilution 100000 Times;

The amount of liquid taken during each dilution step is not less than 3μL , the dilution factor is not more than 100 Times. Each step of dilution should be mixed evenly to avoid foaming.

Preparations before testing:

1 , please advance 10 Minutes remove the kit from the refrigerator and equilibrate to room temperature.

2 、 Standard gradient working solution preparation : join 1mL Universal diluent into lyophilized standard and let stand 15 Minutes until it is completely dissolved and then gently mix ( The concentration is 500pg/mL) And then according to the following concentrations: 500pg/mL 、 250pg/mL 、 125pg/mL 、 62.5pg/mL 、 31.2pg/mL 、 15.6pg/mL 、 7.8pg/mL 、 0pg/mL The dilution was performed.

Double dilution method: Take 7 branch EP Tubes, each tube is added 500μL Universal diluent, 500pg/mL Pipette from the standard working solution 500μL To the first EP Mix evenly in a tube 250pg/mL According to this step, suck and mix the standard working solution in sequence. The last tube is directly used as a blank hole, so there is no need to suck liquid from the penultimate tube, as shown in the figure below.

3 、 Biotin- Preparation of antibody working solution: Before use 15 Minutes will 100× concentrate Biotin- Antibody to 1000× g Centrifugation 1 Minutes, with a universal diluent 100× concentrate Biotin- The antibody is diluted into 1× Working concentration ( Example: 10μL Concentrate +990μL Universal diluent ) , now available for use.

4 、 Preparation of enzyme conjugate working solution: Before use 15 Minutes will 100× Concentrated enzyme conjugate in 1000×g Leave Heart 1 Minutes, in the universal diluent 100× The concentrated enzyme conjugate is diluted into 1× Working concentration ( Example: 10μL Concentrate +990μL Universal diluent ) , now available for use.

5 、 1× Wash liquid preparation : Take 10mL 20× Wash liquid to 190mL In distilled water (the concentrated washing liquid taken out from the refrigerator may have crystals, which is a normal phenomenon. It can be placed at room temperature and prepared after the crystals are completely dissolved ) 。

Operation steps:

1 Equilibration from room temperature 10 After minutes, remove the required slats from the aluminum foil bag, and seal the remaining slats with ziplock bag and put them back 4℃ 。

2 、 Sample addition : The samples or different concentration standards are respectively according to 50μL Each well is added to the corresponding well, and the blank well is added 50μL Universal dilution followed by each well 50μL Biotin- Antibody working fluid. After covering the sealing film 37℃ Incubation 60 Min. ( Recommendation: The sample to be tested is minimized with universal diluent Dilution 1 Then add the enzyme label plate for testing, Thereby reducing the influence of the matrix effect on the test result, Finally, the sample concentration needs to be multiplied by the corresponding dilution factor when calculating it. It is recommended to set up double wells for all samples and standards to be tested during testing).

3 、 Washing plate : Discard liquid and add per well 300μL 1x Wash liquid, stand 1 Minutes, throw away the washing liquid, Pat dry on absorbent paper and repeat plate washing 3 Times (plate washing machine can also be used to wash the plate)

4 、 Adding enzyme conjugate working solution : Added per well 100μL Enzyme conjugate working solution, after covering with sealing membrane 37℃ Incubation 30 Minutes.

5 、 Washing plate : Discard liquid according to step 3 Washing method, wash plate 5 Times.

6 、 Substrate addition : Added per well 90μL Substrate (TMB) Covered with a sealing film, 37℃ Incubation protected from light 15 Minutes.

7 、 Add stop solution : Take out the enzyme label plate and add it directly to each well 50μL Stop solution, immediately in 450nm Wavelength measurement of each well OD Value.

Calculation of experimental results:
Result judgment ：

1 , calculate the average of the standard and sample replica well OD Value. Taking concentration as the abscissa, OD The value is the ordinate, Draw the standard curve of four-parameter logic function on double logarithmic coordinate paper ( Remove the values of blank groups when plotting ) 。

2 If the sample OD If the value is higher than the upper limit of the standard curve, the test should be retested after appropriate dilution and multiplied by the corresponding dilution factor when calculating the sample concentration.

Typical data and reference curves:
The following data and curves are for reference only, and experimenters need to establish standard curves according to their own experiments.

Note: This figure is for reference only, and the sample content should be calculated from the standard curve drawn from each experimental data.

Kit Performance:

1 Repeatability: the coefficient of variation in the plate is less than 10% , the interplate coefficient of variation is less than 10% 。

2 Recovery rate: adding to selected healthy human serum, plasma and cell culture supernatant 3 People with different concentration levels 1,25(OH)2D3 , calculate the recovery.

1. Carry out incubation 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. Refrigerate reagents immediately after use.

2. Incorrect plate washing may 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 micropores to dry for too long throughout the process.

3. Clean 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, and the substrate solution that has turned blue cannot be used.

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

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

7. 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 reagents in the kit.

8. Expired products cannot be used, and components with different item numbers and batch numbers cannot be mixed.

9. Recombinant proteins from sources other than the kit may not match the antibodies in this kit and are not recognized.

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