Sample collection preparation and preservation  

1. Serum: Whole blood sample placed at room temperature 2  Hour or 4°C  Overnight after 1000×g  Centrifugation 20  Minutes, take the supernatant to detect.
The blood collection tubes shall be disposable non-pyrogenic, non-endotoxin tubes.
deposit -20°C  Or -80°C  Storage, avoid repeated freezing and thawing.

2. Plasma: Sample after collection 30  Within minutes 2-8°C 、 1000×g  Centrifugation 15  Minutes, take the supernatant to detect.
Anticoagulants recommended EDTA-Na₂ , avoid using hemolytic, hyperlipidemic samples.
deposit -20°C  Or -80°C  Storage, avoid repeated freezing and thawing.

3. Tissue homogenate: Take an appropriate amount of tissue block and add it to the pre-cooled PBS （ 0.01M ， pH7.0-7.2 ) to remove blood (lysed red blood cells in the homogenate will affect the measurement result), cut the tissue into pieces after weighing, and then mix it with the corresponding volume of PBS (generally according to 1:9 The mass-to-volume ratio, the specific volume can be appropriately adjusted according to the needs of the experiment, and recorded.
It is recommended to PBS Add protease inhibitors) Adding the mixture into a glass homogenizer and thoroughly grinding on ice;
In order to further lyse tissue cells, the homogenate can be subjected to ultrasonic disruption or repeated freeze-thaw treatment (pay attention to ice bath cooling during ultrasonic disruption, and the repeated freeze-thaw method can be repeated 2 Times).
Finally, the prepared homogenate is mixed in 5000×g Centrifugation 5-10 Minutes, take the supernatant to detect.
(Tissue homogenates require simultaneous detection of protein concentrations to obtain a more accurate test substance concentration per milligram of protein.)

4. Cell culture supernatant: Take the cell supernatant from 1000×g Centrifugation 20 Minutes, impurities and cell debris were removed.
Take the supernatant to detect and place it in -20°C Or -80°C Store, but repeated freezing and thawing should be avoided.

5. Urine: Please collect the first urine in the morning (mid-section urine), or 24 Hourly urine, 2000×g  Centrifugation 15  The supernatant was collected after minutes, And save the sample At -20°C And repeated freezing and thawing should be avoided.

6. Saliva: A sample is collected with a saliva sample collection tube, and then 2-8°C, 1000×g  Centrifugation 15  Minutes, take the supernatant to detect, Or after sub-packaging -20°C  Save.
Avoid repeated freezing and thawing.

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

Notes

1. The sample should be clear and transparent, and the suspended solids should be removed by centrifugation.
Hemolysis of the sample will affect the results, so hemolyzed samples should not be used.

2. After sample collection, if 1 Testing within weeks can be stored at 4°C , if it cannot be detected in time, please pack it according to the one-time usage amount and freeze it in -20°C （ 1 Within months), or -80°C （ 3-6 Test within a month) to avoid repeated freezing and thawing.
Keep the sample at room temperature prior to the experiment.

Principles of sample dilution

If your test sample needs to be diluted, refer to the general dilution principles below:

1. Dilution 50  Times: One-step dilution.
Take 5 μL  Sample to 245 μL  Standard & In the sample dilution, is 50  Double dilution;

2. Dilution 100  Times: One-step dilution.
Take 5 μL  Sample to 495 μL  Standard & In the sample dilution, is 100  Double dilution;

3. Dilution 1000  Times: Two-step dilution.
Take 5 μL  Sample to 95 μL  Standard & In the sample dilution, is 20  Dilute, and then take 5 μL
20  Double dilute sample to 245 μL Standard & In the sample dilution, is 50  Double dilution, co-dilution 1000  Times;

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

5. The amount of liquid taken during each dilution step is not less than 3 μL , the dilution factor is not more than 100  Times.
Too small sampling volume can easily cause greater errors in the mixing process, and each step of dilution needs to be mixed evenly to avoid foaming.

6. When the dilution factor is very high, you can use it first PBS  Dilution, last step using standard in kit & Sample dilution.

Sample dilution recommendations

1. Normal fresh serum / Plasma Sample Recommendation (Original solution-1:2) Testing.

2. Due to individual variations, the recommended dilution factor is for informational purposes only.
For actual testing, please estimate the concentration range of the sample in advance, and determine the dilution factor of the sample to be tested through pre-experiments.

Preparation for testing

1. Please advance 30  Minutes remove the kit from the refrigerator and equilibrate to room temperature.

2. Use double distilled water 25× The concentrated wash liquid is diluted to 1× Working fluid, put back unused 4°C 。

3. Standard: Add standard & Sample Universal Diluent 1.0 mL  Into the lyophilized standard, screw the tube cap tightly and let stand 10  Minutes, and after it is fully dissolved, gently mix (concentration of 100 mIU/mL ）。
Thereafter, double dilution is carried out to 100 mIU/mL ， 50 mIU/mL ， 25 mIU/mL ， 12.5 mIU/mL ， 6.25 mIU/mL ， 3.13 mIU/mL ， 1.57 mIU/mL  Standard dilution ( 0 mIU/mL ） Is a blank hole.
Configure the standard according to the amount you need for later use.
The configured standards are recommended in 15  Add the sample within minutes, and it is not recommended to leave it for too long.

4. Biotinylated antibody working solution: calculate the dosage required for the current experiment before the experiment (according to 100 μL/ Hole meter, should be configured more in actual configuration 100-200 μL ), before use 15 Min, concentrated biotinylated antibody was diluted with biotinylated antibody diluent ( 1:100 ) into working concentration, use on the same day.
Dilution principle 1 μL Concentrated biotinylated antibody is added to 99 μL In the biotinylated antibody dilution, mix well with a pipette.

5. Enzyme conjugate working solution: calculate the dosage required for the current experiment before the experiment (according to 100 μL/ Hole meter, should be configured more in actual configuration 100-200 μL ）。
Before use 15  Minutes, dilute and concentrate with enzyme conjugate diluent HRP  Enzyme conjugate ( 1:100 ) into working concentration, use on the same day.
Dilution principle 1 μL  The concentrated enzyme conjugate is added to 99 μL  The enzyme conjugate dilution was mixed with a pipette.

6.TMB  Substrate —— Pipette the desired dose of solution and do not pour the residual solution back into the reagent vial again.

Notes

1. Please make sure that all components are dissolved and mixed before use of the kit.
If the reconstituted standard is not used, please discard it.

2. Concentrated biotinylated antibody, the volume of concentrated enzyme conjugate is small, may be dispersed in various parts of the tube during transportation, please 1000×g  Centrifugation 1  Minutes to allow the liquid of the tube wall or cap to deposit to the bottom of the tube.
Pipette carefully before use 4-5  The solution was mixed once.
Standard, biotinylated antibody working solution and enzyme conjugate working solution should be prepared according to the required dosage, and the corresponding diluent should be use

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.