Mouse HV-Ab ELISA Kit

Plate washing method:

Manual plate washing :
Pour out the solution in the microplate, on absorbent filter paper or other absorbent material Moderate force Slap.
Add to each well 350ul Lotion, and soak 1 To
2 After minutes, the wash liquid is poured out of the plate on absorbent filter paper or other absorbent material Moderate force Slap.

Automatic plate washing :
Use an automatic plate washing machine to wash the plate, and set the washing times, soaking time and the amount of washing solution per well.
The amount of wash liquid added to each well is not less than 350ul 。
After the last plate wash, invert the microplate on absorbent filter paper or other absorbent material Moderate force Remove residual lotion by tapping.
It is recommended that the plate washer be set to soak 1 Minutes.

(Note: Set the needle height of the automatic plate washer to ensure that the liquid can be completely aspirated)

Sample Collection and Storage (General)  

Serum
All serum samples were placed at room temperature 2  Hour or in 2-8°C  Left overnight and at approximately 1000×g  Centrifugation 20  Minutes, the supernatant was collected and assayed immediately.
The blood collection tube is required to be disposable and free of pyrogens and endotoxins.

Sample Requirements:

1.
The test sample was mouse serum.

2.
Freshly collected samples should be fully centrifuged first, and then clear liquid should be taken for testing.
If not sufficiently precipitated, suspended fibrin may cause false positives.

3.
Specimens are stored at 2-8°C , specimens that do not need to be tested within one week should be stored in -20℃ Below, avoid repeated freezing and thawing.

Note:
5 The samples used within days can be used in 2-8℃ Save in the environment, otherwise, it must be -20℃ Or -80℃ Or liquid nitrogen to avoid loss of biological activity and pollution.
Avoid multiple freeze-thaw cycles.
Samples with severe hemolysis are not suitable for this test.

  Reagent Preparation and Storage

Bring all reagents and samples to room temperature before use 30 Minutes. Wash buffer :

Use deionized or distilled water (recommended resistivity is 18MΩ Of ultrapure water) will 50ml Dilute the concentrated wash to 1000ml And mix well.

  Detection procedure

1. Loading:
Take the required number of slats, mark them well, and leave 1  For the well blank control, the slats were fixed on the plate rack, and the remaining slats were stored in sealed bags.
Negative control 3  Well, Positive Control 2  Holes, each 100ul  Control serum.
Blank control 1  Holes are vacant.
The rest is added per well 100ul  Sample dilution, add sample to be tested per well 10ul , gently shake the reaction plate to mix the sample.

2. Incubation:
After sealing the plate with laminating film, place 37℃ Reaction in incubator or water bath 30  Minutes.

3. Washing plate :
After incubation, the coating was removed and the plate was washed with wash buffer 5  Times, each soak 30-60  Seconds.
After the last wash, all wash buffer was removed by aspiration or pouring.

4. Enzyme labeled working solution:
Add to each well 100uL  Enzyme labeled working solution, except blank wells.

5. Incubation:
After sealing the plate with laminating film, place 37℃ Reaction in incubator or water bath 30  Minutes.

6. Washing plate :
After incubation, the coating was removed and the plate was washed with wash buffer 5  Times, each soak 30-60  Seconds.
After the last wash, all wash buffer was removed by aspiration or pouring.

7. Color development:
Substrate added per well A 、 B  Liquid each 50ul Gently shaking and mixing evenly, and sealing the plate with a coating film, 37℃ Hidden incubator 15  Minutes.

8. Termination:
Add stop solution to each well 50ul , gently shake and mix well.

9.OD  Measurement of values :
Single wavelength with microplate reader 450m  Or dual wavelength 450nm/630nm  Measuring each well OD  Value ( When measuring with a single wavelength, it is necessary to zero with a blank control hole ) , and record the results.
Note: At termination of the reaction 30  Readings within minutes.

Positive judgment value

1. Result determination :

1.1.
Each test result is used independently, passing the cut-off value (Cut off) Value determination result.

Cut off (C.0) =0. 10+ Negative control mean (NC) A Value

( When the negative control averaged A Value less than 0.05 When press 0.05 calculate ; When the negative control averaged A Value greater than or equal to 0.05 Calculated according to the actual value ) 。

2. Quality Control :

2.1
Blank hole ( Add only developer and stop solution ) Of A  Value should not be greater than 0.08 。

2.2
Positive control (PC) A  Value greater than 1.0 。

2.3
Negative control mean A  Value less than 0.1 。

If the quality control is valid, the test results are valid.

3. Result determination : 

Positive result :
Sample A Value ≥ Critical value (Cut off) Those are HV Antibody positive.
Negative result :
Sample A Value < Critical value (Cut off) Those are HV Antibody negative.

1. In order to ensure the effectiveness of experimental operation and the appropriateness of sample dilution ratio, it is recommended to use a small amount of sample for pre-experiment.

2. Please keep the microplate dry after opening and before use. Please add desiccant to seal the wells of the enzyme labeled plate that are not in use temporarily to avoid moisture.

3. Centrifuge the reagent to the bottom of the tube by spinning the tube using a centrifuge before using the kit.

4. TMB reagents should be stored in the dark.

5. The washing process is very important. Inadequate washing can easily cause false positives and high background.

6. During the detection process, please prepare the reagents required for the next experiment in advance. After washing the plate, add the reagents to the plate wells in time. Do not allow the microplate to be too dry, because the dry well plate will inactivate the active ingredients on the plate.

7. Do not reuse the gun tip to avoid cross-contamination.

8. The reagents in different kits of our company have different ingredients, so please do not mix them. Do not mix reagents from other manufacturers.

9. In order to ensure the accuracy of the results, the sample is loaded, the plate film is applied to prevent the evaporation of the sample during the incubation process, and then the incubation process is completed at the recommended temperature.