Plate Washing Method:

(Note: Set the needle height of the automated plate washer to ensure complete aspiration of the liquid.)

Sample Collection and Storage (General)

Serum: All serum samples should be placed at room temperature for 2 hours or at 2-8°C overnight. Centrifuge at approximately 1000×g for 20 minutes, collect the supernatant, and assay immediately. Blood collection tubes must be disposable and free of pyrogens and endotoxins.

Sample Requirements:

1. The test sample is mouse serum.

2. Freshly collected samples should be thoroughly centrifuged before the clarified liquid is tested. If not fully precipitated, suspended fibrin may cause false positives.

3. Store specimens at 2-8°C. Specimens not to be tested within a week should be stored below -20°C to avoid repeated freeze-thaw cycles.

Note: Samples used within 5 days can be stored at 2-8°C. Otherwise, they must be stored at -20°C or -80°C or in liquid nitrogen to prevent loss of biological activity and contamination. Avoid multiple freeze-thaw cycles. Samples with severe hemolysis are not suitable for this test.

 Reagent Preparation and Storage

Prior to use, bring all reagents and samples to room temperature for 30 minutes. Wash Buffer

Dilute 50ml of concentrated wash buffer to 1000ml with deionized or distilled water (18MΩ ultrapure water is recommended) and mix thoroughly.

 Test Steps

1.Sample Addition: Take the required number of plates, mark them, and leave one blank control well. Secure the plates to the plate rack and store the remaining plates in a sealed bag. Add 100µl of control serum to each of the three negative control wells and the two positive control wells. Leave the blank control well vacant. Add 100µl of sample diluent to each of the remaining wells. Add 10µl of the sample to be tested to each well and gently shake the reaction plate to mix the samples.

2.Incubation: Seal the plate with film and incubate in a 37°C incubator or water bath for 30 minutes. 3. Washing: After incubation, remove the cover film and wash the plate five times with wash buffer, soaking for 30-60 seconds each time. After the final wash, remove all wash buffer by aspiration or decanting. 4. Adding Enzyme Labeling Working Solution: Add 100 μL of enzyme labeling working solution to each well, excluding blank wells. 5. Incubation: After sealing the plate with the cover film, incubate in a 37°C incubator or water bath for 30 minutes. 6. Washing: After incubation, remove the cover film and wash the plate five times with wash buffer, soaking for 30-60 seconds each time. After the final wash, remove all wash buffer by aspiration or decanting. 7. Color Development: Add 50 μl each of Substrate A and Substrate B to each well, gently shake to mix, seal the plate with film, and incubate in a dark incubator at 37°C for 15 minutes. 8. Stop: Add 50 μl of Stop Solution to each well, gently shake to mix. 9. OD Measurement: Measure the OD value of each well using a microplate reader at a single wavelength of 450 nm or dual wavelengths of 450 nm/630 nm (when using a single wavelength, use a blank control well for zeroing) and record the result. Note: Read the result within 30 minutes of stopping the reaction.

Positive Interpretation Value

1.Result Interpretation:

1.1. Each test result is used independently, and the result is judged by the critical value (Cut off) value.

Cut off (C.0) = 0.10 + negative control average (NC) A value

(When the negative control average A value is less than 0.05, it is calculated as 0.05; when the negative control average A value is greater than or equal to 0.05, it is calculated as the actual value).

2.Quality Control:

2.1 The A value of the blank well (only the color developer and stop solution) should not exceed 0.08.

2.2 The A value of the positive control (PC) is greater than 1.0.

2.3 The average A value of the negative control is less than 0.1.

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

3.Result Interpretation: 

Positive result: If the sample A value is greater than the cut-off value (Cut off), it is HV antibody positive. Negative result: If the sample A value is less than the cut-off value (Cut off), it is HV antibody negative.

1. To ensure the effectiveness of experimental operations and the appropriateness of sample dilution ratios, it is recommended to perform a preliminary experiment using a small amount of sample.

2. Keep the microplate dry after opening and before use. Seal unused ELISA plate wells with desiccant to protect them from moisture.

3. Before using the test kit, spin the tubes in a centrifuge to remove the reagents to the bottom of the tube.

4. TMB reagent should be stored away from light.

5. The washing process is very important; inadequate washing can easily result in false positives and high background.

6. During the assay, please prepare the reagents needed for the next step in advance. After washing, add the reagents to the wells promptly. Do not allow the microplate to dry out excessively, as drying out the wells will inactivate the active ingredients on the plate.

7. Do not reuse pipette tips to avoid cross-contamination.

8. Reagents in different kits from our company have different compositions. Do not mix reagents from other manufacturers. 9. To ensure the accuracy of the results, after adding the sample, seal the plate with film to prevent evaporation of the sample during the incubation process, and then complete the incubation process at the recommended temperature.