Tobacco Mosaic Virus (TMV) ELISA Test Kit Instructions for Use

Detection principle

The kit uses a double antibody sandwich enzyme-linked immunosorbent assay (ELISA). To the coated microwells pre-coated with the tobacco mosaic virus (TMV) capture antibody, a negative control, a positive control, a sample, an HRP-labeled detection antibody were sequentially added, and the cells were incubated and thoroughly washed. Using the substrate TMB to develop color, TMB is converted to blue under the catalysis of peroxidase and converted to the final yellow color by the action of an acid. The color depth is positively correlated with the tobacco mosaic virus (TMV) in the sample. The absorbance (OD value) was measured at 450 nm using a microplate reader to determine whether the sample contained tobacco mosaic virus (TMV).

Sample collection, processing and storage methods

1. Serum: Use a tube containing no pyrogen and endotoxin. Avoid any cell irritation during the procedure. After collecting the blood, centrifuge and centrifuge for 10 minutes at 3000 rpm to quickly and carefully separate the serum and red blood cells.
2. Plasma: EDTA, citrate or heparin anticoagulation. The supernatant was taken by centrifugation at 3000 rpm for 30 minutes.
3. Cell supernatant: Centrifuge at 3000 rpm for 10 minutes to remove particles and polymer.
4. Tissue homogenization: The tissue is mashed by adding appropriate amount of physiological saline. The supernatant was taken by centrifugation at 3000 rpm for 10 minutes.
5. Storage: If the sample is not detected in time after collection, please dispense it once, freeze it at -20 °C, avoid repeated freezing and thawing, thaw at room temperature and ensure that the sample is fully thawed evenly.

Bring your own items

• Microplate reader (450nm)
• High-precision sampler and tip: 0.5-10uL, 2-20uL, 20-200uL, 200-1000uL
• 37 ° C incubator

Operational precautions

1. The kit was stored at 2-8 ° C and equilibrated for 20 minutes at room temperature before use. The concentrated washing liquid taken out from the refrigerator will crystallize, which is a normal phenomenon, and the water bath is heated to completely dissolve the crystals before use.
2. The slats not used in the experiment should be immediately put back into the ziplock bag and sealed (low temperature dry) for storage.
3. The pre-treated sample does not need to be diluted, and 10 μL of the sample can be directly added.
4. Incubation is carried out in strict accordance with the time indicated in the instructions, the amount of liquid added and the order.
5. Shake well all liquid components before use.

Kit composition

Reagent preparation

Dilution of 20× Wash Buffer: Distilled water was diluted 1:20, ie 1 part of 20× Wash Buffer plus 19 parts of distilled water.

Washing method

1. Manually wash the plate: Drain the liquid in the hole, fill each hole with the washing liquid, let stand for 1 min, then drain the liquid in the hole, pat dry on the absorbent paper, and wash the plate 5 times.
2. Automatic washing machine: Inject 350 μL of washing solution into each well, soak for 1 min, and wash the plate 5 times.

Steps

• The required slats were taken out from the aluminum foil pouch after equilibrating for 20 min at room temperature, and the remaining slats were sealed back to 4 ° C with a ziplock bag.
• The negative and positive control wells and the sample wells were set, and the negative control and the positive control were each added with 50 μL of the negative control and the positive control;
• The sample hole to be tested is first added with 10 μL of the sample to be tested, and then 40 μL of the sample diluent is added;
• Then, 100 μL of horseradish peroxidase (HRP)-labeled detection antibody was added to each well of the negative, positive control wells and sample wells, and the reaction wells were sealed with a sealing plate, and incubated at 37 ° C in a water bath or incubator for 60 min.
• Discard the liquid, pat dry on the absorbent paper, fill each well with the washing solution, let stand for 1 min, remove the washing solution, pat dry on the absorbent paper, and repeat the washing 5 times (can also be washed with a washing machine).
• 50 μL of each of the substrates A and B was added to each well, and incubated at 37 ° C for 15 min in the dark.
• 50 μL of the stop solution was added to each well, and the OD value of each well was measured at a wavelength of 450 nm within 15 min.

Result judgment

1. Test validity: the average value of the OD value of the positive control well is ≥ 1.00;
The average value of the OD value of the negative control well was ≤ 0.15.
2. Cut off calculation: critical value = negative control well average + 0.15
3. Negative judgment: sample OD value <Cut off value, sample is negative
4. Positive judgment: sample OD value > critical value (Cut off), sample is positive

Kit performance

• Accuracy: the average value of the OD value of the positive control well ≥ 1.00; the average value of the OD value of the negative control well ≤ 0.15, indicating that the test results are valid.
• Specificity: Does not cross-react with other soluble structural analogs.
• Repeatability: The coefficient of variation between the plates and the plates is less than 15%.
• Storage: 2-8 ° C, protected from light and moisture.
• Validity: 6 months

Disclaimer

• The kit is for research use only and should not be used for clinical trials or human experiments. Otherwise, the consequences will be borne by the experimenter and the company will not be responsible.
• In strict accordance with the instructions, the experimenter violates the instructions, and the consequences are borne by the experimenter.