Human Metapneumovirus (HMPV) ELISA Kit Procedure

This reagent is for research use only: This kit is used to detect human metapneumovirus (HMPV) levels in human serum, plasma, cell culture fluids and related liquid samples.

Experimental principle :

The kit uses double antibody sandwich enzyme-linked immunosorbent assay (ELISA) to determine human metapneumovirus (HMPV) in the specimen. The microporous plate is coated with purified human metapneumovirus (HMPV) antibody to prepare a solid phase antibody, which can be combined with the metapneumovirus (HMPV) antibody in the sample, washed to remove unbound antibody and other components, and then combined with The HRP-labeled metapneumovirus (HMPV) antibody binds to form an antibody-antigen-enzyme-labeled antibody complex, which is thoroughly washed and then added to the substrate TMB for color development. TMB is converted to blue under the catalysis of HRP enzyme and converted to the final yellow color by the action of an acid. The absorbance (OD value) was measured at 450 nm using a microplate reader, and compared with the CUTOFF value to determine the presence or absence of human metapneumovirus (HMPV) in the specimen.

Kit composition :

Sample processing and requirements :

1. Serum: The blood is naturally coagulated for 10-20 minutes at room temperature and centrifuged for about 20 minutes (2000-3000 rpm). The supernatant is carefully collected, and if precipitation occurs during storage, it should be centrifuged again.

2. Plasma: EDTA or sodium citrate should be selected as an anticoagulant according to the requirements of the specimen. After mixing for 10-20 minutes, centrifuge for about 20 minutes (2000-3000 rpm). The supernatant is carefully collected, and if a precipitate forms during storage, it should be centrifuged again.

3. Urine: Collect with a sterile tube and centrifuge for about 20 minutes (2000-3000 rpm). The supernatant is carefully collected, and if a precipitate forms during storage, it should be centrifuged again. The chest and ascites and cerebrospinal fluid are referred to.

4. Cell culture supernatant: When detecting secreted components, collect them in a sterile tube. Centrifuge for about 20 minutes (2000-3000 rpm). Collect the supernatant carefully. When the components in the cells were detected, the cell suspension was diluted with PBS (pH 7.2-7.4), and the cell concentration reached about 1 million/ml. By repeated freezing and thawing, the cells are destroyed and the intracellular components are released. Centrifuge for about 20 minutes (2000-3000 rpm). Collect the supernatant carefully. If a precipitate forms during storage, it should be centrifuged again.

5. Tissue specimen: After cutting the specimen, weigh the weight. Add a certain amount of PBS, pH 7.4. It was quickly frozen and stored in liquid nitrogen for use. The specimen still maintains a temperature of 2-8 ° C after melting. A certain amount of PBS (pH 7.4) was added, and the specimen was homogenized by hand or homogenizer. Centrifuge for about 20 minutes (2000-3000 rpm). Collect the supernatant carefully. One part of the package is to be tested, and the rest is frozen for use.

6. The specimens should be extracted as soon as possible after collection, and the extraction should be carried out according to the relevant literature. The experiment should be carried out as soon as possible after extraction. If the test cannot be performed immediately, the specimen can be stored at -20 °C, but repeated freezing and thawing should be avoided.

7. Samples containing NaN3 cannot be detected because NaN3 inhibits horseradish peroxidase (HRP) activity.

Operation steps :

• No.: The corresponding micropores of the sample are numbered sequentially. Each plate should be set with 2 holes of negative control, 2 wells of positive control and 1 well of blank control (the blank control well is not added with sample and enzyme standard reagent, and the other steps are the same)
• Loading: 50 μl of a negative control and a positive control were added to the negative and positive control wells, respectively. Then, add 40 μl of the sample dilution to the sample well to be tested, and then add 10 μl of the sample to be tested. Add the sample to the bottom of the well of the microplate, try not to touch the wall of the well, and shake it gently.
• Incubation: The plate was sealed with a sealing film and incubated at 37 ° C for 30 minutes.
• Dosing: add 30 (48 times of 20T) concentrated washing solution and distilled water to 600ml for use.
• Washing: Carefully remove the sealing film, discard the liquid, dry it, fill each well with the washing solution, let stand for 30 seconds, then discard it, repeat 5 times, and pat dry.
• Add enzyme: 50 μl of enzyme labeling reagent was added to each well, except for blank wells.
• Incubation: The operation is the same as 3.
• Washing: The operation is the same as 5.
• Color development: Add 50 μl of developer A to each well, then add 50 μl of developer B, gently shake and mix, and develop at 37 ° C for 15 minutes in the dark.
• Termination: 50 μl of stop solution was added to each well to terminate the reaction (in this case, the blue color turned yellow).
• Measurement: The absorbance (OD value) of each well was measured sequentially with a blank air conditioner of zero and a wavelength of 450 nm. The measurement should be carried out within 15 minutes after the addition of the stop solution.

The result is judged:

Test validity: mean value of positive control well ≥ 1.00; mean value of negative control ≤ 0.10

CUT OFF calculation: critical value = negative control well average + 0.15

Negative judgment: Sample OD value < CUT OFF is negative for metapneumovirus (HMPV)

Positive judgment: sample OD value ≥ critical value (CUT OFF) is positive for metapneumovirus (HMPV)

Precautions

1. The operation is carried out in strict accordance with the instructions. The components of the different batches of this reagent shall not be mixed.

2. The kit should be taken out from the refrigerated environment and should be used at room temperature for 15-30 minutes before use. If the enzyme label is unsealed after opening, the slats should be stored in a sealed bag.

3. Concentrated washing liquid may crystallize out. When diluted, it can be heated and dissolved in a water bath. The washing will not affect the result.

• The sealing film is intended for single use only to avoid cross-contamination.

5. Please keep the substrate away from light.

6. The test results must be determined by the microplate reader. When using dual-wavelength detection, the reference wavelength is 630nm.

7. All samples, washings and various wastes should be treated as infectious materials. The stop solution is 2M sulfuric acid and must be used safely.

Storage conditions and expiration date

1. The kit is stored at: 2-8 °C.

2. Validity: 6 months

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