Rat influenza A virus enzyme-linked immunosorbent assay kit instructions

This kit is for research use only.

                                                                     96T

purpose of usage:

This kit allows for determination of serum plasma and other biological fluids expression of influenza virus type A 2,.

Experimental principle

Expression of the influenza virus type A 2 samples rat assay The kit double antibody sandwich method. The microplate is coated with purified rat influenza A virus antibody to prepare a solid phase antibody, which can be combined with the influenza A virus in the sample, washed to remove unbound antigen and other components, and then labeled with HRP. The influenza A virus 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 a wavelength of 450 nm using a microplate reader, and compared with the CUTOFF value to determine the presence or absence of a rat influenza A virus in the specimen.

Kit composition

Specimen requirements

1. 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.

2. Samples containing NaN3 could not be detected because NaN3 inhibited horseradish peroxidase (HRP) activity.

Steps

1.          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)

2.          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.

3.          Incubation: The plate was sealed with a sealing film and incubated at 37 ° C for 30 minutes.   

4.          Solution: 30 times concentrated washing solution diluted with distilled water 30 times and used

5.          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.

6.          Add enzyme: 50 μl of enzyme labeling reagent was added to each well, except for blank wells.

7.          Incubation: The operation is the same as 3.

8.          Washing: The operation is the same as 5.

9.          Color development: Add 50 μl of color developer A to each well, then add 50 μl of color developer B, gently shake and mix, and develop color at 37 ° C for 15 minutes.

10.      Termination: 50 μl of stop solution was added to each well to terminate the reaction (in this case, the blue color turned yellow).

11.      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.

Calculation and result determination:

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 < critical value (CUT OFF) is negative for rat influenza A virus type 2

Positive judgment: the sample OD value ≥ critical value (CUT OFF) is positive for influenza A 2 virus in rats

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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.

4. 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