Monkey B virus enzyme immunoassay

  Kit instruction manual

This kit is for research use only.                                                 96T

Drug Name:

Generic name: Monkey B virus enzyme-linked immunoassay kit

purpose of usage:

This kit qualitatively determines the expression of B virus in monkey blood or other related tissues

Experimental principle:

The kit uses double antibody sandwich enzyme-linked immunosorbent assay ( ELISA ) to determine the monkey B virus in the specimen . Microtiter plate with purified B virus antibody, make solid-phase antibody, the sample may be combined with B virus, and then with the HRP labeled antibody B virus after removing unbound antibodies and other components of the washed, antibody formation - antigen - enzyme-labeled antibody complex, after thorough washing, TMB chromogenic substrate. TMB in HRP enzyme blue, yellow and eventually converted to 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 the monkey B virus in the specimen .

Kit composition:

Specimen requirements:

1 . Specimen processing: serum and plasma samples can be directly detected

2 . The specimens were tested as soon as possible after preparation. If it is not detected in time, the specimen can be stored at -20 °C for one month, but repeated freezing and thawing should be avoided.

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

Steps:

1.          No: Samples corresponding to micropores numbered sequentially, each plate hole 2 should be set negative control, 2 positive control wells, blank control hole (blank control wells do not add sample and HRP, other each step operation)

2.          Loading: Negative control and positive control (standard) 50 μl 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: After sealing plate sealing film and incubated at 37 ℃ board 30 minutes.   

4.          Dosing: add 30 times concentrated washing solution plus distilled water to 600ml and use

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

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

11.      Measurement: The absorbance ( OD value) of each well was measured in sequence 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 ≤ 0.15

  Criteria ( CUT OFF ) calculation: Threshold = negative control well average + 0.15

  Negative judgment: sample OD value < critical value ( CUT OFF ) is monkey B virus negative

  Positive judgment: the sample OD value ≥ critical value ( CUT OFF ) is monkey B virus positive

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 of preservation:; 2-8 ℃.

2 . Validity: 6 months

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