Canine echinococcosis antigen-enzyme immunoassay kit instruction manual

Kit instruction manual

This kit is for research use only.

96T

purpose of usage:

This kit is used to determine the expression of echinococcosis antigens in canine serum, plasma and related fluid samples .

Experimental principle

The kit uses a double antibody sandwich method to determine the expression of canine echinococcosis antigen in the specimen . Purified samples are pipetted into the antibody-coated echinococcosis in dogs, made of solid-phase antibody, the sample can bind to the antigen echinococcosis, after washing to remove unbound antibodies and other components, hydatid With HRP labeled The 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 in HRP enzyme blue, yellow and eventually converted to 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 canine echinococcosis antigen 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: 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: 50 μl of negative control and 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: After sealing plate sealing film and incubated at 37 ℃ board 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 the developer to each well , then add 50 μl of the developer , 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 .

Calculation and result determination:

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

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

  Negative judgment: sample OD value < critical value ( CUT OFF ) is negative for echinococcosis antigen

  Positive judgment: The OD value of the sample ≥ the critical value ( CUT OFF ) is positive for the echinococcosis antigen .

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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