Bovine Foot and Mouth Disease Antigen (FMD) Enzyme Linked Immunosorbent

Enzyme-linked immunosorbent assay for porcine foot-and-mouth disease antigen ( FMD )

Kit instruction manual

This kit is for research use only.

examination range:                                                            96T

10 ng/L - 240 ng/L

purpose of usage:

This kit is used to determine the content of foot-and-mouth disease antigen ( FMD ) in bovine serum, plasma and related liquid samples .

Experimental principle

This kit uses the double antibody sandwich method to determine the level of FMD in specimens. The microplate was coated with purified bovine foot and mouth disease (FMD) antibody to prepare a solid phase antibody, and the foot-and-mouth disease antigen (FMD) was sequentially added to the microcapsule of the coated monoclonal antibody, and then combined with HRP-labeled foot-and-mouth disease (FMD) antibody to form The antibody-antigen-enzyme-labeled antibody complex was thoroughly washed and 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 color depth is positively correlated with the foot-and-mouth disease antigen (FMD) in the sample. The absorbance (OD value) was measured by a microplate reader at a wavelength of 450 nm, and the concentration of the beef foot-and-mouth disease antigen (FMD) in the sample was calculated by a standard curve.

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.          Dilution of Standards: This kit provides a standard of the original standard, which can be diluted in a small tube according to the following chart.

2.          Adding samples: blank holes (the blank control wells are not added with the sample and the enzyme standard reagent, the other steps are the same), the standard holes, and the sample holes to be tested. Coated ELISA plates were standard accurate pipetting 50 μl, the sample holes before adding the sample dilution 40 μ l, was then combined with the test sample 10 μ l (final dilution is 5-fold). Add the sample to the bottom of the well of the microplate, try not to touch the wall of the well, and shake gently to mix.

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

   Taking the concentration of the standard as the abscissa and the OD as the ordinate, draw a standard curve on the coordinate paper, and find the corresponding concentration from the standard curve according to the OD value of the sample ; multiply by the dilution factor; or use the concentration of the standard calculate the standard curve and linear regression equation OD value of the sample OD value in the equation, the sample concentration is calculated, and then multiplied by the dilution factor, is the actual concentration of the sample.

Precautions

1 . 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.

2 . 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.

3 . The sampler should be used for each step, and the accuracy should be corrected frequently to avoid test errors. It is best to control the loading time within 5 minutes. If the number of specimens is large, it is recommended to use a gun.

4.   Please make a standard curve at the same time of each measurement, it is best to make a double hole. If the content of the substance to be tested in the specimen is too high (the OD value of the sample is larger than the OD value of the first hole of the standard well ), please first dilute the sample dilution with a certain multiple ( n times) and then measure it. When calculating, multiply the total dilution by the total dilution. Multiple ( × n × 5 ).

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

6 . Please keep the substrate away from light.

7 . Strictly follow the instructions of the manual, the test results must be based on the microplate reader reading .

8 . All samples, washings and various wastes should be treated as infectious materials.

9 . The different batch components of this reagent must not be mixed.

10. If it is different from the English manual, the English manual shall prevail.

Storage conditions and expiration date

1 . The kit of preservation:; 2-8 ℃.

2 . Validity: 6 months