Tobacco Mosaic Virus (TMV) Enzyme-Linked Immunosorbent Assay (ELISA) Kit Instructions for Use This kit is for research use only: This kit is used to determine the content of tobacco mosaic virus (TMV) in plant tissues, cells and other related samples. Experimental principle: This kit uses the double antibody sandwich method to determine the level of tobacco mosaic virus (TMV) in the specimen. The microporous plate was coated with purified tobacco mosaic virus (TMV) antibody to prepare a solid phase antibody, and tobacco mosaic virus (TMV) was sequentially added to the microcapsule of the coated monoclonal antibody, followed by HRP-labeled tobacco mosaic virus. The (TMV) 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 color depth is positively correlated with the tobacco mosaic virus (TMV) in the sample. The absorbance (OD value) was measured with a microplate reader at a wavelength of 450 nm, and the concentration of tobacco mosaic virus (TMV) in the sample was calculated from a standard curve. Kit composition: Kit composition 48-well configuration 96-well configuration Storage instructions 1 part 1 part sealing film 2 pieces (48) 2 pieces (96) Sealed bag 1 1 enzyme label coated plate 1 × 48 1 × 96 2 -8 ° C preservation standard: 900pg / mL 0.5ml × 1 bottle 0.5ml × 1 bottle 2-8 ° C preservation standard dilution 1.5ml × 1 bottle 1.5ml × 1 bottle 2-8 ° C preservation enzyme standard reagent 3 ml × 1 bottle 6 ml × 1 bottle 2-8 ° C Preservation sample dilution 3 ml × 1 bottle 6 ml × 1 bottle 2-8 ° C Preservation developer A solution 3 ml × 1 bottle 6 ml × 1 bottle 2-8 ° C preservation Reagent B liquid 3 ml × 1 bottle 6 ml × 1 bottle 2-8 ° C Preservation solution 3 ml × 1 bottle 6 ml × 1 bottle 2-8 ° C Preservation concentrated washing solution (20ml × 20 times) × 1 bottle (20ml × 30 times) × 1 bottle 2-8 ° C preservation specimen requirements: 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. Samples containing NaN3 could not be detected because NaN3 inhibited horseradish peroxidase (HRP) activity. Operation steps 1. Dilution and loading of standard products: 10 wells of standard wells are placed on the enzyme labeling board, 100 μl of standard is added to the first and second holes, and then added in the first and second holes. 50 μl of the standard dilution, mix; then add 100 μl from each of the first and second wells to the third and fourth wells, and then add 50 μl of the standard dilution to the third and fourth wells. Then, 50 μl of each of the third and fourth wells is discarded, and 50 μl of each is added to the fifth and sixth wells, respectively, and 50 μl of the standard dilution is added to the fifth and sixth wells, respectively. After mixing, 50 μl from each of the fifth and sixth holes is added to the seventh and eighth holes, respectively, and then 50 μl of the standard dilution is added to the seventh and eighth holes, respectively, and mixed from the seventh. 50 μl of the eighth well was added to the ninth and tenth holes, and 50 μl of the standard dilution was added to the ninth and tenth holes, respectively, and 50 μl of each of the ninth and tenth holes was discarded. (The amount of each well was 50 μl after dilution, and the concentrations were 600 pg/mL, 400 pg/mL, 200 pg/mL, 100 pg/mL, 50 pg/mL, respectively). 2. Adding samples: Set blank holes separately (the blank control wells are not added with the sample and the enzyme standard reagent, the other steps are the same), and the sample holes to be tested. Add 40 μl of the sample dilution to the sample well to be tested on the enzyme-labeled plate, and then add 10 μl of the sample to be tested (the final dilution of the sample is 5 times). 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: The plate was sealed with a sealing film and incubated at 37 ° C for 30 minutes. 4. Solution: 30 (48 times of 20T) concentrated washing solution was diluted with distilled water 30 (20 times of 48T) 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: Add 50 μl of enzyme labeling reagent to each well, except for blank wells. 7. Incubation: operation is the same as 3. 8. Washing: operation is the same as 5. 9. Color development: add 50 μl of color developer A, add 50 μl of color developer B, gently shake and mix, and avoid light at 37 °C. 15 minutes. 10. Termination: 50 μl of stop solution was added to each well to stop the reaction (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. Note: 1. The kit should be taken out from the refrigerated environment and allowed to equilibrate for 15-30 minutes at room temperature. If the enzyme label is unsealed after unsealing, the strip 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 does 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. 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. In the case of an English manual, the English manual shall prevail. Calculation: taking the concentration of the standard as the abscissa and the OD as the ordinate, drawing a standard curve on the coordinate paper, and finding the corresponding concentration from the standard curve according to the OD value of the sample; multiplying by the dilution factor; or using the standard Calculate the linear regression equation of the standard curve by the concentration and OD value, substitute the OD value of the sample into the equation, calculate the sample concentration, and multiply by the dilution factor, which is the actual concentration of the sample. (This figure is for reference only) Kit performance: 1. Sample linear regression and expected concentration correlation coefficient R value is 0.95 or more. 2. Within the batch and batch should be less than 9% and 11% detection range: 15 pg / mL -700 pg / mL Storage conditions and expiration date: 1. Kit storage:; 2-8 ° C. 2. Validity: 6 months
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