Orchid Mosaic Virus (CMV) Enzyme-Linked Immunoassay Kit Instructions for Use This kit is for research use only. Purpose of 96T Use: This kit is used to determine the level of orchid mosaic virus (CMV) in plant tissues, cells and related samples. Experimental principle This kit uses the double antibody sandwich method to determine the expression of orchid mosaic virus (CMV) in the specimen. The microplate is coated with purified orchid mosaic virus (CMV) antibody to prepare a solid phase antibody, which can be combined with orchid mosaic virus (CMV) in the sample, washed to remove unbound antigen and other components, and then combined with The HRP-labeled orchid mosaic virus (CMV) 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 450 nm using a microplate reader, and compared with the CUTOFF value to determine the presence or absence of orchid mosaic virus (CMV) 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. Samples containing NaN3 could not be detected because NaN3 inhibited horseradish peroxidase (HRP) activity. Operation steps 1. No.: The corresponding micropores of the sample are numbered sequentially. Each plate should be set with 2 holes for negative control, 2 holes for positive control, and 1 well for blank control (no blank sample control and enzyme standard reagent, the other steps are the same) 2. Loading: Negative control and positive control 50 μl were added to the negative and positive control wells, respectively. Then, add 40 μl of the sample diluent 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. Gently shake and mix. 3. Incubate: Cover with a sealing plate and incubate at 37 °C for 30 minutes. 4. Liquor: Dilute 30 times concentrated washing solution with distilled water 30 times and reserve. 5. Wash: carefully remove the sealing film, discard the liquid, dry it, fill each well with washing liquid, let stand for 30 seconds and discard it. , repeat this 5 times, 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. 10 min.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. Calculation and result determination: test validity: positive control hole average ≥ 1.00; negative control average ≤ 0.10 critical value (CUT OFF) calculation: critical value = negative control hole average + 0.15 negative determination: sample OD value
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Orchid mosaic virus (CMV) ELISA qualitative detection principle.rar
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