ELISA Direct Method for Measuring Longleaf Plant Mosaic Virus
ELISA Direct Method for Measuring the Content of Long Leaf Plantain Mosaic Virus (RMV)
1. Using different concentrations of RMV solutions to be tested as sample solutions, add 250ul to each well of the assay plate, and incubate in a refrigerator (4 ° C) overnight.
2. After incubation, remove the antigen solution from the hole, wash the hole with PBS-Tween20 solution for 3 minutes, 3 minutes each time, and then remove the clean washing solution.
3. Add 250ul of RMV enzyme-labeled antibody solution (diluted with 1% bovine serum albumin in PBS-ween20) to each well and incubate in an incubator (37 ° C) for 3-6h, or overnight at 6 ° C.
4. Remove the antibody solution of the cave enzyme-labeled antibody, and wash the buffer with PBS-Tween20 solution 3 times for 3 minutes each time, and then remove the washing solution.
5. Add 250ul of freshly prepared substrate solution to each hole, and leave at room temperature for 0.5h, or yellow when it is placed.
6. Add 50ul3N NaOH to stop the reaction.
7. For the yellow solution in each hole, measure the absorbance at a wavelength of 449 nm.
discuss
1. If the polystyrene plastic micro-reaction plate is used for the first time, the standard concentration of RMV solution, RMV antiserum, and the immunoconjugate formed by RMV and its IgG should be used to perform the expected titration to measure The adsorption capacity of styrene plastic micro-reaction plate to RMV antigen and its antibody.
2. Before the formal measurement of samples, use 0.1mol / L pH9.6 carbonate buffer to prepare RMV solutions of various standard concentrations, and determine the A449nm values ​​of various standard concentrations of RMV according to the experimental procedure, with the standard concentration of virus as the abscissa , A449nm value is plotted on the ordinate to draw a standard curve, from which the most suitable concentration of RMV coating can be measured.
3. Measure the absorbance of the original substrate in order to verify the data.
4. With reference to the standard curve, the content of RMV in the sample solution can be calculated.
5. If there is no bovine serum albumin, 0.1% white gelatin can be used instead.
6. For unstable viruses, dilute with neutral buffer.
7. If there is an ELISA tester, it can be tested quickly.
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