Details determine success or failure, and this is also true for laboratory researchers. There are many operational details in the ELISA kit experiment, such as minimizing the use of pipettes, frequently changing pipette heads, and adding samples from low concentration to high concentration, as this can reduce the probability of skipping wells. And the key to the entire experiment is the method of processing the results. In today's technical article, our company brings you the ELISA kit detection result analysis method.
① In ELISA experiments, samples need to be subjected to multiple or triple wells, and the average OD value of each concentration standard should be calculated. The coefficient of variation for multiple wells should be less than 20%.
② Subtract the OD value of the blank hole from the average OD value.
③ Create standard curves.
Using the OD value obtained by subtracting the background as the X-axis and the concentration of the standard as the Y-axis, a suitable calculation method can be obtained using graphical software. It is recommended to use appropriate software for drawing, such as CurveExpert 1.4. This software can provide many methods (such as lines, semi logarithms, logarithms, four parameter equations, etc.) and select the most suitable equation from them. To verify whether a certain curve matches the data in the table, the concentration of the standard sample can be taken as an unknown variable, and the corresponding OD value can be input into the equation to calculate the concentration of the standard sample. The result obtained should be very close to the actual concentration (+/-10%). Choose the curve with the highest fitting degree.
If there is poor linearity or parallelism of the standard curve (OD of the double hole control hole) There may be significant differences in values between the wells of the enzyme-linked immunosorbent assay (ELISA) plate, or the phenomenon of jumping holes, which may be caused by mutual contamination between the wells of the ELISA plate, failure to proceed to the next step as soon as possible after washing the plate, incorrect operation methods when adding samples or other reagents, poor light avoidance of the incubation position or inadequate sealing of the cover film causing water evaporation, and different amounts of reagents added to the wells of the ELISA plate. The corresponding solutions are to be careful not to splash the liquid into another well when adding samples, and also to be careful not to splash the washing solution into another well when manually washing the plate. After washing the plate, please proceed to the next step in a timely manner. When adding reagents, please suspend and drip them, and remember not to touch the gun head into the plate well to absorb different substances. When dealing with the liquid in the reagent bottle, it is necessary to replace the nozzle once, confirm that the incubation environment is opaque, seal the enzyme-linked immunosorbent assay (ELISA) plate with a cover film, and use a calibrated micropipette, If there is a drop of liquid left on the nozzle when adding liquid * times, then the remaining liquid on the nozzle in the future should also be dropped to ensure consistency in the volume of liquid added.