There are various kinds of ELISA kits on sale in the market, the detection range and assay principle of ELISA kits are not the same, especially on different target protein, even for the same target protein from the different manufacturers, the assay principles maybe are different. But the basic principle is consistent, that is an immuno-reaction qualitative and quantitative methods which absorbed soluble antigens or antibodies onto solid matrix (i.e Polystyrene).
In order to completely answer“Why you need to strictly operate ELISA kits according to the delivery manual?”, let’s review the principles of various method of ELISA kits at first.
1. Sandwich ELISA, Double Antibody
2.Sandwich ELISA, Double Antigen
4. Indirect ELISA
After that, we summarized the situation that do not follow up the instructions and unsatisfied results.
Not read the instructions before using it, take “Competitive ELISA” as “Sandwich ELISA”to run the assay, it will lead to dark blue in the whole plate and without any gradient.
Mix use components in different brand，it will cause the kit recovery not as we expected or show strong/weak color on the microplates， because different brand may not have the same ingredient.
Not read literature or not do pre-test, according to the literature you need diluted the samples at 1:10,but you diluted at 1:1000 in the end, it will show the weak color on the microplates.
Not calibrate the pipette and the temperature and humidity of the incubator，it will lead to inaccurate concentration of the buffer and improper incubation temperature, all of these will bring deviation to the test.
Insufficient washing, adding a small amount wash buffer into each wells or still leave residue, it will lead to rapid color development and a higher background.
The tip of the pipe is not suspended to add wash buffer when manual washing, it will lead to contamination of buffer and fail test.
Not put unused microplates into foil bag in time, cause the microplate going to damp，it will lead to weak color, contamination or high CV.
Store our ELISA kits at improper temperature。 All of our ELISA kits need to be stored at 2-8℃,if you stored at -20℃, it will cause the lower sensitivity of our kits.
The above mentioned is the most common operating errors，there are many details that need to be paid attention to to successfully complete an ELISA test, as many operating procedure will affect the final results of the test.
Hence, it is highly recommended to read the instructions carefully and strictly operate ELISA kits according to the delivery manual.