The ELISA enzyme immunoassay in any of its modalities (direct, indirect, competitive, sandwich type …), is a technique widely used in the fields of research and diagnosis to detect and / or quantify biomolecules . Although the protocol for carrying out this type of assay is relatively simple, it is necessary to understand the rationale for the ELISA immunoassay to correctly interpret the results.
As in any other biological test, sometimes the results may not be what you expect. In these cases, it is essential to pay attention to the small details that may have been overlooked, distorting the result and destroying the performance of the immunoassay.
Here we bring you some tips that will allow you to solve problems in an ELISA immunoassay .
How To Optimize Your Elisa Immunoassay And Solve The Most Common Problems
- If the signal is abnormally high and the standard curves have saturated ODs:
- Make sure you have reconstituted the standards in the proper volume
- Respect incubation times
- If you get excessive background noise
- Wash the plates with distilled water
- Make sure the substrate is colorless before adding it to the board (it could have suffered some degradation)
- Correct washing solution
- Check the ambient temperature: the optimum is between 18-25ºC
- Make sure that the reagents have been prepared correctly
- Make sure that the washes are carried out correctly:
- Sufficient number of washes
- Wash solution volume (at least 400ul / well)
- If the OD readings are unexpectedly low
- Make sure the lab, reagents, and / or plates are not too low
- Do not exceed the number of wash cycles
- Make sure the incubation times have been met
- Check that the amounts of antigen and / or antibody used are sufficient.
- If you don’t get coloration
- Check that the reagents have been used in the correct order, and that all steps of the assay have been carried out
- Make sure you have used the correct conjugate
- Check that the solution has been added with the substrate
- Avoid wash buffers containing sodium azide
- If you get low reproducibility within the same board
- Make sure that the reagent addition time does not extend too long until all wells are complete. Always prepare all necessary reagents in advance and use multichannel pipettes
- Make sure the multichannel pipette works correctly
- Check that the washing system works correctly
- Check that the antibody distribution is homogeneous. In case the samples have been frozen or refrigerated, be sure to mix them well before diluting them. Once diluted, mix them again before adding them to the plate.
- If the results are not reproducible between plates
- Maintain the same incubation times for all plates.
- Apply the same number of washes to each plate
- Make sure that the controls and samples are at the same temperature before carrying out the assay.
- Sometimes it could be due to the use of reagents from different lots
The most frequently repeated causes of error in an ELISA usually originate from the instrumentation / washing procedure, the preparation of the reagents and / or the incorrect compliance with the protocol.
If your essay doesn’t deliver the expected results, the tips listed above can provide you with a good starting point for solving problems.
If you need help, contact us .