Is there any approach to reduce inter-assay variability of home-made sandwich ELISA except good pipetting practice?
Thank you for any suggestion!
There is always some variation, but if you include the same standard or control samples in every assay, you can normalize against them. The absorbances can differ but the relative levels of your analyte in a given sample compared to standard/control must be the same in every assay.
It depends quite a bit on the analyte that you are measuring. Some are more prone to ambient disturbances (temperature, buffers etc.), whereas others do not really care.
In general, reduce variables and stick to the letter and not the spirit of the SOP. For me personally, the pipetting is a likely source of variation, especially the sample dilution phase.
- Avoid using any pipetting volume that is below 5ul. For example perform proper dilutions either in tubes or plates with sufficient volumes. If you want 1:10 000 dilution do not use 0.1 ul to 1000ul dilution, use something like 5ul + 500ul -> 5ul + 500 ul instead.
- Do not use the assay plate for dilutions, or if you have to, block the plate to the upper edge.
- Increase wash amount and duration. Especially if you are using nozzle bottle fill to the brim and leave the wash solution there for 5+ minutes. Most importat wash is at the very last one, with polyHRP I usually left the plates for 10 minutes in wash solution and then proceeded to the TMB step.
- Use stopwatch to time your stages properly, and document them. If you have 10+ plates you will start to have a drift where one end of the plate set will be incubated longer.
- Ensure that all plates are exposed to the same incubation temperature, I personally prefer temperature controlled boxes (4C, 20C 37C) that have level shelves.
- Make sure that the assay is not too sensitive. Especially with TMB the reaction is far too violent (
There is always some variation, but if you include the same standard or control samples in every assay, you can normalize against them. The absorbances can differ but the relative levels of your analyte in a given sample compared to standard/control must be the same in every assay.
Thank you Juha and Rojas for your recommendations! I really appreciate them.
Often particles are the cause of ELISA variation. Centrifugation or filtration of samples and reagent stock solutions may be helpful.
- Badges
- Science method
- Similar topics
- Medicine
- Immunology
- Immunology Techniques
- Immunoassay
- ELISA