Hello,

I know this is a simple/mundane ELISA question but appreciate any feedback I could receive. I am a lab tech and have worked other places before, but about half a year ago I started at a diagnostics lab where we run ELISA's daily.

The ELISA is a sandwich ELISA, its not from a kit and we coat our own plates but its a pretty standard test (VWF antigen) and they have used the same tried and true protocols a long time. (Well tested antibodies concentrations, incubation periods, etc. Covers on the plates to reduce evaporation, water bath to reduce edge effect). The long term lab techs have no issues with running the ELISAs.

I, on the other hand, have been having issues.

About half the time my plates look lovely and the control values are fine, the other half the time my control values are too low. (They average the control lot values and they require all controls of that lot to fall within a range of 2 standard deviations from the average, so too low would be falling outside the 2 SD range).

There have been multiple times where I have done the same samples that others in the lab have run and my values always match but my control is low so I am fairly certain it is just an issue affecting the controls.

My CV's are always fine (excluding a random one here and there but they are rare). I plate the diluted samples, standard, and control I have prepared onto multiple plates at once and one plate will end up being low while the other (from the same set of sample dilutions) is absolutely fine so I don't believe it is an error in preparation/dilution of the control or the standard.

Additionally, the curves always look fine as far as I or the higher techs can recognize*, and there is not a high background level.

I have looked at troubleshooting from sites such as bio-rand and thermofisher and they address high controls but not much on low... except to say that the antibody calculation is off which I find hard to believe as, being a diagnostic lab, they use these calculations daily without issues.

I am not the only "new" tech to have had this issue but the higher up techs have walked me through the steps again recently to see if they could figure it out and stated they don't visually see anything I am doing wrong...but they also have stated they aren't very good at troubleshooting the ELISA. This means to me that, while I know how to pipet I cannot rule out if it is technique related (when they eyeball a meniscus are they a little high or do they wash differently or something?) or not (perhaps it is something that is just very sensitive but because I do the majority of the ELISA's I feel the brunt?) as I don't get much feedback.

It is getting very frustrating for me when one plate is fine and the next is not and I am unable to figure out where the difference may be.

If anyone has feedback or even brainstorming ideas as to what may cause my controls to be low it would be greatly appreciated

Thanks!