Efficacy of expired kit since 2011 and troubleshooting - (Jun/19/2013 )

I am extremely new to performing ELISA assays, considering that I used up four days to figure out out to operate a microplate washer and spectrometer.

I am currently using ELISA by Salimetrics to measure for salivary cortisol. Since am a newbie, I have been using old kits which have expired since 2011. I have managed to get some data out of it but the problem is some figures are out of range and CV of wells are extremely high. I am trying to determine if the problem lies with my techniques or does the fault lies with the expired kits.

I have 2 new kits which I am going to use when I do actual runs on my samples. It is just that I have budget constraints so I unfortunately I have to learn ensure that I don't stuff during the use of my actual kits.

Can I run undiluted saliva samples or should I dilute them? I have over 60 samples.

I have attached a pdf file of the run. This run is using undiluted samples.

Any help provided is appreciated.

since you admit that you are new and just learned to use the instruments, did you learn and practice proper pipetting techniques?

are the pipettes properly calibrated?

with an expired kit, you may see loss of linearity but reproducibility should remain if your technique and your pipettes are sound.

Hi mdfenko.

On my fourth try, I did get low CV. I have been practising my pipetting techniques several hours a day for 5 days now. The only problem is that I don't think the pipettes are calibrated. I am requesting to use the ones up in biochem labs so hopefully I get better reproducibility then.

Thanks for your advice.

Hi. I think the kit is likely o.k. The antibody is coated on the plate and the steroid/HRP conjugate is usually pretty stable. The variability between the duplicate samples is likely due to technique. I would practice as much as you can with the standard running in duplicate, triplicate, etc and see if the variability improves.

Most of your samples appear to have very little cortisol in them so diluting them probably will not be helpful. It's important to remember that with a competition ELISA you're looking for a lack of signal instead of signal.

keep in mind that small differences in low readings appear to show high cv

Hey guys, thank you so much for your help.

I did a re-run and I came back with another set of data output. Still I keep get out of range values, even for wells with zero concentration.

However, the mean OD values for some of the unknown samples seem to be within the standard range.>

Sorry for being a noob here.