Blank on ELISA is higher than standard curve - (Oct/05/2010 )

I'm running ELISA plates to look at total IgA in breast milk. I'm using a goat anti human IgA antibody to coat my plates, but I have something very odd going on with my blank and standard curve.

Comparing the duplicates on my samples, the CV is fine (<10%), but the variation on my standard curve is HUGE. In addition, my blank reads higher than most of the plate (the blank is in the bottom left corner.)

I have run this ELISA before without an issue, but this is the second time this week I've had this happen. Someone else in lab is having a similar problem with his blank, the only thing we both use for our assays is the 37C incubator. No one else is running ELISAs at the moment.

Does anyone have any idea what I might be doing wrong???

THANK YOU, I'm desperate and confused

Focus first on your test. It was working perfectly earlier. I am assuming: CV's of both samples and standards were < 10%? and blank was 0.000 or near zero.

What changed between the last working assay and this one?
Have any of the reagents expired? Are you using any different lots of reagents? Are reagents prepared properly? Is any reagent contaminated? Are you using any automated instruments to wash/coat wells? Are the pipettes calibrated?

do you run controls (ie control samples different levels)? If so how do they perform...like the standards or like the samples?

If anything changed can you swap out one component at a time and run (new vs. old component) in parallel? I would start with wells, conjugate, and wash solutions.

With respect to the other person's test. Do they share any components?

Thank you for your suggestions!

The old test had CV <10 and a blank near 0.

I don't share any reagents with the other person.

I haven't run any ELISAs for a couple of months, so I made up and used all new reagents. At least according to what I wrote down in my lab notebook, nothing jumped out as a problem with how I made them, but I will try making up reagents again just to be safe. I can't run old and new in parallel because there's nothing left of the old reagents.

I will check the calibration of pipettes this afternoon.

I do run a control sample, and it behaved like my samples did.

Since I ran the plate twice, I changed the standard I used, with the same problem each time.

Someone mentioned to me that if wells dry out, sometimes this can happen. The standard curve is in the wells that I vaccum aspirate first, do you happen to know if dryed out wells can cause this? And if so, do you or anyone else have any suggestions on how to avoid over drying wells? I use a multichannel pipette and an eight channel vaccum aspirator to wash the plate.

Thanks again!