Titer Plate Reader, ODs off - (May/25/2011 )

For a competitive assay, usually get average blank value of 0.017 but yesterday and today values were at the 0.7 and up range the longer I let the assay develop. We do not have a stop solution so I take the final reading at 1.0 for the lowest standard. The ODs for the samples and the standards all read negative unless I let the assay develop well past the usual time we read it at.

Why have my blanks gone crazy? Why am I getting negative ODs on standards and samples? An OD of 1.0 indicates no protein activity and is the deepest color.

I repeated the assay today to eliminate pipetting errors, only assay buffer/HRP/ABTS in the blank wells as they should be.
Also, used the same buffers as last week when all was going beautifully.

Maybe something is contaminated? Or just a wild guess, has a setting on the plate reader been monkeyed with? Maybe the light source is going out.

Thanks, Lab Rat. I am thinking it was the software also. But also it could be hardware of another sort. I set up a different assay in the software and ran it on plates Friday and I wonder if maybe the filters haven't successfully switched back as the Friday assay was at 430 nm and the problem assay is at 405 nm.

I have a call into the company tech, to go over the hardware checks and will look at the manual today before talking with him to see about checking the light source.

I am just learning the software on this reader, it's a Dynex reader MRX with Revelation software.
Admittedly this is my first experience with immunoassays and plate readers, a learning curve is embedded in all my assays.

To rule out human error, these are the checks I did so far:

plates bad? was from the same lot of plates that worked before.
solutions, any new? just the HRP which I double checked when mixed to be sure I hadn't gotten the two mixed up. But I can check again.
buffers? all the same as before but now I am out of them and will be mixing new, a bad practice because I should only be using a new buffer one at a time, ah well, it's not been the greatest week.
pipettors set correctly? double checked that on the scale with ddH2O to see if they were measuring correctly, all fine there.
plates turned around? double checked that to make sure I was pipetting in the same direction.
samples diluted and stable? I can look today at the Tuesday and Wednesday results and see if they stayed stable between the two days.

any other checks I could be doing?

lab rat on Thu May 26 03:09:22 2011 said: