ELISA Result Interpretation Help! - SoftMax software result interpretation (Dec/02/2008 )
Like many of the posts on here, I am rather new to running ELISAs. We thought we had it figured out but we just got some results that threw us for a loop. We are running a sandwich ELISA with a monoclonal antibody. We are using SoftMax Pro software to calculate our data. It gives; raw OD, calculated result, mean result, std. dev., %CV, Dilution and Adjusted Results. When we set up the template we put in that our samples are in ng/ml, so that is what our results should technically be in, if I am understanding the software correctly. We have multiple dilutions of our sample to make sure 1 falls within the standard range. Where I am confused it if the adjusted result is in ng/ml to get a result in ug/ml I should be able to take that result and divide by 1000, right? What doesn't make sense to me is that when I have two dilutions that are within the standard curve why they don't give around the same result... for example
Dilution 1: Raw OD:0.238 & 0.145, Average Result: 12.287, Dilution: 5120, Adjusted Result: 62908.957
Dilution 2: Raw OD: 0.052 & 0.082, Average Result: 4.886, Dilution: 20480, Adjusted Result: 100066.335
If these are both adjusted results are in ng/ml I would think that their results in ug/ml would be 62.9 and 100.1 respectively.
If this makes sense to anyone I would love some help!
First, I would take a look at the precision of the assay. Secondly, it is not that the results are 'within the standard curve' but are within the linear portion of the dose response curve...the analytical range of the assay. 0.052 'raw OD' may be close to your 0 or background.
I think you are looking at results at the very low end of the curve. The low end and high ends have the highest %CV. Make your sample dilutions to fall within the 0D range of 0.3 to 1.5. Hopefully your curve extends to od 2.0? If your pipetting, dilution, and technique are ok your results should be closer.