High variation among triplicates in ELISA - What I am doing wrong? (Mar/10/2008 )
Hey all,
So I have decided to do an ELISA.
I am using IFNgamma as the capture Ag, anti IFNgamma as the antibody and a 1:6000 diluted Protein-A HRP which serves as the detecting factor. ON top of that, I overlay with a KPL ECL mix (which I know to work excellently in other assays I've done with it).
What I've seen is usually a variation of 1 out of 3 triplicates, though not all wells suffer from this huge variation: 21253, 22494, 13908 (as an example of a 30% variation).
Now, I can do a quadruplicates and even pentaplicates and take out the best 3, but I try to refrain from doing so. Does anyone has a clue as to what be the cause to this problem?
Thanks,
CHG
So I have decided to do an ELISA.
I am using IFNgamma as the capture Ag, anti IFNgamma as the antibody and a 1:6000 diluted Protein-A HRP which serves as the detecting factor. ON top of that, I overlay with a KPL ECL mix (which I know to work excellently in other assays I've done with it).
What I've seen is usually a variation of 1 out of 3 triplicates, though not all wells suffer from this huge variation: 21253, 22494, 13908 (as an example of a 30% variation).
Now, I can do a quadruplicates and even pentaplicates and take out the best 3, but I try to refrain from doing so. Does anyone has a clue as to what be the cause to this problem?
Thanks,
CHG
Could it be a pipetting error maybe? I know we were having trouble with our multichannel pipette (it was a 100 years old and manual
) and even the tiniest variation in OPD solution level gave us CVs of 13% when we did an ELISA on detecting CTGF. (Not to mention it was leaking.. yikes) The other thing is that sometimes when you use tween 20, solution can get stuck inside the pipette tips. When we got a new electronic pipette and low retention tips, the problem went away almost completely. We had the same problem with Collagen detection although the CV was never super high. Have you checked whether the error is evenly between wells that are in the center of the plate, or those in which one of the wells is on the edge of the plate?. If you have big differences, may be as a result of evaporation effect in the wells, and it should be corrected incubating ELISA plates in a moist chamber at 37 ° C.
Hiya,
and to add to this i never use the outside wells of a plate even in a humid chamber as i have noted a marked differnce in readings (cutting the plate down to 60 wells instead of 96).
but it would sound more like a pipetting error, which could be occuring in any of the steps - coating, primary AB, secondary Ab etc etc...
and to add to this i never use the outside wells of a plate even in a humid chamber as i have noted a marked differnce in readings (cutting the plate down to 60 wells instead of 96).
but it would sound more like a pipetting error, which could be occuring in any of the steps - coating, primary AB, secondary Ab etc etc...
wells of a plate have not consistent binding affinities; try to find out the exact binding properties of each well of the lot you use; each new lot must be checked again for binding affinities