[Histonet] ELISA background

[Jeremiah Lyons]

Hi all!

I could do with some advice as regards eliminating background from my ELISA. I am detecting anti-Flk-1 antibody in mouse serum from mice vaccinated against this molecule. I know there is an immune response there but it is quite low, at best around 1:800. I am coating my plates with whole cell lysate which contains the flk-1 protein. I know the antigen is there as my positive control works well. I block for 2 hours using skimmilk 10%. I wash 5 times with a 90 second pause between each wash. My wash solution is PBS plus tween-20. I dilute my sera samples in skim milk 10% and leave at 4 degrees overnight. My secondary antibody is a polyclonal rabbit antimouse-HRP. My signal is positive but is quite weak, and it needs to be double the control absorbance to be conclusively positive. At the moment it is about 1.75 times the control, but the background on my control is quite high. Can anyone offer any suggestions?

Thanking you in advance!