Non-specific binding from plasma in sandwich ELISA
I am having some issues reducing non-specific binding in my sandwich ELISA when assaying human plasma samples. For some background, we use our ELISA for detecting a tumour marker in human plasma. As a quality control we spike the marker into human plasma (pooled from normal, healthy population) at various concentrations to ensure consistency in the assay. However, a recently purchased batch of human plasma is producing a high concentration of the tumour marker without any spiked in. We know this is non-specific as this particular marker should be close to zero in the normal population. We have never had an issue with this before, but it is concerning now knowing that our assay isn't 100% efficient in eliminating interference.
I have tried several things, such as: changing brands of plates (switching from Nunc Maxisorp to Nunc Medisorp plates has improved things slightly), trying different blocking buffers with the primary ab (Tween-20 seems to do a better blocking job than BSA) and optimising the wash buffer. Increasing NaCl concentration in the wash to 0.5M reduced non-specific binding and going up to 1M NaCl reduced it even further, and without compromising binding in the standard curve. Implementing these changes has reduced non-specific binding by about 75%, but of course I would like it to be a close to 100% as possible!
I would greatly appreciate any advice from anyone who has had similar problems or who has greater expertise than myself.
Thanks in advance