Non-specific binding from plasma in sandwich ELISA

Go to the profile of leasky
Jun 12, 2017
0
3

Hello everyone,

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

3 Comments

Go to the profile of relaxin
relaxin 6 months ago

Is it possible the new batch of human plasma "contaminated" with tumor marker? Did you try another batch of human plasma?

Go to the profile of leasky
leasky 6 months ago

Hi Relaxin,

Thanks for your comments, it seems unlikely that it is contaminated with the marker as on one lot of plates we get a high concentration and on another lot of plates we do not. This suggests interference only on certain plates. I understand that it is well known that plate manufacturers can struggle with batch to batch consistency, so there doesn't seem to be a lot I can do about that. My main focus is on getting my assay to the point where it can negate those plate inconsistencies (if that's possible).

To answer your second question, we have multiple batches of plasma that seem to give different baselines of the marker depending on which batch of plates is used (sometimes zero, sometimes in the terminally ill range!). Again, suggesting huge plate inconsistencies. I can't blame everything on the plates though, if my assay was perfect I wouldn't seeing such huge variations.

Go to the profile of relaxin
relaxin 6 months ago

It seems to me that you need to try different blocking buffers rather than optimizing washing solutions. A number of blocking buffers are available from companies such as ThermoFisher, "StartingBlock" is known to be a good blocking buffer. Blocking buffer containing casein may be better than that containing BSA. Washing buffer containing high salt should be avoided.