from plasma not sera !!

In general, I would not expect any problems. What is your plan with the antibodies?

I want to check specific Anti-A27 antibodies in an in-house ELISA but the values obtained are somehow weird.

Are you diluting your samples? Ficoll is increasing the viscosity of your sample, so diffusion might be hindered and a longer incubation times and/or dilution might be required.

If you have heparin as an anticoagulant, it could bind to proteins because it has a strong negative net charge.

What is the pH of your solutions?

Yes, the tubes had heparin...

The pH of my ELISAs protocols buffer are the following ones:

• Coating buffer (CB) = 9.6
• Blocking buffer (BB)= pbs 3%BSA (Should be 7.4 or so)
• Dilution buffer (DB)= pbs 3%BSA + Tween20 0,1%
• Washing buffer (WB)= PBS 0,1% Tween 20