I have been trying to test rhaumatoid arthritis and lupus patient sera alongside normal/healthy patient sera (as controls) for the presence of specific immunoglobulin. I am using NeutrAvidin-coated plates, as from our previous experiments they proved better, and am coating the plate with 8ug/ml biotinilated peptide (in filter sterilized distilled water) then incubate for 2hrs as plate specification sheet advises.
Then I wash the plates with PBS pH7.2 and block with 2% BSA in PBS for 1hr ar 37 degrees C. After 3 washes with PBS/Tween 20 (0.1%), I add patient anti-sera diluted 1:200 in PBS/Tw20 (0.05%)/BSA (2%) for 1hr.
After 3 washes I use two different secondary HRP conjugated antibodies (affinity purified) to compare the reactivity with the antigen-
one is Polyclonal Goat anti-Human IgG F(ab')s/HRP (at 1:1000 dilution) and the other is Polyclonal Rabbit Anti-Human IgA, IgG, IgM Kappa, Lambda/HRP (at 1:8000 dilution).
The concentrations of secondary antibody were optimised according to manufacturer's instructions and they are diluted in PBS/Tw20 (0.1%)/BSA (2%) and incubated for 1hr in the dark. Plates are then washed, TMB is applied for 4mins and reaction stopped with 2N HCl.
My problem is that the normal patient serum keeps giving me high values (as high as diseased sera) even though the reactivity should only be high for RA and lupus patient sera (diseased sera give me high reactivity as expected). Other quality control and blank wells are mostly fine.
Is there anything that you could suggest please? We have tested multiple patient sera before using exactly the same system and have never experienced this kind of problems. I have no clue as to what could have gone wrong this time.. I would be very grateful for any suggestions!