I am using 2 HB Immulon plates, I coat some wells (coated) with bacterial antigen 10^9/ml (60C/1h inactivated bacterial antigen) and I left some wells with no antigen (uncoated). so coated vs uncaoted
I blocked some wells with start block (Thermofisher) vs no blocking in others so blocked vs unblocked. Note: usually when you have high bacterial antigen >/= 10^9, you probably don't need to block, however I tried it anyway
My samples are bovine samples, and my antigen as I mentioned is bacterial antigen that I grow in TSB broth then inactivate either with heating at 60C/1h OR USING 1% phenol for 4 hours in a parallel experiment..
My capture/detection antibody is sheep anti bovine HRP and my substrate is TMB
So My problem is that my negative control sample has signal only on the coated wells (NOT, the uncoated) wither blocked or not, So the blocking is not an issue. Note: My positive control sample > 50 day post vaccination gave almost the same OD value as the negative sample.
My question is what are the possible causes that my negative control is giving a positive signal? Thanks
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