how to reduce the background in the ELISA assay? - using peptide as antigen (Jun/27/2006 )
hi,
The background in my ELISA assay is very strong. Even the blank well and negative control well has positive response using human serum as primary antibody.
the peptide antigen was coated at the final concerntration of 20μg/ml overnight. And then I used skim milk power (containing Tween-20) as blocking buffer (at 37℃ for 2h). The dilution buffer contained BSA.
I feel puzzled about this assay now. Do you have any good suggestions about it?
How high is the OD for the negative control?
The background in my ELISA assay is very strong. Even the blank well and negative control well has positive response using human serum as primary antibody.
the peptide antigen was coated at the final concerntration of 20μg/ml overnight. And then I used skim milk power (containing Tween-20) as blocking buffer (at 37℃ for 2h). The dilution buffer contained BSA.
I feel puzzled about this assay now. Do you have any good suggestions about it?
I've not used peptides before, so this might be a superfluous comment, but 20ug/ml seems very high to me. Have you titrated the coating concentration? I would start at 10 ug/ml and go down to about 2 ug/ml. I also wash the plate with 1% BSA in PBS (300ul per well).
thank you very much!
The OD for the negative control reached at 0.6, which were similar with the target group.
Maybe I should reduce the concentration of the peptide and change the washing buffer, I will try it today.
I would cnsider reducing antibody concentration when bgd is high.
I have tried to reduce the concentration of antibody before, but it didn't work.
I think that the human serum seem to be much more complex than the mouse or rabbit serum.
you can try blocking with the diluent (bsa) instead of milk or dilute with milk. you may be seeing your antibody binding to the blocking agent.
or
you may not be blocking sufficiently. increase concentration and/or incubation time.
Do you think whether BSA would affect the test when human serum is used as primary antibody ?
in some cases if BSA used as a sample diluent, it also gives high back groud. try only PBS as a sample diluent.
try new blocking also. there are many compositions of blocing available on net.