how to reduce the background in the ELISA assay? - using peptide as antigen (Jun/27/2006 )
it shouldn't, unless the human is sensitized to bovine albumin. even so, if you dilute your antibody in it then any binding to the blocking agent will occur in the tube before introduction to the plate.
I have tried to reduce the concentration of peptide and increase the blocking time, but they didn't work either. The blank has high response,too.
This is just a suggestion for you to consider: have you thought about conjugate your peptide onto a carrier protein such as BSA then do the assay. I dont know your peptide design and your biochemical conjugation skill, but that can be done rather easily and it might be a solution for your problem, as you can now reduce the antigen conc low enough to do a decent ELISA. 
I have never done conjugate assay. is that mean to co-incubate my antigen and BSA? what's the proper concerntration of BSA? and how long does it need?
This is just a suggestion for you to consider: have you thought about conjugate your peptide onto a carrier protein such as BSA then do the assay. I dont know your peptide design and your biochemical conjugation skill, but that can be done rather easily and it might be a solution for your problem, as you can now reduce the antigen conc low enough to do a decent ELISA.
I have never done conjugate assay. is that mean to co-incubate my antigen and BSA? what's the proper concerntration of BSA? and how long does it need?
No, you need to link your peptide to a large protein chemically, such as BSA, followed by a column purification to remove unconjugated peptide from the peptide-BSA conjugates. The simpliest way to do is to incorporate a terminal cys residue and conjugate it to BSA previously modified with a thiol reacting group. You can go to this site and read more about it: http://www.piercenet.com/Objects/View.cfm?...85-BA94DCA642F8
No, you need to link your peptide to a large protein chemically, such as BSA, followed by a column purification to remove unconjugated peptide from the peptide-BSA conjugates. The simpliest way to do is to incorporate a terminal cys residue and conjugate it to BSA previously modified with a thiol reacting group. You can go to this site and read more about it: http://www.piercenet.com/Objects/View.cfm?...85-BA94DCA642F8
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Thank you very much!
I changed the blocking buffer and the dilution buffer for the antibody and then the results were ok now!
But I don't know the reason because I don't know the composition of those buffer! They were provided by others.
hi,
i donot think BSA effect ur assay. i wud not expect antibodies against a bovine (bsa) protein.
in general human serum give high back ground in my case it was 0.3-0.4.
another info is i used to coat wells with 5ug of peptide on higher side.
gud luk
payeli.
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.
Hi,
What do you mean by target group? Is it unknown tested sera? So it could be easily negative as well. What are your positive control readings? It is very important to check the positive-negative ratio of the serum while calibrating the assay. I mean that if your positive readings are around 2.5 -3 OD, your BG level is not extremely high.
One way to reduce the BG while working with sera is adding normal sera (in your case human) to the detection solution (usually not more than 10%).
Try to use plates with lower binding capacity (polysorp or medium binding) even though most of the protocols suggesting to use high binding plates for protein coating.
I would not use 20ug/ml for coating because it is usually much more than the saturation concentration of the well.
Good Luck!
Pery