ELISA help needed
Steveh at worldnet.att.net
Tue Oct 15 23:18:57 EST 1996
> On Mon, 14 Oct 1996 10:19:35 -0700, "Martin K. Oaks"
> <moaks at execpc.com> wrote:
> >I am having trouble with high "background" levels in a sandwich elisa
> >assay. My capture antibody is a mouse monoclone, my test substance is
> >human serum, the second ab is a humanized IgG, the reporter ab is
> >peroxidase conjugated anti-human IgG. I get high levels of background
> >regardless of whether the capture ab I use is relevant or not. In other
> >words, ANY mouse ab appears to be reactive. I get no activity without
> >mouse capture ab and/or human serum. In addition, my reporter ab does not
> >react with the capture ab. It would appear that my test sera have
> >anti-mouse antibodies (HAMA) although I have no reason to believe that
> >these people have been exposed to mouse Igs. Should I absorb these test
> >sera (with solid phase mouse Igs) or is there a more straightforward
> >reply here or email me: Moaks at execpc.com
It is quite possible that yoru anti-human IgG is reacting against mouse proteins.
Unless the antibody was specifically adsorbed against mouse proteins, there is a
very high chance of cross-species reactivity. I suggest you look for an anti-human
IgG which has been specifically adosrobed; Jackson Laboratories is a good source
You also didn't explain how your plates were manufactured. If they weren't blocked
with BSA or some other blocker, it is quite likely that you are getting a high
degree of non-specific adsorption on the plates, which no subsequent blocking is
going to help.
Also, it is quite possible your peroxidase antibody is being used at too high a
strength...did you try titrating this down until you get a better signal?
Write me if none of this helps. I've had a bit of experience de-bugging Elisa assays
This process can take a while, but there is usually a way to solve non-specific
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