ELISA help needed

Steve Haas 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
> >approach?
> >
> >reply here or email me: Moaks at execpc.com
> >
> >Thanks.

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
for this.

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
binding problems.

Steve Haas



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