[Protein-analysis] Re: ELISA HELP NEEDED Cant Get anything to Work (Allison)

Dan Guire via proteins%40net.bio.net (by dguire from isurtec.com)
Tue Mar 27 14:32:24 EST 2007

Hi Barbara,

The main point has already been made:  Your anti rabbit alphatase conjugate
can only detect the rabbit antibody down on the Maxisorp plate.  It doesn't
matter whether there is antigen for the capture antibody or not.  No doubt a
negative control, leaving out only the mouse lysate, would show you that. 

The series Allison outlined looks fine; the enzyme-conjugated anti mouse
shouldn't bind unless there is mouse antibody bound to antigen there.  My
point is that if you already have biotinylated anti ENAC of one species, you
can capture any antigen with the non-biotinylated anti ENAC of the other
species, then detect with the biotin and a streptavidin-enzyme conjugate.
And a negative control is a must :-)


P.S. Do you have any affinity column experience by any chance?  I could use
some help in that arena...

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Subject: Proteins Digest, Vol 22, Issue 11

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Today's Topics:

   1. Re: ELISA HELP NEEDED Cant Get anything to Work (Allison)


Message: 1
Date: Mon, 26 Mar 2007 15:10:05 -0500
From: Allison <allison from nospam.com>
Subject: [Protein-analysis] Re: ELISA HELP NEEDED Cant Get anything to
To: proteins from net.bio.net
Message-ID: <CsKdnVlLyK0Oh5XbnZ2dnUVZ_oqmnZ2d from mcgill.ca>
Content-Type: text/plain; charset=us-ascii; format=flowed

Barbara wrote:

> I'm trying to develop an ELISA Assay to measure ENAC & ATPase in
> mouse
> cochlea lysates. I've been able to do so using NFkB, TNF & VEG kits.
> But there are no kits for these ab. I posted initially on the
> wrongforum I think.
> I have tried coating the Nunc maxisporp plates ( at a couple of conc)
> with rabbit anit-mouse ENAC or rabbit anti-mouse ATPase then add my
> antigen (mouse lysate) followed by ALphatase goat anti rabbit IgG. It
> seems that nothing sticks. Given the small amount of lysate &
> variability coating wit hthat is not practical.

If your first Ab is RABBIT anti mouse and your second Ab is goat 
ANTI-RABBIT then all you will detect is the coating primary Ab.

A better series would be:

Rabbit anti-ENAC
blocking protein
Mouse anti-ENAC
Enzyme-conjugated goat anti-mouse IgG

Whatever antibody you decide to use it is important to keep track of the 
animal it was made in as well as its specificity.

I have used the Nunc plates for ELISAs and I find that they work well. NAYY


> I also tried biotynating the anti ENAC & ant ATPAse & then incubating
> with coated plates. Followed by blocking & then my mouse lysate & the
> nthe goat anti rabbit. Every thing is high except fo the ones without
> the biotinalyted AB initially added.
> Anyone have any thoughts or suggstions.
> Thanks
> BL


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