RE: immuno on mouse tissue

From:

Abizar Lakdawalla

Hi Jen, some of the reagents in their (and our) mouse-on-mouse kits are expensive to manufacture. The Fab (Fragment, Antigen binding - a monovalent piece of an antibody) based reagent is pretty hard to make (purify secondary antibody, digest with a protease, isolate the Fab fragments by serial column chromatography, label with biotin, repurify).

There were some other messages in this thread that had mentioned using a secondary antibody (biotinylated, anti-mouse) to bind the primary antibody (mouse monoclonal) in a test tube, blocking, and then placing this antibody-antibody complex on the sections. This will not work as the bivalent secondary antibody (can bind two antigens) will produce more background. How?

1. The bivalent (or multivalent) secondary antibody with multiple Antigen binding sites will act as a bridge between the bound-primary antibody and immunoglobulins normally present in the tissue. So now you may end up with a big complex sitting right on the endogenous immunoglobulins

(Tissue immunoglobulins----Fab1 of Secondary Antibody-Fab2 of Secondary Antibody----Primary antibody). The very thing that you were trying to avoid.

2. Also it is a bad idea to mix a secondary antibody (biotinylated anti-mouse) with its primary antibody (mouse monoclonal antibody) in a vial or test-tube as it may result in a precipitate being formed (more background?).

Abizar

-----Original Message-----
From: Philopena, Jennifer [mailto:jennifer.philopena@canji.com]
Sent: Thursday, March 07, 2002 1:06 PM
To: 'Abizar Lakdawalla'
Cc: 'Histonet' (E-mail)
Subject: RE: immuno on mouse tissue

Abizar, I just used DAKO's mouse on mouse kit, called ARK (Animal Research Kit). It worked beautifully. But it's expensive. Jen

-----Original Message-----
From: Abizar Lakdawalla [mailto:abizarl@innogenex.com]
Sent: Tuesday, March 05, 2002 2:08 PM
To: 'Cathy Gorrie'; histonet@pathology.swmed.edu
Subject: RE: immuno on mouse tissue

Some vendors (including us) sell mouse on mouse kits that work quite well for using mouse monoclonal antibodies on mouse tissues.

Abizar
www.innogenex.com

> -----Original Message-----
> From: Cathy Gorrie [mailto:C.Gorrie@unsw.edu.au]
> Sent: Monday, March 04, 2002 6:03 PM
> To: histonet@pathology.swmed.edu
> Subject: immuno on mouse tissue
>
>
> For all the immunohistologists out there.
>
> This is a pretty general question about immunohistology on mouse
> tissue. When using monoclonal antibodies, what special measures need
> to be taken, or what secondaries need to be used, in order to get
> specific staining. I can imagine if you use an anti-mouse secondary
> everything in the tissue will be targeted, or at least a lot of
> background from serum proteins, immuno cells etc.
>
> or
>
> Can you not use monoclonals on mouse tissue? I have always used other
> species of animal, or human and this has never been a problem before.
>
> or
>
> Is this not as big a problem as I imagine it could be?
>
> Thanks for your help,
>
> Cath
> --
> ---------------------------------------------------
> Cathy Gorrie
> Scientific Officer
> Neural Injury Research Unit,
> School of Medical Sciences,
> University of New South Wales
> Sydney, N.S.W. 2052
>
> Phone: 61-2-9385 2462
> Fax : 61-2-9313 6252
> e-mail: c.gorrie@unsw.edu.au
>
>

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