RE: [Histonet] Human anti Human

From:"Patsy Ruegg"



Andrea,

I agree with your approaches to keep tissue IgG from binding to the anti
human secondary, I especially like the fitc conjugation of the primary then
use rab anti-fitc, then rab labeled polymer.  The reason I avoid SAB is
because of endogenous biotin issues.  I prefer hrp or ap labeled polymers to
avidin biotin systems.

Patsy

Patsy Ruegg, HT(ASCP)QIHC
IHCtech
12635 Montview Blvd. #215
Aurora, CO 80045
720-859-4060
fax 720-859-4110
pruegg@ihctech.net
www.ihctech.net
www.ihcrg.org    
 


-----Original Message-----
From: anh2006@med.cornell.edu [mailto:anh2006@med.cornell.edu] 
Sent: Sunday, October 26, 2008 9:59 AM
To: Patsy Ruegg; histonet@lists.utsouthwestern.edu; johnsobr@zgi.com
Subject: Re: [Histonet] Human anti Human

Very true Patsy, Amos' proposed scheme will not eliminate the problem of
human-on-human.

However, I do not see how using extra host secondary serum will block the
anti-human from binding the endogenous IgG in the tissue. And using human
serum will just make your background worse or eliminate staining altogether
by sopping up the anti-human antibody.

Speaking from experience doing human anti-human many times, you have a
several options some of which are listed below:

1. Preadsorb (in solution in eppendorf) your primary to your bitoinylated
secondary (hopefully one of Jackson IR's highly cross adsorbed Abs, maybe
even Fc specific or an Fab fragment rather than whole IgG) for a few
hours/overnight on a rocker. (Concentrations ratios of both primary and
secondary need to be tested on positive control tissue). Then add excess
human gamma globulin the tube to sop up any extra unbound anti-human IgG (in
fact using a Fab would even be better but more costly than GG). Then add to
regularly blocked tissue and detect with streptavidin. If you want more
abundant amplification you can use a FITC labeled secondary then detect with
rabbit anti-FITC followed by polymer. 

2. Label the human anti-human antibody with FITC (very easy to do!). Then
detect with rabbit anti-FITC and use rabbit polymer.

3. Biotinylate your primary then use strep to detect. I am curious as to why
are you anti-biotinylation? It is your easiest solution to the problem and
is not difficult!

All of the above techniques work with the right reagents in place. Let me
know if you want more information.

Good luck,
Andrea
 
-----Original Message-----
From: Patsy Ruegg 

Date: Sun, 26 Oct 2008 09:03:57 
To: 'Amos Brooks'; ;

Subject: RE: [Histonet] Human anti Human


The only problem I see with this is similar to running an anti mouse ab on
mouse tissue.  The secondary link anti human IgG will bind to all IgG in the
human tissue, not just the primary antibody, so you would need to use serum
blocks to prevent that.  I would use 10% serum from the host of the
secondary before the primary and then again before the secondary as a block.
I would also think about using some human serum in the diluents but you have
to be careful not to block all human IgG.

Patsy

Patsy Ruegg, HT(ASCP)QIHC
IHCtech
12635 Montview Blvd. #215
Aurora, CO 80045
720-859-4060
fax 720-859-4110
pruegg@ihctech.net
www.ihctech.net
www.ihcrg.org    
 


-----Original Message-----
From: histonet-bounces@lists.utsouthwestern.edu
[mailto:histonet-bounces@lists.utsouthwestern.edu] On Behalf Of Amos Brooks
Sent: Saturday, October 25, 2008 10:19 AM
To: johnsobr@zgi.com; histonet@lists.utsouthwestern.edu
Subject: [Histonet] Human anti Human

Hi,
   I should start by saying: I have not done this, but this is the way I
would approach this problem if I were to encounter it.
   First I assume you have an human antibody raised in humans right? So, you
need an antibody that will detect the human antibody like "Anti-Human IgG
(-chain specific) antibody produced in rabbit" or "Monoclonal Anti-Human
IgG (-chain specific) antibody produced in mouse". (As seen here:
http://www.sigmaaldrich.com/life-science/cell-biology/antibodies/antibody-pr
oducts.htm?TablePage=14574992.
Then you can detect the Rabbit or Mouse secondary with your usual anti
Rabbit or Mouse detection. (I would use a polymer just to minimize biotin
background.)
   If anyone sees any holes in that line of approach, I'd love to hear other
ideas. It's an interesting problem.

Best of luck,
Amos Brooks


 Message: 22
Date: Fri, 24 Oct 2008 09:57:24 -0700
From: "BJON (Brian Johnson)" 
Subject: [Histonet] Human anti Human
To: histonet@lists.utsouthwestern.edu
Message-ID: <746E68D5CBB205409B12EC32A61EE4010A2FE8E7@ned.zgi.com>
Content-Type: text/plain; charset=us-ascii

Hello

I am wondering if anyone has found, and or used, an IHC polymer kit for
a human anti human monoclonal antibody.  Beyond the polymer kit, does
anyone have any tips for using a human anti human antibody for IHC
without biotinylating it?

Thank you.

Brian Johnson


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