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 [EMAIL PROTECTED] www.ihctech.net www.ihcrg.org -----Original Message----- From: [EMAIL PROTECTED] [mailto:[EMAIL PROTECTED] Sent: Sunday, October 26, 2008 9:59 AM To: Patsy Ruegg; histonet@lists.utsouthwestern.edu; [EMAIL PROTECTED] 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 <[EMAIL PROTECTED]> Date: Sun, 26 Oct 2008 09:03:57 To: 'Amos Brooks'<[EMAIL PROTECTED]>; <[EMAIL PROTECTED]>; <histonet@lists.utsouthwestern.edu> 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 [EMAIL PROTECTED] www.ihctech.net www.ihcrg.org -----Original Message----- From: [EMAIL PROTECTED] [mailto:[EMAIL PROTECTED] On Behalf Of Amos Brooks Sent: Saturday, October 25, 2008 10:19 AM To: [EMAIL PROTECTED]; 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 (γ-chain specific) antibody produced in rabbit" or "Monoclonal Anti-Human IgG (γ-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)" <[EMAIL PROTECTED]> Subject: [Histonet] Human anti Human To: histonet@lists.utsouthwestern.edu Message-ID: <[EMAIL PROTECTED]> 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 _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet