Re: NCAM/CD56

Florence Harrod (fharrod@pharmingen.com)
13 Aug 1996 15:53:43 -0700

Reply to: RE>NCAM/CD56
Dear Jim, and other members of this forum who use 2nd step antibodies:
I'll leave the main questions to others in this forum who are more expert on the
antibody titration and blocking issues.
Regarding the naming of the anti-IgG antibody: Usually an antibody is named for
the immunogen that was used in preparing that antibody, regardless of the
cross-reactivity. So, if the goats were injected with mouse IgG, the antibody
is properly named "anti-mouse IgG". This name is not at all misleading. Since
IgG contains light chains, and also since there are probably some epitopes on
IgG heavy chains which are shared with other Ig heavy chains, it is likely that
this polyclonal antibody will cross-react with other immunoglobulin isotypes.
Similarly, the proper name for the anti-NCAM antibody that you're using would be
"anti-mouse NCAM" because mouse NCAM was the antigen. The name, anti-mouse IgG
or anti-mouse NCAM is not meant to imply or deny cross-reactivity with other
antigens or other species. That is why the supplier often adds relevant
information, such as "H & L chain specific" or "gamma chain specific" for
anti-IgG antibodies.
Theoretically, an anti-mouse IgG which is H & L chain specific would react with
all mouse immunoglobulins -- A, E, D, M, and the various G isotypes. In
practice, the reactivity with IgG may be stronger than the reactivity with one
or more of the other isotypes. In our labs, we have found that anti-mouse IgG
(H & L chain specific) may not adequately detect IgM antibodies against antigens
which are expressed at low levels. Therefore, we routinely try out anti-mouse
IgG and anti-mouse IgM as a second step for IgM primaries to determine which
will work best for us. Researchers who use antibodies as tools in their work
would be wise to learn about immunoglobulin structure, isotypes, and epitopes to
avoid possible problems in their staining protocols which are due to the
inherent characteristics of the reagents. There is a wide variety of very fine
second step antibodies available from several companies, and those companies
make every reasonable effort to supply the customer with the information needed
for choose the correct reagent for their use. But the companies must assume
that the customer has a basic understanding of the tools that he/she is planning
to use.
Jim, thanks for asking the same questions about second-step antibodies that we
often receive from our own R&D staff and from our customers, so that I was given
a chance to attempt to clarify some common misconceptions.
Florence Harrod
Manager, Immunology Research Products
PharMingen

--------------------------------------
Date: 8/13/96 12:36 PM
To: cyto-inbox
From: Jim Zanghi

Here's a problem that has been puzzling me, and after going through
Carleton Stewart's lecture at the recent Clinical Course in Flow Cytometry,
I'm even more weary. It also raises a very general question about purified
antibodies:

I am trying to use flow cytometry to measure a cell surface antigen (NCAM
or CD56, which belongs to the Ig superfamily) on a CHO (hamster) cell line.
The primary antibody is purified from a hybridoma (Immunotech), and it is
a rat IgG2a raised against mouse NCAM. In general, there is cross
reactivity between chicken, mouse, and rat NCAM, so I have assumed that
this will also bind CHO NCAM. My secondary antibody is goat F(ab')2
anti-rat IgG (H+L) FITC conjugate (Caltag), affinity purified by column
chromatography and adsorbed to remove cross reactivity to mouse
immunoglobulins". I'm getting 100% reactivity, as expected, but when I
titer it, the reactivity keeps going up and up without ever plateauing as I
increase antibody concentration. I have not been blocking my cells, and I
have been carrying out the incubations in PBS containing 1% BSA, which I've
now been told is not a sufficient blocking reagent.

Any recommendations? Is this a bad antibody or am I getting some sort of
non-specific binding that I can avoid if I block properly? My isotype
control doesn't react, but I'm told not to trust isotype controls.

Also, it seems that most, if not all, of the flow cytometry references I've
read in the literature do not block with non-specific immunoglobulins and
use only 1% BSA in PBS. Are they all wrong, or is Dr. Stewart being overly
cautious, or is it implied that they do this?

--


This may be a side issue:  What does "anti-rat IgG (H+L)" mean?  Will this
bind to _all_ rat immunoglobulins because it reacts to the light chain?
That's the impression I get from Carleton and Sigrid Stewart's comments on
page 43 of "Cell Preparation for the Identification of Leukocytes" (Ch. 3,
Methods in Cell Biology, v.41, 1994), quoted here:


"A typical second antibody might be labeled "FITC conjugated goat F(ab')2
anti-mouse IgG antibody (gamma- and light-chain specific, purified by
affinity chromatography)."  This label contains considerable
information...[next paragraph]...  Since this goat polyclonal second
antibody was prepared using a mouse IgG affinity column, this preparation
contains antibodies that are specific for the heavy chain of mouse IgG.
Because all isotypes found in serum...have light chains, they will also
bind this second antibody because it is also "light-chain specific".
Therefore, this polyclonal second reagent is not specific for mouse IgG at
all.  In order for a second antibody to be specific for IgG is must have no
light-chain activity.  If the antibody were only heavy-chain specific the
label would read 'gamma specific'. "


Are the antibody descriptions misleading information, or is it assumed that
everyone already knows this?  I did not, and I thought I was buying
antibody that reacted only to IgG, although I don't think it really matters
in my case.   On the otherhand, if it read  "goat anti-rat Ig", you
wouldn't know for certain if it reacts to IgG, so I suppose what is written
is appropriate.


Thanks,
Jim


(PS:  Thanks for all the help & tips from my first post entitled "BD
software"!  I clarified my email signature file since it apparently was
misinterpreted by some as arrogance on my part.  Actually, it is the title
of a free jazz tune that is very inspiring to me.  Sorry I clarified my email signature file since it apparently was
misiabout any
confusion.)












...truth is marching in


                        - Albert Ayler, a free jazz musician






------------------ RFC822 Header Follows ------------------
>From flowcyt.cyto.purdue.edu!owner-cyto-sendout Tue Aug 13 11:50:51 1996 remote
from donews
Received: from flowcyt.cyto.purdue.edu by donews.cts.com with smtp
	(Smail3.1.29.1 #5) id m0uqOYD-0000MNC; Tue, 13 Aug 96 11:50 PDT
Received: by flowcyt.cyto.purdue.edu (940816.SGI.8.6.9/930416.SGI.AUTO)
	for cyto-sendout id KAA24868; Tue, 13 Aug 1996 10:30:01 -0500
Received: from merle.acns.nwu.edu by flowcyt.cyto.purdue.edu via ESMTP
(940816.SGI.8.6.9/930416.SGI.AUTO)
	for <cytometry@flowcyt.cyto.purdue.edu> id UAA22082; Mon, 12 Aug 1996 20:02:56
-0500
Received: from [129.105.205.221] (e234mac221.chem-eng.nwu.edu) by
merle.acns.nwu.edu with SMTP
	(1.40.112.4/16.2) id AA220537986; Mon, 12 Aug 1996 19:59:46 -0500
Message-Id: <v01530501ae357d1fe68c@[129.105.205.221]>
Mime-Version: 1.0
Content-Type: text/plain; charset="us-ascii"
Date: Mon, 12 Aug 1996 20:04:01 -0600
To: cyto-inbox
From: zanghi@merle.acns.nwu.edu (Jim Zanghi)
Subject: NCAM/CD56

Home Page Table of Contents Sponsors Web Sites
CD ROM Vol 2 was produced by staff at the Purdue University Cytometry Laboratories and distributed free of charge as an educational service to the cytometry community. If you have any comments please direct them to Dr. J. Paul Robinson, Professor & Director, PUCL, Purdue University, West Lafayette, IN 47907. Phone:(317) 494-0757; FAX (317) 494-0517; Web http://www.cyto.purdue.edu EMAIL robinson@flowcyt.cyto.purdue.edu