Nora,

Normal serums have a lot more protein and antibodies than the antibody
you buy. Most commercial antibodies have been affinity purified, which
means that almost all that is left is the antibody of interest and some
proteins. If you are using monoclonal Fab fragments you should see no
background at all. Ascitic fluids will have polyclonal antibodies and
some proteins, but not as much as serum. So commercial preps are usually
very clean.

The control serum is intended to show non-specific staining (NSS) that
could be caused by the serum in the primary. If the pattern is totally
different it is not necessarily bad, especially if the pattern is
different. If the NSS is too heavy, however, you may want to check your
serum protein concentration (may be too high) and/or filter the serum to
take out complexes of protein that may have formed (use a .22 um
filter).

Another thing to do is use non-specific antibodies in the same form
(poly, mono, ascitic fluid) and from the same animal species as
negatives. This may give you a truer picture of NSS.

If you still have problems, review your blocking procedures.

Tim Morken, B.S., EMT(MSA), HTL(ASCP)
Infectious Disease Pathology
Centers for Disease Control
MS-G32
1600 Clifton Rd.
Atlanta, GA 30333
USA

email: tim9@cdc.gov
       timcdc@hotmail.com

FAX:  (404)639-3043


----Original Message Follows----
Date: Fri, 23 Oct 1998 14:52:13 -0700
From: Nora Fox <nefox@leland.Stanford.EDU>
Subject: negative controls
To: histonet@pathology.swmed.edu

Up until recently, I have used my primary Ab diluent as my negative
control.  All of the buzz a few weeks ago convinced me that it was a
good
idea to use sera from the animal the primary was made in.  Using the
control antibody at the same dilution as my primary I am now seeing
staining.  However, I see only what I would expect to see labelled with
my
primary Ab.  Does this mean that I have to futz around with my
conditions
until I see no labelling using my negative control antibody even though
I
see a total different staining pattern than that of my primary?

Thanks again!!!!

Nora

Nora Fox
Division of Orthopaedic Surgery
Stanford University
Edwards R144, MC 5341
300 Pasteur Drive
Stanford, CA 94305
phone: (650)725-2746
fax:  (650)723-6396






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