conjugation vs. sandwich

Kevin Holmes (KHOLMES@atlas.niaid.nih.gov)
Fri, 28 Mar 1997 16:26:07 -0500

The easiest way to go is using FITC-anti-IgG. This works very well,
usually gives some amplification of signal, and there are plenty of
commercially available anti-Ig's. However, dependent upon the cells
you are using or the experiment, there are several important
considerations.
1) If you are looking at cells that are or contain B lymphocytes, you
need to make sure that your sandwich antibody is not recognizing the
surface Ig on the B cells. So if you are looking at mouse spleen
cells and you use a anti-rat IgG-FITC, then you have to make sure that
it is specific for rat Ig and not mouse. This is difficult to obtain,
unless you use a monoclonal mouse anti-rat.

2) Be very careful using sandwich antibodies when you are doing
multicolor flow cytometry. Often the other antibody you are using may
be of the same strain as the first and you will get cross reactions.
Blocking with unlabeled antibodies may only partially alleviate those
cross reactions.

If the above issues are difficult to solve in your particular
experimental situation, then you can label the antibody yourself, but
again there are issues to be aware of:
1) You need to determine whether a stabilizing protein has been added
to the antibody you bought. If so, it will interfere with labeling
and you cannot do the conjugation, unless the antibody is repurified.

2) Labeling small amounts of antibody can be done, but you need to
use microdialysis chambers (Pierce sells these) in order to perform
the dialysis steps, or you will loose too much protein. Also, it is
more critical to have the antibody at a higher concentrations, i.e.. 1
mg/ml, than to have a lot of protein.

Hope this helps.

Kevin L. Holmes, Ph.D.
Head, Flow cytometry Unit
Office of the Scientific Director
Bldg 7, Room 01
NIAID, NIH

Email: kholmes@atlas.niaid.nih.gov

----------
From: Dave Novo[SMTP:novod@muss.cis.mcmaster.ca]
Sent: Thursday, March 27, 1997 12:06AM
To: cyto-inbox
Subject: conjugation vs. sandwich

Hello everyone,

I have to buy an antibody for which it seems that there is no
company selling one pre-conjugated to a dye. I was wondering what
people
thought of the relative merits of
conjugating FITC to the antibody myself vs.
buying a generic anti IgG-FITC premade conjugate and using a sandwich
type technique.

What are the pro's and cons of the various ideas?

Thanks in advance,
Dave

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CD-ROM Vol 3 was produced by Monica M. Shively and other 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: (765)-494-0757; FAX(765) 494-0517; Web http://www.cyto.purdue.edu , EMAIL cdrom3@flowcyt.cyto.purdue.edu