Re: conjugation vs. sandwich

Florence Harrod (
31 Mar 1997 10:41:45 -0800

Reply to: RE>conjugation vs. sandwich
I would like to clarify a couple of the points made by Kevin Holmes (reproduced
below) regarding 2nd-step antibodies:
1) FITC anti-rat Ig 2nd-step antibodies which do not cross-react with mouse Ig
are EASY to find. Several companies supply FITC conjugates of polyclonal mouse
anti-rat Ig 2nd-step antibody, which do not cross-react with mouse Ig (these
tend to be expensive). The other alternative, which is widely available and
generally less expensive, is a polyclonal anti-rat Ig 2nd-step made in donkey,
goat, or rabbit and which has been adsorbed with mouse Ig. The same is true for
anti-mouse Ig 2nd-step antibodies: FITC conjugates which do not cross-react with
Igs of other species can be easily obtained from reputable commercial suppliers.

The more difficult issue is when you are using first-step antibodies which were
made in the same species as the cells you want to stain, for example, a mouse
anti-mouse cell surface monoclonal antibody. Then you should not use a
polyclonal anti-mouse Ig 2nd step, unless you are absolutely sure that the cells
you are staining do not have any cell-surface Ig. To get around this problem,
an anti-IgG subclass-specific 2nd step can be used. You must know the isotype
of your first-step antibody, then obtain a second-step antibody to match it.
FITC-conjugated Isotype-specific 2nd-step antibodies (adsorbed polyclonal sera
or monoclonal anti-Ig antibodies) are available from several commercial
2) Isotype-specific 2nd-step antibodies can be effectively used to avoid
cross-reactions between your second-step antibody and the various first-step
antibodies in multicolor experiments.
Any of the suppliers of the fluorochrome-conjugated 2nd-step antibodies should
be able to help you choose the appropriate reagents for your experiments.
If a second-step antibody simply will not work in a particular situation, there
are several companies which will perform custom conjugations of your antibody to
a variety of fluorochromes.
Here at PharMingen, we have alot of experience using 2nd-step antibodies with
cells of a variety of species and in multicolor applications. We also conjugate
customers' antibodies on a special-order basis. If you need advice for your
particular situation, you may contact us through our Web Site at, or call us from the US and Canada at 1-800-825-5832.
I hope this information is helpful to some of you!
Florence Harrod, Ph.D.
Manager, Research Immunocytometry Products

Date: 3/28/97 6:20 PM
To: cyto-inbox
From: Kevin Holmes

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
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


From: Dave Novo[]
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
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,

Deep thought of the day:
If the local 7-11 is open 24 hours a day - why are there locks on the

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