steric hindrance?

Houston, Jim Jim.Houston at
Fri Sep 22 16:48:37 EST 2000

The question is whether closely bound reagents are absorbing light from
their neighbors ( there is a term for this)or are the antibodies competing
for sites.  I have seen cases of CD8 antibodies blocking CD3 sites.  To
solve the problem of competition you have to stain one antibody at a time.
You do not have to wash between staining.  In a three color stain you will
have to try the different combinations.
On the kappa, lambda 19 stain.  Try 19 in fitc since it is the common on
both kappa and lambda.  this will cut down on the PE/PE competition.

Jim Houston

-----Original Message-----
From: Tom McHugh [mailto:mchugh at]
Sent: Thursday, September 21, 2000 3:05 PM
To: cyto-inbox
Subject: steric hindrance?

With 3-color we see significant reductions in staining intensity in the
PE-Cy5 or PerCP-Cy5 signal (FL3) when there is also a strong PE signal. I
assume this is steric hindrance. The antibody combinations of "kappa FITC,
lambda PE and CD19 PE-Cy5 or PerCP-Cy5" and "CD10 FITC, CD34 PE and CD45
PE-Cy5 or PerCP-Cy5" show this reduction in the staining in FL3. We see this
from different vendors and with a high % of samples which have the right
antigen mix. Diluting the antibodies does not help eliminate this problem.
Are others having this problem? Any practical suggestions as to how to avoid

Thomas M. McHugh
Technical Director, Clinical Laboratories
UCSF Medical Center
mchugh at

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