Hi,
My name is Gloria Juan and I am part of the research group who has been
"successfully" using the monoclonal antibody specific for the
underphosphorylated form of the Rb protein, not only in the Leukemia paper,
also in several other publications: Exp. Cell Res, 239: 104-110,1998; Exp.
Cell res., 244: 83-92, 1998; Clin. Immunol. Newsletter, 18:89-94, 1998,
among others.
We were not aware that Pharmingen decided no to market the FITC-conjugated
antibody. In fact, we are in the process of patenting together (NYMC and
Pharmingen) this new approach. The antibody G99-549 is specific and you
could very well use it (purified or conjugated). Of course, there is a lost
of resolution but it is a matter of compromise: the use of conjugated
antibodies allows us to study simultaneously many more parameters and this
is a great feature of flow cytometry.
Tanks and good luck,
Gloria
-----Original Message-----
From: Kevin Waddick [mailto:waddi002@tc.umn.edu]
Sent: Thursday, July 15, 1999 11:26 AM
To: cyto-inbox
Subject: Underphosphorylated Rb detection by FCM
In a report appearing in Leukemia 12: 1241-1248, 1998, a monoclonal
antibody specific to the underphosphorylated form of the retinoblastoma
(Rb) protein was shown able to define by flow cytometry a population of
cells in the G0/1 compartment of the cell cycle. In the Materials and
Methods section, this was described as a FITC conjugate of the G99-549
MoAb clone obtained from PharMingen.
Has anyone else out there used this MoAb clone (or any other MoAb)
to specifically detect underphosphorylated pRb by flow cytometric
analysis?
I ask because when I contacted PharMingen I was informed that the
FITC-conjugate of this anti-Rb MoAb was a test batch provided to the
research group doing the study referenced above. PharMingen claims it
was unable to duplicate the results due to a high level of
nonspecific/background staining. Therefore, PharMingen decided not to
market the FITC-conjugate and does not list flow cytometry in their
catalog as one of the applications for this MoAb clone. However, just
because PharMingen doesn't make any claims for its performance in flow
cytometric analysis doesn't necessarily mean people have not
successfully used it for this purpose. That is why I am asking everyone
if they may have achieved decent flow cytometric results detecting
underphosphorylated pRb by indirect staining using the PharMingen
G99-549 MoAb clone (or by any other means).
Dr. Kevin G. Waddick
Hughes Institute
2657 Patton Road
St. Paul, MN 55113
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