Happy Wednesday everyone!
We have an electrofusion where half the antigen specific hybridomas we have
secrete both IgG and IgG base on 2 sandwich ELISAs:
* IgG ELISA
* Capture goat anti-mouse Fc gamma specific
* Detect goat anti-mouse F(Ab’)2
* IgM ELISA
* Capture goat anti-mouse IgM, mu specific
* Detect goat anti-mouse F(Ab’)2
The other half only secrete IgG.
The only thing I can think of the hybridomas have 2 B cells – one IgG and 1
IgM. We have not subcloned yet … but these cultures did come from a ClonePix
run.
Thoughts ???
Frances
Frances Weis-Garcia, Ph.D.
Head
Bi-Institutional Antibody and Bioresource Core Facility
(RRID: CR 017691)
Associate Laboratory Member
Immunology Program
Memorial Sloan Kettering Cancer Center
1275 York Avenue, New York, NY 10065
T 646.888.2354 C 516.967.0656
ABCF-MSKCC
website<https://www.mskcc.org/research-advantage/core-facilities/monoclonal-antibody-core-facility>
Adjunct Faculty
Center for Clinical and Translational Science
The Rockefeller University
1230 York Avenue, New York, NY 10065
T 212.327.7030
ABCF-RU website<https://www.rockefeller.edu/monoclonal/>
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Hi Frances,
I'm not sure what stage you're at with screening these hybridomas, but a
possibility could be that you're picking up on "contaminating" antibody from
antibody diffused from a different hybridoma in the methylcellulose in the
proximity of the chosen clone when the clones are picked. If this is the first
screen of these hybridomas after clone picking that might be the case. We've
found that with several passages of the clones the non-specific antibody signal
goes away. Are the IgGs and IgMs specific to the target with an isotype
specific secondary in an indirect ELISA?
Just a thought.
Thanks,
Kurt
Kurt Christensen
Lab Director
Protein and Monoclonal Antibody Production Core
Web Page:
https://www.bcm.edu/centers/cancer-center/research/shared-resources/antibody-based-proteomics/recombinant-protein-monoclonal-antibody
iLab Requests:
https://bcm.corefacilities.org/account/login
Office: 713-798-2325
Lab:713-798-2327
Email: <email obscured>
Baylor College of Medicine
Margaret M. Alkek Building for Biomedical Research, Room R551E, Mail Stop
BCM929
1 Baylor Plaza
Houston, Texas 77030
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-----Original Message-----
From: Antibody Discussion Forum <email obscured>> On Behalf Of
Weis-Garcia, Frances/Sloan Kettering Institute
Sent: Tuesday, July 27, 2021 10:32 PM
To: ABRF Antibody Discussion Group <email obscured>>
Subject: [ABRF Ab Forum] Hybridoma sups scoring positive for IgG and IgM
Happy Wednesday everyone!
We have an electrofusion where half the antigen specific hybridomas we have
secrete both IgG and IgG base on 2 sandwich ELISAs:
* IgG ELISA
* Capture goat anti-mouse Fc gamma specific
* Detect goat anti-mouse F(Ab’)2
* IgM ELISA
* Capture goat anti-mouse IgM, mu specific
* Detect goat anti-mouse F(Ab’)2
The other half only secrete IgG.
The only thing I can think of the hybridomas have 2 B cells – one IgG and 1
IgM. We have not subcloned yet … but these cultures did come from a ClonePix
run.
Thoughts ???
Frances
Frances Weis-Garcia, Ph.D.
Head
Bi-Institutional Antibody and Bioresource Core Facility
(RRID: CR 017691)
Associate Laboratory Member
Immunology Program
Memorial Sloan Kettering Cancer Center
1275 York Avenue, New York, NY 10065
T 646.888.2354 C 516.967.0656
ABCF-MSKCC
website<https://urldefense.proofpoint.com/v2/url?u=https-3A__www.mskcc.org_research-2Dadvantage_core-2Dfacilities_monoclonal-2Dantibody-2Dcore-2Dfacility&d=DwIFaQ&c=ZQs-KZ8oxEw0p81sqgiaRA&r=_dqqB8TU9vN7Hm15OCeZVQ&m=Cq-Nv9S-ZYzO9JkVdbbaJW231UrAb7laQwd2Y5diEXQ&s=-g9lJvrqUJEPum7Vtra94yqlTuEf-HIOAf7jJCf5WGg&e=
>
Adjunct Faculty
Center for Clinical and Translational Science
The Rockefeller University
1230 York Avenue, New York, NY 10065
T 212.327.7030
ABCF-RU
website<https://urldefense.proofpoint.com/v2/url?u=https-3A__www.rockefeller.edu_monoclonal_&d=DwIFaQ&c=ZQs-KZ8oxEw0p81sqgiaRA&r=_dqqB8TU9vN7Hm15OCeZVQ&m=Cq-Nv9S-ZYzO9JkVdbbaJW231UrAb7laQwd2Y5diEXQ&s=L04n0wmp7XTkxI7aCThBxnecYhaiODn1-OBRmgje-OQ&e=
>
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other use of this communication or any of its attachments is strictly
prohibited. If you have received this communication in error, please notify
the sender immediately by replying to this message and deleting this message,
any attachments, and all copies and backups from your computer.
=====================================================================
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Memorial Sloan Kettering Cancer Center may be privileged, confidential,
and protected from disclosure under applicable law. If the reader of
this message is not the intended recipient, or an employee or agent
responsible for delivering this message to the intended recipient,
you are hereby notified that any reading, dissemination, distribution,
copying, or other use of this communication or any of its attachments
is strictly prohibited. If you have received this communication in
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