Posts in Antibody Discussion Forum

Hi Ed,
Let me tell you some of our approaches:

1) Transduce/transfect murine fibroblast with the construction (TCR,
HLA/peptide), any other ECD). Immunize with those cells and screen against
those and parental mock. Do Dry cells ELISA for screening that pretty match the
FACS and is less work. In our hands dry cells are 99 % similar to FACS data,
and we can test 20 ELISA plates. Cells have to be coated in suspension in PBS
100k cells per well and dry on the hood. You can save the plates frozen until
use.  The background in serum titration is kind of high for parental mock, but
as soon as you see a difference you can proceed to fusion and  you will find
positive clones.

2) immunize with the TCR or complex receptor, or HLA/peptide. Don’t need KLH if
it is human and big. Screen against same protein biotinylated in an ELISA plate
coated with SV. This gives you a 3D aspect of the target. Anyway we screened
2,000 to get a couple.
I hope this helps!
Laura

Laura Bover, PhD
Professor
Director Monoclonal Antibody Core Facility
University of Texas M.D.Anderson Cancer Center
Associate member of The University of Texas Graduate School of Biomedical
Sciences at Houston
Member of the Clinical  Research Committee 2
Member of the Steering Committee of the Immunology Program GSBS
Immunology Department/ Genomics Medicine Department
7455 Fannin St Room 1SCR4.3015
Houston- TX 77054
T  713-563-3301 <tel:713-563-3301>
F 713-563-3276 <tel:713-563-3276>
<email obscured>

In order to continue obtaining Core support in a challenging environment, it is
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(see following text).  Thank you!
 “This work was supported in part by CCSG grant number P30 CA016672.”

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On 4/11/19, 12:45 PM, "Antibody Discussion Forum on behalf of Greenfield,
Edward A.,Ph.D." <email obscured> on behalf of
<email obscured>> wrote:

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    Hi Frances -  Thanks for the advice.  I agree it is exactly like making TCR
mAb.  The PI is reluctant to try new things.  I suggested the subtractive
approach and cDNA but they are too far off what he is used to doing.  I was
thinking of immunizing with cells expressing the intact receptor.  I am trying
to get him to change the screening assay from FACS to a blocking assay, which
may be more reflective of the antibody titer we are looking for.  Ed.

    -----Original Message-----
    From: Antibody Discussion Forum <email obscured>> On Behalf Of
Weis-Garcia, Frances/Sloan Kettering Institute
    Sent: Thursday, April 11, 2019 1:30 PM
    To: <email obscured>
    Subject: Re: [ABRF Ab Forum] Novel heterodimer receptor mAb

            External Email - Use Caution

    I think you are playing numbers game ... if the mouse contains a specific
antibody at all.  I view it like making a TCR specific antibody, which is not
trivial but can be found.  In the past we tried with purified protein and
failed after 4 attempts / strategies.

    This year we just succeeded against another TCR.  The mice were immunized
with primary DCs engineered to express the target TCR.  If you cannot do that,
might want to try genetic immunization so the only thing that is different is
the heterodimer.  I have been told by people who used a gene gun approach to
drive a T cell response that the endogenous DCs express the target.  We have
genetically good humoral responses with both the gene gun as well as high
pressure tail vein injection (inject 2 mls plasmid in Ringers in 5 - 10
seconds) techniques ... so I would try this.

    Have you tried subtractive immunization?  Subtracting with cells expressing
one or the other and then driving the response you want with cells expressing
both.

    If you try either of these approaches, it would be great if you could share
with group.

    Frances

    -----Original Message-----
    From: Antibody Discussion Forum <email obscured>> on behalf of
Edward Greenfield <email obscured>>
    Reply-To: <email obscured>>
    Date: Thursday, April 11, 2019 at 1:06 PM
    To: <email obscured>" <email obscured>>
    Subject: [ABRF Ab Forum] Novel heterodimer receptor mAb

        Has anyone had any experience making antibodies specific for a
heterodimeric receptors?  The target receptor is composed of 2 different
chains,  It is a novel receptor so antibodies are not available to either chain
individually.  The desired antibody needs to be specific for the complete
receptor and not recognize either chain individually.  We have immunized double
knock-out mice (for both chains) and have been able to get a titer of 1:8000
but cannot separate the individual responses from the intact receptor response.
Anyone have any suggestions for immunizing or screening?  Ed.


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I think you are playing numbers game ... if the mouse contains a specific
antibody at all.  I view it like making a TCR specific antibody, which is not
trivial but can be found.  In the past we tried with purified protein and
failed after 4 attempts / strategies.

This year we just succeeded against another TCR.  The mice were immunized with
primary DCs engineered to express the target TCR.  If you cannot do that, might
want to try genetic immunization so the only thing that is different is the
heterodimer.  I have been told by people who used a gene gun approach to drive
a T cell response that the endogenous DCs express the target.  We have
genetically good humoral responses with both the gene gun as well as high
pressure tail vein injection (inject 2 mls plasmid in Ringers in 5 - 10
seconds) techniques ... so I would try this.

Have you tried subtractive immunization?  Subtracting with cells expressing one
or the other and then driving the response you want with cells expressing both.

If you try either of these approaches, it would be great if you could share
with group.

Frances

-----Original Message-----
From: Antibody Discussion Forum <email obscured>> on behalf of Edward
Greenfield <email obscured>>
Reply-To: <email obscured>>
Date: Thursday, April 11, 2019 at 1:06 PM
To: <email obscured>" <email obscured>>
Subject: [ABRF Ab Forum] Novel heterodimer receptor mAb

    Has anyone had any experience making antibodies specific for a
heterodimeric receptors?  The target receptor is composed of 2 different
chains,  It is a novel receptor so antibodies are not available to either chain
individually.  The desired antibody needs to be specific for the complete
receptor and not recognize either chain individually.  We have immunized double
knock-out mice (for both chains) and have been able to get a titer of 1:8000
but cannot separate the individual responses from the intact receptor response.
Anyone have any suggestions for immunizing or screening?  Ed.


    The information in this e-mail is intended only for the person to whom it
is
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e-mail
    contains patient information, please contact the Partners Compliance
HelpLine at
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Thanks ... I am building my referral list!

-----Original Message-----
From: Antibody Discussion Forum <email obscured>> on behalf of Robyn
Cassan <email obscured>>
Reply-To: <email obscured>>
Date: Wednesday, April 10, 2019 at 12:43 PM
To: <email obscured>>
Subject: Re: [ABRF Ab Forum] Sequencing the HC from a mouse hybridoma

    I have had success with sequencing hybridomas with Absolute Antibodies (NGS
sequencing) and also Lake Pharma (5'RACE). The majority of our hybridomas are
generated with Sp2 myelomas as the fusion partner.
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Thanks so very much!

-----Original Message-----
From: Antibody Discussion Forum <email obscured>> on behalf of Mary Ann
Pohl <email obscured>>
Reply-To: <email obscured>>
Date: Wednesday, April 10, 2019 at 12:39 PM
To: <email obscured>" <email obscured>>
Subject: [ABRF Ab Forum] Sequencing the HC from a mouse hybridoma

    Hi Frances,

    Yes this happens often and has been documented in the literature. It's not
that the myeloma HC has paired with an antigen specific LC, rather that the
hybridoma cells can express a non-productive variable gene transcript from the
fusion partner. So when you do PCR, that non-expressing allele can overwhelm
the DNA population in the PCR amplicon and the actual HC encoding for the
antibody is not picked up when sequenced because it is in such low abundance.

    One way to overcome this issue is to do N-terminal amino acid sequencing of
the antibody, then based on that design a specific primer to amplify your
variable gene. Lake Pharma has this capability.

    Another option is to try 5'RACE as opposed to traditional RT-PCR which may
give more specific amplification.

    Mary Ann


    --
    Mary Ann Pohl, PhD
    Associate Director, Antibody Generation
    Tri-Institutional Therapeutics Discovery Institute
    408 E. 69th St. Lab 1925
    New York, NY 10065
    646.888.3849



    On 4/10/19, 12:10 PM, "Antibody Discussion Forum on behalf of Frances
Weis-Garcia" <email obscured> on behalf of
<email obscured>> wrote:

        Dear Experts ...

        An MSKCC researcher sent a hybridoma to GenScript for sequencing. The
only heavy chain they were able to sequence was the myeloma heavy chain
identified.  I do not know the fusion partner, but am hunting now.

        Does this happen with some relative frequency?
        I find it statistically impossible that the HC from the myeloma would
pair off with a B cell LC to make an antigen specific antibody.

        Is there a different company you would recommend?
        Syd Labs has gotten sequences for hybridomas other researchers have
sent.

        Thanks for any advice you have.

        Frances

        P.S. - If you have some words of wisdom to share, would you please
reply to the Antibody Discussion Forum <email obscured>> so we can all
be more well rounded experts ;)
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Boy the antibody engineering questions are rolling in today ..

A researcher at the Virginia Commonwealth University School of Medicine would
like to contract out the sequencing of the VH and VL from a hybridoma and the
subsequent  construction of an scFv a vector.

As always ... thanks.

I have had success with sequencing hybridomas with Absolute Antibodies (NGS
sequencing) and also Lake Pharma (5'RACE). The majority of our hybridomas are
generated with Sp2 myelomas as the fusion partner.

Hi Frances,
If the fusion partner is Sp2/0 , there is an aberrant light chain that makes
difficult the cloning and sequencing.
We use to send our users to Vanderbilt for sequencing (when Robert was at the
Core). Good services good price.

Now we are doing it ourselves and it took us a while to design the correct
primers to avoid the aberrant.
No experience with other fusion partners.

Laura Bover, PhD
Professor
Director Monoclonal Antibody Core Facility
University of Texas M.D.Anderson Cancer Center
Associate member of The University of Texas Graduate School of Biomedical
Sciences at Houston
Member of the Clinical  Research Committee 2
Member of the Steering Committee of the Immunology Program GSBS
Immunology Department/ Genomics Medicine Department
7455 Fannin St Room 1SCR4.3015
Houston- TX 77054
T  713-563-3301 <tel:713-563-3301>
F 713-563-3276 <tel:713-563-3276>
<email obscured>

In order to continue obtaining Core support in a challenging environment, it is
crucial that our users acknowledge the following grants in all publications
(see following text).  Thank you!
 “This work was supported in part by CCSG grant number P30 CA016672.”

iLab-Solutions links:
https://mdanderson.ilabsolutions.com/service_center/show_external/3631
or:  https://mdanderson.ilabsolutions.com/account/login
If you do not already have an iLab account please create one prior to sample
submission


On 4/10/19, 11:10 AM, "Antibody Discussion Forum on behalf of Frances
Weis-Garcia" <email obscured> on behalf of
<email obscured>> wrote:

    WARNING: This email originated from outside of MD Anderson. Please validate
the sender's email address before clicking on links or attachments as they may
not be safe.

    Dear Experts ...

    An MSKCC researcher sent a hybridoma to GenScript for sequencing. The only
heavy chain they were able to sequence was the myeloma heavy chain identified.
I do not know the fusion partner, but am hunting now.

    Does this happen with some relative frequency?
    I find it statistically impossible that the HC from the myeloma would pair
off with a B cell LC to make an antigen specific antibody.

    Is there a different company you would recommend?
    Syd Labs has gotten sequences for hybridomas other researchers have sent.

    Thanks for any advice you have.

    Frances

    P.S. - If you have some words of wisdom to share, would you please reply to
the Antibody Discussion Forum <email obscured>> so we can all be more
well rounded experts ;)
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I think his previous one was called The Cell and they had a fake Cell Journal
cover with themselves on it.

I like The Checkpoints better.

-----Original Message-----
From: Antibody Discussion Forum <email obscured>> on behalf of Laura
Bover <email obscured>>
Reply-To: <email obscured>>
Date: Friday, March 29, 2019 at 5:20 PM
To: <email obscured>" <email obscured>>
Subject: Re: [ABRF Ab Forum] [EXT] Re: complex carbohydrate antigens

    Thanks! Have a great weekend all of you too!
    As I said I will pass more details next week. We obtained both IgM and IgG
but don’t remember the conjugation details.

    You know that Jim Allison has a music band named The Checkpoints.
    We may create something similar (any musicians? I could do vocals an
percussion!) and named it The MAbs, (LOL) and then play in our meetings!
    Rehearsals might be complicated though...
    😊
    Laura

    Laura Bover, PhD
    Professor
    Director Monoclonal Antibody Core Facility
    University of Texas M.D.Anderson Cancer Center
    Associate member of The University of Texas Graduate School of Biomedical
Sciences at Houston
    Member of the Clinical  Research Committee 2
    Member of the Steering Committee of the Immunology Program GSBS
    Immunology Department/ Genomics Medicine Department
    7455 Fannin St<x-apple-data-detectors://5/1> Room 1SCR4.3015
    Houston- TX 77054<x-apple-data-detectors://7/0>
    T  713-563-3301<tel:713-563-3301>
    F 713-563-3276<tel:713-563-3276>
    <email obscured><email obscured>>

    Sent from my iPhone

    On Mar 29, 2019, at 4:02 PM, Weis-Garcia, Frances/Sloan Kettering Institute
<email obscured><email obscured>>>
wrote:

    WARNING: This email originated from outside of MD Anderson. Please validate
the sender's email address before clicking on links or attachments as they may
not be safe.

    Boy I love this group.
    We may be a quite bunch but when one of us needs some tactical support we
band together ...

    Have a great weekend all.

    -----Original Message-----
    From: Antibody Discussion Forum
<email obscured><email obscured>>> on behalf of Edward
Greenfield
<email obscured><email obscured>>>
    Reply-To: <email obscured><email obscured>>>
    Date: Friday, March 29, 2019 at 5:00 PM
    To: <email obscured><email obscured>>"
<email obscured><email obscured>>>
    Subject: Re: [ABRF Ab Forum] complex carbohydrate antigens

       Hi David -

       I have limited experience but of the 3 projects, we got antibodies to
two carbohydrate targets.  Immunizations were done with material provided by
the investigator (purified carbohydrate linked to a small 8-mer peptide and
conjugated with KLH or OVA for alternating immunizations.  Conjugating on the
N-term. & C-term (individually) and boosting together helped.  Freund's
adjuvant was used and I doubled the amount of immunogen for immunization to 100
ug/mouse.  After repeated immunizations, we got a moderate response and the
fusion yielded mostly IgMs and a few IgG3's.

       Best,
       Ed.



       -----Original Message-----
       From: Antibody Discussion Forum
<email obscured><email obscured>>> On Behalf Of David Blum
       Sent: Friday, March 29, 2019 2:56 PM
       To: <email obscured><email obscured>>
       Subject: [ABRF Ab Forum] complex carbohydrate antigens

               External Email - Use Caution

       Anybody have experience generating murine mAbs against complex
carbohydrates and have any suggestions on best practices?

       David

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Thanks! Have a great weekend all of you too!
As I said I will pass more details next week. We obtained both IgM and IgG but
don’t remember the conjugation details.

You know that Jim Allison has a music band named The Checkpoints.
We may create something similar (any musicians? I could do vocals an
percussion!) and named it The MAbs, (LOL) and then play in our meetings!
Rehearsals might be complicated though...
😊
Laura

Laura Bover, PhD
Professor
Director Monoclonal Antibody Core Facility
University of Texas M.D.Anderson Cancer Center
Associate member of The University of Texas Graduate School of Biomedical
Sciences at Houston
Member of the Clinical  Research Committee 2
Member of the Steering Committee of the Immunology Program GSBS
Immunology Department/ Genomics Medicine Department
7455 Fannin St<x-apple-data-detectors://5/1> Room 1SCR4.3015
Houston- TX 77054<x-apple-data-detectors://7/0>
T  713-563-3301<tel:713-563-3301>
F 713-563-3276<tel:713-563-3276>
<email obscured><email obscured>>

Sent from my iPhone

On Mar 29, 2019, at 4:02 PM, Weis-Garcia, Frances/Sloan Kettering Institute
<email obscured><email obscured>>>
wrote:

WARNING: This email originated from outside of MD Anderson. Please validate the
sender's email address before clicking on links or attachments as they may not
be safe.

Boy I love this group.
We may be a quite bunch but when one of us needs some tactical support we band
together ...

Have a great weekend all.

-----Original Message-----
From: Antibody Discussion Forum
<email obscured><email obscured>>> on behalf of Edward
Greenfield
<email obscured><email obscured>>>
Reply-To: <email obscured><email obscured>>>
Date: Friday, March 29, 2019 at 5:00 PM
To: <email obscured><email obscured>>"
<email obscured><email obscured>>>
Subject: Re: [ABRF Ab Forum] complex carbohydrate antigens

   Hi David -

   I have limited experience but of the 3 projects, we got antibodies to two
carbohydrate targets.  Immunizations were done with material provided by the
investigator (purified carbohydrate linked to a small 8-mer peptide and
conjugated with KLH or OVA for alternating immunizations.  Conjugating on the
N-term. & C-term (individually) and boosting together helped.  Freund's
adjuvant was used and I doubled the amount of immunogen for immunization to 100
ug/mouse.  After repeated immunizations, we got a moderate response and the
fusion yielded mostly IgMs and a few IgG3's.

   Best,
   Ed.



   -----Original Message-----
   From: Antibody Discussion Forum
<email obscured><email obscured>>> On Behalf Of David Blum
   Sent: Friday, March 29, 2019 2:56 PM
   To: <email obscured><email obscured>>
   Subject: [ABRF Ab Forum] complex carbohydrate antigens

           External Email - Use Caution

   Anybody have experience generating murine mAbs against complex carbohydrates
and have any suggestions on best practices?

   David

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Hi David -

I have limited experience but of the 3 projects, we got antibodies to two
carbohydrate targets.  Immunizations were done with material provided by the
investigator (purified carbohydrate linked to a small 8-mer peptide and
conjugated with KLH or OVA for alternating immunizations.  Conjugating on the
N-term. & C-term (individually) and boosting together helped.  Freund's
adjuvant was used and I doubled the amount of immunogen for immunization to 100
ug/mouse.  After repeated immunizations, we got a moderate response and the
fusion yielded mostly IgMs and a few IgG3's.

Best,
Ed.



-----Original Message-----
From: Antibody Discussion Forum <email obscured>> On Behalf Of David
Blum
Sent: Friday, March 29, 2019 2:56 PM
To: <email obscured>
Subject: [ABRF Ab Forum] complex carbohydrate antigens

        External Email - Use Caution

Anybody have experience generating murine mAbs against complex carbohydrates
and have any suggestions on best practices?

David

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