Posts in Antibody Discussion Forum

Stability comes from the drug selection post transfection not the transfection
method, which addresses efficiency of getting the DNA into the cells.  You can
use the same transfection protocol, just put a drug marker in the vector and
select for those that grow in the drug.

For me the key is to find the best expressing clones ... that can be done in
the ClonePix.

There are DHFR deficient /CHO cells (made at Columbia many moons ago) that when
you use a vector with DHFR and culture the cells in increasing concentrations
of methotrexate, you select for ones that have amplified the that region(s)
where the vector has inserted ... getting more copies in an active area of the
chromatin and increasing expression.

There are a few protein production experts in this discussion forum who will
likely be able to contribute more to this discussion than I.  I look forward to
listening and learning.

-----Original Message-----
From: Antibody Discussion Forum <email obscured>> on behalf of Laura
Bover <email obscured>>
Reply-To: <email obscured>>
Date: Tuesday, July 16, 2019 at 8:22 PM
To: <email obscured>" <email obscured>>
Subject: [ABRF Ab Forum] ABRF Ab Forum

    Hi guys, we were using the Invivogen kit to make mouse human chimeras and
so far so good, but it only give us the transient transfection and an amount
that need to be larger for in vivo experiments.
    Any experience with the kit and making stable transfectants? Any
recommendation? We are going to contact Invivogen anyway, but you may know
better.
    Thanks!

    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>

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    On 7/16/19, 3:43 PM, "Antibody Discussion Forum on behalf of Weis-Garcia,
Frances/Sloan Kettering Institute" <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.

        Ugh ... no fun.



        Many moons ago we always purified all our MAb with Anion Exchange
Chromatography (HiTrap Q) from bioreactor supernatant (CELLine) and if we could
not get it to 95% purity we would then polish it off with Ceramic
Hydroxyapatite.  This approach was taken to avoid denaturing the antibody.



        If you need more specifics, I’ll need to dig them up.

        -----Original Message-----

        From: Antibody Discussion Forum <email obscured>> on behalf of
Scott Brown <email obscured>>

        Reply-To: <email obscured>>

        Date: Tuesday, July 16, 2019 at 4:16 PM

        To: <email obscured>" <email obscured>>

        Subject: [ABRF Ab Forum] Purification of IgM



            Good afternoon all,

            I have run into a bit of a snag trying to purify IgM antibodies
from hyrbridoma supernatant.  I don't typically select the IgM clones, but one
of my labs is interested in studying the IgM response, so I need to purify
them.  I've tried GE Lifesciences  HiTrap IgM Purification HP.  While I
purified it, I lost activity.  I haven't tried size exclusion chromatography,
as I wanted to purify from larger volumes of supernatants.  If anyone purifies
IgM routinely and can send me some suggestions, I would be most appreciative.

            Thanks,



            Scott



            Scott A. Brown, Ph.D.

            262 Danny Thomas Place

            Immunologic Reagent Facility

            Department of Immunology

            St. Jude Children's Res. Hosp.

            Memphis, TN 38105

            w. (901) 595-4067

            F. (901) 595-3107

            c. (901) 674-9960





            ________________________________



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GE will have a protocol for the HiTrapQ (Pretty sure we did 5% steps of NaCl
and most came out between 15 and 30% salt) ... it is the CHT do not remember
well at all.

-----Original Message-----
From: Antibody Discussion Forum <email obscured>> on behalf of Scott
Brown <email obscured>>
Reply-To: <email obscured>>
Date: Tuesday, July 16, 2019 at 4:54 PM
To: <email obscured>" <email obscured>>
Subject: Re: [ABRF Ab Forum] Purification of IgM

    Ugh is right!  IgGs are so much fun!   I will see if I can find a resource!
    Many thanks Frances!
    Scott

    -----Original Message-----
    From: Antibody Discussion Forum <email obscured>> On Behalf Of
Weis-Garcia, Frances/Sloan Kettering Institute
    Sent: Tuesday, July 16, 2019 3:43 PM
    To: <email obscured>
    Subject: Re: [ABRF Ab Forum] Purification of IgM

    Caution: External Sender


    Ugh ... no fun.



    Many moons ago we always purified all our MAb with Anion Exchange
Chromatography (HiTrap Q) from bioreactor supernatant (CELLine) and if we could
not get it to 95% purity we would then polish it off with Ceramic
Hydroxyapatite.  This approach was taken to avoid denaturing the antibody.



    If you need more specifics, I’ll need to dig them up.

    -----Original Message-----

    From: Antibody Discussion Forum <email obscured>> on behalf of Scott
Brown <email obscured>>

    Reply-To: <email obscured>>

    Date: Tuesday, July 16, 2019 at 4:16 PM

    To: <email obscured>" <email obscured>>

    Subject: [ABRF Ab Forum] Purification of IgM



        Good afternoon all,

        I have run into a bit of a snag trying to purify IgM antibodies from
hyrbridoma supernatant.  I don't typically select the IgM clones, but one of my
labs is interested in studying the IgM response, so I need to purify them.
I've tried GE Lifesciences  HiTrap IgM Purification HP.  While I purified it, I
lost activity.  I haven't tried size exclusion chromatography, as I wanted to
purify from larger volumes of supernatants.  If anyone purifies IgM routinely
and can send me some suggestions, I would be most appreciative.

        Thanks,



        Scott



        Scott A. Brown, Ph.D.

        262 Danny Thomas Place

        Immunologic Reagent Facility

        Department of Immunology

        St. Jude Children's Res. Hosp.

        Memphis, TN 38105

        w. (901) 595-4067

        F. (901) 595-3107

        c. (901) 674-9960





        ________________________________



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Ugh ... no fun.



Many moons ago we always purified all our MAb with Anion Exchange
Chromatography (HiTrap Q) from bioreactor supernatant (CELLine) and if we could
not get it to 95% purity we would then polish it off with Ceramic
Hydroxyapatite.  This approach was taken to avoid denaturing the antibody.



If you need more specifics, I’ll need to dig them up.

-----Original Message-----

From: Antibody Discussion Forum <email obscured>> on behalf of Scott
Brown <email obscured>>

Reply-To: <email obscured>>

Date: Tuesday, July 16, 2019 at 4:16 PM

To: <email obscured>" <email obscured>>

Subject: [ABRF Ab Forum] Purification of IgM



    Good afternoon all,

    I have run into a bit of a snag trying to purify IgM antibodies from
hyrbridoma supernatant.  I don't typically select the IgM clones, but one of my
labs is interested in studying the IgM response, so I need to purify them.
I've tried GE Lifesciences  HiTrap IgM Purification HP.  While I purified it, I
lost activity.  I haven't tried size exclusion chromatography, as I wanted to
purify from larger volumes of supernatants.  If anyone purifies IgM routinely
and can send me some suggestions, I would be most appreciative.

    Thanks,



    Scott



    Scott A. Brown, Ph.D.

    262 Danny Thomas Place

    Immunologic Reagent Facility

    Department of Immunology

    St. Jude Children's Res. Hosp.

    Memphis, TN 38105

    w. (901) 595-4067

    F. (901) 595-3107

    c. (901) 674-9960





    ________________________________



    Email Disclaimer: www.stjude.org/emaildisclaimer

    Consultation Disclaimer: www.stjude.org/consultationdisclaimer



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Hi Angel,
We do this all the time. We inject sometimes 3, sometimes 2 peptides KLH. When
we perform serum titration we test the 3 peptides separated, KLH and peptide
alone.
Interestingly, and of course depending on the project and peptides similarity
(some are very different) we obtain sometimes response against one or two and
not the third.
Sometimes we boost with peptides alone (2 last injections) because the response
to KLH if much higher since you have the three KLH.
Doing screening with peptides BSA is not useful for us because BSA always give
us high background.
Let me know if you have more questions.
Thanks!
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 Jun 28, 2019, at 6:24 PM, Greenfield, Edward A.,Ph.D.
<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
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Hi Angel -

We have immunized a library of peptides for obtaining a methyl group or
sulfhydryl group specific mAb.  In addition to using multiple peptides, we also
conjugated KLH to the N-term. of one set of the peptides and the C-term of a
second set of the peptides.  This was helpful as well.

Best,
Ed.

-----Original Message-----
From: Antibody Discussion Forum
<email obscured><email obscured>>> On Behalf Of
angel.sampson
Sent: Friday, June 28, 2019 5:29 PM
To: <email obscured><email obscured>>
Subject: [ABRF Ab Forum] Question about conjugating peptide pools to carrier
proteins

       External Email - Use Caution

Dear ARG Members,

I have a question about using peptide pools conjugated to carrier protein for
immunization. I have an investigator who wants to produce a monoclonal antibody
targeting a specific histidine residue in/near the active site of a particular
membrane protein to block enzymatic activity.

The PI identified 3 peptides that were predicted to be immunogenic.  Rather
than pick one, I suggested using all three peptides. Also, I suggested that
equimolar amounts of the three peptides could be conjugated to the same carrier
molecule, since the peptides are identical except 3-4 amino acid differences. I
spoke with a technical rep from Thermo Scientific, and this person assured me
that this is a common practice. However, I have only been able to find one
example of a somewhat similar approach in the literature
(https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5015836/). Is this approach
commonly used? Do you know of a publication that has used peptide pools to
produce site directed antibodies?

Thank you for your advice!

Angel


Edith “Angel” Sampson, Ph.D.
Monoclonal Antibody Scientific Director

UF ICBR
2033 Mowry Road, Gainesville, FL 32610
http://Biotech.ufl.edu | Tel: 352.273.8039| Fax: 352.273.8070

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Dear ARG Members,

I have a question about using peptide pools conjugated to carrier protein for
immunization. I have an investigator who wants to produce a monoclonal antibody
targeting a specific histidine residue in/near the active site of a particular
membrane protein to block enzymatic activity.

The PI identified 3 peptides that were predicted to be immunogenic.  Rather
than pick one, I suggested using all three peptides. Also, I suggested that
equimolar amounts of the three peptides could be conjugated to the same carrier
molecule, since the peptides are identical except 3-4 amino acid differences. I
spoke with a technical rep from Thermo Scientific, and this person assured me
that this is a common practice. However, I have only been able to find one
example of a somewhat similar approach in the literature
(https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5015836/). Is this approach
commonly used? Do you know of a publication that has used peptide pools to
produce site directed antibodies?

Thank you for your advice!

Angel


Edith “Angel” Sampson, Ph.D.
Monoclonal Antibody Scientific Director

UF ICBR
2033 Mowry Road, Gainesville, FL 32610
Biotech.ufl.edu | Tel: 352.273.8039| Fax: 352.273.8070

I always spot 5 ul of diluted peptide on the top corner of the blot before
blocking just to make sure the westerns are working.
Is it known if the protein has a short half-life?  You may need to add some
proteasome inhibitors. If your anti-gfp is working in your controls, that
shouldn't be the problem.
How strong is your anti-gfp band?  And is it the correct size with your fusion?
Any chance for post translational modifications in the region of the protein
where the peptides were selected?  Unlikely this would occur in all three
regions you picked.

Best,
Scott

-----Original Message-----
From: Antibody Discussion Forum <email obscured>] On Behalf Of
Frances Weis-Garcia
Sent: Tuesday, May 28, 2019 10:48 AM
To: <email obscured>
Subject: [ABRF Ab Forum] Anti-peptide respond that does not work in WB

Caution: External Sender


Happy Tuesday ...

Before we go and sequence the peptide, I want to float the following challenge
past everyone ...

Objective:  to make MAbs that work in WB

Approach:  Peptide immunization - 3 peptides (N, C and internal) conjugated to
KLH

Screening:  ELISA with Peptides conjugated to BSA and WB using whole cell
lysate from a DOX induced expression system

Results:
> Half Max in ELISA is 1:20,000
> Does not work in WB on overexpressed protein for which the protein
> sequence has been confirmed WB controls are anti-GFP since it is a
> fusion protein is fused to the N terminus

Next:
> Get more protein on the WB by IPing out with an anti-GFP antibody

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Sorry I was in 3 days conference and I missed this. Although I partially
replied another of your mails.
Probably you already solved it.
1) We use L cells murine fibroblast overexpressing the target.
2) We prefer the cells to be transduced (lentivirus) than retro or
transfection. For some reason we see less background.
3) We ask for the mock cells as control.
4) We inject BalbC
5) We don’t need substractive
6) The serum titration will show background on the Mock or parental. As far as
you see some difference in a bell-shape curve titration of serum on your target
cells, is OK to proceed to fusion.
7) All the selection will be shown in the screening.
8) Be sure that the homology human-mouse is less than 90%.
9) If the cells express the murine protein and the homology is less than 90
(enough to trigger immune resppnse), you may see background on the L cells if
the final selected antibodies cross react with the murine protein.

I think I summarized every aspect.
Thanks, and call me if you need help Frances!
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
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
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If you do not already have an iLab account please create one prior to sample
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On 5/1/19, 7:24 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.

    For cell based immunizations of mice, do any of you have a particular
favorite?

    Considering immunizing BAlb/c with NIH/3T3 (Swiss background)
overexpressing the cell surface target.  We will also do DNA immunizations in
parallel.

    Also, how often have you found it necessary to do subtractive immunization
to eliminate the "distracting" immune response to the irrelevant immunogens in
the cell line itself?

    Thanks for sharing your experience and opinions.

    Frances
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Thanks Laura.
Love this group!

-----Original Message-----
From: Antibody Discussion Forum <email obscured>> on behalf of Laura
Bover <email obscured>>
Reply-To: <email obscured>>
Date: Thursday, May 2, 2019 at 3:06 PM
To: <email obscured>" <email obscured>>
Subject: Re: [ABRF Ab Forum] [EXT] Re: Cell based immunizations

    We use L cells, murine fibroblasts, phenotype not BalbC mice and we inject
BalbC. No tumor development,  no irradiation needed. In fact you will have some
background when you test serum for immune response against parental cells vs
transfected.
    Then testing clones, you will select the ones that has no background with
parental.
    I injected before 3T3. No tumor and no irradiation.
    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<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 May 2, 2019, at 1:10 PM, Frances Weis-Garcia
<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.

    Those of you have immunize with cells 3T3 cells ... did you inject them as
is or irradiate them.  Cannot find the MHC phenotype of Swiss (Webster?) mice
so I am not sure irradiation is necessary to prevent tumor formation.

    Frances
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Hi Frances -

I do not irradiate 3T3 cells before immunizing with them.  I did not observe
any tumor growth at the injection sites (SC & IP).  They originated from NIH
Swiss albino outbred mice, which I believe are H-2q haplotype and will only
grow in SWR  and the Swiss NIH mice.

Best,
Ed.

-----Original Message-----
From: Antibody Discussion Forum <email obscured>> On Behalf Of Frances
Weis-Garcia
Sent: Thursday, May 2, 2019 2:10 PM
To: <email obscured>
Subject: Re: [ABRF Ab Forum] Cell based immunizations

        External Email - Use Caution

Those of you have immunize with cells 3T3 cells ... did you inject them as is
or irradiate them.  Cannot find the MHC phenotype of Swiss (Webster?) mice so I
am not sure irradiation is necessary to prevent tumor formation.

Frances
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dispose of the e-mail.

Those of you have immunize with cells 3T3 cells ... did you inject them as is
or irradiate them.  Cannot find the MHC phenotype of Swiss (Webster?) mice so I
am not sure irradiation is necessary to prevent tumor formation.

Frances