Posts in Antibody Discussion Forum

Yes, I agree about Ni plates, we use them only for special projects.

Regarding boiled protein , we didn't have issue on loosing the protein in
solution.

Rest of post


Laura


________________________________
From: Antibody Discussion Forum <email obscured>> on behalf of Frances
Weis-Garcia <email obscured>>
Sent: Tuesday, May 26, 2020 12:29 PM
To: <email obscured>
Subject: [EXT] Re: [ABRF Ab Forum] Denature proteins

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.

Thanks everyone ...

I am looking NOT to immunize with denatured protein, but rather to screen with
denatured protein via ELISA.  IF I boil the protein we are concerned the
protein will fall out of solution, which would be fine for immunization but not
coating an ELISA plate.

Laura ....

With respect to Ni plates ... they are great ... but expensive (10x more than a
regular plate).  We use them when we want the target to be as native as
possible, we have a HIS tag, and we do not have an antibody / tag combo for a
sandwich ELISA.  I am not at all surprised the ELISA results are different.
Binding a well formed protein to plastic will alter it's structure.  Had not
thought of using it as a screening protocol ... thanks.



On 5/25/20, 2:28 PM, "Antibody Discussion Forum on behalf of Bover,Laura"
<email obscured> on behalf of <email obscured>> wrote:

    Hi Frances! Hope you and the group are well and hanging there!


    Same than Ed. Denatured by boiling and use for injection and coat.
Sometimes I alternate injections: boil or not, in case I want to get the two
options.


    One interesting alternative that I found for protein coated directly on the
plate (meaning "flat"?) is to use the his tag version of the protein and side
by side regular plates and Niquel coated plates (commercial).


    The first time I asked  my technician to do it, he was panicking because
although some of the wells showed identical OD, other showed very different and
some were negative/positive depending on the plate.

    So, I use those plates for some projects, not as an alternative but
complementary.


    Laura Bover


    ________________________________
    From: Antibody Discussion Forum <email obscured>> on behalf of
Greenfield, Edward A.,Ph.D. <email obscured>>
    Sent: Monday, May 25, 2020 8:22 AM
    To: <email obscured>
    Subject: [EXT] Re: [ABRF Ab Forum] Denature proteins

    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.

    Hi Frances -

    The last few projects I have had to make antibodies to native (for FACS)
and denatured (for WB) protein I have found immunizing with the protein after
boiling it for about 1 minute has been effective for denaturing.  The boiled
protein can be bound to ELISA plates or used in Dot-Blots.

    Best,
    Ed.

    -----Original Message-----
    From: Antibody Discussion Forum <email obscured>> On Behalf Of
Frances Weis-Garcia
    Sent: Friday, May 22, 2020 3:09 PM
    To: ABRF Forum <email obscured>>
    Subject: [ABRF Ab Forum] Denature proteins

            External Email - Use Caution

    Yes. … it’s me again 😉

    Looking for a protocol by which to screen denatured proteins.  I have a
really nicely folded one and would like to see the antibody response against it
and a denatured version of it.  Anyone have protocol they like.

    I know an antibody core used to use a Urea protocol but they closed ☹.

    Frances



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    Yes. … it’s me again 😉 Looking for a protocol by which to screen denatured
proteins. I have a really nicely folded one and would like to see the antibody
re



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Thanks everyone ...

I am looking NOT to immunize with denatured protein, but rather to screen with
denatured protein via ELISA. IF I boil the protein we are concerned the
protein will fall out of solution, which would be fine for immunization but not
coating an ELISA plate.

Laura ....

With respect to Ni plates ... they are great ... but expensive (10x more than a
regular plate). We use them when we want the target to be as native as
possible, we have a HIS tag, and we do not have an antibody / tag combo for a
sandwich ELISA. I am not at all surprised the ELISA results are different.
Binding a well formed protein to plastic will alter it's structure. Had not
thought of using it as a screening protocol ... thanks.

On 5/25/20, 2:28 PM, "Antibody Discussion Forum on behalf of Bover,Laura"
<email obscured> on behalf of <email obscured>> wrote:

Hi Frances! Hope you and the group are well and hanging there!

Same than Ed. Denatured by boiling and use for injection and coat.
Sometimes I alternate injections: boil or not, in case I want to get the two
options.

One interesting alternative that I found for protein coated directly on the
plate (meaning "flat"?) is to use the his tag version of the protein and side
by side regular plates and Niquel coated plates (commercial).

The first time I asked my technician to do it, he was panicking because
although some of the wells showed identical OD, other showed very different and
some were negative/positive depending on the plate.

So, I use those plates for some projects, not as an alternative but
complementary.

Laura Bover

________________________________
From: Antibody Discussion Forum <email obscured>> on behalf of
Greenfield, Edward A.,Ph.D. <email obscured>>
Sent: Monday, May 25, 2020 8:22 AM
To: <email obscured>
Subject: [EXT] Re: [ABRF Ab Forum] Denature proteins

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.

Hi Frances -

The last few projects I have had to make antibodies to native (for FACS)
and denatured (for WB) protein I have found immunizing with the protein after
boiling it for about 1 minute has been effective for denaturing. The boiled
protein can be bound to ELISA plates or used in Dot-Blots.

Best,
Ed.

-----Original Message-----
From: Antibody Discussion Forum <email obscured>> On Behalf Of
Frances Weis-Garcia
Sent: Friday, May 22, 2020 3:09 PM
To: ABRF Forum <email obscured>>
Subject: [ABRF Ab Forum] Denature proteins

External Email - Use Caution

Yes. … it’s me again 😉

Looking for a protocol by which to screen denatured proteins. I have a
really nicely folded one and would like to see the antibody response against it
and a denatured version of it. Anyone have protocol they like.

I know an antibody core used to use a Urea protocol but they closed ☹.

Frances

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urldefense.com
Yes. … it’s me again 😉 Looking for a protocol by which to screen denatured
proteins. I have a really nicely folded one and would like to see the antibody
re

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Hi Frances,
I hope all is going well and you are safe in New York - Maine is socially
distanced by nature but still we are taking precautions, although relaxing
those a little now as we head into a “Phase 1” return to the lab, meaning wet
bench scientists are the first back. Core Facilities have worked all the way
through this using a shift system to minimize contact and we have managed to
get on-campus presence down to 25% of normal. The place feels empty!

We also boil the protein prior to injection. We mix 10ug per animal of boiled
(and cooled) with 10ug of undenatured with adjuvant for injecting.
All the best and stay safe everyone.
Alan

Rest of post

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On May 25, 2020, at 14:28, Bover,Laura
<email obscured><email obscured>>> wrote:

Hi Frances! Hope you and the group are well and hanging there!


Same than Ed. Denatured by boiling and use for injection and coat. Sometimes I
alternate injections: boil or not, in case I want to get the two options.


One interesting alternative that I found for protein coated directly on the
plate (meaning "flat"?) is to use the his tag version of the protein and side
by side regular plates and Niquel coated plates (commercial).


The first time I asked  my technician to do it, he was panicking because
although some of the wells showed identical OD, other showed very different and
some were negative/positive depending on the plate.

So, I use those plates for some projects, not as an alternative but
complementary.


Laura Bover


________________________________
From: Antibody Discussion Forum
<email obscured><email obscured>>> on behalf of
Greenfield, Edward A.,Ph.D.
<email obscured><email obscured>>>
Sent: Monday, May 25, 2020 8:22 AM
To: <email obscured><email obscured>>
Subject: [EXT] Re: [ABRF Ab Forum] Denature proteins

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.

Hi Frances -

The last few projects I have had to make antibodies to native (for FACS) and
denatured (for WB) protein I have found immunizing with the protein after
boiling it for about 1 minute has been effective for denaturing.  The boiled
protein can be bound to ELISA plates or used in Dot-Blots.

Best,
Ed.

-----Original Message-----
From: Antibody Discussion Forum
<email obscured><email obscured>>> On Behalf Of Frances
Weis-Garcia
Sent: Friday, May 22, 2020 3:09 PM
To: ABRF Forum <email obscured><email obscured>>>
Subject: [ABRF Ab Forum] Denature proteins

       External Email - Use Caution

Yes. … it’s me again 😉

Looking for a protocol by which to screen denatured proteins.  I have a really
nicely folded one and would like to see the antibody response against it and a
denatured version of it.  Anyone have protocol they like.

I know an antibody core used to use a Urea protocol but they closed ☹.

Frances



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urldefense.com<http://urldefense.com>
Yes. … it’s me again 😉 Looking for a protocol by which to screen denatured
proteins. I have a really nicely folded one and would like to see the antibody
re



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Hi Frances! Hope you and the group are well and hanging there!


Same than Ed. Denatured by boiling and use for injection and coat. Sometimes I
alternate injections: boil or not, in case I want to get the two options.


One interesting alternative that I found for protein coated directly on the
plate (meaning "flat"?) is to use the his tag version of the protein and side
by side regular plates and Niquel coated plates (commercial).


The first time I asked  my technician to do it, he was panicking because
although some of the wells showed identical OD, other showed very different and
some were negative/positive depending on the plate.

So, I use those plates for some projects, not as an alternative but
complementary.


Laura Bover

Rest of post

________________________________
From: Antibody Discussion Forum <email obscured>> on behalf of
Greenfield, Edward A.,Ph.D. <email obscured>>
Sent: Monday, May 25, 2020 8:22 AM
To: <email obscured>
Subject: [EXT] Re: [ABRF Ab Forum] Denature proteins

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.

Hi Frances -

The last few projects I have had to make antibodies to native (for FACS) and
denatured (for WB) protein I have found immunizing with the protein after
boiling it for about 1 minute has been effective for denaturing.  The boiled
protein can be bound to ELISA plates or used in Dot-Blots.

Best,
Ed.

-----Original Message-----
From: Antibody Discussion Forum <email obscured>> On Behalf Of Frances
Weis-Garcia
Sent: Friday, May 22, 2020 3:09 PM
To: ABRF Forum <email obscured>>
Subject: [ABRF Ab Forum] Denature proteins

        External Email - Use Caution

Yes. … it’s me again 😉

Looking for a protocol by which to screen denatured proteins.  I have a really
nicely folded one and would like to see the antibody response against it and a
denatured version of it.  Anyone have protocol they like.

I know an antibody core used to use a Urea protocol but they closed ☹.

Frances

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urldefense.com
Yes. … it’s me again 😉 Looking for a protocol by which to screen denatured
proteins. I have a really nicely folded one and would like to see the antibody
re

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Hi Frances -

The last few projects I have had to make antibodies to native (for FACS) and
denatured (for WB) protein I have found immunizing with the protein after
boiling it for about 1 minute has been effective for denaturing.  The boiled
protein can be bound to ELISA plates or used in Dot-Blots.

Rest of post

Best,
Ed.

-----Original Message-----
From: Antibody Discussion Forum <email obscured>> On Behalf Of Frances
Weis-Garcia
Sent: Friday, May 22, 2020 3:09 PM
To: ABRF Forum <email obscured>>
Subject: [ABRF Ab Forum] Denature proteins

        External Email - Use Caution

Yes. … it’s me again 😉

Looking for a protocol by which to screen denatured proteins.  I have a really
nicely folded one and would like to see the antibody response against it and a
denatured version of it.  Anyone have protocol they like.

I know an antibody core used to use a Urea protocol but they closed ☹.

Frances

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Yes. … it’s me again 😉

Looking for a protocol by which to screen denatured proteins.  I have a really
nicely folded one and would like to see the antibody response against it and a
denatured version of it.  Anyone have protocol they like.

I know an antibody core used to use a Urea protocol but they closed ☹.

Rest of post

Frances

Happy Friday everyone.

There are 2 open positions here is NY at the Tri-Institutional Therapeutics
Discovery Institute.

Associate Director, Antibody
Generation<https://www.linkedin.com/jobs/view/1840882309/>
Associate Director, Assay Development and
Screening<https://www.linkedin.com/jobs/view/1840882107/>
If you are interested … give me a call … I know the group well.

Stay well and have a great albeit unusual weekend to kick off the summer.

Rest of post

Frances

I love this company ... the only brand I buy form.
Working with a group who does human antibody ELISAs all the time ... and they
are wed to Southern Biotech.

On 4/28/20, 2:42 PM, "Antibody Discussion Forum on behalf of Bover,Laura"
<email obscured> on behalf of <email obscured>> wrote:

    We were using secondaries from Jackson Immunoresearch, minimal cross
reactivity with bovine and many other species.
    HRP.
    There are a bunch of  varieties, I like donkeys for lower background and
there are also fragments in case you need (F(ab)2) fragments and I think that
Fab also never tried the last one

    Laura Bover


    On 4/28/20, 1:33 PM, "Antibody Discussion Forum on behalf of Brown, Scott
A" <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.

        Yes.  Hrp and sigmafast.

        Scott

        Scott Brown, Ph.D.
        Director of Immunologic Reagent Facility
        Department of Immunology
        St. Jude Children’s Research Hospital
        262 Danny Thomas Place
        Memphis, TN 38105
        ________________________________
        From: Antibody Discussion Forum <email obscured>> on behalf of
Frances Weis-Garcia <email obscured>>
        Sent: Tuesday, April 28, 2020 1:29:30 PM
        To: <email obscured> <email obscured>>
        Subject: Re: [ABRF Ab Forum] BG from human sera / plasma

        Caution: External Sender


        David and Scott,

        Were you using HRP and something like TMB?
        We are using a fluorescent reagent called AttoPhos which is supposedly
more sensitive.

        Frances

        On 4/25/20, 9:08 PM, "Antibody Discussion Forum on behalf of Brown,
Scott A" <email obscured> on behalf of <email obscured>> wrote:

            Yep.  Same here.  We are using PBS with 0.1% Tween 20 and 1%
non-fat milk.   Looks great.  Even at 1:50.
            Best,


            Scott

            Scott Brown, Ph.D.
            Director of Immunologic Reagent Facility
            Department of Immunology
            St. Jude Children’s Research Hospital
            262 Danny Thomas Place
            Memphis, TN 38105
            ________________________________
            From: Antibody Discussion Forum <email obscured>> on behalf
of David Blum <email obscured>>
            Sent: Saturday, April 25, 2020 1:58:42 PM
            To: <email obscured> <email obscured>>
            Subject: Re: [ABRF Ab Forum] BG from human sera / plasma

            Caution: External Sender


            When I was at Vanderbilt I worked on a project looking for
autoantibodies
            directed against therapeutic antibodies.  We used human sera
diluted in
            PBST and it worked fine.  I can dig up my notes if you want more
details.

            On Sat, Apr 25, 2020 at 12:40 AM Frances Weis-Garcia <
            <email obscured>> wrote:

            > Have any of you run human sera or plasma in an ELISA?
            > I have been told BG is a notorious problem … Samples diluted to
1:100 in
            > block (5% BSA).
            > Pre-adsorption on BSA blocked wells is not sufficient to get rid
of BG
            > coming from human sample.
            >
            > Have any of you run into this problem?
            > As a Hail Mary pass … Thinking about stripping the diluted sample
with
            > activated charcoal because I have nothing to loose.
            >
            > Thankfully mice do not have this problem.
            >
            >
            >
            > ――
            > View topic
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We were using secondaries from Jackson Immunoresearch, minimal cross reactivity
with bovine and many other species.
HRP.
There are a bunch of  varieties, I like donkeys for lower background and there
are also fragments in case you need (F(ab)2) fragments and I think that Fab
also never tried the last one

Laura Bover


On 4/28/20, 1:33 PM, "Antibody Discussion Forum on behalf of Brown, Scott A"
<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.

    Yes.  Hrp and sigmafast.

    Scott

    Scott Brown, Ph.D.
    Director of Immunologic Reagent Facility
    Department of Immunology
    St. Jude Children’s Research Hospital
    262 Danny Thomas Place
    Memphis, TN 38105
    ________________________________
    From: Antibody Discussion Forum <email obscured>> on behalf of
Frances Weis-Garcia <email obscured>>
    Sent: Tuesday, April 28, 2020 1:29:30 PM
    To: <email obscured> <email obscured>>
    Subject: Re: [ABRF Ab Forum] BG from human sera / plasma

    Caution: External Sender


    David and Scott,

    Were you using HRP and something like TMB?
    We are using a fluorescent reagent called AttoPhos which is supposedly more
sensitive.

    Frances

    On 4/25/20, 9:08 PM, "Antibody Discussion Forum on behalf of Brown, Scott
A" <email obscured> on behalf of <email obscured>> wrote:

        Yep.  Same here.  We are using PBS with 0.1% Tween 20 and 1% non-fat
milk.   Looks great.  Even at 1:50.
        Best,


        Scott

        Scott Brown, Ph.D.
        Director of Immunologic Reagent Facility
        Department of Immunology
        St. Jude Children’s Research Hospital
        262 Danny Thomas Place
        Memphis, TN 38105
        ________________________________
        From: Antibody Discussion Forum <email obscured>> on behalf of
David Blum <email obscured>>
        Sent: Saturday, April 25, 2020 1:58:42 PM
        To: <email obscured> <email obscured>>
        Subject: Re: [ABRF Ab Forum] BG from human sera / plasma

        Caution: External Sender


        When I was at Vanderbilt I worked on a project looking for
autoantibodies
        directed against therapeutic antibodies.  We used human sera diluted in
        PBST and it worked fine.  I can dig up my notes if you want more
details.

        On Sat, Apr 25, 2020 at 12:40 AM Frances Weis-Garcia <
        <email obscured>> wrote:

        > Have any of you run human sera or plasma in an ELISA?
        > I have been told BG is a notorious problem … Samples diluted to 1:100
in
        > block (5% BSA).
        > Pre-adsorption on BSA blocked wells is not sufficient to get rid of
BG
        > coming from human sample.
        >
        > Have any of you run into this problem?
        > As a Hail Mary pass … Thinking about stripping the diluted sample
with
        > activated charcoal because I have nothing to loose.
        >
        > Thankfully mice do not have this problem.
        >
        >
        >
        > ――
        > View topic
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Yes.  Hrp and sigmafast.

Scott

Scott Brown, Ph.D.
Director of Immunologic Reagent Facility
Department of Immunology
St. Jude Children’s Research Hospital
262 Danny Thomas Place
Memphis, TN 38105

Rest of post

________________________________
From: Antibody Discussion Forum <email obscured>> on behalf of Frances
Weis-Garcia <email obscured>>
Sent: Tuesday, April 28, 2020 1:29:30 PM
To: <email obscured> <email obscured>>
Subject: Re: [ABRF Ab Forum] BG from human sera / plasma

Caution: External Sender


David and Scott,

Were you using HRP and something like TMB?
We are using a fluorescent reagent called AttoPhos which is supposedly more
sensitive.

Frances

On 4/25/20, 9:08 PM, "Antibody Discussion Forum on behalf of Brown, Scott A"
<email obscured> on behalf of <email obscured>> wrote:

    Yep.  Same here.  We are using PBS with 0.1% Tween 20 and 1% non-fat milk.
Looks great.  Even at 1:50.
    Best,


    Scott

    Scott Brown, Ph.D.
    Director of Immunologic Reagent Facility
    Department of Immunology
    St. Jude Children’s Research Hospital
    262 Danny Thomas Place
    Memphis, TN 38105
    ________________________________
    From: Antibody Discussion Forum <email obscured>> on behalf of David
Blum <email obscured>>
    Sent: Saturday, April 25, 2020 1:58:42 PM
    To: <email obscured> <email obscured>>
    Subject: Re: [ABRF Ab Forum] BG from human sera / plasma

    Caution: External Sender


    When I was at Vanderbilt I worked on a project looking for autoantibodies
    directed against therapeutic antibodies.  We used human sera diluted in
    PBST and it worked fine.  I can dig up my notes if you want more details.

    On Sat, Apr 25, 2020 at 12:40 AM Frances Weis-Garcia <
    <email obscured>> wrote:

    > Have any of you run human sera or plasma in an ELISA?
    > I have been told BG is a notorious problem … Samples diluted to 1:100 in
    > block (5% BSA).
    > Pre-adsorption on BSA blocked wells is not sufficient to get rid of BG
    > coming from human sample.
    >
    > Have any of you run into this problem?
    > As a Hail Mary pass … Thinking about stripping the diluted sample with
    > activated charcoal because I have nothing to loose.
    >
    > Thankfully mice do not have this problem.
    >
    >
    >
    > ――
    > View topic
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David and Scott,

Were you using HRP and something like TMB?
We are using a fluorescent reagent called AttoPhos which is supposedly more
sensitive.

Frances

On 4/25/20, 9:08 PM, "Antibody Discussion Forum on behalf of Brown, Scott A"
<email obscured> on behalf of <email obscured>> wrote:

    Yep.  Same here.  We are using PBS with 0.1% Tween 20 and 1% non-fat milk.
Looks great.  Even at 1:50.
    Best,


    Scott

    Scott Brown, Ph.D.
    Director of Immunologic Reagent Facility
    Department of Immunology
    St. Jude Children’s Research Hospital
    262 Danny Thomas Place
    Memphis, TN 38105
    ________________________________
    From: Antibody Discussion Forum <email obscured>> on behalf of David
Blum <email obscured>>
    Sent: Saturday, April 25, 2020 1:58:42 PM
    To: <email obscured> <email obscured>>
    Subject: Re: [ABRF Ab Forum] BG from human sera / plasma

    Caution: External Sender


    When I was at Vanderbilt I worked on a project looking for autoantibodies
    directed against therapeutic antibodies.  We used human sera diluted in
    PBST and it worked fine.  I can dig up my notes if you want more details.

    On Sat, Apr 25, 2020 at 12:40 AM Frances Weis-Garcia <
    <email obscured>> wrote:

    > Have any of you run human sera or plasma in an ELISA?
    > I have been told BG is a notorious problem … Samples diluted to 1:100 in
    > block (5% BSA).
    > Pre-adsorption on BSA blocked wells is not sufficient to get rid of BG
    > coming from human sample.
    >
    > Have any of you run into this problem?
    > As a Hail Mary pass … Thinking about stripping the diluted sample with
    > activated charcoal because I have nothing to loose.
    >
    > Thankfully mice do not have this problem.
    >
    >
    >
    > ――
    > View topic
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Thanks Abdul.
Thankfully we have nice signal ... but our BG is hurting our sensitivity and
very erratic ... hate when I cannot control or at least understand variables in
an assay.  We can definitely live with it ... as we get hit with a standard and
even a high cut off (Average normal sera + 3 standard deviations vs 6 standard
deviations).  Guess one has to live with this when dealing with human samples.


On 4/26/20, 8:32 PM, "Antibody Discussion Forum on behalf of Abdul Khan"
<email obscured> on behalf of <email obscured>> wrote:

    With my limited experience with human ELISA, I remember the problem is
mostly the detection antibodies. In past we tried few HRP conjugated and only
one from GE was the best with minimum BG.

    Best
    Abdul


    On 4/25/20, 12:40 AM, "Frances Weis-Garcia"
<email obscured>> wrote:

        Have any of you run human sera or plasma in an ELISA?
        I have been told BG is a notorious problem … Samples diluted to 1:100
in block (5% BSA).
        Pre-adsorption on BSA blocked wells is not sufficient to get rid of BG
coming from human sample.

        Have any of you run into this problem?
        As a Hail Mary pass … Thinking about stripping the diluted sample with
activated charcoal because I have nothing to loose.

        Thankfully mice do not have this problem.



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With my limited experience with human ELISA, I remember the problem is mostly
the detection antibodies. In past we tried few HRP conjugated and only one from
GE was the best with minimum BG.

Best
Abdul


On 4/25/20, 12:40 AM, "Frances Weis-Garcia" <email obscured>>
wrote:

    Have any of you run human sera or plasma in an ELISA?
    I have been told BG is a notorious problem … Samples diluted to 1:100 in
block (5% BSA).
    Pre-adsorption on BSA blocked wells is not sufficient to get rid of BG
coming from human sample.

    Have any of you run into this problem?
    As a Hail Mary pass … Thinking about stripping the diluted sample with
activated charcoal because I have nothing to loose.

    Thankfully mice do not have this problem.



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Yep.  Same here.  We are using PBS with 0.1% Tween 20 and 1% non-fat milk.
Looks great.  Even at 1:50.
Best,


Scott

Scott Brown, Ph.D.
Director of Immunologic Reagent Facility
Department of Immunology
St. Jude Children’s Research Hospital
262 Danny Thomas Place
Memphis, TN 38105

Rest of post

________________________________
From: Antibody Discussion Forum <email obscured>> on behalf of David
Blum <email obscured>>
Sent: Saturday, April 25, 2020 1:58:42 PM
To: <email obscured> <email obscured>>
Subject: Re: [ABRF Ab Forum] BG from human sera / plasma

Caution: External Sender


When I was at Vanderbilt I worked on a project looking for autoantibodies
directed against therapeutic antibodies.  We used human sera diluted in
PBST and it worked fine.  I can dig up my notes if you want more details.

On Sat, Apr 25, 2020 at 12:40 AM Frances Weis-Garcia <
<email obscured>> wrote:

> Have any of you run human sera or plasma in an ELISA?
> I have been told BG is a notorious problem … Samples diluted to 1:100 in
> block (5% BSA).
> Pre-adsorption on BSA blocked wells is not sufficient to get rid of BG
> coming from human sample.
>
> Have any of you run into this problem?
> As a Hail Mary pass … Thinking about stripping the diluted sample with
> activated charcoal because I have nothing to loose.
>
> Thankfully mice do not have this problem.
>
>
>
> ――
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When I was at Vanderbilt I worked on a project looking for autoantibodies
directed against therapeutic antibodies.  We used human sera diluted in
PBST and it worked fine.  I can dig up my notes if you want more details.

On Sat, Apr 25, 2020 at 12:40 AM Frances Weis-Garcia <

Rest of post

<email obscured>> wrote:

> Have any of you run human sera or plasma in an ELISA?
> I have been told BG is a notorious problem … Samples diluted to 1:100 in
> block (5% BSA).
> Pre-adsorption on BSA blocked wells is not sufficient to get rid of BG
> coming from human sample.
>
> Have any of you run into this problem?
> As a Hail Mary pass … Thinking about stripping the diluted sample with
> activated charcoal because I have nothing to loose.
>
> Thankfully mice do not have this problem.
>
>
>
> ――
> View topic http://list.abrf.org/r/topic/5ERnD68oB1r5NpoeDnbcVD
> Leave group <email obscured>?Subject=Unsubscribe
>
>

Yes, we set up running serum human samples when we were working with anti OX40
years ago and now one group in Argentina is producing the kit, RUO Of course
but they don’t have the problem
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 Scientific Review Committee 2 (former CRC 2)
Member of the Steering Committee of the Immunology Program GSBS
Immunology Department/ Genomics Medicine Department
7455 Fannin St<x-apple-data-detectors://1/1> Room 1SCR4.2021
Houston- TX 77054<x-apple-data-detectors://2/0>
T  713-563-3301<tel:713-563-3301>
F 713-563-3276<tel:713-563-3276>
<email obscured><email obscured>>

On Apr 24, 2020, at 11:40 PM, Frances Weis-Garcia
<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.

Have any of you run human sera or plasma in an ELISA?
I have been told BG is a notorious problem … Samples diluted to 1:100 in block
(5% BSA).
Pre-adsorption on BSA blocked wells is not sufficient to get rid of BG coming
from human sample.

Have any of you run into this problem?
As a Hail Mary pass … Thinking about stripping the diluted sample with
activated charcoal because I have nothing to loose.

Thankfully mice do not have this problem.



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Have any of you run human sera or plasma in an ELISA?
I have been told BG is a notorious problem … Samples diluted to 1:100 in block
(5% BSA).
Pre-adsorption on BSA blocked wells is not sufficient to get rid of BG coming
from human sample.

Have any of you run into this problem?
As a Hail Mary pass … Thinking about stripping the diluted sample with
activated charcoal because I have nothing to loose.

Thankfully mice do not have this problem.

I can see it.


On 30.03.2020 14:26, Greenfield, Edward A.,Ph.D. wrote:
> I can see it too.
>
> -----Original Message-----
> From: Antibody Discussion Forum <email obscured>> On Behalf Of Frances
Weis-Garcia
> Sent: Friday, March 27, 2020 10:17 PM
> To: ABRF Forum <email obscured>>
> Subject: [ABRF Ab Forum] Test 2
>
>          External Email - Use Caution
>
> Test 2
> If anyone sees this please reply back so I know I am having problems with the
system

Rest of post

>
> ――
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>
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--
Goran Mitulović, Ph.D.
ORCID 0000-0003-1964-3965
Head Proteomics Core Facility
Medical University of Vienna
Clinical Department of Laboratory Medicine
Proteomics Core Facility, E5, LS5H
Waehringer Guertel 18-20
A-1090 Wien, Austria
Tel: +43 1 40400 53750
Mail: <email obscured>
Web: www.meduniwien.ac.at/proteomics-core

I can see it too.

Rest of post

-----Original Message-----
From: Antibody Discussion Forum <email obscured>> On Behalf Of Frances
Weis-Garcia
Sent: Friday, March 27, 2020 10:17 PM
To: ABRF Forum <email obscured>>
Subject: [ABRF Ab Forum] Test 2

        External Email - Use Caution

Test 2
If anyone sees this please reply back so I know I am having problems with the
system

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I see it

Scott

Scott Brown, Ph.D.
Director of Immunologic Reagent Facility
Department of Immunology
St. Jude Children’s Research Hospital
262 Danny Thomas Place
Memphis, TN 38105

Rest of post

________________________________
From: Antibody Discussion Forum <email obscured>> on behalf of Frances
Weis-Garcia <email obscured>>
Sent: Friday, March 27, 2020 9:48:40 PM
To: <email obscured> <email obscured>>
Subject: Re: [ABRF Ab Forum] [EXTERNAL] Test 2

Caution: External Sender


Thanks.
Guess it is working now.
Stay well.

On 3/27/20, 10:32 PM, "Antibody Discussion Forum on behalf of Alan Sawyer"
<email obscured> on behalf of <email obscured>> wrote:

    I'm seeing it

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    On 3/27/20, 22:17, "Antibody Discussion Forum on behalf of Frances
Weis-Garcia" <email obscured> on behalf of
<email obscured>> wrote:

        Test 2
        If anyone sees this please reply back so I know
        I am having problems with the system

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