I have a riddle / puzzle for everyone on this Friday before Thanksgiving ...
In an ELISA, why would a MAb bind a target, lets call it "A," NOT bind a
similar entity we will call "A2" prepared with the same protocol, and bind an
uncoated well?
So
Target "A" ... SIGNAL
Similar "A2" ... no signal
Empty well ... SIGNAL
All wells were incubated with binding buffer over night with Target "A",
Similar "A2" or NOTHING and then blocked with 1% BSA in binding buffer.
I would expect these options but not the one above.
Specific MAb
Target "A" ... SIGNAL
Similar "A2" ... no signal
Empty well ... no signal
Cross reactive MAb
Target "A" ... SIGNAL
Similar "A2" ... SIGNAL
Empty well ... no signal
Non-Specific / Sticky MAb
Target "A" ... SIGNAL
Similar "A2" ... no signal
Empty well ... no signal
In the interest of reasonable transparency, the target is a proteoglycan ...
but not sure it really matters.
Looking forward to thinking this through with everyone who likes a challenge ;)
99% sure ... because we have different combinations of results with different
new MAbs ... Just to sweeten the pot ... Antigen A is prepared in to different
manners and there is "similar" lets call A3. So I question our negative
control since it should not bind the negative and the target but not related
targets prepared in the same manner.
See attached for actual data to help make things clear ... kept some of the
negative cultures.
Hi Frances -
I have seen unbound (empty) ELISA plate wells look positive before. Often this
seems to be an edge effect or a bubble in the well. I have seen BSA blocking
buffer coat an unbound well sufficiently well to be "sticky". Try Non-fat
powdered milk buffer or Superblock and see if the problem goes away. I find
using a different irrelevant protein bound to the wells can help. I have even
seen negative controls like scrambled peptides being picked up by some mAbs. I
would test the mAbs in some other assay like FACS, IHC, WB and see how it
fares.
Projects like these do help keep things interesting!
Rest of post
Best,
Ed.
-----Original Message-----
From: Antibody Discussion Forum <email obscured>> On Behalf Of Frances
Weis-Garcia
Sent: Friday, November 22, 2019 12:46 PM
To: <email obscured>
Subject: Re: [ABRF Ab Forum] Do we trust the Negative Control?
External Email - Use Caution
β 1 file link β
99% sure ... because we have different combinations of results with different
new MAbs ... Just to sweeten the pot ... Antigen A is prepared in to different
manners and there is "similar" lets call A3. So I question our negative
control since it should not bind the negative and the target but not related
targets prepared in the same manner.
See attached for actual data to help make things clear ... kept some of the
negative cultures.
β 1 file β
β‘ TrustTheNegative.xlsx (13kb)
http://list.abrf.org/r/file/ApTjzBtFUr7Ei6HzoSGr1rm2UZc-3pg-2LRaZb1/
ββ
View topic http://list.abrf.org/r/topic/3Sonzhjb0Lb6e7i9ipTFfq
Leave group <email obscured>?Subject=Unsubscribe
The information in this e-mail is intended only for the person to whom it is
addressed. If you believe this e-mail was sent to you in error and the e-mail
contains patient information, please contact the Partners Compliance HelpLine
at
http://www.partners.org/complianceline . If the e-mail was sent to you in error
but does not contain patient information, please contact the sender and
properly
dispose of the e-mail.
Alan and Ed ... You 2 always make me feel less dumb. Thank you ...
So we have tried NF dry milk ... do difference
Will likely try Superblock. Never knew it existed ... "kits" for everything
these days __
We first saw this confusing data using a Rat IgG as the negative control ...
nothing really good for a peptidoglycan ... then we thought and empty well
would be better ... not the case this time.
If it were a protein, changing assays would be easy. They are trying IF ...
but for the peptidoglycan it is tricky. We are also considering attaching the
peptidoglycan to a bead and using it to IP the MAb out of the media ... not
trivial either.
Frances
ZRC 1563
Office 646-888-2354
Mobile 516-967-0656
ο»ΏOn 11/22/19, 1:33 PM, "Antibody Discussion Forum on behalf of Greenfield,
Edward A.,Ph.D." <email obscured> on behalf of
<email obscured>> wrote:
Hi Frances -
I have seen unbound (empty) ELISA plate wells look positive before. Often
this seems to be an edge effect or a bubble in the well. I have seen BSA
blocking buffer coat an unbound well sufficiently well to be "sticky". Try
Non-fat powdered milk buffer or Superblock and see if the problem goes away.
I find using a different irrelevant protein bound to the wells can help. I
have even seen negative controls like scrambled peptides being picked up by
some mAbs. I would test the mAbs in some other assay like FACS, IHC, WB and
see how it fares.
Projects like these do help keep things interesting!
Best,
Ed.
-----Original Message-----
From: Antibody Discussion Forum <email obscured>> On Behalf Of
Frances Weis-Garcia
Sent: Friday, November 22, 2019 12:46 PM
To: <email obscured>
Subject: Re: [ABRF Ab Forum] Do we trust the Negative Control?
External Email - Use Caution
β 1 file link β
99% sure ... because we have different combinations of results with
different new MAbs ... Just to sweeten the pot ... Antigen A is prepared in to
different manners and there is "similar" lets call A3. So I question our
negative control since it should not bind the negative and the target but not
related targets prepared in the same manner.
See attached for actual data to help make things clear ... kept some of the
negative cultures.
β 1 file β
β‘ TrustTheNegative.xlsx (13kb)
http://list.abrf.org/r/file/ApTjzBtFUr7Ei6HzoSGr1rm2UZc-3pg-2LRaZb1/
ββ
View topic http://list.abrf.org/r/topic/3Sonzhjb0Lb6e7i9ipTFfq
Leave group <email obscured>?Subject=Unsubscribe
The information in this e-mail is intended only for the person to whom it
is
addressed. If you believe this e-mail was sent to you in error and the
e-mail
contains patient information, please contact the Partners Compliance
HelpLine at
http://www.partners.org/complianceline . If the e-mail was sent to you in
error
but does not contain patient information, please contact the sender and
properly
dispose of the e-mail.
ββ
View topic http://list.abrf.org/r/topic/6UsrB73tdo6jNms5MDv25I
Leave group <email obscured>?Subject=Unsubscribe