Do we trust the Negative Control?: Antibody Discussion Forum: ABRF Discussion Forums

Do we trust the Negative Control?

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 ;)

Rest of post

Frances

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.

TrustTheNegative.xlsx 12.8kb

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

― 1 file link ―

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.
― 1 file ―

  □ TrustTheNegative.xlsx  (13kb)
      http://list.abrf.org/r/file/ApTjzBtFUr7Ei6HzoSGr1rm2UZc-3pg-2LRaZb1/

――
View topic http://list.abrf.org/r/topic/3Sonzhjb0Lb6e7i9ipTFfq
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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


On 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

    ― 1 file link ―

    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.
    ― 1 file ―

      □ TrustTheNegative.xlsx  (13kb)
          http://list.abrf.org/r/file/ApTjzBtFUr7Ei6HzoSGr1rm2UZc-3pg-2LRaZb1/

    ――
    View topic http://list.abrf.org/r/topic/3Sonzhjb0Lb6e7i9ipTFfq
    Leave group <email obscured>?Subject=Unsubscribe



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