Antibody-binding to DNA

Greg Denomme denomme at FHS.CSU.MCMASTER.CA
Thu Jul 8 14:15:04 EST 1993

On 8 Jul 1993 GRGGTA at wrote:

> Dear All,
>    I recently ran a series of standard PAGE-retardation analyses using
> 20mer oligonucleotides labelled with 32P. The Oligo's contain the sequence
> for the binding of AP-1 (fos/jun). I retarded these oligo probes with various
> fibroblast cell nuclear extracts and got specific bands. To show the presence
> of specific transcription factors in this complex I added antibodies to c-fos
> and c-jun (ie in seperate reactions) and got what I thought were appropriate
> supershifted complexes. I have now had it suggested to me that these complexes
> may be due to the antibody serum binding the oligonucleotide probes directly.
> Does anyone know anything more about this phenomenum? It is new to me
> Thanks Graham Atherton
  The phenomenon you are describing is real.  Some monoclonal antibodies 
isolated by hybridoma technology have been shown to have polyreactivity to
DNA, both double-stranded and single-stranded. I think this was first
demonstrated by Avrameas in 1982 and has been studied in immunology especially
for natural (auto)antibodies.  The problem you are refering to is that during
the development of antibodies in an immune reponse (like when one is
immunizing an animal with a specific antigen) there is also the production
of autoantibodies some of which are polyreactive.  These antibodies can
bind to DNA among other antigens like myosin and the Fc protion of IgG. 
So, not only do you have antibodies to c-fos but also weaker titers of
polyreactive antibodies.  Now these polyreactive antibodies shouldn't be a
problem because normally you would used the antisera at a titer far beyond
their reactivity range.  If you are concerned try and ELISA in which you
coat a microtiter plate with single-stranded DNA.  If your antisera binds
to single-stranded DNA at the dilution of your other assay then I would be
concerned about the results.  You can find ELISAs for DNA in Cairns et al.
J Clin Invest 74:880-887, 1984 and Cairns et al. J Immunol 135:3839-3844,
Good Luck,
denomme at FHS.McMaster.CA

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