Hi,
I just wanted to add a few things to what Graham Sheperd wrote. First,
when the terms on-rate and off-rate are used, it usually indicates that the
binding kinetics, and ultimately the affinity constant, are measured in real
time by an instrument like the BIAcore that measures surface plasmon
resonance. In these studies, the target antigen is immobilized on a chip and
the antibody (or scFv) is flowed accross the chip and the amount of antibody
binding over a short period is measured. Buffer without antibody in it is
then flowed across the chip and the rate that the antibody dissociated from
the antigen is measured. This is performed at multiple concentrations to
determine accurate rates of association and dissociation. There are also a
number of factors that must be considered such as rebinding of the antibody
to antigen, etc.
Second, while affinity maturation usually results in a better fit between the
antibody and the antigen and hence an increase in the energy required for the
dissociation (resulting in decreased rate of dissociation or Koff), changes
can be made to the rate of association or on-rate. This is not as common as
changes to the off-rate since the on-rate is dictitated by a number of
factors including movement of parts of the antibody or antigen. For example,
with one scFv we work with, it appears that the heavy chain CDR3 actually
"flips" over like a door opening to allow the binding to antigen. Therefore,
the structural changes required for the scFv to have a different on-rate
would entail wholescale redesign of the CDR3. Mutations to this region are
unlikely to lead to this without changing the scFv's antigenic specificity.
Finally, many of the affinity maturation techniques employ phage display
selection steps that are biased to result in slower off-rates rather than
faster on-rates. Faster on-rates are found by decreasing the incubation
periods such that only the scFvs that bind quickly are isolated, while slower
off-rates are found by decreasing the antigen concentration and increasing
the incubation times to allow the stronger binding clones to out compete the
weak binders. I hope this helps.
Good Luck,
Greg Adams
Graham Shepherd wrote:
> Esbjorn Fiers <esfie at gengenp.rug.ac.be> wrote in message
> news:3896D799.237755F1 at gengenp.rug.ac.be...> > Dear all,
> > I am working with scFv phage display libraries. In some articles I come
> > across the words on- and off-rate, being the rate at which an antibody
> > binds its antigen and the rate at which an antibody dissociates from its
> > antigen. Furthermore, they say that when antibody affinities become
> > higher this is mostly due to a lower off-rate, with the on-rate being
> > less important. I kind of have problems to completely understand this.
> > Is there anybody who could explain this to me, or has anyone a reference
> > in which this is clearly explained?
> > Thanks!
> >
> > Esbjorn Fiers
> >
> > --
> > ==================================================================
> > Esbjorn Fiers
> > DEPARTMENT OF GENETICS Fax:32 (0)9 2645349
> > UNIVERSITY OF GENT, K. L. Ledeganckstraat 35, B-9000 Gent, Belgium
> > Vlaams Instituut voor Biotechnologie VIB
> > mailto:esfie at gengenp.rug.ac.behttp://www.plantgenetics.rug.ac.be> >
> > ==================================================================
> >
> >
>> The on-rate depends on the antigen and antibody coming into close
> association spatially to form a complex, and the rate is mainly influenced
> by diffusion of the entities in the surrounding medium. Once the
> antigen-antibody complex has formed, the off-rate depends mainly on the
> strength of the association, which is basically the antibody affinity. The
> higher the affinity, the lower the off-rate. There is data somewhere that
> I've seen which measures the length of time that the complex persists; for
> low affinity antibodies the time was measured in milliseconds; for high
> affinity antibodies times were up to 35 seconds. These data relate to
> solid-phase bound antigen complexing and decomplexing with soluble phase
> antibody. Rates for soluble antigen and antibody will be different.
>> GS
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