Antibody's specifity...

Mike Clark mrc7 at
Thu Jul 24 07:22:10 EST 2003

In article <3F1F1222.9000403 at>, Bryan Heit
<URL:mailto:bjheit at> wrote:
> Comments within:
> Mike Clark wrote:
> ><snip>
> >
> > Antibodies may or may not be "specific" depending upon how they are
> > selected and how in practice you define specificity. Specificity is not
> > an absolute universal concept. In most cases antibody specifity is
> > defined for a narrow application based on emprical observation. e.g.
> > "In ELISA assays my antibody only binds to antigen A but not to control
> > antigens B, C, or D."
> >
> But how many times have you run a cell lysate blot with an antibody and 
> seen greater then 1 line?  It's rare, and when it does occur it's 
> usually human error or a closely related protein.  In all of the blots 
> and elisa's I've done (that's a lot) I've never once encountered an 
> antibody with cross reactivity for a non-related protein, and that 
> includes some "dirty" polyclonal serums I derived myself.

My answer is based on a lot of experience of working with monoclonal
antibodies and polyclonal antibodies, going right the way back to my PhD
project in the laboratory of Cesar Milstein in which I was looking at the
properties of monoclonal antibodies made and selected in different ways.

"Antibodies in Somatic Cell Hybrids" 1978-1981, University of Cambridge

Over the years several monoclonal antibodies have been reported in the
literature with biological cross reactions and many more have gone
unreported because it is not considered worthy of repeated publication of
a similar trivial observation. One of the first I can recall was an
antibody to human CD4 which cross reacted with an antigen in the brain of

The point is that most antibodies are only defined as "specific" when they
fail to show detectable cross reactions in the assay in which they are put.
If you buy antibodies out of a catalogue advertised for one purpose and
then put them into a different assay system you frequently find a change in
the absolute specificity because of a change in the absolute sensitivity of
the system.

> > The physiological specificity of an antibody usually results from both
> > a positive selection of B-cells on one antigen and negative selection
> > on others. The most commonly encountered negative selection in-vivo is
> > negative selection for autoreactivity.
> >
> You are mixing up B-cell development with germinal centre formation. 
>  During B-cells development the pre-B-cells undergo immunoglobin gene 
> rearrangement.  Cells which fail rearrangement die, a process known as 
> positive selection.  Once the gene rearrangement is complete the cells 
> then undergo negative selection (how this occurs is not clear).  During 
> negative selection B-cells which react with self-proteins are 
> eliminated.

Whilst I agree that my reference to "B-cells" was a simplification of the
process I stand by the statement and I am not failing to understand
different developmental stages of the B-lineage. When I said positive and
negative selection I was using the word selection in the broadest sense,
not as a description of a single developmental step as you are trying to
interpret it. Apologies if I misled through my simplification.

> But this process has almost nothing to do with the  maturation of an
> antibody you would buy or isolate.

Multiple steps of selection have everything to do with what you buy and or
isolate. I will mention a few of the many that there might be.

[1] What was the nature of the immunogen? Was it a pure antigen or was it a
mixture of antigens? Was it in adjuvant? How closely related is the antigen
to any similar host antigen in the animal immunised?

[2] How many times was the animal immunised? Was the same form of immunogen
or antigen used every time?

[3] Am I using a polyclonal antisera from the animal? If so has it been
purified in any way?

[3b] Has my antisera been affinity purified on antigen?

[3c] Has my antisera been purified so that it represents a restricted
portion of the antisera such as the IgG fraction purified on protein-A?

[3d] Has my antisera been adsorbed for removal of "non-specific"
or "cross-reacting" antibodies?

[4] Am I using a monoclonal antibody? If so how was it selected for further
study? How many monoclonals were rejected because they failed to satisfy
the selection criteria for further study?

I could probably go on for hours about point 4. Indeed one of the first
lessons we all learnt about monoclonal antibodies was the following 

 "What you get is frequently what you selected for, but not necessarily
  what you wanted"
I attribute my learning of that lesson to one of my tutors whilst I was  in
the Milstein laboratory, Giovanni Galfre, who was in the late 70s and early
80s one of the most prolific of scientists making monoclonal antibodies
and collaborating on the production of many of the first monoclonal
antibodoies to human cell surface antigens, to rat cell surface antigens,
to mouse cell surface antigens, to neurotransmitters, to purified proteins
etc etc.

> When a B-cell becomes activated by an antigen (i.e. when you immunize 
> the animal to raise your antibody) you are only activating the cells 
> which have undergone negative and positive selection.  Chances are that 
> some of these cells will weakly recognize the antigen you inject and 
> start to divide.  As these cells divide they deliberately mutate their 
> immunoglobin gene to make "new" versions of their original immunoglobin. 
>  Occasionally this process results in an immunoglobin with higher 
> affinity, and there are mechanisms within the lymphoid tissues to select 
> for these higher affinity cells (a process called affinity maturation). 
>  The end effect of this is after we immunize an animal a limited number 
> of B-cells will be formed.  All will express antibodies with high 
> affinity and specificity to the antigen you immunize the animal with.

But you forget to point out that at each stage of somatic mutation there is
the potential for a different affinity on a range of different antigens
including the immunogen, but also including other self-antigens. Thus a
process of negative selection is required following each cycle of somatic

> > So the answer to the original question posed by Nikolai above is, yes,
> > under some circumstances.
> >
> Can you cite a single occurrence where antibodies show cross-specificity 
> with a protein only 50% identical, unless it is recognizing a shared 
> epitope?  I did a quick check of pubmed and could not (not to say there 
> isn't, I didn't look too closely).

I'll refer you again to my thought experiment. If you raise a monoclonal
antibody to any antigen you can subsequently raise an anti-idiotype to that
same antibody. That anti-idiotype is as much of an antigen as the first
antigen was. It would be quite reasonable for different anti-idiotypes to
differ by 50% homology in their V-region sequences, and if you selected
anti-idiotypes of different subclasses or classes you could have a
difference in the C-regions of approximately 50%. So I don't need to hunt
in the literature for what is obvious from first principles.

And if you don't like the idea of anti-idiotypes because you think I am
cheating (which I don't think I am because the concept of antibody and
antigen in the case of idiotype and anti-idiotype is only one of arguing
semantics), I'll bring up another example.

In the early days of making monoclonal antibodies scientists frequently
wanted to isolate the genes encoding the antigen recognised by the
antibody. They did this by selection of random shot-gun clones using
monoclonal antibodies. Many, many scientists ended up cloning and
expressing the "wrong" genes based on reactivity with their antibody! Few
of these scientists wrote up their failure to clone and select the right

Finally have you never read the extensive literature on "mimitopes"?
Mimitopes are selected antigens, often from peptide libraries, which show
reactivity with a selected antibody. However they are called mimitopes
because they mimmick an epitope of the antigen, rather than having identity
to the antigen.

> > Also all antibodies have the potential to bind a large number of
> > different antigens and thus specificity can easily be seen to be  a
> > "relative" concept.  
> >
> Specificity is not a relative concept, at least not in any of the lab's 
> I've worked with.

Specificity is a relative concept and it is a pity that the labs ypu have
worked in haven't appreciated this fundamental scientific principle. If you
increase the sensitivity of an assay procedure you often reduce the
specificity because you start to see low affinity cross-reactions which
were originally below your threshold of selection. Specificity is entirely
dependent on a definition of thresholds at which you regard a result as
negative. If you change the threshold then defined negatives and positives
in the system change as well.

I'll give you another thought experiment. Take an antibody-antigen crystal
structure. Now make selected mutations in the contact residues for
antigen one at a time. Conservative changes are likely to make small
changes in the affinity of interaction, whilst non-conservative changes are
likely to have a big effect.

How specific is you antibody if you define specificity in terms of ability
to descriminate different mutated forms of your antigen? 

Specificity IS defined by the system, it is NOT an absolute concept.

> Antibody specificity is well demonstrated in the  literature - it is very
> rare for an antibody raised against an antigen  to identify other
> antigens, even when closely related.  For example, in  my lab we do a lot
> of work looking at the adhesion and signalling  molecules involved in
> leukocyte recruitment.  We use both murine and  human models, and we need
> separate antibodies for both systems, even  though it's not uncommon for
> the murine and human forms of these  proteins to be greater then 95% at
> the amino acid level, and almost  identical structurally.  

Ah! But return to my numbered points above! Are your monoclonal antibodies
to human adhesion and signalling molecules by any chance mouse monoclonal
antibodies? If so then were they made by immunising a mouse with human
antigen? Have you tried them out on other species such as dog, rat,
non-human primate, rabbit etc etc? What then of specificity?

Are the anti-mouse antibodies also mouse monoclonals? Were these raised by
immunising mice with mouse antigen? Are the monoclonals auto-reactive or
allo-reactive in specificity?

Of course you may be working with another species of antibody such as rat
monoclonal antibodies. But them of course the immune response in a rat has
undergone negative selection on rat antigens, so you need to know how the
similar human, mouse and rat antigens might be related by sequence and

> > You can illustrate this principle with a simple thought experiment. For
> > example if you take an antibody to any given antigen, and then you
> > raise a series of different anti-idiotype antibodies to that first
> > antibody, you essentially have a whole family of different antigens all
> > with binding affinity to the same antibody.
> >
> Or you could look at it as a group of antibodies all recognizing the 
> same antigen.  Idiotype antibodies are a special case, as it is hard to 
> identify what is the "antigen" and what is the "antibody".  As I said 
> previously, I am unaware of any study showing cross reactivity between 
> proteins with little/no homology.

Anticipating you might reject my anti-idiotype system I also brought up the
examples of shotgun cloning and selection, and mimitope selection, both of
which are well covered in the literature and also unpublished laboratory


Mike Clark,                        <URL:>
M.R. Clark, PhD. Division of Immunology
Cambridge University, Dept. Pathology
Tennis Court Rd., Cambridge CB2 1QP
Tel.+44 1223 333705  Fax.+44 1223 333875

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