Antibody's specifity...

Bryan Heit bjheit at
Thu Jul 24 10:32:24 EST 2003

Sorry this took so long, I ran some of this by one of the B-cell people 
here, and I think our "conflict" is more a matter of semantics and our 
background.  Most of the B-cell/Ab material I am familiar with are from 
the labs here who study the development of antibody repertoire, which 
apparently has it's own definitions for things like affinity and 
sepcificty, and these are not widely used outside of their field.  Also, 
I snipped a lot, not trying to hide anything, my server doesn't let me 
post more old text then new...

Mike Clark wrote:

>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
Which, assuming we heard the same story, was cross reacting with CD4 on 
the microgleal cells.  If this is the case then you're talking about a 
cross-species reaction reaction between the same gene product.

>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.
Depends on your definition of specific.  This of course depends on where 
you are coming from, but the labs here have some very specific 
definitions of specificity, including ranges of on/off rate, Kd's, etc, 
that they use to define specificity for an antigen.  Obviously this 
doesn't have much impact on doing a western blot, but when you are 
looking an repertoire development, etc, you need a way to quantify 
"specificity" and apparently there way is commonly accepted.

>>But this process has almost nothing to do with the  maturation of an
>>antibody you would buy or isolate.
Bad wording on my part.  What I was trying to say is that the initial 
stages of B-cell development (i.e. positive and negative selection) will 
generate antibodies that are generally of: low affinity for any 
particular antigen, and will bear little resemblance to the antibodies 
generated after an immune response.

>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?
But irregardless of the vaccination method all of the antibodies will 
have some general characteristics:

1) as a group they will identify a limited range of antigen epitopes.
2) The recognized epitiopes will be derived from the antigen(s) 
administered.  This may be only one epitope (i.e. in the case of an 
injected peptide), or multiple epitopes (in the case of an injected 
protein or group of proteins)
3) each individual altibody will liekly only recognise 1 epitope in the 
origonal antigen
4) There are exceptions for 1&2, for example if you were to "vaccinate" 
for anergy you will not expand/develop the B-cell repertoire, but lets 
just assume you are vaccinating to develop an immune response.

>[2] How many times was the animal immunised? Was the same form of immunogen
>or antigen used every time?
Just one point, if you're using a different immunogen you are going to 
generate a new response that will have nothing to do with the previous 
response.  A different group of naive cells will be expanded and 
selected upon, whereas the cells from your original will be unaffected.

>[3] Am I using a polyclonal antisera from the animal? If so has it been
>purified in any way?
But in the case of the original question a polyclonal serum isn't really 
applicable here.  The question was about 1 AB recognizing >1 Ag, with 
low homology between the antigens.  After all, you could take serum from 
you or me and find antibodies what react with pretty much anything under 
the sun - but it isn't one antibody doing these interactions, it's 
thousands of different antibodies, each reacting with one (or a few) 

>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
I've seen a few people who would disagree with this point - I've heard a 
few people argue that self mimicry may work because there is a lack of 
negative selection during affinity maturation.  I don't know if I buy 
this argument, but epitope spreading does occur during affinity 
maturation, a process driven by forming "new" antibodies that react with 
things other then then original antigen.  And from what I understand of 
the process the selection that does occur during this process is diven 
by affinity - B-cells with low affinity for the antigens present in the 
GC get outcompeted and die.  I am unaware of any proof that negative 
selection against self antigens occurs at this phase (not to say there 
isn't, B-cell development is not my area of specialty).

>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
Not to be rude to those scientists, but that suggests an error on their 
part.  You mention that this was a problem a while ago - and we all know 
that even today antibody purification from sera is plagued with all 
kinds of problems, including pulling out other antibodies that aren't 
specific to your antigen.   Before I would accept that there results 
represent cross reactivity of a single antibody I would ask:

1) were they truly working with a monoclonal?
2) how did they select/purify their antibody?
3) how pure was their original antigen used for immunization?
4) how similar were the proteins that they identified in comparison to 
the protein the antibody actually identified.

I would argue that their results were more likely a result of poor 
antibody purity, rather then cross-reactivity between dissimilar epitopes.

>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.
But they mimic the structure of the epitope, which is the point I was 
trying to argue.  Most cross reacting antibodies I have run into cross 
reacted because they were identifying a shared epitope on different 
proteins (i.e. have the same amino acid sequence and identical 3D 
structure).  A perfect example of this is the phospho-tyrosine antibody. 
 This antibody identifies epitopes on many different proteins, but it is 
the SAME epitope - phorphylated tyrosine.  So at the point of 
identification there is "100% homology" between the proteins.  As I said 
previously, I've never heard of an antibody cross reacting with another, 
completely different epitope.

>>>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.
See, this is where we're arguing over definitions.  As I mentioned at 
the beginning most of the interactions I've had with B-cell people is 
with groups who look at repertoire development.  They have very specific 
definitions of affinity and specificty, as they need a "ruler" to 
measure the development of the immune response.  

I will concede that specificity can vary depending on the assay you put 
an antibody into, or the conditions you use it at, but I would argue 
that these variations in specificity are more a product of the assay's 
rather then the antibodies.  For example, in a western blot your 
proteins will be partially denatured, even if you use a "non-denaturing" 
gel.  This is simply a product of the solvents and gels we use for 
blotting.  So an antibody designed for use with ELISA, which "never" 
sees denatured protein (assuming you're better at ELISA's then I am), 
may not work in a western.  But this variation in "specificity" is due 
to environment, rather then an intrinsic characteristic of the antibody. 
 Likewise, differences in temperature, osmolarity, pH, etc can affect 
protein conformation and thus antibody affinity, but this once again is 
due to the assay rather then the antibody.

>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? 
Ahh, but you could easily define the specificity as the affinity for the 
now mutated antigens.  You could easily determine the Kd, on/off rates, 
etc for each antigen.  From this data you could state the specificity 
for each antigen empirically.  But you seem to be arguing my point (or 
maybe I missed yours).  I was arguing (apparently badly) that a single 
antibody tends to recognize a single epitope.  You can modify this 
epitope in minor ways and still have some antibody binding to the 
epitope, but it is highly unlikely that the antibody will identify an 
dissimilar eptiope - like the one in the original question which only 
had 50% homology to the original epitope.  It is conceivable that you 
could "build" a similarly shaped/charged epitiope from a completely 
different amino acid sequence that would be recognized, but I would 
argue this would arise naturally very rarely, and even if done 
deliberately would be difficult.

>Specificity IS defined by the system, it is NOT an absolute concept.
Once again, depends on how you are defining specificity.  I tend to 
think in vivo, and in vivo specificity is very well defined.  But we're 
arguing semantics and definitions, which no one will ever win.

>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?
Many of the antibodies we used origonally were raised in rabbits because 
we hoped they would cross react with both human and mouse.  In rabbits 
we could raise both anti-human and anti-mouse antibodies, but they 
rarely cross reacted.  In hind sight these are bad example, as many 
adhesion molecules are highly glycosylated and these glycosylation 
states are variable between species.  

A better example would be the TLR4 antibody I'm fighting with right now. 
 It was raised using a peptide, rather then the whole molecule, and the 
portion used has near perfect a.a. homology between mice and humans 
(this was done with the hopes of creating a Ab that cross reacts b/w 
species).  The antibody binds beautifully to human TLR4 and shows little 
cross reactivity on westerns or FACS (you need to use massive amounts of 
antibody to get non-specific bands on western).  We can only get random 
binding to mouse cells.  

>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?
Varies, depending on the antibody.  All are monoclonal (nothing else 
would be publishable), most from rats or rabbits (we have one raised in 
a donkey, god only knows why).  Since many are purchased I don't know 
about auto/allo reactivity.

>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
This is often a problem with mice, but it kinda proves the point I was 
trying to make (apparently badly): antibodies recognize a single 
epitope, and rarely will identify >1 protein unless the proteins share 
identical or nearly identical epitopes.  I still think we're arguing 
about semantic/definitions.  I seem to be taking a more biochemical view 
of things - defining specificity as binding to only one epitope, 
irregardless of the location of the epitope.  You seem to be arguing 
from a techniques point - antibodies can and do react between proteins 
and species, but going back to my point, this usually occurs as a result 
of shared epitopes rather then specificity for another epitope.


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