Low-affinity MAbs in flow cytometry?

Frederick Garbrecht FRED at bmt.mcw.edu
Wed Oct 26 10:35:02 EST 1994

> To:            immunology at net.bio.net
> From:          ladasky at leland.Stanford.EDU (John Ladasky)
> Subject:       Low-affinity MAbs in flow cytometry?
> Date:          25 Oct 1994 19:28:56 GMT

> Greetings, fellow colleagues (my, doesn't that sound pompous),
> 	I was just wondering.  If you had an antibody that bound an
> epitope on a cell, but the affinity of that binding was not so high,
> what would you expect to see on a flow cytometer?  I got into a debate
> with someone at a recent lab meeting about this issue.  One of my 
> *real* fellow colleagues thinks that a cell with a low-affinity epitope
> for a given MAb would show a decreased *level* of staining when com-
> pared to a cell with the same number of binding sites, but a higher 
> affinity.  I say that, using a "normal" antibody titer, and washing
> the sample several times before analysis, a low-affinity binding would
> exhibit either saturating fluorescence or no fluorescence.  My reason-
> ing is that the half hour or so between staining and analysis, plus the
> wash steps, should be adequate to resolve any intermediate levels of
> fluorescence.
> 	Comments?  
> -- 
> Unique ID : Ladasky, John Joseph Jr.
> Title     : BA Biochemistry, U.C. Berkeley, 1989
> Location  : Stanford University, Dept. of Structural Biology, Fairchild D-105
> Keywords  : immunology, music, running, Green
We have routinely used various anti-CD8 antibodies of varying 
affinity, and I can tell you what we see.  Most of the antibodies are 
of reasonably high affinity, and they all show a characteristic 
staining pattern, and of course all stain the same number of cells in 
any given population.  The antibody with lousy affinity stains the 
same cell population (same % positive) but the distribution of the 
histogram is shifted to the left in all cases.  Presumably the low 
affinity antibody does bind to all of the cells that carry the 
recognized antigen, but because the Keq is low, the number of binding 
sites occupied at equilibrium is lower, hence the number of photons 
emitted by the fluorochrome is lower and the spectrum shifts 

Frederick C. Garbrecht
fred at bmt.mcw.edu
Bone Marrow Transplant Program
Medical College of Wisconsin
phone 414 257 5053
fax   414 257 7994

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