Antibody titering for flow cytometry

Rachel Caspi rcaspi at helix.nih.gov
Thu Oct 6 18:42:18 EST 1994


In article <36uq0b$58m at nntp.Stanford.EDU>, ladasky at leland.Stanford.EDU
(John Ladasky) wrote:

  > Greetings, everyone,
  > 
  >         Using a flow cytometer and an antibody at various dilutions, I've
  > just analyzed the fluorescence intensity of a particular cell-antibody 
  > combination.  I now have a nice, asymptotic curve.
  > 
  >                             Mean
  >         ug MAb added    Fluorescence
  >         ----------------------------
  >             0.00               8
  >             0.63             364
  >             1.25             541
  >             2.50             812
  >             5.00            1050
  >         
  >         Now what?  At what level is an antibody considered to be "titered"?
  > Any advice or references would be greatly appreciated.  Thanks!
  > 
  > 
  > -- 
  > Unique ID : Ladasky, John Joseph Jr.
  > Title     : BA Biochemistry, U.C. Berkeley, 1989
  > Location  : Stanford University Dept. of Cell Biology, Fairchild D-105
  > Keywords  : immunology, music, running, Green

Theoretically, antibody is titered when increasing the concentration does
not increase the mean fluorescence, i.e., you have saturated all the
binding sites. In practice, increasing the concentration to that point
sometimes results in unacceptably high backgrounds, particularly with
lower affinity antibodies. The best concentration of antibody to use can
only be decided by looking at the distribution plots. What you are looking
for is maximal peak separation between positive and negative cells, with
minimal background in the negative population.

-- 
   Rachel R. Caspi, Ph.D.                         phone: 301-496-6409 
   Laboratory of Immunology, NEI                         301-496-6394 
   NIH Building 10, Room 10N222                   fax:   301-402-0485
   Bethesda, Maryland 20892               email: rcaspi at helix.nih.gov 



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