Affinity Constant

Simon Easterbrook-Smith sbe at biochem.usyd.edu.au
Sun Jun 1 22:02:49 EST 1997


Hi Chris

(1)  Make Fab fragments from your Ab (I assume MAb, if not then you will
not get  a unique binding constant) and label the Fab with 125I.

(2)  Titrate labelled Fab into suspension of your ag-positive cells.

(3)  Measure free and bound Fab (from 125I radioactivity of cells and S/N)
under equilibrium conditions (ie when [bound] is constant with time).

(4)  Plot [bound] vs [free], fit appropriate eqn to data (usually
non-cooperative binding).

(5)  Best to correct for non-specific binding using labelled irrelevant
(but isotype-matched) Fab.

(6)  Good luck.

 


In article <5CAC1671BB5 at rna.bio.mq.edu.au>, cweir at RNA.BIO.MQ.EDU.AU ("Chris
Weir") wrote:

|| Hi all,
||    Could someone please be of assistance!
|| How do you determine the affinity constant of an antibody, when we 
|| are unable to purify the antigen (as it is expressed on the surface 
|| of the cell1)???
|| 
|| Thanks in advance
|| 
|| Chris Weir
|| Macquarie University
Simon
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