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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