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The approach of using an antibody to a drug to explore intra-cellular
distribution will only generate reliable data if (1) the antibody enters
the cell, (2) antibody binding does not relaese the drug from the
target(s), (3) the antibody-drug complex has 'the same' affinity for the
target(s).
Permeabilization of the cells after the drug incubation will most likely
destroy the drug-target complexes and thus yield unreliable data. However,
you may consider permeabilization/antibody loading of the cells prior to
the drug incubation. If you trap a sufficient number of antibody molecules
and they distribute randomly in the cell, this procedure may yield reliable
data.
Another approach would be to tag the drug with FITC.
And finally, you could tag the drug with an azido group, which allows you
to cross-link the drug to the target(s) after the appropriate incubation
time. You can then permeabilze the cells and use the antibody to analyze
the drug distribution.
Jan.
>----------
>From: Richard K. Meister
>Sent: Wednesday, May 28, 1997 3:20 PM
>To: cyto-inbox
>Subject: Drug studies
>
>
>Hi, everyone:
>
>I have an investigator who wants to look at intracellular distribution of a
>drug using confocal laser scanning microscopy. The drug is not fluorescent,
>so he has a monoclonal antibody (FITC conjugate) to the drug. Our question
>is, during permeablization to allow the antibody into the cells, what is the
>best way to prevent the drug from leaching out of the cells? I suppose a
>pre-permeablization paraformaldehyde fixation step would be helpful, but I
>would like to hear from anyone who has done such drug studies and knows if
>this is an effective approach.
>
>Thanks in advance.
J.F. Keij
Los Alamos National Laboratory
LS-5, MS M888
Los Alamos, NM 87545
tel (505)-667-3526
fax (505)-665-6894
e-mail keij@lanl.gov
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CD-ROM Vol 3 was produced by Monica M. Shively and other staff at the
Purdue University Cytometry Laboratories and distributed free of charge
as an educational service to the cytometry community.
If you have any comments please direct them to
Dr. J. Paul Robinson, Professor & Director,
PUCL, Purdue University, West Lafayette, IN 47907.
Phone: (765)-494-0757;
FAX(765) 494-0517;
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