I don't know what the effect of temperature on antibody
binding is, but I would not add the antibody to the
phagocytosis, only afterwards as done in the Phagotest. Some
of the preservatives may interfere with the assay and if the
cells are fit enough they would internalize the antibody.
If that organism would compete with the binding site it
should also occur at 4oC.
Gerhard.Nebe-von-Caron@unilever.com
______________________________ Reply Separator _________________________________
Subject: phagocytosis
Author: paukovic@mbcmr.unimelb.edu.au at INTERNET
Date: 04/02/97 01:34
Hey ho Flow-ers,
We have an established phagocytosis protocol, which HAS worked well for us.
The organism being used is Mycobacterium avium-FITC labelled (MA-FITC),
which is phagocytosed by HIV- and HIV+ monocytes. (a commercial quenching
agent is being used.) Of recent times we have switched to a Becton Dickinson
(BD) CD14-PE antobody (Ab) from a DAKO CD14-PE. The problem encountered is
that where as once I used to get staining at 37C with DAKO CD14, the BD Ab
fails to do this at the same temperature, however the BD Ab stains
beautifully at 4C.
So the dilemma?
Has anyone come across inhibition of binding of BD Abs, (or any other Ab for
that matter) when working with MA at 37C (or any temp). Is it common for BD
Ab to become inactive if kept at 37 for only 10mins (in our protocol) with
MA or any other organism?? Does MA have properties which inactivate this
particular BD CD14-PE...at 37C?? Is there a chance of MA digesting PE at
37C, rendering it non fluorescent but still bound??
(and oh yes, the antibody hasn't expired yet)
Fanx... In advance
Geza Paukovics - Flow Laboratory - AIDS Pathogenesis Research Unit
Macfarlane Burnet Centre .***. .***. .**
PO Box 254 _--_|\ * | | | * * | |
Fairfield, VIC 3078 / \ * * | | | * * | | |
AUSTRALIA. \_.--._/ * * | | | * | | | *
ph. (+61 3) 9282 2132, O '***' '***'
FAX (+61 3) 9282 2100
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