I am trying to set up a screening method for antibodies to galectin-3 to block galectin-3 function (galectin-3 sourced from Peprotech). So far I have tried to use neutrophil activation, monocyte chemotaxis and red blood cell haemaglutination.
The neutrophils appear not to be activated by galectin-3 (as measured by ROS production with a luminol assay). Similarly, galectin-3 doesn't appear to induce chemotaxis in monocytes- either with primary monocytes or THP-1 cells.
The only test thus far is that galectin-3 induces red blood cell haemaglutination. This can be blocked using an inhibitor, but doesn't appear to be blocked by a galectin-3 anitbody (a B2C10 clone which has been shown to have function blocking effects).
If anyone has any other alternate assays or advise on these it would be greatly appreciated!
Hello Ross,
According to published papers, neutrophil and moncyte are known to secrete Gal3 protein themselves. The logic behind Gal3 activation experiment is justifiable? Is your inhibitor used glycoconjugate?
I found in our many experiments that intracellular accumulation of ROS is also caused by non-genetic components present in the cell (no receptor and ligand necessary). Those cells are not immune cells by the way. Thus ROS is a validated final scoring point in Gal3-treated cells?
Gal3 appears to be involved in glycosylation of terminal oligosaccharides of interacting cell glycoproteins, thus I would think a-D-GalNAc and a-D-Gal containing sugars may be an option to get a clear meaning of inhibition although addition of these sugars together needs attention in pH change of medium. Since neutrophil and monocyte show intrinsically secretion of Gal3, I am not sure whether you would get clear inhibition. But try for the selection of a specific antibody or something else.
Best wishes.
Hi,
Thanks for the answers, they're helpful. In part what we have been trying to do is two fold
Look at the ROS production of neutrophils from activation with Gal-3, since this is such a short assay (
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