Re: Non-specific binding
Karel Drbal (drbal@leuko.biomed.cas.cz)
Wed, 24 Apr 1996 14:36:35 -0600
On Apr 23, 4:19pm, Peter Chapple wrote:
> Subject: Non-specific binding
> I'm sure that I am not alone in noticing that some antibody clones have a
> higher level of background staining than others. In my case, I have a
> particular FITC conjugated CD19 (gamma 1) which appears to have a
> substantially higher background labelling than my gamma1 negative control
> (approx half a decade higher).
>
> My question is what explaination of this phenomenon do people offer to end
> users (most notably Pathologists) who want to know what the fundamental
> biology of this interaction is ??.
>
> I have shown with various gating demonstrations that the background level is
> constant for different sub-populations of cells (lymphoid, granulocytic,
> monocytic and *blast cells*) - so it is apparently unrelated to Fc receptor
> presence/density.
>
> A plausible explaination will save me many hours of talking
>
> Thanks in advance.
>
>
> Peter Chapple
> Peter MacCallum Cancer Institute
> Melbourne AUSTRALIA
>
>
>-- End of excerpt from Peter Chapple
Hi Peter,
I have seen that phenomenon quite often during testing different batches (one
batch originate from one mouse) of ascites from the same hybridoma cells. The
background fluorescence was sometimes one decade higher (max.). I also realized
it has nothing to do with Fc receptors.
There are two additional questions:
1/ Does your mAb concentration match your isotypic control conc.? > If yes,
than we can't speak about "non-specific" binding but this is rather specific.
2/ Is your particular mAb an ascites fluid or a culture supernatant? > If it is
an ascites fluid, my interpretation would be that the higher background comes
from natural antibodies (usually low affinity Ab against carbohydrates,
cross-reactive) and their levels could be different in a particular mouse,
therefore different batches of mAb could give you various background staining.
I am not sure whether this applies also to culture supernatants (I have not
tested large number of supernatants) but I have not seen such a variability in
their background staining.
Hope this helps.
--
_______________________________________________________________
Karel Drbal
Institute of Molecular Genetics
Academy of Sciences of the Czech Republic
Videnska 1083
142 20 PRAHA 4
Czech Republic, Europe
voice: +42-2-4752589
fax: +42-2-4727979
e-mail: drbal@biomed.cas.cz
WWW: http://leuko.biomed.cas.cz
CD ROM Vol 2 was produced by 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:(317) 494-0757; FAX (317) 494-0517; Web http://www.cyto.purdue.edu
EMAIL robinson@flowcyt.cyto.purdue.edu
