Cell ELISA - No differences between cells expressing and not, the receptor (Mar/03/2011 )

Hello

I have been trying to get a cell ELISA right for about 6 months without success and would greatly value any advice.

I have some MDCK cells expressing a chemokine receptor which binds IL8, as well as control cells without the receptor (also MDCK). I have an anti-human IL8 (mouse and goat types), and have tried both traditional HRP optical density and fluorescence to detect a signal.

The problem is that I get essentially equivalent signal strength from the cells bearing the IL8 receptor and those not, both by HRP and fluorescence. However when I did a fluorescent stain with anti-IL8 after incubating it, and looked under confocal microscopy I can see membrane fluorescence on the cells with the receptor and not in those which don't have it.

Also my results reveal higher and equivalent values in wells where I am or not adding IL8, primary antibody or not, secondary antibody or not, and only the cells have have only had washes give much lower signal.

Procedure: Cells are seeded and checked to be confluent, I incubate the IL-8 for 45 min on ice, wash off x3 with PBS, then fix with 4% paraformaldehyde, wash x3, block with avidin and biotin if using HRP method, and then wash with PBS-tween 0.05% followed by a block step with PBS-tween and BSA 1% (Fraction V Cohn). Then I proceed to primary antibody (goat anti human IL8 but similar problem with a different mouse anti-human), wash 3 times and secondary (fluorescent or HRP conjugate), followed by development if appropriate.

I don't understand why we are having these issues. In particular what appears to be significant background and fluorescence given off even if no secondary antibody (conjugated) is being used (??).

I can post excel graphs of optical density results and my plate layout scheme if this would be useful.

I would really appreciate anyone's comments.

Thanks for reading this

inov

Hi inov:)
I don't think that I can really help but I read your post because I'm gonna try cell ELISA these days and I am just checking for more information here and there. I can only help you by thinking on your problem.

Looking at what you've written it seems that your problem comes from the primery/secondary Ab-because you have either one of them in all controls with high absorbance values (and you don't have neither in the clear control-control with the cells only). What is the secondary Ab specific for-whole molecule or just Fc-specific?
You can try dilute them more (but I think you have already tried this). Also you can try to block the plate with higher concentration of your blocking agent. Or try different blocking buffer.
Wish you success with future work!

Best, Vera

Seems the primary or secondary are sticking or binding non-specifically. Read about heterophile antibodies and blocking reagents and you may find a path toward resolving your problem (suggested by Vera)