Methods and Techniques Discussion => Immunocytochemistry (ICC) => Topic started by: MFeirer on January 26, 2012, 06:15:54 PM

Title: Issue with secondary antibody binding to B/Plasma Cells
Post by: MFeirer on January 26, 2012, 06:15:54 PM
Another antibody question for the group:

I'm staining canine lymph nodes (imprints) for MUM1,using a mouse monoclonal antibody.  For my detection, I stain with a F(ab')2 Goat x Mouse HRP antibody, followed by DAB treatment.  For the last several months, I haven't had any issues with my negative controls (PBS in place of my primary), but several days ago my negative controls have been very positive.  There's intense cytoplasmic staining in plasma cells and what I presume to be activated B's.  No other lymphocytes or any other cell stains.  While I haven't used this secondary in several weeks, what's strange is that I haven't had issues like this before.  I've gone back and made up new buffers, gotten new pipettes, etc, and it's still happening.

I've ruled out issues with inadequate peroxidase quenching, so it has to be the secondary.  What's odd is that I also have a F(ab')2 Goat x Rabbit HRP secondary, purchased at the same time (5 months ago) and stored the same way, which doesn't have these issues.  My only guess is that this secondary has degraded inappropriately.  Has anyone else experienced a problem like this?  Could the antibody be good, and for some reason, these 3 dogs are specifically binding the secondary for some reason?

I thought about my goat secondary being non-specifically bound by Fc receptors on the dog lymphocytes, but then again, being a F(ab')2 antibody, my secondary lacks Fc, so I don't think it can be that.  My latest nodes have been more reactive than those in the past, so there are more plasma cells present, which could also be a factor (although I did have nodes with significant plasma cells in the past, and this inappropriate staining wasn't present).

Thanks for any answers/insights anyone can give.  I've got a new secondary coming tomorrow, so I'll be able to compare old vs. new batch.
Title: Re: Issue with secondary antibody binding to B/Plasma Cells
Post by: CanuckPhD on January 27, 2012, 02:17:05 AM
Your goat anti-mouse secondary may be crossreacting with the dog IgG in the plasma cells. Is it highly absorbed against other species? To test it add some dog plasma to your secondary IgG let it incubate for 30 minutes and then apply it to your sections. If there is no more staining then you know it is crossreacting with dog IgG. To avoid this in the future add some dog plasma to your secondary antibody (1-5% should suffice I think).

Good luck