Regeneration issues

I am trying to set up an assay to look at IgM interaction with another protein. I amine couple and anti-IgM antibody to the surface of a CM5 chip. When I capture IgM (purified from serum by chromatography), I am able to get on the the order of 900-1000RU captured. However, I am unable to succesfully remove the captured IgM from the chip. I have tried High pH, Low pH, high salt (NaCl), MgCl2, ethylene glycol, Tween (up to 0.5%). I tried to look at the interaction from the reverse approach by coupling my protein of interest to the chip (neutravidin-biotin interaction) and then passed my IgM over the chip and noticed that in the channel with only neutravidin (as well as the channel containing my protein of interest) I am nonspecifically getting IgM on the chip which will not come off with low pH glycine. I am wondering if the IgM may be precipitating on the chip? Any thoughts? FYI, my running buffer is HBS-EP+ and I always dilute my proteins in this buffer. Should I up the Tween concentration to perhaps prevent IgM precipitations, or maybe up the salt? Anyone have any experience with polyclonal Ig preparations in Biacore?
rhaspel

Hi,

Regeneration of antibodies can be difficult when the interaction is very strong. Did you check the regeneration page ( www.sprpages.nl/kinetics/regeneration.html ) and tried the cocktails method of Andersson (Andersson, K. et al Identification and optimization of regeneration conditions for affinity- based biosensor assays. A multivariate cocktail approach. Analytical Chemistry 71: 2475-2481; (1999).)?

Sometimes it is better to use an antibody which interacts less strong.

The reasons of non-specific binding of IgM can be many. It is often difficult to find out what is causing the problem. I would increase the salt concentration (200 -250 mM) in stead of the detergent to avoid precipitation of the IgM. It is worth to look at other buffers like PBS or TBS if they give the same problems.
Spin down your IgG stock (5' hard) prior to dilution to remove precipitates in the stock.

regards

Arnoud

Thanks...I am trying all those suggestions. I have also switched to a CM4 chip which has decreased background substantially so I think my antibody prep is cross reacting with th e dextran. I am now trying to add cm-dextran to my samples as well as working with other buffers. On another note, I have not been preconditioning my chips before immobilizing protein....is this recommended (I was never instructed to do this with the BiaCORE x100)?

Thanks

Robyn

I'am not used to precondition CM5 sensor chips. I cannot help you there.

Maybe you can pre-incubate the antibodies with dextran and then spin them down to get rid of the species binding dextran.

Arnoud