Has anyone metal-labeled an IgE class monoclonal for CyTOF?

[mds4z]

I need to label an IgE-class mAb (not anti-IgE) for IMC. Standard Maxpar conjugation requires partial TCEP reduction, but IgE has a more complex disulfide structure, so I’m concerned about disrupting the antibody.

I'm unsure if the standard Maxpar labeling is compatible. Two approaches I’m considering: Amine-reactive DTPA/DOTA (no reduction) then load the metal or very mild TCEP reduction to allow maleimide-polymer conjugation without disrupting the IgE structure.

Has anyone tried a method with an IgE class? Any feedback would be appreciated.

Thanks
Mike

[mleipold]

Hi Mike,

A couple comments:

1. Years ago, I did reverse engineer a way to use Sulfo-SMCC to add a maleimide to a protein of interest, then use DTT to recreate a thiol on MAXPAR. This was an early attempt to MAXPAR-label things that don't contain thiols (eg, streptavidin). It worked (I got signal when using it as a secondary stain on a biotin-labeled antibody), but I didn't pursue it further.
- you can find my protocol here (MAXPAR-DTT-SSMCC-amine protocol.docx): viewtopic.php?p=3022#p3022

2. Most antibodies we label are IgG. However, HCD57 was an IgM CD57 antibody clone (now discontinued) that was commonly used by many labs, including my own. We never looked at gel electrophoresis or size exclusion to see whether the IgM pentamer was falling apart, but the staining was fine (bright on "right" cells, negative on "wrong" cells)
- so, I'd recommend just trying it on your IgE- class antibody; I'd probably try that first, before reverse engineering


Mike