ELISAs are probably your best bet here. Depending on the species, there are likely commercial kits available that can give you actual concentrations per antibody isotype based on standard curves. If testing a non-standard species, you can always use a home made ELISA as long as you have anti-Ig antibodies or sera you can use for probing.

If these are murine B cells, there are a number of commercial kits sold by BioLegend for basically all isotypes of mouse immunoglobulins

Arlind Mara Thanks for the suggestion! These are indeed murine B-cells, but they were exposed to wild-type cancer cells rather than a specific antigen. Since I’m not targeting a particular antigen-antibody interaction, I’m just looking to measure whether the B-cells are producing antibodies at all - and if so, quantify the total antibody concentration (regardless of specificity).

Hi Erica Weber

You should still be able to use a direct ELISA to do so, simply coat the plate with purified protein from the supernatants. Just do a buffer exchange to a carb-bicarb buffer, coat the plate overnight, and probe with anti-mouse Ig antibody. I think there are some clones that recognize both pan IgG and IgM, but you’ll likely have to probe IgE and IgA separately. You can also use a molecular weight cut off filter to concentrate first and then do the buffer exchange.

if you find you have trouble with coating the plates, you can also try a dot blot or a western blot and do densitometry analysis for a semi-quantitative look. As long as you compare to a medium only control, or unstimulated cell control, that should give you what you need.

Good luck!

Thank you for the details Arlind Mara

Just to make sure we’re on the same page - I’m trying to measure total antibody secretions from these B cells, regardless of specificity. Since the wild-type cancer cells overexpress a bunch of antigens (like MAGE, CEA, HER2, and p53), I don’t want to limit detection to just one antigen (e.g., coating with p53 and missing antibodies against the others).

Just one more thing - since i'm working with limited sample volumes, coating with all the different cancer antigens isn't really practical for this experiment. I'm really just trying to get a basic readout: are these B cells producing antibodies at all, and if so, what's the total concentration

Hi Erica Weber

That is fine, you can still just coat with the supernatant from the B cell culture which will contain your total antibody/immunoglobulin.

This does not restrict you to antigen specific cells, and is a standard assay by which researchers can measure total immunoglobulin in serum samples for example. Should work just as well on the supernatants

Start with ELISA for accurate, antibody-specific quantification.

Hello Arlind Mara, is there a better way to purify antibodies from the supernatant without losing so much protein? I tried using MWCO spin columns, but I ended up with barely anything - just 0.07 mg/ml after an A280 check. This was from supernatant cultured from 1 million plasma cells (B cells), which came from like 20 mouse spleens. Any recommendations to improve recovery?