Antibody clean up prior to conjugation

Does this antibody work? Designing a new panel?
Antibodies, isotopes, etc. for Imaging Mass Cytometry applications
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Akil
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Antibody clean up prior to conjugation

Post by Akil » Wed Feb 28, 2018 5:02 pm

I know the requirement for MaxPar conjugation is to have carrier free antibodies, but has anybody determined a minimal concentration level that is permissible? What I mean is that other primary amine based conjugations allow, for example, up to 0.1% gelatin or 0.1% BSA and still seem to work.

Also, is there is an option to “concentrate” rather than purify (essentially a buffer exchange) antibodies which greatly reduces but does not 100% clean up the other proteins? This option is nice because loss of antibody is less.

Are there preferred antibody clean up kits/methods that people on the group would recommend that work best upstream of MaxPar labeling? Or for specific carriers like BSA/glycerol or Gelatin etc.
mleipold
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Re: Antibody clean up prior to conjugation

Post by mleipold » Wed Feb 28, 2018 6:27 pm

Hi Akil,

Gelatin does not have any Cys, therefore it won't interfere in the conjugation reaction (ie, won't react with the MAXPAR maleimide instead of your antibody). The main issue with gelatin is that if it's not removed, it might affect your protein concentration determination (Nanodrop reading) at the end of the prep.

Glycerol, trehalose, and azide are also fine to have around: they're small molecules, so they'll get washed away during the R buffer exchange step. Also, they don't have any thiols, so they wouldn't interfere anyway.

I do know that BSA can interfere (33 Cys, as 16 disulfides and 1 free), but at what point the BSA conc is low enough to not *significantly* interfere, I don't know. At the very least, it would decrease your signal intensity if some of the BSA got labeled rather than your antibody.

I know some people have used BSA removal kits (https://www.cytobank.org/nolanlab/conju ... tocol.html), but you'll have some loss of your antibody as well.

Most standard IgG antibodies have MW in the 120-150kDa range. BSA is *primarily* a monomer at about 66kDa, so if you had a 100kDa MWCO spin filter, you might be able to get rid of *most* of the BSA with repeated washes. However, BSA can also form dimers (~120-130kDa), which wouldn't be efficiently removed with a 100kDa MWCO filter.

You could also try concentrating/purifying the antibody with a protein A/G column, but that's generally done at the preparative scale when you're purifying your own antibodies from a hybridoma or something; I'm not sure it's really worth it for 100ug at a time.


Mike
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bernd.bodenmiller
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Re: Antibody clean up prior to conjugation

Post by bernd.bodenmiller » Tue Mar 13, 2018 2:48 pm

Hi Akil,

My co-workers Andrea Jacobs and Stefanie Engler once looked into this. They found that for suspension MC up to 0.1 % BSA (depending on the antibody concentration) is tolerated. They also tested the same antibodies for IMC and found that these conjugates produce a higher background staining compared to the purified antibody. Since the conjugations with BSA are not as reproducible as the ones without BSA and the often associated higher effort (e.g. antibody titrations for each new antibody lot since in many cases the antibody concentration is not known) we stopped using BSA containing antibodies. In case an antibody is only available in BSA, we purify it before labeling. We either use Magne™ Protein A/G Beads from Promega or the Pierce™ Antibody Clean-up Kit. For both techniques we calculate a 50 % antibody loss if you are in the low 100s microgram range of starting material. If you have lots of starting material losses go down, of course.

Best,
Bernd
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Akil
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Re: Antibody clean up prior to conjugation

Post by Akil » Tue Mar 13, 2018 4:26 pm

Thanks to all those that replied. Very helpful.
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