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Number of Metals Per Antibody

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nameerabaig

Participant

Posts: 3

Joined: Wed Nov 20, 2019 5:20 pm

Post Thu Feb 27, 2020 10:43 pm

Number of Metals Per Antibody

Hi all,

Do you determine the number of metal atoms per antibody for custom conjugated antibodies? If so what procedure do you use and what is the typical number of metal atoms per antibody that you observe? Literature and Fluidigm says that there ought to be 100-200 metal atoms per antibody but, I am observing a wide range depending on the antibody (as low as 30 and as high as 105). Does anyone have any input on this? Thank you!
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mleipold

Guru

Posts: 2095

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Wed Mar 04, 2020 5:01 pm

Re: Number of Metals Per Antibody

Hi Nameera,

In short: in my experience, different antibodies (or, more generally, probes) label with different amounts of metal. This is generally reproducible for a given *clone*, which means that a given antibody clone may have more metal (more signal) or less metal (less signal) than a different antibody clone, even targeting the same marker and using the same polymer+metal.

Assuming 100% loading of the MAXPAR polymers the amount of metal signal that you see is based on the *distribution* of the number of polymers covalently attached to the antibody molecules in your stock solution. There's not likely to be an exact amount (eg, not likely to have 3 and only 3 X8 per antibody, on every single antibody molecule).

This can be seen in the "smear" on the SDS-PAGE gels:
Fig 4 and S1: https://doi.org/10.1002/cyto.a.23283
Fig 1: https://doi.org/10.1021/acs.molpharmaceut.7b01000

I've also heard of people HPLC-purifying their conjugates to tighten this up (and also remove any unreacted/free polymer or antibody).


Mike

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