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Bismuth conjugataion

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GregHopkins

Participant

Posts: 12

Joined: Tue Apr 11, 2017 8:39 pm

Post Tue May 07, 2019 5:55 pm

Re: Bismuth conjugation

Hello all,

I would like to resurrect this old post to get some new feedback.

Recently, we have had some issues with the 209Bi CD8 conjugates we have been getting done externally for us. It appears that either the titre we get is highly variable, or that the antibodies don't work well, or die off very quickly (the intensity of staining goes down and background creeps up to almost "swallow" the positive) I understand the challenges in the conjugation chemistry, so am certainly not saying this is unexpected.

We are looking to potentially start doing the 209Bi conjugations ourselves and wanted to get overall (updated) opinion on what the best source was for 209Bi and in what formats (Trace Sciences/Sigma, Chloride/Acetate?). I see some responses below but hope to get some updated opinions and sharing of experiences as mentioned.

If someone has a protocol too that is more specific to conjugating to Bi209, it would be very much appreciated to set us up for successful conjugations in the future.

Greg
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mleipold

Guru

Posts: 1751

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Tue May 07, 2019 6:06 pm

Re: Bismuth conjugataion

Hi Greg,

Since this thread began, the Han et al paper with 209Bi conjugation came out: http://dx.doi.org/10.1038/s41596-018-0016-7

They used Bismuth (III) nitrate pentahydrate (Sigma-Aldrich, cat. no. 254150)


Mike
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GregBehbehani

Master

Posts: 78

Joined: Tue Apr 12, 2016 10:17 pm

Location: The Ohio State University, Columbus, Ohio

Post Tue May 07, 2019 6:23 pm

Re: Bismuth conjugataion

Hi Greg,

We follow the Han et al. protocol as well and get good results. We used to perform 209 labeling using the standard X8 labeling protocol, but the problem that arises is the Bismuth will precipitate during the polymer loading. This is somewhat variable, and is likely to lead to contamination of your antibody with free metal precipitate (which might explain the high background and low positive signal). The Han protocol performs the polymer loading in acid which keeps the Bismuth soluble, resulting in more consistent labeling. We've had three different people in our lab perform Bismuth labeling and all have gotten good results.

best of luck,

Greg
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nkhanbham

Master

Posts: 51

Joined: Wed Feb 25, 2015 3:03 pm

Post Tue Sep 17, 2019 1:06 pm

Re: Bismuth conjugataion

Can I just ask if anyone has tested whether Bi209-loading onto the MaxPar polymer is better done at room temperature for 1hr (Han paper) or at 37C for 30min?
Thanks
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Jahangir

Contributor

Posts: 34

Joined: Sun Oct 29, 2017 6:34 pm

Post Tue Sep 17, 2019 3:23 pm

Re: Bismuth conjugataion

Hi,

I've always done my Bi-209 loading onto the polymer for 45 mins at 37c and I've got antibodies which are more than 1 year old in that channel which still work perfectly!

Best,

Jahangir
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hartmannfj

Participant

Posts: 7

Joined: Fri Oct 31, 2014 3:51 pm

Post Sun Sep 22, 2019 5:57 pm

Re: Bismuth conjugataion

Just to add to this: we have recently published the Bendall lab's conjugation protocols that also includes a section on Bismuth conjugation.

Its similar to Guojun's approach with some differences: we use 1% HNO3 during the chelation step (typical 40min at RT) and afterwards, wash the polymer with a "modified" C-Buffer which is 150 mM tris, 150 mM NaCl, 1 mM EDTA, pH 7.5 (instead of water I believe). Everything else is the same as for lanthanides. Works great for us!

You can find it here: https://doi.org/10.1007/978-1-4939-9454-0_5

Best, Felix
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