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Re: Bismuth conjugation

PostPosted: Tue May 07, 2019 5:55 pm
by GregHopkins
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.


Re: Bismuth conjugataion

PostPosted: Tue May 07, 2019 6:06 pm
by mleipold
Hi Greg,

Since this thread began, the Han et al paper with 209Bi conjugation came out:

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


Re: Bismuth conjugataion

PostPosted: Tue May 07, 2019 6:23 pm
by GregBehbehani
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,


Re: Bismuth conjugataion

PostPosted: Tue Sep 17, 2019 1:06 pm
by nkhanbham
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?

Re: Bismuth conjugataion

PostPosted: Tue Sep 17, 2019 3:23 pm
by Jahangir

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!



Re: Bismuth conjugataion

PostPosted: Sun Sep 22, 2019 5:57 pm
by hartmannfj
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:

Best, Felix