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Alternative sources for isotopes

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Jahangir

Contributor

Posts: 36

Joined: Sun Oct 29, 2017 6:34 pm

Post Tue Jan 23, 2018 11:18 am

Re: Alternative sources for isotopes

Hi Guys,

Thanks a lot for all your replies on my previous queries, I have another question, this is a big and strange one and is related to antibody conjugation.

The range of atomic masses that the Helios, at least, can measure is between the range of 73-209 and I've wondered for quite a while that why is the CyTOF community not using the range to it's maximum potential. I do understand there are some metals that you cannot conjugate antibodies to such as Gold, Arsenic (for obvious reasons) and so on. Then there are some metals which you can't use because they don't come in a stable enough monoisotope. Then there are others which are unavailable as they cannot be sourced and purified to a high enough enrichment to be used, and if they can, they're usually super expensive (e.g. Gd157). Then there are those which can be sourced to a high enough purity and is relatively well priced, but need different conjugation chemistry (e.g. Bi209).

But there are a huge number of metals which I don't think have ever been used for some reason or other. I would like to know these reasons, if anyone has ever tried them and if they could kindly share any information whatsoever they have on them. I've listed all these metals below in order of their atomic mass. For some elements, some of their isotopes overlap, so I chose the element which has the highest abundance for that particular atomic mass. Lastly, I've also written their relative abundance of that particular isotope as a percentage... Here we go:

Rubidium-85 -> 72%
Strontium-88 -> 83%
Niobium-93 -> 100%
Cesium-133 -> 100%
Barium-138 -> 72%
Lanthanum-139 -> 99.9%
Cerium-140 -> 88.4%

(Fluidigm commercially sells metals from the atomic mass of 141 to 176 (minus the Gd157) but they can also be found elsewhere)

Hafnium:
-177 -> 18.5%
-178 -> 27.1%
-179 -> 13.8%
-180 -> 35.2%

Tantalum-181 -> 100%

Tungsten:
-182 -> 26.3%
-183 -> 14.3%
-184 -> 30.7%
-186 -> 28.6%

Ruthenium:
-185 -> 37.4%
-187 -> 62.6%

Osmium:
-188 -> 13.3%
-189 -> 16.1%
-190 -> 26.4%
-192 -> 41%

Platinum:
-194 -> 33%
-195 -> 33.8%
-196 -> 25.3%
-198 -> 7.2%

Thalium:
-203 -> 29.5%
-205 -> 70.5%

Lead:
-206 -> 24.1%
-207 -> 22.1%
-208 -> 52.4%

Bismuth-209 -> 100%

I excluded Mercury (Hg) from the above list, as I'm quite certain we can't use it for antibody conjugation, which occur in the:
(-196 -> 0.2%)
-198 -> 10.1%
-199 -> 16.9%
-200 -> 23.1%
-201 -> 23.2%
-202 -> 29.7%
-204 -> 6.8%


It would be amazing if the CyTOF community could come together with all their brains so that we could form and share a Master List of all the different metal isotopes which can be used for CyTOF and which can't be used and why and where they can be sourced with their prices as well so that we can all benefit from it.

Many thanks for all your help guys and I'm definitely looking forward to the responses!!

Jahangir
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ChristophS

Contributor

Posts: 21

Joined: Thu May 07, 2015 2:11 pm

Location: Switzerland

Post Tue Jan 23, 2018 3:00 pm

Re: Alternative sources for isotopes

Hi Jahangir,

the entire theoretical range (75-209) has been explored in our lab.
Some interesting additions may be feasible soon (the Grapevine has it that Fluidigm is doing their fair share of development with some metals, we focus on others).
We are preparing a publication but still have some work to do on stability and reproducibility.

Best regards

Christoph
Christoph Schwärzler
Director Cytometry
Flow Cytometry Facility (https://www.cytometry.uzh.ch/en.html)
University Zürich
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mleipold

Guru

Posts: 2086

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Tue Jan 23, 2018 4:02 pm

Re: Alternative sources for isotopes

Hi Jahangir,

Ru and Os have been used in CyTOF. One example: http://dx.doi.org/10.1002/cyto.a.22848
There are some concerns about the Os toxicity, in the procedures we would be using it for staining......something to be discussed further.

So has Pt: among others,
http://dx.doi.org/10.1002/cyto.a.22778
http://dx.doi.org/10.1002/cyto.a.22067

La139, frankly, I've had some trouble with. I can get it into a polymer (or DOTA-maleimide chelator for live-dead), but it never seems to work as well as the other lanthanides: higher background, conjugates seem not to last as long as the others, etc. We've had enough trouble with it that we try to avoid using it.

Thallium's pretty toxic: https://en.wikipedia.org/wiki/Thallium#Toxicity
- also has at least 2 oxidation states

Several of the other metals in the mass range such as Tin have multiple oxidation states (http://eesemi.com/ox_potential.htm), which may not remain chelated in the same way. For example, for charge reasons alone, a 2+ oxidation state would probably not bind into a chelator in the same way as the 4+ oxidation state.


I'm not saying it's impossible to make probes across the entire CyTOF mass range. But for cost, purity, and chemistry reasons, most of the low-hanging fruit have already been taken.


Mike
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Jahangir

Contributor

Posts: 36

Joined: Sun Oct 29, 2017 6:34 pm

Post Tue Jan 30, 2018 10:09 am

Re: Alternative sources for isotopes

Hi Christoph and Mike,

Many thanks for your replies on this. @Christoph, I can't wait for your publication to be released! When do you think you'll have a preprint of it available on bioRchive? I would love to take a look at it. @Mike, thanks for all those papers and info, will definitely have a read.

Jahangir
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Kjwaller

Participant

Posts: 11

Joined: Sat Aug 27, 2016 6:56 am

Post Wed Jan 31, 2018 4:06 pm

Re: Alternative sources for isotopes

Hi,

I just wondered if anyone could help. I need an 89Y conjugation which fluidigm will do in house but I don't think the turn around time will work for me. Does anyone know where I can purchase 89Y from?

Thanks,
Kate
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mleipold

Guru

Posts: 2086

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Wed Jan 31, 2018 4:21 pm

Re: Alternative sources for isotopes

Hi Kate,

Yttrium is naturally monoisotopic (ie, only stable isotope is 89, at 100% natural abundance). Therefore, like Tb159, Ho165, and Tm169, you can buy highly-pure 89Y chloride from places like Sigma-Aldrich.

In all cases, look for "trace metals basis" salts, which will be highly purified (ie, not contain anything *not* YCl3). For example, Sigma #204919 is 99.999% Yttrium (III) chloride hexahydrate (YCl3 * 6H2O). When calculating the amount of salt you need for 50mM, remember to include the 6 waters of hydration into the molar mass.

Dissolve the salt into L buffer or slightly acidic MilliQ (eg, 100uL 3% nitric per 10mL MIlliQ), and use as any other salt in MAXPAR conjugations.


Mike
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Kjwaller

Participant

Posts: 11

Joined: Sat Aug 27, 2016 6:56 am

Post Wed Jan 31, 2018 5:40 pm

Re: Alternative sources for isotopes

Hi Mike,

Thanks so much for getting back to me so quickly, it sounds fairly straight forward, my only concern is the response I saw on a previous post:

"Thanks to Mike and Adeeb for bringing up an important topic.
At Fluidigm we have attempted to use several chemistries to load 89-Y. While possible, we are not recommending using either X8 or DN3 with 89-Y because the loading is inefficient. Inefficient loading plus a relatively weak signal range will prove inadequate and frustrating for most targets.
For 89Y-CD45 we are using a new chemistry that we are still optimizing, and therefore we do not offer it as a labeling kit yet. We encourage anyone desiring an 89Y conjugation to contact us and we can discuss your needs and options".

Do you have any experience with labelling this particular isotope or what adjustments may be best? I'm labelling CD3, so at least it's a fairly robust antibody.

Thanks again,
Kate
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mleipold

Guru

Posts: 2086

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Wed Jan 31, 2018 6:06 pm

Re: Alternative sources for isotopes

Hi Kate,

We haven't seen any issue with the 89Y.

However, remember: 89Y is a very "dim" metal, as it's far to the left of the mass optimum for most instruments. I think it's assumed to be 8-10x lower in signal than, say, Tb159 or Tm169.

Therefore, I would recommend only putting very bright/high abundance markers out there: CD57, CD45, etc....kinda like using a dim fluorophore in flow.


Mike
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Chowduck

Contributor

Posts: 24

Joined: Wed Nov 19, 2014 4:39 pm

Location: Toronto, Canada

Post Wed Jan 31, 2018 6:29 pm

Re: Alternative sources for isotopes

Hi Kate,

Something to consider is I believe FLDM is not using the Maxpar X8 polymer in their 89Y conjugations. 89Y will certainly work with the Maxpar conjugation kit but you will not achieve the same number of metal ions per antibody; the result will be a dimmer but still useable signal. Please see the attached comparison of FLDM’s CD45/89Y (labelled: old lot) vs a titration of the same clone conjugated with the Maxpar kit using 89Y from Sigma.

CD45-89Y.jpg


Regards,
-Greg Chang
The SickKids - UHN FMCF
Toronto, Canada
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GregBehbehani

Master

Posts: 80

Joined: Tue Apr 12, 2016 10:17 pm

Location: The Ohio State University, Columbus, Ohio

Post Wed Jan 31, 2018 10:19 pm

Re: Alternative sources for isotopes

Hi Kate,

I can agree with what Mike and Greg Chang have posted. 89Y from Sigma works fine, we dissolve it in L-Buffer and use it just like any other metal in labeling reactions using the X8 polymer.

I can confirm Greg's comment that Fluidigm uses a different polymer that allows more metal to be loaded per antibody for their 89Y products, and that's why their loading protocol is different.

Similar to Mike's experience, we only get about 10% as much metal signal from 89Y antibodies as we would from an antibody in the "sweet spot" of the mass range, so you can only use this for extremely high abundance antigens (even CD3 might be a little dim if you think you might see any changes in CD3 expression levels in your samples or are studying cells that might have lower levels of expression).

Best of luck with your work!

Greg
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