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Stability of Maxpar conjugated antibodies

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JamesW

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Joined: Tue Nov 21, 2017 6:59 am

Post Fri Feb 02, 2018 2:34 am

Stability of Maxpar conjugated antibodies

Hello all,

Does anyone have an idea of how long I can expect an average antibody to be stable after maxpar labelling, assuming that I store it in 100% Candor antibody stabilizer in the fridge? Fluidigm suggest that their antibodies will be ok for a long time but only actually guarantee them for 6 months. I have no concerns using flow antibodies well past their use by date but have not been doing Cytof long enough to have a feel for this yet.

Also, does this vary at all dependent on the metal used? Particularly now I am making some 139 Lanthanum and non-lanthanide conjugates such as Bismuth, Indium and Yttrium.

Thanks,
James
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cguidos

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Post Fri Feb 02, 2018 2:53 am

Re: Stability of Maxpar conjugated antibodies

Hi James

In our experience, the stability of metal tagged Abs is highly variable. We've had many that continue to perform well after 2 or 3 yrs, and others that die in less than 1 year. Even different conjugations of the same Ab can show variable longevity, which I suspect reflects the quality of the primary Ab. But overall we find most Abs to work well for more than 1 year.

Cindy
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mleipold

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Post Fri Feb 02, 2018 4:05 pm

Re: Stability of Maxpar conjugated antibodies

Hi James,

I would largely agree with Cindy: in my experience, different antibody conjugates have different stabilities, even in 50-100% stabilizer (though the stabilizer definitely at least doubles lifetime relative to only W buffer). For example, the CD38 clone I use is stable for >1yr (maybe even 2yr), whereas the CD85j clone I use sometimes starts fading around 5mo even in 50:50 stabilizer:W buffer.

I have seen *some* prep to prep variability in lifetime, but the qualitative trends are always the same: CD85j fades early, CD38 lasts longest, etc.


Regarding metal selection:
1) I've only worked with the Fluidigm 209Bi conjugates, which have been fine for >6mo (longest I've had a given tube....I haven't done actual lifetime studies).
2) La139 on the other hand, I personally try to avoid using: I have had some problems with it. I can definitely get antibodies labeled with it, but I usually see higher background (streaking) and often reducing signal intensity over the order of months. I'm not sure if La139 just isn't as stable somehow in the DTPA chelator on the MAXPAR polymer, or what, but it's been a lot touchier for me than any of the other Ln metals.
3) Indium has been no problem....goes in easily, is stable. But remember that it's "dimmer" than the Lanthanides: you'll want something fairly abundant like CD45, CD57, CD8, or HLADR, for best resolution. I haven't measured O16 oxide signal directly, but it would spill into the Xe region where you don't have any probes anyway.
4) Yttrium also hasn't been a problem for us, the few times we've labeled with it. Again, 89Y is even dimmer than Indium, so take that into account with your marker choice. Also, see this recent discussion on in-house vs Fluidigm 89Y conjugates: viewtopic.php?f=7&t=3


Mike


Mike
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GregBehbehani

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Posts: 85

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Location: The Ohio State University, Columbus, Ohio

Post Tue Feb 06, 2018 4:33 pm

Re: Stability of Maxpar conjugated antibodies

Hi James,

We don't have as much experience as Mike, but we have several antibodies that have been generally stable for up to 5 years. While it's hard to say for sure (I've used these antibodies on 4 CyTOFs now: early CyTOF1, later CyTOF1, CyTOF2, Helios), I do think the antibodies very slowly loose metal over time, so the antibody still works, it just doesn't give signal that's quite as bright.

If the antibodies aren't processed correctly to begin with, however, you can have an antibody go bad in just a few months. We noticed this very clearly in the Nolan lab, when a group created a big panel of mouse antibodies and didn't put them into stabilizer for a week or so. These almost all went bad quickly, in spite of these same antibody-metal combinations being stable for years in other preps.

I can also confirm that and 113In and 115In are very stable, I have antibodies 4-5 years old labeled with this that still work fine. We have less experience with 89Y, but just like Mike; no problems so far. We have also been doing 209Bi labeling, but I can't really comment on stability yet.

Best of luck,

Greg
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mleipold

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Post Tue Feb 06, 2018 4:47 pm

Re: Stability of Maxpar conjugated antibodies

Hi Greg,

You said "I do think the antibodies very slowly loose metal over time, so the antibody still works, it just doesn't give signal that's quite as bright". How have you tested this?

The reason I ask, it's not easy to separate issues of metal loss (metal falling out of the chelator and being rinsed away in subsequent washes) from issues of loss of antibody binding activity (the metal is still there, but the antibody doesn't efficiently bind to the antigen anymore due to progressive misfolding over time, etc).

In theory, you could run some diluted in liquid mode, wash your antibody again in a spin filter to remove unbound metal ions and then run some diluted again in liquid mode to look at signal intensity. But especially if you have stabilizer around, the error in your protein concentration would probably be high enough to make those calculations semi-quantitative at best.

Another way would be to wash and then try to load in a second metal (which would bind into the "empty" chelators). But since you already have an antibody-conjugate, the known issue of protein nonspecific metal binding would again cause problems in your calculation.....probably even if you did the binding in the presence of a weak Ln chelator like citrate to suppress or remove the nonspecific binding.


Do have a good idea on how to rigorously examine this? I do get this question from time to time, and it would be nice to have a way to test it.....


Mike
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GregBehbehani

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Posts: 85

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Location: The Ohio State University, Columbus, Ohio

Post Tue Feb 06, 2018 4:55 pm

Re: Stability of Maxpar conjugated antibodies

Hi Mike,

I haven't rigorously tested this, in large part because I haven't had the same machine for an extended period of time until just now.

Since coming to OSU, we have been performing a labeling index on every antibody we make (binding the antibody to IgG capture beads under saturating conditions) and this works great and should allow one to separate binding activity from metal chelation. (I actually got this idea from Jonathan Irish, who also does this at Vanderbilt.) We've only compared old antibody to new ones once or twice, so I can't really comment much (one old one was dimmer than the new one; the other was the same).

If you have some new and old antibodies around, I would be interested to know how their labeling indexes look. Feel free to shoot me an email if you want the details. You may also want to reach out to Jonathan, as he has probably been doing this even longer than we have.

best,

Greg
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mleipold

Guru

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Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Tue Feb 06, 2018 5:05 pm

Re: Stability of Maxpar conjugated antibodies

Hi Greg,

Interesting idea on the beads. To confirm: the idea would be that the binding to the IgG capture beads shouldn't change significantly even if antigen-binding activity goes down?

Or, in other words, if the beads retain their signal but the cell signal starts going down, then it's the antibody activity (antigen-binding) that's decreasing; but if the beads start losing their signal, it's metal loss?


Side note: how much variation in signal intensity do you get with the antibodies on the capture beads under saturating conditions? The reason I ask, when I've tried it with BD's kappa capture beads, I've seen signals from ~150 Dual to ~1400 Dual, depending on the metal+clone. In the above comparison for activity vs metal, you would of course be comparing Day 0 to Day X for a given antibody prep, not between clones.


Mike
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tomaskalina

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Joined: Fri Dec 20, 2013 9:19 am

Post Tue Feb 06, 2018 5:14 pm

Re: Stability of Maxpar conjugated antibodies

Hi Mike,

on capture beads: they are quite diverse, so be careful - most are polyclonal, so expect signal variation on staining time, concentration and temperature (and cost to impossible to reach saturation for all clones in that polyclonal mixture); some are monoclonal capture (anti mouse) these shall perform more reproducibly. Stating the obvious for a sake of completeness: you saturate the capture, so ideally always with the same conc of the tested Ab, irrelevant of the optimal titer of your Ab in your assay. Tomas
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mleipold

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Posts: 5792

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Tue Feb 06, 2018 5:19 pm

Re: Stability of Maxpar conjugated antibodies

Hi Tom,

Thanks for that insight into the capture beads. Most of the antibodies I work with are mouse-anti-human; is there a particular monoclonal anti-mouse that you might recommend?


Mike
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tomaskalina

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Joined: Fri Dec 20, 2013 9:19 am

Post Tue Feb 06, 2018 5:41 pm

Re: Stability of Maxpar conjugated antibodies

Hi Mike,

I did some (fluorescent) signal comparisons over few dozens of music-ant-humans and got best data (reproducibility and CV) with eBio UltraComp beads. This contain a mixture of monoclonals anti mouse, hamster and rat.
How do you threshold on beads on CyTOF? E.g. if there will be no metal signal? How do you tell a result where the captured Ab had no signal from result with no beads (lost in centrifuge)?
Cheers Tomas
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