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Antibody stability over time

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javells

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Joined: Mon Mar 28, 2016 4:58 am

Post Fri Jan 06, 2017 6:08 am

Antibody stability over time

Dear all

I came across some problem with some antibodies over time.

Some antibodies shows more background over time and together with some orphan signals.

I use Candor PBS based antibody stabilizer supplemented with NaN3. They claim that the antibody should be good for long term storage in this stabilizer. However the more I use the antibodies the more I encounter these problems. So I thought these temperature changes (getting the antibodies from from fridge for aliquoting) may be the culprit

Here is what it says in antibody datasheet "After 251 days incubation at 45°C with Antibody Stabilizer the antibody retained around 73% binding activity. This would correspond to a shelf life at 4°C with 73% binding activity of more than 11 years by conservative correlation according to Arrhenius."

I havent come across this issue with any flow antibodies before.

Please see attached the plots plots. Has anyone else came across the same problem ?

Image signal shift
Image increased background
Image orphan signals

Thanks

Muharrem
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mleipold

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Location: Stanford HIMC, CA, USA

Post Fri Jan 06, 2017 4:15 pm

Re: Antibody stability over time

Hi Muharrem,

A couple points, and a couple questions:

1. Yes, the antibodies lose activity over time. This is an antibody-by-antibody (clone-by-clone) thing. In my experience, this is highly reproducible for a given clone, but I haven't been able to predict which ones are more likely to fail earlier rather than later (isotype, source organism, etc). Initially, in just W-buffer (no stabilizer), I had a couple clones that started losing activity by 2 months, while others were good for over a year. Adding stabilizer to a final 50% concentration at least doubled lifetime for the most sensitive clones; I'm now almost always using up my stocks before they start to go off. When I spoke with Candor, they recommended as much stabilizer as possible (ideally, 100% stabilizer, 0% W buffer). My understanding is that Fluidigm antibodies are typically supplied in 100% stabilizer....whether this has changed, I don't know.

In your case, you don't mention how long your antibodies have been stored.....what is the earliest you have had a typical antibody start failing?

Note: accelerated aging lifetimes don't always work out in practice. Again, sometimes this is due to clone-specific stability issues, other times due to non-linearities in the extrapolation.


2. Even flow antibodies from BD, Biolegend, etc *do* go off over time. They may generally be good for longer than CyTOF antibodies, but then again the manufacturers have had a couple decades more to work on the stability problems.

3. In your plots, the 153Eu streak appears to be a washing or titer issue to me. When I have antibodies starting to fail, the most typical sign is a decrease in signal intensity on the positive cells: it is very rare for me to see increase in background on nonspecific cells.

4. I'm not sure what you mean by "orphan signals" in your third plot: do you mean the 153Eu-bright but Ir-neg event?
a) That could just be some debris.
b) It could also be an antibody aggregate in your 153Eu antibody (do you 0.1um spin-filter your cocktail before use?).
c) Finally, there are some cells such as platelets that might legitimately be DNA-neg...which antibody is on 153Eu in this experiment?


Mike
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javells

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Posts: 12

Joined: Mon Mar 28, 2016 4:58 am

Post Thu Jan 19, 2017 8:31 pm

Re: Antibody stability over time

Hello

I also emailed to Candor bioscience regrading stabilizer performance for metal conjugated antibodies.

This is what they said "It could be possible that some labeled antibodies aggregate especially after multiple temperature cycles. Proclin 300 is normally stable but we don’t know if metal could have a catalytic effect on proclin which leads to a premature degradation........ If you suppose that aggregation could be a problem it could be useful to add [b]Tween 20 (e.g. 0.05-0.2%)[/b] that should be able to reduce such aggregation. But we have not tried out this with metal-labeled antibodies but with antibodies labeled with fluorescence-dyes. When we know the reason fort he high background and bad performance of your antibodies (e.g. aggregation or degradation or bacterial growth) then it should be possible to improve the antibody stabilizer especially for the metal-labeled antibodies. "

I did some experiment to see whats going on with some antibodies

I had anti-eomes antibody conjugated to 162Dy at different time points : 1 week old, 6-month old and 14 month old. the first 2 antibodies work well except the 14-month old one causing significant signal shift and messing up with other channels
So I used BD comp bead and stain with just these antibodies separately. one looks the best but with the third one there is considerable background suggesting that those are aggregates.
And when I use linear scale there is a considerable difference between first 2 working antibodies and third non-working antibody as is seen in the figure below. I believe those are many aggregates with varying numbers of antibodies per aggregate.
Image

I also ran these three antibodies on western blot to see if there is a degradation and also included stabilizer alone and pure antibody

It reveals at least there is no degradation. All there antibodies seem similar but the older antibodies show bigger band size. Supposedly all antibodies plus stabilizer must have the same protein concentration. (I store the antibodies at 0.5 mg/ml concentration in stabilizer.)

the third antibody (and also the second one, although very weak) has a band more than 250kDa in size.
Image


to my experience making a master mix of antibodies and using an antibody having those aggregates in that mix causes aggregation of other antibodies and leads to signal shift and increase nonspecific signals in some channels. And when we use all the antibodies except the faulty one it helps to get rid off those signals.

My question is :

- How good is the current antibody stabilizer for long term storage of metal conjugated antibodies ?

- Do you think adding tween, as suggested by company, would have any adverse effect on metal conjugated antibodies ? Is it safe to add it ?

Thanks

Muharrem
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mleipold

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Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Thu Jan 19, 2017 9:05 pm

Re: Antibody stability over time

Hi Muharrem,

Thanks for the followup; I know I haven't done this sort of detailed time-course check, adn I doubt many people have.

Regarding your questions:
- How good is the current antibody stabilizer for long term storage of metal conjugated antibodies ?

I would say that it depends on what you mean by "long term storage". If you're asking on the few-months level, then I would say things are good with it. If you're talking the two-year level, I would say no, very few if any MAXPAR antibodies retain activity after a couple years. The difficulty with trying to develop a general stabilizer is that not every protein "likes" the same conditions, and it's often cost- and/or-time-prohibitive to develop multiple stabilizers.

- Do you think adding tween, as suggested by company, would have any adverse effect on metal conjugated antibodies ? Is it safe to add it ?
I'm not aware of any reason why Tween 20 would cause a problem. Especially since you would be adding it to your master stock, and then diluting beyond that in your day-of cocktail. The only thing I would advise is that you take a small aliquot of your Tween stock, dilute it, and run it in liquid mode on the CyTOF. That way, you can catch any potential metal impurities *before* you would contaminate your antibody stocks.



Have you tried spinning your stocks at high speed (12K x g or something), and pipetting off the top supernatant? Depending on the size of the aggregates, this can sometimes remove (or at least diminish) them....basically pelleting out the aggregates. You could also try filtering the stock itself, but the volume loss you would encounter would probably not make that worthwhile.


Mike
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fcl54643

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Joined: Mon Nov 02, 2015 2:13 pm

Location: Greater Philadelphia Area

Post Wed Feb 01, 2017 4:15 pm

Re: Antibody stability over time

Has anyone encountered issues reading the antibody concentration using Nanodrop at 280nm once the antibody has been diluted in antibody stabilizer supplemented with 0.05% sodium azide? While still in W-Buffer, I obtain expected values (e.g. 0.85 mg/mL). However, as soon as I dilute the antibody with antibody stabilizer/0.05% sodium azide (e.g. to 0.5mg/mL), I obtain negative values. I have tried to both blank with antibody stabilizer/0.05% sodium azide or W-Buffer, but I keep having the same problem.

Note that the metal-conjugated antibodies are perfectly active when I use them in CyTOF experiments, so I did not lose them during spinning steps nor did they degrade. I am just no longer able to read their concentration by nanodrop 280nm.

Is it possible that the sodium azide interferes with the nanodrop reading?

Any insight would be appreciated. Thank you,

Florence
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mleipold

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Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Wed Feb 01, 2017 4:41 pm

Re: Antibody stability over time

Hi Florence,

When you are reading the Nanodrop, are you looking at the *whole spectrum trace*, or are you just taking the 280nm reading from a table (or from the cheapest Nanodrop model, which doesn't show the trace)? And how negative a value are you getting? The reason I ask, your reading could be an artifact of a jagged trace from Azide absorption.

Azide *does* have absorbance in the 280nm range: http://dx.doi.org/10.1063/1.1673134 (see Fig 1, trace "c" for NaN3; x-axis wavelength units are in m-u, or millimicron, which is the same as nanometer).


Between the Azide absorption and the presumable protein content of the stabilizer, I wouldn't suggest even trying to spec an antibody once it's in stabilizer+azide.


Mike
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AdeebR

Grand master

Posts: 171

Joined: Thu Mar 13, 2014 5:58 pm

Location: NYC

Post Wed Feb 01, 2017 4:51 pm

Re: Antibody stability over time

Hi Florence,
The azide may be contributing a little, but the bigger issue is likely the stabilizer, which is packed full of protein that's going to interfere with your reading at 280nm. You're best off reading your antibodies while they're in W buffer and then just calculating the concentration based on your dilution ratio with stabilizer.

Adeeb
Adeeb Rahman
Icahn School of Medicine at Mount Sinai, NYC
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fcl54643

Contributor

Posts: 21

Joined: Mon Nov 02, 2015 2:13 pm

Location: Greater Philadelphia Area

Post Wed Feb 01, 2017 5:31 pm

Re: Antibody stability over time

Thank you, Mike and Adeeb!

I am reading the value from a table, and it is usually around -0.1mg/mL. It makes sense that the high protein content in stabilizer interferes with the reading at 280nm... I will just continue to calculate my concentration based on the 280nm reading in W-buffer and the amount of stabilizer added.

Thanks again!

Florence

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