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Low recoveries after Maxpar labeling

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Kaliong

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Joined: Wed Sep 28, 2016 5:05 pm

Post Tue Oct 04, 2016 6:50 pm

Low recoveries after Maxpar labeling

After Maxpar labelling, I realised some antibody yields were too low (<0.20mg/mL) to be used. I have been pondering back and forth upon this issue. As I am working in multiple antibodies labelling, some of the antibodies just could not be recovered.

I conjugated one antibody (at the amount of 100ug) per day (to be careful). The recoveries were as below
Antibody A = 0.81mg/mL
Antibody B = 0.65mg/mL
Antibody C = 0.61mg/mL
Antibody D = 0.10mg/mL, *0.12mg/mL
Antibody E = 0.00mg/mL, *0.01mg/mL
*Second trial with 20 minutes partial TCEP reduction, instead of 30 mins.

I read some threads here. And I put more concerns in work:-
1) To solve over reduction: I reduced the length of incubation upon partial TCEP reduction, for I thought the over reduction that causes antibody aggregations.

2) To solve technical problems including pipetting errors: The protocols were followed religiously. At the last step, I resuspended the solution at least ten times, pipetting at least 80% of the volume, being careful not to introduce bubbles. I also wash down the sides of the spin filter.

3) Suspecting the bad lot of spin?: I believe it is unlikely given the satisfying recoveries of antibody A, B and C.

4. All the antibodies are BSA- and gelatin-free.

5. Suspecting the bad lot of antibodies?: I am not sure of how this bad lots could affect the recoveries of antibody D and E. I guess it is all about the stability of antibody? It is unlikely to change the antibody clone for it has the validated specificities (in-house). Would you try some other lot/batch of the same clone? I need to work a bit more to justify the problems.

This post a shot in the dark. And I do hope I can get some constructive feedbacks. :)
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BjornZ

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Joined: Fri Jul 10, 2015 1:04 am

Post Thu Oct 06, 2016 2:54 am

Re: Low recoveries after Maxpar labeling

Hi Ka-Liong,

I assume A-E are different clones?

1. Did you measure the antibody concentration before you started to confirm that you started with the amount you expected?

2. What isotype and subclass are the antibodies? Every IgG3 clone that I've tried precipitated significantly and consistently during conjugation.

3. What species? Some species conjugate poorly.

Zach
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Kaliong

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Posts: 3

Joined: Wed Sep 28, 2016 5:05 pm

Post Thu Oct 06, 2016 10:28 am

Re: Low recoveries after Maxpar labeling

Thanks for the reply. I like it you share your experience in your lab.
I afraid they are not of different clones- just different antibodies targeting different proteins.

Q1. Did you measure the antibody concentration before you started to confirm that you started with the amount you expected?
A. I did measure before labelling. That's how I calculated the volume of 100ug antibodies to begin with.

Q2. What isotype and subclass are the antibodies? Every IgG3 clone that I've tried precipitated significantly and consistently during conjugation.
A. They are all of IgG isotype. Antibody B, C and D are IgG without no information on subclass. Antibody E is IgG2A whereas antibody A is IgG1k. I am confused with your precipitation step. May you clarify what step is that? And if I understand correctly, you said IgG3 antibodies yield consistent recovery?

Q3. What species? Some species conjugate poorly.
A. Antibody A is raised from mouse while the rest of them are from rabbit. All the antibodies are against human proteins. I haven't checked the quality of conjugation JUST yet. This post is to ask why I could not recover some antibodies properly. (You could still share with me of your conjugation experiences.)

PS:
I read this viewtopic.php?f=7&t=360&p=1124&hilit=30k#p1124 There are a lot of things to consider, really.
*I believe my recovery problem is not uncommon, if any of you could advise it would be highly appreciated.
*It is almost impossible (in my case) to changing the clone because those antibodies are the gold standard in the corresponding proteins - they are validated in WB, IHC, IF in a strict fashion.
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mleipold

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Posts: 5796

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Thu Oct 06, 2016 3:21 pm

Re: Low recoveries after Maxpar labeling

Hi Kaliong,

2. I believe that Zach is saying that he has *problems* with IgG3 antibodies: they precipitate into an unusable form, rather than staying in solution and getting labeled. Precipitation is *not* desirable.

3. I have had at least two experiences with bad lots of antibodies. In one case, it was from Biolegend, and in another it was from BD. In the case of the Biolegend incident, I was using an aCD20 clone I had successfully conjugated at least ten times before. I then had three failed attempts to conjugate, from the same lot number: super-low yield (<0.2mg/mL), and what I did recover wasn't active in staining tests. I contacted Biolegend, and got replacements from another Lot of the same clone, and everything was back to normal (>0.6mg/mL recovery, full activity at the regular titer, etc). This is one reason why it's important to track lot numbers....

However, I have also received a bad box of spin filters (Amicon Ultra 0.5mL 30K). Again, using my regular clones, I had at least 5 labelings out of 10 under 0.2mg/mL recovered protein yield. I reported it to Fisher/Millipore, and got a replacement box (had to send them the bad box for QC testing); my problems went away.


Regarding your other Cytoforum link: some antibodies, even within the same class (eg, IgG2a), are more problematic to couple than others. As far as I know, there's no way to fully predict which will succeed and which won't. A rule of thumb is that if you can buy that clone with a wide variety of fluorophores, there's a good chance it will retain activity after you do the MAXPAR conjugation. However, if a clone is available only on 1-2 fluorophores, then there may be a problem with conjugation (or, at least a given type of chemistry, such as amine-linkage vs thiol-linkage).

Along those same lines, regarding your "gold standard" clones: unfortunately, you may not have a choice. If your "gold standard" antibodies refuse to couple and retain staining activity, then your choices are limited to:
1. Finding another clone, even if it's not quite as good
2. Not staining for that marker
3. Doing some kind of secondary staining (ie, stain with your "gold standard" fluorescent or biotin-labeled clone in a primary fashion, then come back with a MAXPAR-labeled anti-biotin or anti-fluorophore antibody).


Regarding TCEP and reduction: the reason for using TCEP is that it's a phosphine-based reducing agent. Unlike a thiol-based reducing agent like DTT or BME, the TCEP won't interfere in the conjugation to the MAXPAR polymer (ie, thiols like DTT and BME will compete with the thiols from your partially-reduced antibody and reduce your polymer coupling efficiency).


Mike
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mleipold

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Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Thu Oct 06, 2016 7:34 pm

Re: Low recoveries after Maxpar labeling

Hi Zach,

Your comment about problems with IgG3 antibodies made me curious about the differences in sequence and structure between the different IgG classes.

Here are some useful papers:
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4202688/
http://dx.doi.org/10.1016/j.molimm.2015.03.255

Not being overly familiar with antibody structures, I didn't know that IgG3 class antibodies have a MUCH longer hinge region, with a lot more thiols in it. "IgG3 has a much longer hinge region than any other IgG subclasses or Ig human isotype, i.e., about four times as long as the IgG1 hinge, containing up to 62 amino acids (including 21 prolines and 11 cysteines), forming a poly-proline helix with limited flexibility."

So, I could see how they might be prone to aggregation upon TCEP reduction. In a few papers I found on conjugating them, most used amine chemistry (FITC or dansyl chlorides, or biotin usually using an NHS ester), and a few others engineered another Cys into the sequence as a point mutation from Ser (http://www.pnas.org/content/95/22/13206.full.pdf)


In short: there may be IgG3's that you can do regular thiol reduction chemistry on (for example: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2768258/). But the literature I can find seems to be steering away from it.


Mike
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Chowduck

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Joined: Wed Nov 19, 2014 4:39 pm

Post Thu Oct 06, 2016 8:06 pm

Re: Low recoveries after Maxpar labeling

Hello Ka-Liong,

On two occasions I have also encountered the situation you described with Antibody D and E with almost no recovered antibody and a few more where the recovery has been poor (<50%). I suspect the condition of the antibody is the culprit in a vial specific manner, such that most of the UV absorbing peptides are smaller than 50kDa and washing through the filter. The vendors have been quite supportive of replacing defective vials.

I’ve attached a non-reducing SDS-PAGE of a particularly terrible antibody, 4 different vials all the same lot to illustrate an extreme example of antibody degradation where the nano-drop would yield an expected concentration but everything would wash through the 50kDa filter for vial 2.

For other antibodies where the recovery is not satisfactory if you use a 50kDa filter to wash the unconjugated antibody (spin, add PBS, spin then elute) when you re-quantify and start the conjugation again the recovery is usually quite high since the smaller UV absorbing peptides that skewed the initial quantification have been washed out. This in my opinion is the largest value of “Maxpar ready” antibodies as the vendor is performing the first spin.

Zach, regarding your comment about some species conjugating poorly, could you please elaborate? My experience has been limited to Hamster, Mouse and Rag IgG1 and IgG2 and I would be curious to know about other potential situations to watch out for.

The IgG3 precipitation is news to me. Thank you bringing it up and Mike, thank you for the paper links!

-Greg
UHN/Sickkids FCF
Toronto, Canada
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BjornZ

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Posts: 43

Joined: Fri Jul 10, 2015 1:04 am

Post Fri Oct 07, 2016 5:37 pm

Re: Low recoveries after Maxpar labeling

Thanks Mike for posting that info.

Greg - A long time ago folks in our lab struggled with Armenian hamster IgG. Since then folks have avoided it, but as this was early-on, there could very well have been other factors that happened to correlate with the species. I hate to spread rumors, and as you specifically mentioned that you've used it, I should temper my original suggestion. :)

Ka-Liong, it might be worth titrating your conjugates anyway. On occasion when I've had low recovery, I've also had phenomenal labeling efficiency and been able to use the conjugates at <0.1 ug/ml.

Best,
Zcah
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mleipold

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Posts: 5796

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Fri Oct 07, 2016 6:26 pm

Re: Low recoveries after Maxpar labeling

Zach: Thanks for that comment about occasional phenomenal labeling when it has worked: I wonder whether using a lower TCEP *concentration* rather than shorter TCEP *incubation time* could make a difference (or combining the two). Assuming aggregation is caused by inter-antibody disulfide crosslinking, it would probably be a function of the number of free thiols generated. It might be better to generate fewer (and/or more slowly) to keep too many of them from forming at one time.

You could even go to the point of having the MAXPAR polymer around during the TCEP step, to "capture" free thiols as they're generated: if they're covalently linked to the polymer, then by definition they can't covalently link to something else. In that case, I guess the workflow would be to pre-load the polymer with Ln, wash away excess Ln, then add the polymer to the antibody and then add the TCEP.


Depends on how far people want to chase this. :)


Mike

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