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problem with metal conjugation

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robem01

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Posts: 12

Joined: Wed Apr 23, 2014 5:18 pm

Post Fri Jul 17, 2015 6:28 pm

Re: problem with metal conjugation

Hi Adeeb,

I am not aware of any adverse consequences of using the 30K filter. We have done a fair amount of conjugating and have not found any thus far. One of my colleagues here at Penn had a similar experience where about a quarter of the antibodies he was conjugating, simultaneously, had very poor recovery. A loss of total protein to that degree, he felt, might have been from antibody passing through the filter pores. Thus he repeated the conjugation of those clones with poor yield on the 50K filters, over the 30K filters. He found that the use of 30K filters on these clones resulted in a yield similar to the other antibodies, about 75-80% of input protein. We are all using the 30K filters here now for all antibody conjugations.
Hope this helps,
Emily
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BjornZ

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Posts: 43

Joined: Fri Jul 10, 2015 1:04 am

Post Fri Jul 17, 2015 8:14 pm

Re: problem with metal conjugation

That's interesting. Wonder if that means that the antibodies are getting over-reduced and split in half or that the light and heavy chains are separating? I haven't found a consensus about the structure of Igs after labeling (if they become monomeric or not) after good recovery, although I think the supposition is that most of them remain intact.
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hluche

Participant

Posts: 9

Joined: Tue Nov 26, 2013 12:25 am

Post Sun Jul 19, 2015 8:58 am

Re: problem with metal conjugation

Hi all,

Wonder if that means that the antibodies are getting over-reduced and split in half or that the light and heavy chains are separating

It probably does for some antibodies:
It happens that we ran some antibody before and after MAXPAR conjugation on a native and denaturing PAGE gel as a control for labeling by MAXPAR kit of another class of molecule.

We see at least four forms of polymer grafted onto the antibody after conjugation. On the native gel, we also see some light poly-peptide that could possibly be the light chain released from the full antibody (although we had no size marker for native gel) due to the reduction of S-S disulphydie bridge between H and L chain. On the gel, we also see a band of a slightly higher mass that could be due to polymer engraftment on some free L-chain.

The experiment is n = 1 and I don't intend to do this all the time to QC our antibodies as empirically labeling does work well for most of our antibodies. However, we did not pick this antibody for known poor protein recovery: it is an antibody that showed good labeling efficiency and that we used as a control to monitor labeling of another class of molecule. I have been told this effect is also seen for antibodies labeled to fluorochromes for conventionnal flow but there is certainly a chromatography step after labeling and before conditioning in tubes that ensure you just get the labeled antibody without free floating L-chain. It certainly explain why we need to add way more antibody in ug for mass cytometry than in conventional flow to reach labeling in saturating conditions: some mAb of the pool are just not fonctionnal anymore due to altered structure upon conjugation. One of the key point to limit this is to strickly follow the protocol and do not incubate more than 20-30 min for the reduction phase with TCEP.

For that exact reason, I would not try to change the size of the spin column from 50K to 30K even though you increase your yield of protein.
You will certainly retain in your conjugation prep some free floating L-chain fragment conjugated to polymer that will increase your yield in protein but will not help as not linked to your antibody anymore. However, this could increase background in your assay so I am not sure if it is worth it. For these particular mAbs, you should test it of course and let us know if reducing the size of spin column to recover more protein is increasing background staining.


Hervé
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MaxPar labeling.pdf
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komalkumaralienboy

Contributor

Posts: 44

Joined: Tue May 20, 2014 2:24 pm

Location: Linkoping University, Sweden and Finnish Institute of Molecular Medicine, Finalnd

Post Mon Jul 20, 2015 5:29 am

Re: problem with metal conjugation

Hi Adeeb,

Initially I test purified antibody by flowytometry using Secondary antibody, after conjugation I test them again by flowcytometry to see the signal intensity using secondaries. Later I do it in CyTOF.

Thnaks
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komalkumaralienboy

Contributor

Posts: 44

Joined: Tue May 20, 2014 2:24 pm

Location: Linkoping University, Sweden and Finnish Institute of Molecular Medicine, Finalnd

Post Thu Jul 23, 2015 10:19 am

Re: problem with metal conjugation

I have still 2 vials of antibody that contain BSA
Nolan lab use Buffer exchange (Zeba Desalt Spin Columns - order #89889) BSA removal (Melon Gel IgG Purification- order #45206) from life technologies: expected recovery is only 50%.
abcam also have BSA removal kit.

guys suggest me the kit based on your experience.

thanks
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Kaliong

Participant

Posts: 3

Joined: Wed Sep 28, 2016 5:05 pm

Post Thu Oct 06, 2016 12:32 pm

Re: problem with metal conjugation

Just a question to hluche, hope that he read this...
Do you think TCEP is too strong to partially reduce the antibodies? So what have you done to recover the antibody?
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