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Alternative Conjugation Protocol

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ichbintony911

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Posts: 2

Joined: Fri Aug 04, 2017 12:40 am

Post Thu Aug 10, 2017 1:25 am

Alternative Conjugation Protocol

Hi, I am a newcomer in Cytof.

I am recently conjugating metals to antibodies. And I wondered if I can conjugate only the polymer to antibody first and store it. Then conjugate metal atom to the polymer on the antibody before use.

I am planing an alternative protocol of conjugation. Had anyone here tried to do this before?
Can we communicate some details about this?
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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 Thu Aug 10, 2017 5:17 pm

Re: Alternative Conjugation Protocol

Hi,

Conjugating the antibody to the polymer and then loading the metal ion in has some issues.

That was the original protocol for labeling the antibodies: for example, see here: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2600572/

However, many proteins, including most antibodies, have regions of their structure that can non-specifically bind metal ions. Since they are low-affinity, they often keep "bleeding"/streaking metal ions throughout all of your staining and washes, and running on the instrument. This gives higher background in your sample.

This is one of the main reasons why the procedure was changed to load the polymer first, wash away all excess metal, and only *then* conjugate to the antibody. This way, the antibody never "sees" free metal.


If you do choose to test this, it may be clone-by-clone whether you can do this with low enough background. I would strongly recommend doing the comparison of the "antibody+polymer then load metal" (new) with the current MAXPAR "load metal into polymer then conjugate to antibody" so you have a rigorous test of the success.

I would also strongly recommend including EDTA or citrate into your washes, to hopefully pull off some of the non-specifically bound metal from your antibody.


Mike
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ichbintony911

Participant

Posts: 2

Joined: Fri Aug 04, 2017 12:40 am

Post Fri Aug 11, 2017 6:41 am

Re: Alternative Conjugation Protocol

Thanks for the information.

That is what I found from my result, large amount of metals.
Now I am planing to wash with EDTA and see how many will fall apart from antibody, comparing with the standard protocol.
This may be a dead end but let's see what will happened.

Thank you.



Tony
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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 Fri Aug 11, 2017 2:59 pm

Re: Alternative Conjugation Protocol

Hi Tony,

You might try the Ln binding to polymer in the presence of citrate: it's a weak chelator of Ln, so it shouldn't interfere with binding to the high-affinity DTPA moieties on the polymer. But it might be a strong enough chelator to compete well with the nonspecific binding sites on the antibodies.

I'd probably recommend putting the antibody+polymer conjugate into buffer containing citrate, then adding the Ln salt also in citrate-containing buffer: that way, the conjugate should never "see" Ln without the competitor.

When I did some similar washes, I used 25mM citrate pH 7.0 (10.1021/pr800645r). For ITC measurements of Gd plus citrate, "sodium citrate (10 mM) in MES buffer (100 mM)" was used (10.1021/jp311812a).


Keep us posted: it would be far easier to make the antibody+conjugate and then load, if the background can be kept low!


Mike

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