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metal conjugation to polyclonal antibody

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It is fine to promote your company's reagents. Just make sure they are relevant to CyTOF, and do so in moderation and style :-)
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CyTOFP4Hb

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Posts: 3

Joined: Tue Sep 22, 2015 3:17 pm

Post Thu Sep 24, 2015 3:44 pm

metal conjugation to polyclonal antibody

I'd like to label my antibodies with metal by using MaxPar Reagents. Two of my Ab are IgG which I can follow Nolan Lab's protocol. But one of my antibody is actually commercial whole goat antiserum which I bought from Sigma and it is already delipidized. My question is do I need to purify IgG from serum before metal labeling? Or I can label metal by using the serum (anyhow IgM can not be labeling based on the protocol) then purify metal-labeling IgG? Which way is more efficiency for labeling and less for losing my IgG?
I am new to CyTOF, any suggestion counts! Thank you so much in advance!!

Mei-Ling
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ErinSimonds

Master

Posts: 50

Joined: Tue May 13, 2014 8:04 pm

Post Sun Sep 27, 2015 7:21 am

Re: metal conjugation to polyclonal antibody

Whole serum contains other proteins (mostly albumin) that will compete with IgG during the MaxPar labeling process. If you put the serum directly into the MaxPar labeling protocol, you would get a very small amount of labeled IgG and a ton of MaxPar-labeled albumin.

You need to isolate the IgG before labeling using the Thermo Melon kit. Take a look at the protocol here:
http://www.cytobank.org/nolanlab/conjug ... tocol.html

With the Melon kit, you can start with as little as 10 uL of serum, which typically contains 100-150 ug of IgG. However, you should assume that you're going to lose 50% of the IgG during the purification. So start with at least 20 uL serum in Step 6 of the protocol linked above. You can purify up to 250 uL of whole serum in the protocol linked above. Good luck!
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CyTOFP4Hb

Participant

Posts: 3

Joined: Tue Sep 22, 2015 3:17 pm

Post Mon Sep 28, 2015 3:50 pm

Re: metal conjugation to polyclonal antibody

Hi, Erin,
Thank you SO much!! I will try your recommendation!!
Mei-Ling
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ghowell

Participant

Posts: 19

Joined: Wed Nov 26, 2014 3:18 pm

Post Fri Oct 13, 2017 10:49 am

Re: metal conjugation to polyclonal antibody

Dear all,
I have a user who is interested in conjugating a polyclonal antibody and i picked up this post. What have the current experiences been like performing conjugations to these antibody formats? I haven't got any details as yet (species, Ig class, purified or not, etc) but was just wondering how people had found it. Thanks Gareth
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mleipold

Guru

Posts: 5796

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Fri Oct 13, 2017 4:16 pm

Re: metal conjugation to polyclonal antibody

Hi Gareth,

People definitely have used polyclonals in CyTOF experiments: for example, https://doi.org/10.4049/jimmunol.1502005


I guess the thing to be cautious about is potential lot-to-lot variation in polyclonal activity. For example, I think this is the goat antisIgM antibody from the above paper (authors: correct me if I'm wrong): https://www.southernbiotech.com/?catno= ... Polyclonal

There, the product description states "Pooled antisera from goats hyperimmunized with human IgM". While it's purified by affinity chromatography and supplied in borate saline (thereby avoiding the serum protein reaction problem Erin mentioned), it's still *not* just one antibody, purified from a hybridoma like a monoclonal is. Different batches of goats (or whatever host organism) will probably differ.


Mike

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