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New conjuate doesn't work, now what?

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ianfrank

Contributor

Posts: 29

Joined: Thu Nov 21, 2013 11:59 pm

Location: Tampa, FL

Post Thu Dec 05, 2013 5:55 pm

New conjuate doesn't work, now what?

Dear All,
I would like to get some opinions on what to do if a metal conjugate doesn't work. Do you troubleshoot the antibody, scrap it and try another conjugation, try a new clone or manufacturer? It seems to take fiendishly long to develop a panel in which all of the antibodies are working at the same time. Has anyone developed a clever system for handling this? Any opinions??

Ian
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mleipold

Guru

Posts: 5796

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Thu Dec 05, 2013 7:58 pm

Re: New conjuate doesn't work, now what?

Hi Ian,

It *does* take a fiendishly long time to develop a robust panel. Ofir and I are trying to find ways to set up a database of working conjugates for general knowledge, but we're not there yet.

To answer your question more directly: When you say it doesn't work, what do you mean by that? No significant signal on your expected cells?

This could come from a number of things.

1. The prep itself is bad.
a. Poor antibody recovery (low Nanodrop A280): if the recovery is less than 20% or so, I throw out the prep, usually untried. Even if there is some activity, there are usually antibody aggregates or other problems that will affect staining. Not to mention, with a really low yield, you're going to be remaking the conjugate again soon anyway. It could be a bad lot of antibody, a hole in the spin filter, or overreduction causing aggregation.

b. Poor metal loading. If your protein concentration is OK but you don't see signal, try making a dilution of your antibody stock and running it in liquid mode (like tuning solution). I would recommend serial dilutions down to 1/100,000x and then work your way up. Most antibodies will give you detectable signal at about 1/10,000x.
This could be caused by a bad metal stock (or pipetting error), a bad polymer stock (if the maleimide has been hydrolyzed, it can't covalently link to the antibody free thiol after reduction), or under-reduction (too dilute or expired TCEP; though TCEP should be good for at least a year, it *does* go bad eventually). All of these could lead to the antibody just not having much metal.

c. Overreduced antibody, due to too high [TCEP] or too long spent with TCEP.

In all these cases, trying the particular prep *again* is what I usually do (ideally, with a different lot number of antibody). Only if it fails a second time do I move on to trying other clones. I personally haven't seen much difference in quality between the same clone from different vendors.


2. Try another clone. I had to try at least 4 CD85j antibodies before I found one that worked well enough in CyTOF.

3. Make sure that the epitope you're testing for survives experimental conditions. CD62L doesn't survive freeze/thaw during cryopreservation, but does come back at least somewhat after resting. Fixation drastically affects the binding of most anti-CD16 clones (drops to almost nothing) and many anti-CD56 clones (often get a lot of false-positives). Healthy resting cells may not make a lot of an activation marker: you may need to stim them, or use a cell line for validation (proteinatlas.org can help you find a cell line).

These questions can be worked out on standard flow, *as long as you exactly follow the CyTOF staining protocol* for consistency.


4. There are some epitopes we just haven't been able to find a good CyTOF clone for yet. Using a FITC/APC/PE/biotin-labeled primary and doing secondary staining with a MAXPAR-labeled anti-FITC/APC/PE/biotin/etc adds a step, but has been shown to work in many cases where the primary antibody lost activity upon direct MAXPAR labeling. This also has the advantage of repurposing flow antibodies you *know* work for you. Of course, validate the MAXPAR-labeled secondary with something specific and abundant like FITC/APC/PE/biotin-CD3/CD4/CD8/CD20 beforehand.

This is generally the last thing that I try.


Mike
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robs

Contributor

Posts: 40

Joined: Mon Dec 02, 2013 3:42 pm

Location: University of Connecticut

Post Fri Dec 06, 2013 9:13 pm

Re: New conjuate doesn't work, now what?

Hi Ian,
If you are trying to figure out at what stage things are failing here are two things to try, the first I tried and the second I picked up at a meeting:

1. Stain standard flow compensation beads with your antibody and run them on CyTOF. The beads will bind the antibody so if you get a signal on the CyTOF then the metal loading worked but there is an issue with epitope binding. No signal may mean the metal/polymer was never loaded. Just make sure the beads will bind your antibody whether it happens to be mouse (kappa or lambda) or a different species. This is especially helpful for determining if conjugations worked on low expressing markers, or markers that only appear after stimulation, because the positive beads will be "brighter" than the cells

2. Use a fluorescent antibody (e.g. goat anti-mouse IgG PE) against your conjugated CyTOF antibody and run the doubly stained cells on regular Flow. If there is a fluorescent signal then the issue is with the metal loading. If there is no signal, then something is wrong with the antibody from the start or with the epitobe binding after conjugation procedure.

-Rob

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