IgG control - (Jan/28/2012 )

Hello,

I have already posted this question in the Flow Cytometry forum, but haven't heard from anyone... I thought it might be a good idea to post it here as the principle is the same, and this forum seems to be more active. Hope I can get some answers from you!

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I just wanted to be sure I understand this correctly. So, if your primary antibody is anti-human IgG1, raised in mouse (mouse monoclonal), and APC-conjugated, then you would want to use anti-human IgG secondary antibody conjugated to the same fluorochrome (APC), correct? Then does this mean we need a negative control for every flurochrome we will be using?

e.g.
sample 1: anti-human mouse monoclonal (IgG2) CD49f-APC
neg control: anti-human mouse monoclonal IgG-APC

sample 2: anti-human mouse monoclonal (IgG1) CD34-biotin (with SA-PE secondary Ab)
neg. control: anti-human mouse monoclonal IgG-PE

sample 3: anti-human mouse monoclonal (IgG2a) CD24 (with anti-mouse secondary Ab-FITC)
neg. control: anti-human mouse monoclonal IgG-FITC

Correct?

Also, how do you choose a company when you buy your IgG control? It probably won't matter too much. Just curious.

Thanks!

I myself don't quite get what you are saying. And I suppose that's why you're not getting many replies.
For starters, you're saying your primary antibody is anti-human IgG1, but then later on you say: sample 1: anti-human mouse monoclonal (IgG2) CD49f-APC. Which is which?
And also about the anti-human IgG secondary antibody conjugated to the same fluorochrome (APC)... Why would you need a secondary antibody if your primary is already conjugated?
I think if you explain a bit more, you'll get a lot of replies.

BioMiha on Sun Jan 29 09:15:52 2012 said:

This was just meant to be an example... Let's say it's IgG1, then.

Oh, thanks for pointing that out. I meant IgG negative control conjugated to the same fluorochrome, NOT IgG secondary Ab...

Hope this is clear!

So, if I understand this correctly your antibody is mouse anti-human CD49 and it is isotype IgG1 (or IgG2 or whatever...). THIS is very different to what you said earlier when you said anti-human IgG1. The latter means that your primary antibody binds to human antibodies of the IgG1 class.
OK, now to the point. The isotype control, which is not exactly a negative control should be of the same isotype and conjugated with the same fluorochrome as your antibody of interest, however specific for a different surface marker that cannot be found on your cells of interest. So if your anti-CD49f-APC is a murine IgG2, then as your isotype control you should use CDXY-APC (raised in mouse) of the class IgG2. And for all the other Abs you are using, you should do the same. This gives you an idea whether the signal you are seeing is specific for your cell type or not.

Ok... so does that mean I need a different IgG control for each of IgG1, IgG2a and IgG2b antibodies even when they are labeled with the same fluorochrome? (I won't be running them all at the same time, of course.... but in terms of whether or not to buy them). I was wondering because someone told me IgG control (without any number attached) can be used as a control for most IgGx primary antibodies. And in many cases, companies don't specify which isotype their IgG control is - they only indicate what it is conjugated to (IgG-APC, IgG-PE, etc.). Can you please clarify this?

Thanks!

If you want to do things by the book, yes, you need controls that match the subclass that you are using. Non-target signals can also arise because of the primary Ab binding to various receptors on the cells and this is subclass dependent. Therefore if you need to control for this, it's worth investing in the various isotype controls. However, a non-subclass specific IgG control is of course better than no control at all.

I see. Thanks!

One more question... Have you ever used FACS-tested, fluorochrome-labeled antibody (that has not been validated for IHC or ICC) for staining FFPE tissue or fixed cells?