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Category 'Protocols'
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Positive isotype control

Sofia Omari (Author)

Tuesday, April 12, 2011 2:39 AM

Hello, I have problem with the isotype control. I'm trying to detect intracellular antigen in the leukocytes. I fixed, permed  blocked the cells to get our antibodies into the cells. I used isotype control to distinguish positive and negative signal, however the isotype control gives always the same pattern as my sample. I tried several times with different procedures and it's the same. What do think the problem is?

Stephanie Foan

Tuesday, November 8, 2011 12:12 PM

Hi Sofia,

Could you be a bit more specific - what antigen are you staining for? I can see that you posted this some time ago so I imagine you have an answer by now but all the same! :-)

Steph

Sofia Omari (Author)

Tuesday, November 8, 2011 1:29 PM

Hi Steph,

Thanks for your reply. I'm trying to detect TRPV1 antigen, the receptor of the hot chilli, inside the leukocytes. I have not get an answer yet, so I did not complete the experiment yet. Thanks again

Sofia

Stephanie Foan

Tuesday, November 8, 2011 2:18 PM

Hi,

Just a bit of background of what I do - I'm staining T lymphocytes for extra- and intracellular markers such as transcription factors and cytokines. I do it routinely and successfully (now!) but I've had a few things to overcome along the way. I'll suggest a few things that maybe you can try.

Firstly, are you sure you're getting adequate permeability? I started off trying self-made saponin/methanol/tween preparations etc and didn't get anywhere - I now use a BioLegend permeabilising buffer (it's actually marketed for Foxp3 staining but I use it whenever I'm doing intracellular staining and it's great). I guess it depends where your TRPV1 antigen is in the cell - if it's nuclear it might be worth investing in a commercial buffer, if it's cytoplasmic then saponin or something may well work fine. Try also running a control antibody to a nuclear or cytoplasmic antigen depending on where your antigen of interest is, to see if it's permeabilisation that's the problem.

Re: isotype binding - do you expect that TRPV1 is in all leukocytes (sorry - I really know nothing about this molecule)? Or is it only on a very tiny population such that you might not see an appreciable difference when overlaying histograms for example? If you compensate another channel and look on a two colour dot plot, can you see anything at all that is positive relative to isotype control?

Let me know if these suggestions are useful or if you think it might be something else. If you give me more details I might be able to suggest something else.

Kind regards,

Steph

Sofia Omari (Author)

Thursday, November 10, 2011 1:10 AM

Hi Steph,

TRPV1 receptor is placed across the cytoplasmic membrane. The primary antibody binding sites are either on the C- or N- terminus of the receptor (intracellularly). I tried different fix/permeabilise methods such as using tween 20 in PBS, Caltag TM invitrogen and Cytofix/CytopermTM BD Kits. I did blocking steps with BSA, human albumin and human AB blood serum and going no where. We did the compensation optimisation. We used secondary antibody control and it always gives negative results, however the isotype control for primary antibody was positive. 

There is lackage in the literature regarding TRPV1 expression in leukocytes, so it may not be expressed strong enough to get a strong signal. Even though, isotype control should give negative result if the procedure is right. However, it did not.  Here is our overlay histogram. Green colour is for primary+ secondary antibodies, pink colour is for isotype control with secondary antibody and blue colour is for secondary antibody control. I wish this will give an idea about my work. Thanks, Sofia

Sofia Omari (Author)

Thursday, November 10, 2011 1:10 AM

Hi Steph,

TRPV1 receptor is placed across the cytoplasmic membrane. The primary antibody binding sites are either on the C- or N- terminus of the receptor (intracellularly). I tried different fix/permeabilise methods such as using tween 20 in PBS, Caltag TM invitrogen and Cytofix/CytopermTM BD Kits. I did blocking steps with BSA, human albumin and human AB blood serum and going no where. We did the compensation optimisation. We used secondary antibody control and it always gives negative results, however the isotype control for primary antibody was positive. 

There is lackage in the literature regarding TRPV1 expression in leukocytes, so it may not be expressed strong enough to get a strong signal. Even though, isotype control should give negative result if the procedure is right. However, it did not.  Here is our overlay histogram. Green colour is for primary+ secondary antibodies, pink colour is for isotype control with secondary antibody and blue colour is for secondary antibody control. I wish this will give an idea about my work. Thanks, Sofia

AnisL

Thursday, November 10, 2011 2:13 AM

Hi there,

For such weakly expressed antigens, I would recommend you use the brightest secondary antibody you could. Using the same sample use as control a highly expressed antigen, such as CD45 which you will also stain with the same secondary antibody. This will tell you if it is a dye/antibody or other issue. Anis

Ruud

Thursday, November 29, 2012 4:19 PM

Clearly, there's a problem with the isotype control - or maybe not ... Do these cells express Fc-recpetors ? If so, your isotype control is doing what it is supposed to do: it is the ligand for Fc-receptors. But if you used blocking buffer to avoid this type of binding, you should optimize the blocking procedure and see no isotype binding. Isotype controls can really only be used to asess Fc-receptor binding and are controls for Fc-receptor blocking procedures. They are not controls for so called 'non-specific' antibody binding or background signal. For this, the internal 'negative' cell population (the cells that don't express the target antigen) is the appropriate control. But first of all, an antibody titration needs to be performed. The antibody titration will show at which antibody amount and concentration the target cells are 'brightest', while the internal negative population remains as 'negative' as the unstained control. If the sample contains target cells only (i.e. no internal negative population present), prepare your own mixture of target and non-target cells using a cell type that is known not to express the target antigen. Hope this helps. Ruud

Sofia Omari (Author)

Sunday, December 2, 2012 12:42 PM

Hi Rudd,

Thank you for your reply. My cells are white blood cells ( monocytes and lymphocytes mainly). I tried to block with FcR blocking reagent, BSA and 10% goat serum. The isotype control keeps giving positive signal. When I tried to push it toward the second quadrant ( to be negative), my sample signal become negative as well. Can you recommend me some literature about FC receptor relation with the isotype control. As the literature contents of the isotype control in general is not informative enough. I got some negative cells that Im going to try this week as a negative control. Otherwise, I still cant get a negative results using the isotype control making my results unrepresentitive.

Thanks! Sofia

Elnaggar

Wednesday, January 2, 2013 5:55 AM

I think that you need to try onther new isotype control in parallel with the old one

 

Elnaggar

Wednesday, January 2, 2013 5:55 AM

I think that you need to try onther new isotype control in parallel with the old one

 

Ruud

Wednesday, January 2, 2013 2:26 PM

Indeed there's not much literature on this topic out there. I recently reviewed most if it in here: Cytometry B Clin Cytom. 2009: Hulspas R et al. Considerations for the control of background fluorescence in clinical flow cytometry. Nov;76(6):355-64. Again, the isotype control is only useful to determine Fc-receptor binding - not to determine the level of so called non-specific binding of the antibody of interest.

Sofia Omari (Author)

Thursday, January 3, 2013 12:51 PM

Thanks every one for your post. Elnaggar, I tried different isotype control and it give the same or slightly better results. However, I tried new unconjugated primary Ab which was 'affinity purified' so supposidly its more specific for the my Ag and FINALLY I could get separation from the isotype control. The primary Ab was more specific that it gives brighter results. Ruud, thank you so much, you were such a help for me and opened my eyes on the role of the isotype control. As I said before, no much help in the literature. I still have not checked the 'negative cells'. But I will.

Thanks you,

Sofia

Sofia Omari (Author)

Thursday, January 3, 2013 1:19 PM

Rudd, thanks for the paper. I have been searching for long for such a piece. Its remarkable, well done. Keep up the good work for people like us to learn from you.

Thanks again,

Cheers,

Sofia

Creative Animodel Brook

Monday, October 19, 2015 8:54 AM

Hi, Sofia,

The main aim of the same type of control is to determine the binding specificity of a resist, rather than a nonspecific Fc receptor or the interaction with other proteins. It can also be used to compete for the binding of antibodies, with function blocking antibodies playing the same function.

 

Hope it can help you!

Creative Animodel