Hello

Many labs use only the secondary ab as negative control, if you have no isotype control you have no choice. Good luck

Hi, I get problems only with primary Ab from rabbit when staining human cells. With mouse Ab the staining is quite clean. I' m persuaded that rabbit Ig binds human Fc receptors with high affinity. Thus, in this case the staining with goat anti rabbit is specific (for the primary rabbit anti-human) but the problem in this case is the primary, and normal serum control (rabbit are policlonal) is a good option. As always, however, proper controls are helpful. I'm not referring to Iso controls. Look at your staining pattern (if using mixed cell populations such as splenocytes or blood). There are populations to be used as internal negative and/or positive controls? Or alternatively you can use another Ab not reacting for shure with your population...

Mario Picozza

Dear Mario

I am using PBMC from CLL (a B cell malignancy) patients. When I use Isotype control with the same concentration of my test mAb, I will get negative results. But if I use less mouse Isotype control or only secondary FITC conjugated Ab, I always get very nice results with a separate and sharp pick.

Have you titrated your test Ab (and used isotype accordingly)? Is your primary a mouse rat or other? Are your CLL cells positive for CD32 or CD64 or CD16 or other Fc receptors? Are there few monocytes or lymphocytes in your PBMCs to compare the staining? Try to use normal mouse serum to pre-block (if the primary is from mouse). Sorry for not giving you an answer, only suggestions, but I have limited informations about. Mario

We use isotype controls at the same concentration of our primary as this is the only way to demonstrate there is no non-specific Fc binding by your target antibody. Secondary alone, is not an isotype control, but controls for non-specific binding of your secondary. In other words both your primary and secondary may non-specifically bind your cells of interest. However, you say that secondary alone is negative- so no problem there!

If you are getting a lower background with a lower concentration of isotype, then this only confirms that you are getting non-specific Fc binding, as you are able to titrate down the effect.

A serum blocking step may reduce the non-specific Fc binding, but as Mario says without knowing more it is difficult to suggest anything else.

Isotype controls are not recommended for routinely determining positive from negative in flow cytometry as it is almost impossible to have the isotype at the same concentration and fluorescence to protein ratio as the antibody of interest. For indirect staining using just the secondary antibody is the standard used for negative. If doing multicolor, fluorescence minus one is what is used to set negative gates.

Dear Doris

Would you please give me a proper referance.

Thanks

The Purdue Cytometry list serve has an ongoing thread about this. Here is a link to the site and thread. let me know if it helps.

http://www.google.com/cse?cx=008655903951728234384%3Ap00kvkbsavm&ie=UTF-8&q=isotype+controls&hl=en&siteurl=www.google.com%2Fcse%2Fhome%3Fcx%3D008655903951728234384%3Ap00kvkbsavm%26hl%3Den&ref=&ss=2576j522198j16

Many thanks. I will read them

I think pre-block is a selection. At the same time you must titer you primary antibody.

I have found this article very enlightening for the things to consider to control for background in flow cytometry. I think it may help you. Good luck!

Hi Mohammad

Doris is correct. We routinely use single stained controls to set our compensation and FMOs to set our gating - we moved away from isotype controls to set our gating some years ago for the above (and other) reasons.

Good luck

Al

Hi Mohammad,

The isotype control is essential in determining Fc receptor binding especially in cells expressing so much of them, i.e. monocytes. The isotype control should be of the same concentration and type as the primary Ab (Try 1 ug of the Abs for 1 x 10^6 cells as a start). Blocking step is essential for blocking the Fc receptors; this needs to be optimized. Try to choose the same serum of your secondary Ab species. Example, if your secondary is goat anti- rabbit, try to obtain a goat serum and block with 10% goat serum +0.1% BSA+0.05% sodium azide. You can use human AB serum instead. The isotype may not give you absolute negative result, but if you choose a good primary Ab (preferred affinity purified), you will get a nice separation between the 3 histograms. If you want to detect 'unspecific' binding caused by the primary Ab, the only way is to use 'negative cells' which are cells that do not express the target Ag of interest.

I have been working on this problem since a long time, and this is what I finally found.

Hope this helps!

I would go by text books. You need to use the proper isotype matched control antibody. If you do not have that and can not buy it, in this case it is better to use the secondary antibody only as a control rather than not using any control antibody. If you want to read a book in Persian about flow cytometry, then I recommend my own book in flow cytometry (please see my website: http://hamid-zarkesh.staff.shef.ac.uk).

Dear friends

My main problem is that when I use secondary Ab as negative control for staining the lymphocytes, my test mAb will work very nice. But as soon as I add isotype mached with the same concentration of my test, the results will become negative. As most of you suggested, probabely blocking can be a good option.

This is most likely not a blocking issue but the fluorescence/protein ratio of the isotype is higher than your test antibody. If each molecule of isotype antibody has twice as many fluorochrome molecules as your test one, then binding of 1 molecule of isotype will have twice the fluorescence of your test one.

From what I've heard and seen, the best option is actually to change your primary antibody. Rabbit polyclonals often do not give meaningful results due to this aweful background you describe. Your primary will have it also, not just your isotype control.

you may face a dim expression of the antigen which make isolating the signal from control an issue. My suggestions as I said before, try different blocking protocols. secondly, change your primary Ab to another that can pick much Ag than you previous one. The isotype control should be put as an alternative to the primary Ab NOT with it. I mean, in different tube.

Dear Mohammed..as you already suggested proper blocking might lead to successful results..on the other hand, if, by using a proper isotype control your actual staining turns to be negative, this could simply mean that the staining does not work..either the antibody itself is crappy or the molecule itself is not suitable being detected by flow cytometry (due to low expression, difficult fixing/permeabl. etc)...unfortunately, simply using the second antibody alone is pretty common today, it is in fact not correct..

i disagree with the opinion with the upper comments about protein/fluorochrome ration..since u use an INDIRECT staining you use the same secondary (fluorescent ab) for both, control and actual staining..so there cannot be a difference in the ab/fluoroch. ratio between your control and staining approach!

..if the usage of a proper isotype control (and this includes isotype for the actual epitope, which is the primay ab!!!) does not yield to successful results (meaning a detectable difference between the isotype and the staining ab) I would think about switching the detection method..maybe its works for IF or western blot etc...

if you dont see any difference in staining of your lymphocytes when you compare your actual staining (on lymphocytes) with a proper isotype (primary ab plus the secondary, fluorescent ab..of course also on lymphocytes!), how can you say that the difference you saw by only using the secondary is really based on the actual staining of your molecule of interest...in fact you can not..

nevertheless..we all struggle too!

cheers & regards!

Good point. I forgot this was an indirect staining. Blocking would be the first thing to try.

Doris is right. The main problem with isotype controls is that you cannot know the protein: fluorochrome ratio. Even given that information, it is difficult to use the right concentration of the isotype. BTW, what is the right concentration of the isotype? How can you titrate something that should not bind to cells? In my opinion, and also in the opinion of many people routinely using flow cytometry, isotypes should be avoided unless there is no other way to have controls. In your case, the secondary Ab alone can be a good thing. Is your antigen expressed only by a subset of cells, for example by the CLL and not by the normal PBMC? Therefore, the best control to determine positivity is a population that is not supposed to express your marker vs. another one that does. We routinely co-stain PBMC with multiple markers and then titer the antibody of interest. We generally see differential expression between populations in the PBMC (for example between naive and memory cells). If the concentration of the Ab you are using is to high, you would have increase of the background in the whole population, thus telling you that your staining is, most likely, unspecific.