Bookmark with More
Category 'Protocols'
Status: Unanswered
Views: 4015

Antibody Titration - Help Needed

LucyT (Author)

Saturday, January 5, 2013 9:01 AM

Hi All,

I am pretty new to flow cytometry and would greatly appreciate advice.

I am trying to titrate a new antibody but can't seem to get it to work out no matter what concetration I start with. The company that I purchased it from recommened that I begin with 1ug/100ul and go downward serially diluting 1:2. I, instead, started with 2ug.

My main difficulty is that the receptor I am staining for is expressed in the vast majority (almost all) of cells, so I don't have a traditional positive vs negative population to compare. I have also been titrating the isotype alongside the receptor antibody, to use the isotype as my "negative" but no matter what concentration I start off at the histograms for the isotype and the receptor anitbody overlap almost perfectly. I ran an unstained and there is a clear difference between my unstained vs the isotype and receptor anitbody. This leads me to conclude I just have cross reactivity in my isotype that I am not appropriately getting rid of?

I have increased the amount of Fc block that i use as well as my staining time, but this has not helped.

I greatly appreciate any advice of how to proceed to get my antibody titered. Thanks so much!

antigoni060

Saturday, January 5, 2013 8:45 PM

Hi Lucy, 

Here are some things I would consider:

1)The number of cells you stain. I use between 100.000-500.000 cells/tube in 100ul. 

2) The amount of 2ug/sample looks ok to me. Actually I never had to use that much. 250ng is the usual or 1ug maximum. But that depends on the antibody used so it is great that you titrate yours first .

3)Is the receptor constitutively expressed on your cells or do you need to upregulate somehow its expression? If it's an inducible receptor then I would incubate my cells with the factor that upregulates the receptor's expression and later stain as a postivie control.

4) Is the antibody appropriate for flow cytometry?

5) Do you probably fix the cells before staining?Fixing before staining may interfere with your staining results.

6)Isotypes might sometimes give some background staining but your positive antibody should give you much higher signal than that. Is the signal of isotype control too high or the signal of the anti-receptor Ab too low? 

7) Do you see any dilution effect in the samples where you titrated your Ab? In other words is there any decrease in the signal as you dilute the Ab concentration while titrating? If you don't see any dilution effect it seems to me that the Abs stick to your cells non specifically or they are in too much excess. 

8)Fc blocker is a good idea for decreasing non-specificity (as long as it per se does not interfere with your staining. When I first started Flow I compared Fc blocker and noc Fc blocker staining to make sure that the Fc blocker actually made a difference).Also try to incubate your cells on ice. It decreases non specificity and keeps your cells healthy. 

Good luck!

cabr0022

Monday, January 7, 2013 4:53 AM

It would help to know the receptor. Having some experience with flow (10 years using machines here and there), I could let you know if the antibody you are using (what clone, what receptor, what fluorochrome) can really affect the results. 

The staining time, temperature and concentration should always be kept constant, but my experience an antibody for a receptor/antigen widely available (let's say CD3, CD4, B220 etc) will stain at almost any condition (ie I've stained "compensation controls" at the last time in a hurry, incubating them for 5 min while holding the tube in my hand :).

My guess is that as the person above pointed out, you might be fixing the cells and staining after fixation. The fixation process uses formaldehyde which will crosslink any proteins in the surface of the cell, rendering the epitopes useless in many cases. Also, if the antibody is a tandem dye, you will be subject to increased signal spillover into another channel, since the fixation will also render the tandem inert (people call this "tandem breakdown" but it's more like the double bonds in the tandem molecule are disrupted, making it non functional. The original dye (APC, PE) the tandem was supposed to "piggyback" onto (ie, PE-Cy7, APC-Cy7, etc) will then be the channel where your signal appears.

i have experiments where I stain 5-10 million cells in 100-200μL without problems. You have to realize that the amount of antibody you add is way way way more than you need for a million cells. And 1μg is alot of antibody. Companies will ask you to stain with that much because 1) they make more money that way and 2) you want to saturate your sample with antibodies and get the most binding per cell, at an overkill concentration of antibody per cell. But for example, staining mouse splenocytes for CD4 on FITC (a relatively weak fluorophore) you can dilute down to 1:800, even 1:1600 without losing signal. The eBioscience antibody (http://us.ebioscience.com/media/pdf/tds/11/11-0042.pdf) says .125μg to be optimal, and this can probably be titrated to 1/4 that amount to get similar results. Sometimes, using too much antibody will shift the positive signal closer to the negative/unstained events, so finding the optimal titration is not only useful but might save you antibody and get you cleaner looking results.

Isotype controls are usually immunoglobulins with no specificity. If you're using a rat anti-mouse antibody, then the rat isotype should be suitable, but make sure there's no cross species reactivity.

But again, knowing the receptor/epitope, the clone, and the fluorochrome, as well as the fixation/processing steps involved, would be useful in helping you out.

Michael Daws

Monday, January 7, 2013 2:02 PM

As the person above said, we need more details about the antibody. Some antibodies are not very good. If your antigen is expressed at high levels on all cells, and you can't see any difference from the isotype, then it may be that your antibody is junk. If you tell us what the antibody is then maybe someone can recommend another that works better.

There are other possibilities though. You may not be blocking Fc receptors sufficiently (although if your antigen is expressed on T cells, then this shouldn't be a problem - just titrate on T cells). Are you using enough Fc block? You can include 10% host serum to block Fc receptors too.

Are you using a secondary antibody? If so, it may be cross-reacting with your Fc block.

Chloe Mica

Monday, October 19, 2015 7:32 AM

I have tried for several times but always failed. I can't reduce the levels of bacterial substance. Maybe I should read the materials here again. 

Chloe Mica

Monday, October 19, 2015 7:33 AM

I have tried for several times but always failed. I can't reduce the levels of bacterial substance. Maybe I should read the materials here again.