Most antibodies have affinities that are in the micromolar* to nanomolar  range (Kd) and to get an optimal staining of antigen (i.e. high saturation) you would need to be in antibody excess, probably by many orders of magnitude. The Kd is approximate to the concentration of antibody that would give you 50% maximal saturation of your antigen. Thus the optimal dilution of antibody specified in catalogues for such staining use usually ends up being in huge antibody excess over antigen. Your question above is only proposing to change the antigen concentration in the system by about 5 fold providing that you keep the volumes in the procedure the same, so according to the law of mass action and with knowledge of the likely antibody affinity it is unlikely to have a big effect on staining.

There is a secondary point to your question which is that it is of course perfectly possible for an antibody to bind with different affinities to different antigens (e.g. consider the thought experiment of where you make minor modifications to the structure of a given antigen such that those modifications change the affinity). If the affinities for the two antigens differ by several orders of magnitude or more e.g. it binds to one with micromolar affinity and the other with nanomolar affinity, then it is possible when titrating your antibody to encounter concentrations at which the antibody would be giving significant saturation of the higher affinity antigen, but not of the lower affinity antigen.

* You can do a quick back of the envelope calculation to estimate what this means: Thus micromolar is 10^-6 * 6x10^23 molecules of antibody per litre i.e. approximately 10^18 molecules per litre or 10^14 molecules in 0.1ml  Thus if you're staining 10^6 (or fewer cells) in that volume and with a micromolar concentration of  antibody you have more than 10^8 molecules of antibody available per cell in the procedure. Clearly if you have a higher affinity nanomolar Kd antibody you can in theory get away with a smaller excess of antibody and still get good staining.

Using excess antibody is very unlikely to lead to non-specific binding. However, too little antibody will certainly result in reduced fluorescence intensity. It is best to titrate each new antibody in your cells of interest to determine the optimal concentration. Personally, I always error on the side of excess antibody.

Thank for your answer Justin

I'll titrate the antibody. Well, I used the antibody at high concentration that usually need to be, so I found positive cells, but I did not know whether these were specifically to the target that I was looking for. I'm according to you, thanks.

in order to check the specificity of your signal, use in parallel isotope control.

Alexis is correct that an isotype control will also ensure specificity. Also if your cells have a lot of Fc receptors (Macs/DCs) be sure to use an Fc blocking ab to reduce background.

Most antibodies have affinities that are in the micromolar* to nanomolar  range (Kd) and to get an optimal staining of antigen (i.e. high saturation) you would need to be in antibody excess, probably by many orders of magnitude. The Kd is approximate to the concentration of antibody that would give you 50% maximal saturation of your antigen. Thus the optimal dilution of antibody specified in catalogues for such staining use usually ends up being in huge antibody excess over antigen. Your question above is only proposing to change the antigen concentration in the system by about 5 fold providing that you keep the volumes in the procedure the same, so according to the law of mass action and with knowledge of the likely antibody affinity it is unlikely to have a big effect on staining.

There is a secondary point to your question which is that it is of course perfectly possible for an antibody to bind with different affinities to different antigens (e.g. consider the thought experiment of where you make minor modifications to the structure of a given antigen such that those modifications change the affinity). If the affinities for the two antigens differ by several orders of magnitude or more e.g. it binds to one with micromolar affinity and the other with nanomolar affinity, then it is possible when titrating your antibody to encounter concentrations at which the antibody would be giving significant saturation of the higher affinity antigen, but not of the lower affinity antigen.

* You can do a quick back of the envelope calculation to estimate what this means: Thus micromolar is 10^-6 * 6x10^23 molecules of antibody per litre i.e. approximately 10^18 molecules per litre or 10^14 molecules in 0.1ml  Thus if you're staining 10^6 (or fewer cells) in that volume and with a micromolar concentration of  antibody you have more than 10^8 molecules of antibody available per cell in the procedure. Clearly if you have a higher affinity nanomolar Kd antibody you can in theory get away with a smaller excess of antibody and still get good staining.

Thank you for all answers

That´s correct, as Alexis and Justin said, I used 5 fold concentration of isotype control and I did not show positive staining, so probably the concentration does not have a big negative effect in the specificity of the antibody.

Thank you Mike R. Clark for your full explanation, you are an expert in this field.

Another point to remember is: manufacturers antibodies have practiced safe: 1 antibody it is calibrated in slight excess to ensure a longer life of the compound. 2 The antibody contains a stabilizer (BSA) which, when used at high concentrations, may result steric hindrance, .3- most antibodies are purified by affinity, which may lower the Kd.

three reasons always find the optimal dilution of the antibody from which the manufacturer has a range of use.

A final point concerns the isotype control: always use a control from the same manufacturer, because of three reasons outlined above, but also whether an antibody linked to a fluorophore is used: it strongly affects the Kd of the antibody.

One word of caution re control antibodies. In many instances the control antibodies are produced from pooled sera from different animals and it is possible that one or more of the animals might have some antibody activity to antigens on the cell population under test. Therefore test the control reagent on your specific cell population under investigation. Ideally the best isotype control antibody would be from a monoclonal antibody. Since most murine monoclonal antibodies are IgG1 then this would be simple but of course it would be more difficult for other isotypes.