I agree with Vivek, but would like to add a few points to think about if you move forward with this.

See this paper for an example of how it is done from patient sera.

(http://www.sciencedirect.com/science/article/pii/S0022175912002360) For the 2D gels, they leave a halo of the spot after picking it for mass spectrometry so they can still transfer and probe by western to make sure they picked the correct spot.

Any method you use to try to determine the antigen will be biased in some way. Protein Arrays will not have every protein present, just as tissues and cell lysates will not have every protein present. It will be important to try several approaches that can support each other and validate, validate, validate.

Co-IP and mass spec is useful for validation of the target after you have identified the antigen and can obtain appropriate antibodies to perform the tests. Your antibody may work well for western, but may be lousy for IP if the epitope is lost or changed during the processes.

Finally, this will likely take a long time. If this is work you are doing for someone else, make sure they understand that it will not be finished in 6 months. More like 1-3 years. You don't want to invest 8 months in the project only to have them pull the plug.

Good luck!

1st of all you must know the nature of the antibody (isotype). In your case I can see that both ends are open like you don't know about the antibody as well as the antigen. But you must be having some kind of a control subject too. So, I suggest you to do 2-D electrophoresis with your samples and compare the pattern with the control ones. You would get to know about the alien spots (antibody and antigen). After comparing the spots then you must proceed for MALDI-TOF or other proteomic technique suitable to you.

Secondly, you may also proceed for co-immunoprecipitation using secondary antibody labeled beads (magnetic) or chromatography. But the thing is if your antigen is attached with the cell surface that would create problem because antibody-antigen interactions would be lost during the process and you probably get only the antibody not the antigen which would get washed away with the flow through during chromatography. Use of magnetic beads for the purpose might help because its not harsh and quick retrieval happens. But the contradiction is whatever secondary antibody you will choose that will interact non-specifically to all the other immunoglobulins, creating a mess with the situation.

I would prefer the 1st option of 2-D electrophoresis. Good luck

A protein array (Similar to DNA microarray) might be a choice if you have enough antibody to spare.You can sreen the antibody sample on protein array to identify the antigen it binds.

I agree with Vivek, but would like to add a few points to think about if you move forward with this.

See this paper for an example of how it is done from patient sera.

(http://www.sciencedirect.com/science/article/pii/S0022175912002360) For the 2D gels, they leave a halo of the spot after picking it for mass spectrometry so they can still transfer and probe by western to make sure they picked the correct spot.

Any method you use to try to determine the antigen will be biased in some way. Protein Arrays will not have every protein present, just as tissues and cell lysates will not have every protein present. It will be important to try several approaches that can support each other and validate, validate, validate.

Co-IP and mass spec is useful for validation of the target after you have identified the antigen and can obtain appropriate antibodies to perform the tests. Your antibody may work well for western, but may be lousy for IP if the epitope is lost or changed during the processes.

Finally, this will likely take a long time. If this is work you are doing for someone else, make sure they understand that it will not be finished in 6 months. More like 1-3 years. You don't want to invest 8 months in the project only to have them pull the plug.

Good luck!

IN order to address this question properly, it would help to know a bit about how you got the antibody? is this a monoclonal from screening hybridomas, or is it a polyclonal serum? In the old days, we used antibodies to screen expression libraries in phage or bacteria. A library was plated out and proteins bound to filters. The antibody was then used to probe the filters and the spots picked, amplified and rescreened. The inserts in the library where then cloned and sequenced to get the coding regions that bound the antibody.

Dear Gregory,

It is the swimming antibody in the serum, thought to be involved in autoimmunity.

Just another method: Bind the antibody on a solid phase (CNBr-Sepharose, Latex with active groups, ...). So you can run a small scale affinity chromatography to enrich your antigen for later detailes analysis with several methods (2D-EP, MS, HPLC-MS, ...).

The coupling of the antibody can be carried out even in small volumes (1 ml Latex, best with Fe-core to use magnetic separations, 1 mL CNBr-Sepharose). Small volumes can be handled for separation also by centrifugation. The elution of the antigen from the Solid phase can be done best by

- 3 mol/L KSCN or 8 mol/L Urea. Both require intensive dialysis afterwards or by

- 0,005 mol/L Glycin/HCl pH 2.0. This requires only a neutralization. You have to check the required volume of your basic buffer in advance.

Ip followed by sds-page stain coommassie blue excise bands send for c- and or -n sequencing. Less than a month.

In order for the affinity methods suggested above to work, the antibody must be pure. It is not clear from your description that you have a pure antibody. What is your assay for the antibody? In other words, how do you know it is enriched? Even if the antibody is pure, it is extremely unlikely that an IP will give you a clean enough sample for MS without some prior purification by biochemical methods. Furthermore, for unknown protein identification you may need 10-100 ng or protein, so you may have to start with several hundred plates of cells if the antigen is in a cell line, for example.

Hi Raul,

I have the same idea to do for now :)

You can get a semiquantitative answer as well as excellent localization using immunogold against the proteins of interest in tissue sections. This reagent looks red under light microscopy, but at the EM level the marker is intensely black and beautifully localized. This seems a good alternative to grind and find, considering the variety of tissues, treatments and potentially minor but significant differences that may be in play.

For what I see, you have a raw serum containing auto-antibodies of unknown specificity. How do you know your antibody is enriched? Do you have an idea of which might be the antigen? I would however try to perform a Western Blot from a 2-D gel and challenge it with your enriched serum at an appropriate dilution (I would try 1:100), if you have an idea of what might be the target tissue, to look for the putative antigen. I would develop the blots with a broadly reactive, non subtype-specific secondary antibody. You should also run a control blot challenged with a serum which does not contain your antibody, as well as a blot developed with just your secondary, labelled Ab,compare the blots and pick up the specific spots (from the stained gel) to identify the putative antigen(s) by MS. Good luck!

I agree with you Alberto, sure I do blots in parallel with healthy serum and autoantibody containing serum. lets see what it tells :) thanks for your expert comments!

Ananda, do you have an idea of which could be the tissue against which the auto-Ab is directed? Is it a human or an animal antigen? In this latter case (for example, mouse) you should use the organ as the source of Ag, separate the proteins by 2D electrophoresis and challenge the blots with your Ab, a control serum and the secondary Ab alone. If the tissue is of human origin, it might be more complex to do, obviously, but you could use extracts from cell lines.

Ya mine is obviously a human serum and I am gonna work with cell line lysates. Hope to see some Ag binding to them!

I have done some autoimmunity work. If you have an unknown antibody in serum, it woudl be good to know where it roughtly binds. in Immunopathology diagnostiscs, you woudl do some immunohistochemistry with cryosections of tissue to see which tissue, which cells and perhpas, even compartiment is is stained wtih an anti human IgG, or IgM (do you know the isotype ? is it an agglutinin ?), is a disorder involved. I then woudl go and work with cell lysate(but you need to know from what tissue, unless you think its an ubiquitous antigen). One of my papers is describes and autoantibody against intercalated cells in the kidney, but we never came around to identify the antigen..... though..

exactly we are going for a suspected tissue corresponding cell line, and gonna interact the serum containing antibody with these cell lysates. I would look for your paper now :)

thanks!