I'm not quite sure whether this is practical or not but I am looking for a method to separate antigen-antibody complexes without denaturing them, especially the antigen (which is a protein).
I've looked for some methods such as using acid/ bases/ detergents but I think that it will completely denature the protein.
Does anyone have any ideas?
I will really appreciate all the answers. Thank you in advance.
How about chopping the antibody with a selective protease like papain? You will still have binding of the scFv, but it will kill the avidity, which makes your separation task easier by probably a few orders of magnitude. That is, if the protease leaves your antigen alone.
I would try gel filtration using for example Sepharose 4B or CL-4B. Saline buffer should be used. Antibody and antibody-antigen complexes should separate depending on antigen MW. Then you may concentrate the sample using amicon. A disadvantage is the dillution.
I don't know what kind of antibodies you use, but you could possibly bind them to protein A beads/column and elude the protein with high salt concentration (for example with immidazole)? I am just a Master student though so no true expert.
John: I'm afraid the protease will degrade both the antigen and antibody. Actually I want to obtain the antigen (which is a globular protein) without altering its conformation.
Michal: Thank you very much. Oh I forgot to say, the antigen-antibody complex are in patient serum, which is quite challenging.
Yaro: Yeah, I kinda agree with you, but lemme check if there are those antihuman antibodies :)
How about having the antibody covalently linked to beads / column and elute the antigen through the classical pH shift (0.2M Glycine-HCl pH 2.6)? When the eluted antigen is caught into neutralizing Tris buffer, the conformation of the antigen should not be severely affected.
I'd also try low pH first, then high salt (NaCl or MgCl2), then Urea, then Guanidinium salts. If you have antibodies against your antigen that still bind it when complexed, you could try to covalently immobilize this antibody, bind the complex and then split the complex as suggested by a gradient elution, maybe the antibody comes off first. At least you'd get rid of unrelated material this way. For further resolution gel filtration (SEC) might be the best aproach.
We do it almost exactly as Jan Voskuil recommends (0.1 M Glycine, pH 2.7, for 4 column volumes), and it works great! We just follow this protocol: https://www.funakoshi.co.jp/data/datasheet/PCC/20384.pdf
Some procedures are in attached publication
Article A surface plasmon resonance immunosensor for human growth ho...
Hi,
I am looking for sOME strategy to seperate antgen from antibody because i want to do MS/MS. I have done ms/ms but problem is high with heavy chain contamination in ip sample
Since all the trypsinised IgG fragments are known, you should be able to identify the antigen fragments without having to separate.
While I can't say that it won't denature your antigen (this will depend on how labile the particular protein is), but my research group was successful in regenerating antibodies immobilized on glass columns using the chaotropic agent 0.1 M glycine-HCl buffer (pH 2.4). We were able to use this reagent numerous times without degrading the binding activity of the antibodies.
I have used a system similar to Richard's, but instead of a buffer, I used RPMI 1640 medium at pH = 3.0 using acetic acid to adjust the pH. I use this to release antibodies from cells while keeping the cells viable. The procedure is simple, pellet the cells, remove the supernatant, resuspend the pellet with a pastuer pipette full of the RPMI pH=3, then dilute with ~10 ml D-PBS pH 7.5. Viability is typically >95%.
Can anyone help me with a similar kind of problem. I have a recombinant his-tagged protein for which I have to purify antibodies from sera of immunised rabbit. I have tried 0.1M glycine at low as well as high pH but overtime the whole complex is getting eluted.
Can anyone suggest me a way where I can break the bond and get purified antibodies without affecting their activity.
Your his-tag protein does not retain to your Nickel column at 0.1M glycine. Try cross-linking the protein to a new column. Only use the nickel column to purify your protein, not for the antibody retrieval from sera. Then, you should be fine.
Thanks Jan.
I have tried to bind the complex to Protein G and then remove the bound antigen from the antibody which is bound to the resin but then also the antigen is getting eluted with the antibody.
You need a cross-linker to prevent the antigen from getting detached from the column during elution.
Himani,
If you have enough of your recombinant protein, you can covalently link your protein to sepharose by CNBr method .you can then use it as a column or in batch for purifying cognate antibodies from sera.
A change in pH or salt concentration (maybe try a mild chaotropic) might work, but it's a question of trial and error. For separation, try gel filtration / SEC. Regarding your attempt with an antibody binding resin (Protein A, G), a gradient with increasing or decreasing pH might do the trick. Or an immobilized antibody against the Fc of your complexing antibody.
Is this antibody mono or polyclonal?
I agree with most of the answers regarding changing pH and increasing ionic strength by increasing salt concentration gradually. Increasing temperature of sample can help a lot (but it depends on how labile the protein would be). Immobilization of antibody can be done by low pressure chromatography using previous parameters.
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