Compatibility issues with ELISA and a 6xhis tag?

[Compton]

We're a group developing serodiagnostic assays for novel viruses. For this purpose, we express a lot of proteins and use them to detect antibodies in human sera. Recently we have tried using histidine tags for protein purification, mainly under denaturing conditions. The purity and yield has been extremely good.

However, when applying these proteins in ELISA, we get a lot of background and crazy results with signals jumping up and down almost independent of the amount of antigen used. Traditionally we have used Nunc polysorb for the assays which, until now, has worked well for us. The previous antigens working well with the polysorb plates consist e.g. of virus like particles expressed in insect cells and purified under native conditions and GST proteins.

Is there any kind of known compatibility issue with the hexa-histidine tag and certain ELISA plates? Considering the small size of the tag, I would regard this unlikely but there seems to be no explanation. One would perhaps assume that the problem might be caused by the proteins being transferred from high concentration urea to milder concentrations (diluted in PBS) with this resulting in protein aggregation and precipitation. However, virus like particles expressed in insect cells and purified under native conditions using Ni-NTA gave almost the similar kind of results with high background (described above) compared to similar particles purified from insect cells by gradient centrifugation.

Any ideas? Thanks.

[relaxin]

The presence of His-tag in the antigen should not affect the signal of the ELISA, if you have adequate blocking. Perhaps, you can try nickel coated plates. The His-tag should be tied up with the plate and will not interact with other proteins in the human serum.

For information of nickel coated plates, please click the following link:
http://wolfson.huji.ac.il/purification/ ... Plates.pdf

[Compton]

The presence of His-tag in the antigen should not affect the signal of the ELISA, if you have adequate blocking. Perhaps, you can try nickel coated plates. The His-tag should be tied up with the plate and will not interact with other proteins in the human serum.

For information of nickel coated plates, please click the following link:
http://wolfson.huji.ac.il/purification/ ... Plates.pdf

OK, thanks. I had considered these plates but they are very expensive for large scale ELISA. Good to hear that the tag itself is not known to cause problems. I'll test the nickel coated plates and try to continue testing also with other buffers and plastic types.

[relaxin]

I just have an idea, but I do not know if it works. You can add 10 mM nickel chloride or sulphate in your blocking solution. This may block the His-tag from binding to other protetns.

[protold]

Hola, In my Knowledge the problem is an excess of antibody which increase unspecific unions. Try to analize 3or4 samples of different concentration of your protein with higher dilutions of antibody . -What do you think? Good luck.

[Compton]

The presence of His-tag in the antigen should not affect the signal of the ELISA, if you have adequate blocking. Perhaps, you can try nickel coated plates. The His-tag should be tied up with the plate and will not interact with other proteins in the human serum.

For information of nickel coated plates, please click the following link:
http://wolfson.huji.ac.il/purification/ ... Plates.pdf

Because you seem to know pretty much everything related to proteins, I might as well ask you how go about setting up an ELISA from the starting point of having a protein in 8 M urea. It seems that the protein is pretty insoluble. Our standard protocol for virus like particles has been that the proteins have been diluted in PBS to the required concentration and then coated on the plates. I would assume that the assay fails if the protein becomes insoluble and aggregates due to being diluted directly from 8 M urea to pure PBS. One would think that the protein needs to soluble for uniform binding to the plate.

Should I start coating the plates using e.g. the maximum concentration of a suitable denaturing agent as the diluting buffer and possibly go down from there? Thanks for your previous replies.

[relaxin]

Protein should be diluted with coating buffer (50 mM sodium carbonate buffer, pH 9.6) and add to the plate.

I have not tried coating the plates with protein in 8M urea. I would expect the proteins are denatured. Even if they are coated on the plate, they may not be function, i.e. they do not bind the viral particles.

If your recombinant protein is in 8M urea, you may need to refold and dialyse out the urea.

[Compton]

Protein should be diluted with coating buffer (50 mM sodium carbonate buffer, pH 9.6) and add to the plate.

I have not tried coating the plates with protein in 8M urea. I would expect the proteins are denatured. Even if they are coated on the plate, they may not be function, i.e. they do not bind the viral particles.

If your recombinant protein is in 8M urea, you may need to refold and dialyse out the urea.

I didn't actually spesifically mean 8 M urea. I think that might not be compatible with the plate. What I meant was the minimum amount of urea required to keep the protein soluble (e.g. 4 M urea).

What we are trying to do is to detect human antibodies against the viral capsid proteins. The antibodies can detect linear epitopes, that has been ascertained with western blotting so the protein being denatured shouldn't be a problem per se.

[relaxin]

I see, the protein you want to coat the plate is recombinant viral capsid protein. Since you are sure the epitope is linear sequence, then I will say give it a try. It may work. You may still need the high pH carbonate buffer for the protein to coat on the plate.

[Compton]

You may still need the high pH carbonate buffer for the protein to coat on the plate.

Sorry but one final question: we have previously used PBS for most proteins although they have been mostly native proteins without denaturing agents. What's with the high pH? Will it be worth trying this high pH carbonate buffer in combination with urea (referring to my earlier point about keeping the protein soluble with the necessary amount of a denaturing agent)?

[relaxin]

Carbonate buffer, pH 9.6, promotes the binding of protein to the plate. You can simply dilute your sample with carbonate buffer instead of PBS. If you do that immediately before adding to the plate, mixing carbonate buffer with urea should not be a problem.

[ticirocha]

Hi! I know that this is an old post, but I will give it a try! I'm having the same problem... I have a denature protein in 8M urea that works really fine in the western blot but for ELISA, any good results... I'd like to know if the coating with it in solution in the buffers (carbonate or 8M urea) worked... thanks!

[relaxin]

Even if you are able to bind your protein on the plate in the presence of urea, the urea will prevent binding of your antibody to your protein. If you change to a buffer without urea, you may face an insoluble protein, that may not be recognized by the antibody.

Is it possible to refold the protein and remove the urea?