If the epitopes are linear and not conformational (the antibody should work in WB) there shouldn't be any problems unless the denatured target tends to aggregate.

Antibodies are directed against proteins that are not presumed to be in the native state, most often they will be denatured. So the denaturing on itself is not the issue. SDS however will hamper binding because of steric and charge interference. You can get rid of SDS, yet as far as I know this is laborious and difficult to guarantee completeness. I think the best to do is running a native gel and to do western blots.

You can resolve the sample on SDS native page and purify your protein of interest from the gel by using columns available with commercial suppliers. Then you coat this antigen on plates and perform ELISA.

ALL THE BEST

Dear Raheleh, the protein which is separated by SDS PAGE, can be eluted and use for coating Ag in ELISA method. Some antigenic properties of the protein is related to conformational and some sequential epitop. As you know the second is not changed by SDS PAGE, so I suggest you, elute the protein from the gel with suitable buffer. Then dialyze the eluted solution, contained the protein. Finally you will have a close natural structure protein, which after protein content determination, you can use it for coating Ag in microtiter wells, in Bicarbonate or Phosphate coating buffer.

best

mehdi

As Mehdi said, if the epitope is sequential you'll have no problems, although the structure in the SDS micelle might be different than in solution and so that, the affinity of the antibody can change. How where the antibodies in the serum generated? Is this protein the immunogen used? If this is the case, was used in native state or denatured?

Thank you all for your kind responds.

Dear Beatriz,

I used the protein in its native form for immunization. Does it matter in Elisa test?

Dear Raheleh, if the epitope that generated the elicited antibody is conformational, and when you plate the protein this epitope changes its conformation, then yes, you may not see a positive result. You might try different ways to plate your protein to maintain the conformation If it were the case. Good luck!

Luckily all epitopes are linear, so I think it wouldn't  be a problem.

Thank you Beatriz, Best regards!

If the epitopes are linear and not conformational (the antibody should work in WB) there shouldn't be any problems unless the denatured target tends to aggregate.

You must first check whether your antibody recognizes a linear or conformation epitope.

If linear, I think that it could be used :)

If the antibody/serum works in a western blot, then it should also work in an ELISA when eluted from the gel. We elute from the SDS-gels with 10 mM Ammonium-hydrogen-carbonat (NH4HCO3).

I've use this method to isolating protein - antigen from SDS-gels and used it to ELISA with success. 

If the epitopes of the protein can not be destoried ,you can certainly use if for ELISA

It would work well if much of the epitopes of the protein is linear.

You could always try isolating protein using Native PAGE?

bw

Ian

i have used a antibody for Eliza, on wb, it works but with different dillution