ELISA working for peptide, but not working for protein - (Sep/21/2016 )

Hi,

We are trying to optimize ELISA protocol for the first time in lab. Experts please help me.

We purified antibodies that were raised using a peptide. When we do ELISA with peptide, it gives dose dependent signals, but with protein it does not give any signal .

Earlier we were using native protein, we thought may be the epitope is hidden, then we tried denaturing the protien using 2% SDS, however the attempt was unsuccessful. I also tried acetone precipitation after denaturing protein to remove SDS. But that also dint work.

Concentration of protein and peptide was same, tried with higher conc. of protein also, but all failed. 

Please give tips/ideas, what should we do. 

We know the protein works, as we have seen the band in western blot.

western blot (from sds-page) is denatured protein. elisa is native protein, unless specifically denatured.

although you think you ruled it out, your problem is that the epitope is hidden in the native protein, exposed in the denatured protein (as evidenced by the western blot).

you can try denaturing the protein the same way you do for page and using it without removing sds or you can try preparing antibody to a different epitope (one that is exposed in the native protein).

Denaturing buffer that we use for western blot is having SDS+beta mercaptoethanol. Will it not affect the primary antibody binding? 

you denature with sds and mercaptoethanol then bind to the elisa plate. as long as you don't use a non-ionic surfactant, the sds will remain bound and the protein will remain denatured. there will be no sds in solution to prevent binding to the primary antibody.

you may be able to denature with urea but it may renature when diluted.

It is not clear from the question which exact peptide sequence (from the full-length protein) used to raise the Abs? are these peptides tagged? are these Abs monoclonal or polyclonal? 

We learnt that raising Abs against peptides is unlikely would give Abs binding with the native protein. But if you clearly provide more details about your issue, then we might be able to address the challenge well.  

There is always a risk when raising antibodies against a peptide that it will not make the jump to recognizing native protein or a properly folded recombinant protein. If the peptide was somewhat linear in the native protein then the chances are better. If it has structure in the native protein then the chances are not good. Your western blot results support that either 1) your epitope is hidden as mdfenko suggested or 2) structurally not present in the native protein (part of a coil, etc). Functionally, if the epitope is hidden maybe you can be successful partially denaturing but if it is not present as a linear epitope in the native protein full denaturation is likely necessary. I have never been successful at creating a good ELISA using a denatured antigen let alone an optimized one. You may want to consider a different assay format. Do you have any other antibody candidates you can look at?

Hi Everyone,

Thanks a lot for your replies. Peptide sequence was from the tag. We were using antibody against the tag. Peptide was used as positive control. We figured out, in that particular protein the tag was not exposed well so we were not getting any signal. However when we used some other protein, it gave nice signal.

Hopefully it is optimized now.. .

One more question, what is the minimum concentrations of antigen and antibodies in direct ELISA? Will that vary according to the lab and experiments?

Glad it is working for you now! Unfortunately, there is no minimum concentrations for antigen and antibodies in direct ELISA's. It will vary slightly from lab to lab and experiment to experiment (hopefully!) but will vary incredibly widely between different antigens and different antibodies. Some antibodies will have good sensitivity down to the single nanogram levels or even below while others need to be used much higher. It is all dependent on the characteristics of your antigen-antibody pair.