What are the ODs of test and control groups. Is it similar to blank. How is your E coli expressed protein purified? With coli expressed proteins high non specific background is common. Go for a western to find out background if any?

DNA-based vaccines are considered to be potentially revolutionary due to their ease of production, low cost, long shelf life, lack of requirement for a cold chain and ability to induce good T-cell responses. Please remember that intramuscular DNA vaccination by needle injection fails  to induce detectable antigen-specific antibodies

I'm suspecting about the antigenicity of your protein. We have encountered the same problem with some proteins. How many immunizations have you done? You might need to booster two to three times to see if you can detect any antibody. DNA vaccination has been shown to target T-cell mediated immune responses and delivery route plays an important role in the outcome of vaccination. If you have purified protein, you might want to booster your animals with protein as well. Good luck!

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Dear Panduranga Rao the ODs of test and control groups are approximately the same. I purified the protein by Ni column. 

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Dear Prof. Rasooli I want to know why is this the case about my DNA vaccination, while there are plenty of studies used needle injection for their DNA immunization and they get proper responses. moreover, i have added several elements in my gene to elicit better responses.

best regards

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Dear Jiafen Hu i have suspected that either, however the sequences a have used are the regions of best immunogenecity properties. moreover, the commercially produced antibodies against DKK1 used these regions (not exactly the same) for their immunizations.

best regards

DNA-based vaccines induce good T-cell responses. Despite availability of studies with good results from injections, there are also strudies with similar results of yours. You should either give longer time or more boosters OR go through your methods. If you still do not get results, then arrive at a disappointing conclusion.

Hello Saeed, 

I hope you have solved your problem by now- but if not- please consider that you are immunizing in a way that like results in antibodies against conformation epitopes, and by screening with ELISA you may not be able to detect antibodies against conformation epitopes, on linear. Testing may be more relevant by screening against transfected cells (or finding a cell line that naturally expresses the protein - if it is a cell surface protein).