Some antibodies are just used for western blotting, and some antibodies are just used for ELISA. So what is the difference? I producted serum antibody with Virus particle which has several major capsid proteins such as VP1 to VP4, and I also expressed one kind of protein such as VP1. But when I want to use the antibody to detect the VP1 protein with ELISA, I failed. So I want to know if I sould do western blotting to detect the VP1 protein?
Thanks for anyone's advice.
Dear Yingji,
I think if you consider difference between WB and ELISA you can find answer of your question.
At first similarity: WB and ELISA both can analyze proteins, but WB qualitative and some time semi quantitative analysis, ELISA can analyze proteins in three mode: qualitative, semi quantitative and quantitative.
WB and ELISA both can use serum, plasma, tissue culture supernatant, tissue homogenate, and other biological samples
WB need to more steps and need more time but ELISA is more simple and rapid.
WB and ELISA both are sensitive and ultrasensitive method based on their substrate and signal generation and determination.
In WB at first we need a electrophoresis technique which increase specificity.
ELISA deal with a complex molecule solution and it may has cross reactivity based on epitope which its antibody detect
ELISA is cheaper than WB.
So, the main difference between ELISA and WB related to their specificity.
Both can use the same detection antibody, but in WB after an electrophoresis step, and ELISA lack this step.
Finally based on Antibody and its specificity, we can use the antibody only in WB, in ELISA or in both techniques.
But for your intent I suggest you, using WB for lowering false positive results.
best
Mehdi
An ELISA antibody detect generaly a protein folded in its native form. A WB antibody detect a protein that is denatured during the process of SDS-PAGE and transfer.
You could try. Are you sure that the antibodies generate by the immunisation (with the particle ?) are directed against the VP1 protein. In WB, you could test the 4 proteins.
Thanks Stéphane, and I am not sure the antibodies generate by the immunization are directed against the VP1 protein. I just know that the virus particles contains four kinds of protein, and the VP1 is one of them. So I am not sure the virus particles could induce the antibodies for VP1.
I do agree with Stéphane Roche. Antibodies used in western blot can detect and bind to denatured protein but antibodies used in ELISA must be able to bind to the target protein in native form.A good way to obtain antibodies for the purpose of detection of native proteins is the use of Genomic Antibody Technology.
The structure of the epitope is one very important part as already said. In ELISA you need th e have a pure antibody (monoclonal) or at least an immunoglobulin fraction of a specific antiserum for coating the microtiterplates surface. In western blots the antigen is already separated so you can work with any kind of antibody containing preparations: cell culture supernatants, ascitic fluid, antiserum, immunoglobulin fractions, ... all unbound proteins will be removed during washing. Also the detecting antibody hasn't to be a purified one. I've already biotinlylated whole crude cell culture supernantants to see in a very early stage of the experiment in a monoclonal antibody was suitable as a primary or secondary antibody in ELISA or Western Blot.
Dear Yingji,
I think if you consider difference between WB and ELISA you can find answer of your question.
At first similarity: WB and ELISA both can analyze proteins, but WB qualitative and some time semi quantitative analysis, ELISA can analyze proteins in three mode: qualitative, semi quantitative and quantitative.
WB and ELISA both can use serum, plasma, tissue culture supernatant, tissue homogenate, and other biological samples
WB need to more steps and need more time but ELISA is more simple and rapid.
WB and ELISA both are sensitive and ultrasensitive method based on their substrate and signal generation and determination.
In WB at first we need a electrophoresis technique which increase specificity.
ELISA deal with a complex molecule solution and it may has cross reactivity based on epitope which its antibody detect
ELISA is cheaper than WB.
So, the main difference between ELISA and WB related to their specificity.
Both can use the same detection antibody, but in WB after an electrophoresis step, and ELISA lack this step.
Finally based on Antibody and its specificity, we can use the antibody only in WB, in ELISA or in both techniques.
But for your intent I suggest you, using WB for lowering false positive results.
best
Mehdi
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