I want to make a sandwich ELISA for the detection of antigen X. I want to know if I can use the same antibody as detection and coating (detection antibody will be conjugated to HRP).
If your antigen is repetitive regarding the epitope recognized by the monoclonal antibody the assay can work with the same monoclonal antibody for capture and detection. For instance, monoclonal antibodies against major repetitive viral capside proteins or against homopolymeric compounds can work. While some of the epitopes are bound by the coating antibody, the remaining apitopes in the repetitive antigen can be recognized by the detection antibody. But this is the only chance that one monoclonal antibody is enough. If the epitope is present only once in the antigen, then you have to use two different mAbs or a polyclonal or a polyclonal/monoclonal combination. In the latter case, you can also consider using the monoclonal for capture and the polyclonal Ab for detection.
It won't work if your antibody is monoclonal. If one molecule of antigen X only has one epitope that can be recognized by your antibody, that epitope will first stick to the coated antibody. The epitope will then not be available for the detection antibody to stick to. It could work if your antigen X is a dimer or multimer though.
If you are talking about using a polyclonal antibody, it should work, but your specificity may not be so good (higher background).
I agree with Shanshan and would suggest to use polyclonal antibody as capture and HRP conjugated monoclonal antibody for detection if both type could be available for a high specificity.
If your antigen is repetitive regarding the epitope recognized by the monoclonal antibody the assay can work with the same monoclonal antibody for capture and detection. For instance, monoclonal antibodies against major repetitive viral capside proteins or against homopolymeric compounds can work. While some of the epitopes are bound by the coating antibody, the remaining apitopes in the repetitive antigen can be recognized by the detection antibody. But this is the only chance that one monoclonal antibody is enough. If the epitope is present only once in the antigen, then you have to use two different mAbs or a polyclonal or a polyclonal/monoclonal combination. In the latter case, you can also consider using the monoclonal for capture and the polyclonal Ab for detection.
Could try inhibition assay with enzyme labeled antigen being inhibited by the soluble un labeled antigen if no interfering serum antibodies are present in whatever your sample is
Of course! If it's a polyclonal antibody, it's no sweat. If your antigen is a homo-multimer, it should work with monoclonals too.
Your question is not clear. You are going to develop sandwich assay against which antigen? It is a hapten (low molecular weight antigen) or immunogen (high molecular weight antigen). For hapten one can develop direct competitive and indirect competitive type of ELISAs whereas for high molecular weigh antigen one can develop direct competitive, indirect competitive, indirect sandwich and sandwich type of ELISAs. This depends on availability of polyclonal and monoclonal antibody against antigen and labelled antigen and antibody immuno-reagent.
If you are developing sandwich ELISA for high molecular weight antigen and you have polyclonal antibody against it then you may use your polyclonal antibody for both the purpose that is for coating and labelling. You may get good or bad assay depends on against how many antigenic epitope polyclonal antibody has been developed and present in an antiserum. Antibody should be pure for conjugation.
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