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Would using same antibody as coating antibody and detection antibody (biotinylated) possible have a potential to cause competition for the antigen epitope in ELISA assay


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Guest Sudheer

HI All,

 

I am new to this forum and i have a rather basic question and will appreciate your view on this:

 

My question really is - We have an in-house HCP ELISA method in place. The antibody generated against the HCP is being used as coating antibody and biotin conjugation product of the same antibody is used as biotinylated Ab. I would like to you know from you all experts that would using the essentially same Ab (non-conjugated and conjugated with Biotin) could cause any sort competition for the antigen epitope ??

 

Thanks all in advance 

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Welcome Sudheer!

Using the same antibody clone for both capture and detection is possible in ELISA, but only in situations where your antigen is of the correct "format" and it comes with a caveat!:

1. The antigen must be in the form of homodimer so that each molecule has one epitope avaible for binding to the capture, and the same epitope available for binding to the detection mab. It will then work, and homodimer production is not a rare occurance in nature. However, many analytes are heterodimers and then it is gameover. Furthermore, an analyte can be reported to exist in the form of a homodimer, but unknown to current research is that it also comes in a monomer (in addition to the homodimer). You then will miss the monomer format. 

2. In all situations where our analyte of interest is reported as being secreted as a homodimer, the setup using the same capture and detect have worked. However, it is always less sensitive compared to using two different monoclonals against seperate epitopes. Why? We do not know 100%, but maybe the phenomenon of unknown monomer prodoction is more common in nature then we think, but that is just a guess on my side. There are probably other reasons as well, I would not claim to be a super expert here.  

Take home message: it can work, but most likely you will have a suboptimal assay. 

The info above is based on your ELISA system being based on monoclonal antibodies.  

 

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