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Abhishek Singh
Abhishek Singh's picture

I want to start ELISA set for a petide antigen. so which should be coated antibody or antigen

Sami Tuomivaara
Sami Tuomivaara's picture


There are several things you need to take into account when designing an ELISA experiment. First, the coating agent needs to adhere to the microplate surface. The peptide immobilization can be done by incubating the peptide solution in the well on a covered plate (usually overnight at +37 degC) and aspirating the supernatant and proceeding to blocking, etc.. Success is not totally guaranteed with this approach since not all peptides stick to the microplate surface. Further possibility is to use covalent chemistry for the immobilization but this could destroy the epitope. Several chemistries exist, if you have unique thiol, carboxyl or amino functionality, you can for example tag it with X-linker-biotin moiety (where X is some reactive group, NHS for amino, maleimide for thiol, etc.) and bind the peptide to avidin coated plate.

If you immobilize the antibody (so called sandwich approach), you need another antibody with different epitope for detection, or use a polycolonal antibody for both so that both immobilization and detection are possible on the same peptide. Depending on the peptide size, this might not work very well because of mutual blocking of the binding sites by the two antibodies.

So, I'd try to immobilizing the peptide, google (or google scholar) "peptide immobilization ELISA" or something to that effect and you'll probably find tons of protocols for peptide ELISAs. In clinical work, this is the norm approach.

In any case, you need to try and see what works the best.