Direct ELISA
a) Antigen is added in buffer. The protein attaches passively to the plastic surface of microtitre plates well. After a period of incubation the non-adsorped protein is washed away.
b) Antibodies with enzyme covalently linked (conjugate) is added in a solution containing inert protein and detergent (to prevent non-specific attachment of the antibodies to plastic wells). The antibody binds to the antigen on well surface. After incubation, non-bound antibodies are washed away.
c) Add substrate and chromogenic dye solution. Substrate interacts with enzyme to affect dye solution to give a colour reaction.
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