In Sandwich ELISA, what is required from the antigen?
Sandwich configuration captures antigen between two antibodies recognizing different regions. Capture antibody immobilized on plate holds antigen, while detection antibody binds elsewhere to generate signal. For simultaneous binding without steric hindrance, antigen must display at least two distinct epitopes spatially separated. A molecule possessing only single epitope cannot be bridged by two antibodies at once, causing assay failure. Larger proteins and multimeric antigens naturally provide multiple determinants, enabling high specificity and sensitivity, whereas haptens require competitiv
Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.