The diagram illustrates the molecular components of an indirect Enzyme-Linked Immunosorbent Assay (ELISA), which is widely used in global health monitoring to screen blood samples for specific antibodies against pathogens.
Which of the following represents the correct sequence in which components must be added to the wells of the microtitre plate to successfully detect the presence of the patient's antibody?
Pre-coated antigen →\rightarrow→ Patient serum →\rightarrow→ Enzyme-conjugated secondary antibody →\rightarrow→ Substrate
Pre-coated antibody →\rightarrow→ Patient serum →\rightarrow→ Antigen →\rightarrow→ Substrate
Pre-coated antigen →\rightarrow→ Enzyme-conjugated secondary antibody →\rightarrow→ Patient serum →\rightarrow→ Substrate
Pre-coated substrate →\rightarrow→ Patient serum →\rightarrow→ Enzyme-conjugated secondary antibody →\rightarrow→ Antigen