You are given a blank 96-well plate and asked to run a sandwich ELISA. Which sequence below correctly orders the five general steps and gives the primary purpose of the blocking step?

Coat with capture antibody → block remaining surface to prevent nonspecific binding (e.g., with casein) → incubate patient sample to allow target antigen binding → add enzyme-linked detection antibody → add substrate to produce color; blocking prevents antigens/antibodies from adsorbing to plastic and reducing background.

Coat with capture antibody → add enzyme-linked detection antibody immediately to saturate capture sites → block remaining surface later to maximize signal → incubate patient sample → add substrate; blocking here is used mainly to enhance enzyme activity by providing cofactors.

Add substrate to prime wells → coat with capture antibody → incubate sample → block with casein to remove unbound antigen → add enzyme-linked detection antibody; blocking is meant to enzymatically digest nonspecific proteins so only specific antigen remains.

Incubate patient sample directly in uncoated wells to capture antigens → block with casein to fix bound antigens → coat with capture antibody to stabilize complex → add detection antibody → add substrate; blocking step here stabilizes protein conformation for antibody recognition.