A technician observes high background in many wells of an ELISA plate. Which change to the protocol most directly addresses nonspecific binding and why?

• A. Eliminate the blocking step entirely and instead reduce the concentration of capture antibody so fewer binding sites are available for the target antigen; removing blocking actually forces better specificity by lowering antibody availability.
• B. Increase blocking time using casein or BSA and add more thorough wash steps between incubations; blocking occupies uncoated plastic and washes remove unbound reagents, both reducing nonspecific adsorption that produces background signal.
• C. Switch to a fluorescent substrate without changing blocking or washes; fluorescence detection eliminates the need for blocking because fluorescent dyes are not affected by nonspecific protein binding on the plastic surface.
• D. Replace washes with a single brief rinse in distilled water between steps because detergent-containing wash buffers are known to permanently inactivate antibody binding, and water will remove all loosely bound proteins without altering antibody-antigen affinity.

Answer: (B)