You need to ensure genuine monoclonality and production of an antibody with the desired specificity. Which combined screening strategy best guarantees selection of a single hybridoma clone producing the correct monoclonal antibody?

• A. Select the largest colonies visually under a microscope without any screening assays, because colony size correlates perfectly with production of the desired antigen-specific antibody in every experimental system.
• B. Pool supernatants from multiple untested wells and assay the pooled mixture by ELISA for activity; if the pool tests positive, bulk expand the entire pool to increase antibody yield and assume monoclonality.
• C. Randomly pick any rapidly growing colony and sequence its whole genome; if the genome has no known contaminants, assume the culture is monoclonal and proceed directly to clinical production without antigen-specific testing to save time.
• D. Perform limiting-dilution cloning to obtain wells with a single cell, screen supernatants from those wells by antigen-specific ELISA and functional blocking assays, then expand a confirmed single-well clone and re-clone if necessary to verify monoclonality.

Answer: (D)