BM9 and BM10 scFvs bind to the N-terminal CBS domain of LANA1 with low but specific binding. (A) Representative enzyme-linked immunoassay activities for 10 Fab clones isolated by phage display from LANA1-immunized rabbits. Clones BM9 and BM10 having highest immunoreactivity were subcloned into single-chain Fv (scFv) and examined for LANA1 binding. (B) HA epitope-tagged BM9, but not BM10, scFv binds LANA1 on direct immunoblotting. FL-9/8TR and FL-9 are BJAB cell lines constitutively expressing LANA-1 protein, and pSG5 is a BJAB cell line transfected with empty vector alone. (C) In vitro binding of BM9 scFv to a GST-LANA1 fusion protein. In this assay, [³⁵S]methionine-labeled BM9 scFv but not BM10 scFv directly interacted with GST-LANA1 fusion protein isolated with glutathione-linked beads. (D) Human serum competition assay: BM9 was mixed with the indicated dilutions of KSHV LANA1-seropositive human serum, and ELISAs were performed using plates coated with peptides P7, P8, P9, P19, and P43. BM9 competes with hyperimmune sera for peptide binding with P7, P8, and P43 but not with P19. Each point represents the value of binding minus nonspecific binding of scFv or human serum to uncoated wells blocked with 3% BSA/PBS. (E) Schematic representation of LANA1 and binding epitopes of BM9, BM10, and other cellular proteins that bind to its N-terminal domain.