title: Molecular characterization of a single-chain antibody variable fragment (scFv) specific for PspA from Streptococcus pneumoniae.
abstract: Streptococcus pneumoniae is a major infectious agent responsible for pneumonia, otitis media, sepsis and meningitis. Pneumococcal surface protein A (PspA) is a well-characterized virulence factor localized on the surface and a target for vaccine development. In this study, we screened a single-chain antibody variable fragment (scFv) using phage display from a human synthetic library to select a clone 2B11. Affinity (Kd) of 2B11 was measured to be 5 nM using biolayer interferometry. 2B11 exhibited a dose-dependent recognition of recombinant PspA with no cross-reactivity towards pneumococcal antigens. The epitope on PspA was defined to residues 231-242 by mutational analysis. Molecular docking analysis supported the experimentally determined epitope, suggesting that the helix spanning residues 231-242 can bind to 2B11 with residues in the CDR-H3 (complementarity determining region 3 in the heavy chain) actively participating in the molecular contacts. Comparison of 2B11 with a commercial PspA antibody revealed that 2B11 exhibited a better specificity towards recombinant PspA antigen. 2B11 was capable of detecting endogenous PspA from pneumococcal lysates with affinity similar to that of the commercial antibody. Our study provides a molecular tool for biosensors detecting pneumococcal diseases.
authors.affiliation: Department of Biological Sciences, Sungkyunkwan University, Suwon, 16419, South Korea.
authors.forename: ShinA
authors.initials: S
authors.lastname: Jang
authors.affiliation: Department of Biological Sciences, Sungkyunkwan University, Suwon, 16419, South Korea.
authors.forename: Gyuhee
authors.initials: G
authors.lastname: Kim
authors.affiliation: Department of Biological Sciences, Sungkyunkwan University, Suwon, 16419, South Korea.
authors.forename: Jihye
authors.initials: J
authors.lastname: Oh
authors.affiliation: School of Pharmacy, Sungkyunkwan University, Suwon, 16419, South Korea.
authors.forename: Seungyeop
authors.initials: S
authors.lastname: Lee
authors.affiliation: Department of Biological Sciences, Sungkyunkwan University, Suwon, 16419, South Korea.
authors.forename: Dongho
authors.initials: D
authors.lastname: Kim
authors.affiliation: SKKU Advanced Institute of Nanotechnology (SAINT), Sungkyunkwan University, Suwon, 16419, South Korea.
authors.forename: Kook-Han
authors.initials: KH
authors.lastname: Kim
authors.affiliation: SKKU Advanced Institute of Nanotechnology (SAINT), Sungkyunkwan University, Suwon, 16419, South Korea.
authors.forename: Yong Ho
authors.initials: YH
authors.lastname: Kim
authors.affiliation: School of Pharmacy, Sungkyunkwan University, Suwon, 16419, South Korea.
authors.forename: Dong-Kwon
authors.initials: DK
authors.lastname: Rhee
authors.affiliation: Department of Biological Sciences, Sungkyunkwan University, Suwon, 16419, South Korea. Electronic address: sangholee@skku.edu.
authors.forename: Sangho
authors.initials: S
authors.lastname: Lee
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000276:immunology* / Q000302:isolation & purification; D001665:Binding Sites; D015374:Biosensing Techniques; D007118:Immunoassay; D062105:Molecular Docking Simulation; D011485:Protein Binding; D057127:Single-Chain Antibodies / Q000737:chemistry* / Q000276:immunology* / Q000302:isolation & purification; D013296:Streptococcus pneumoniae / Q000737:chemistry / Q000276:immunology*
chemical_list: D001426:Bacterial Proteins; D057127:Single-Chain Antibodies; C051592:pneumococcal surface protein A
keywords: 2B11; Epitope; Phage display; PspA; Single-chain antibody variable fragment; Streptococcus pneumoniae