title: Generation and Characterization of Human Single-Chain Antibodies Against Polycyclic Aromatic Hydrocarbons.
abstract: Polycyclic aromatic hydrocarbons (PAHs) are widely distributed and relocated in the environment as a result of the incomplete combustion of organic matter. Many PAHs and their epoxides are highly toxic, mutagenic and/or carcinogenic to microorganisms as well as to higher systems including humans. BP is one of the most toxicologically active PAHs and is often used as a prototype for this entire class of contaminants. In order to select anti-BP antibodies, the conjugate of BP with BSA (BP-BSA) was used to screen naïve combinatorial phage library of human scFvs. Seven unique scFvs against BP-BSA were selected after three rounds of selection. Analysis of the genes encoding the scFvs subdivided them to gene families and subfamilies. Homology with the closest germline ranged from 80.21% to 97.57% for heavy chains and 88.89% to 98.57% for the light chains. Four of the seven scFv amino acid residues sequences without stop codons in frame were selected for proteomic analysis with each other. Four scFvs encoded unique non-related proteins with low-sequence identity among them. All CDRs and the boundaries in the CDR3 formation were carried out. Two of the scFvs (T68 and T72) with the highest binding capabilities to PAHs were expressed in E. coli and purified using a nickel resin. The KDs of T68 to BP-BSA, chrysene, pyrene, and benzo[a]anthracene were almost similar, approximately 10(-7 )M. The KDs of T72 to benzo[a]anthracene and chrysene were 9.42 × 10(-8 )M and 2.63 × 10(-7 )M, respectively. The computational models of T68 and T72 active centers were different.
authors.affiliation: The Institute of Human Ecology of Siberian Branch of Russian Academy of Science , Kemerovo , Russia and.
authors.forename: Valentin A
authors.initials: VA
authors.lastname: Ustinov
authors.forename: Artem E
authors.initials: AE
authors.lastname: Studennikov
authors.forename: Vitaliy A
authors.initials: VA
authors.lastname: Vavilov
authors.forename: Marina A
authors.initials: MA
authors.lastname: Tyumentseva
authors.forename: Vera V
authors.initials: VV
authors.lastname: Morozova
authors.forename: Nina V
authors.initials: NV
authors.lastname: Tikunova
authors.forename: Andrey N
authors.initials: AN
authors.lastname: Glushkov
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D001564:Benzo(a)pyrene; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D057127:Single-Chain Antibodies / Q000235:genetics / Q000276:immunology*
chemical_list: D004269:DNA, Bacterial; D057127:Single-Chain Antibodies; D001564:Benzo(a)pyrene
keywords: antibody; benzo[a]pyrene; immunology; phage display; polycyclic aromatic hydrocarbons; single-chain antibody