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Human Single‐chain Antibodies against Polycyclic Aromatic Hydrocarbons.
Author(s) -
Ustinov Valentin,
Studennikov Artem,
Vavilov Vitaliy,
Tyumentseva Marina,
Morozova Vera,
Tikunova Nina,
Glushkov Andrey
Publication year - 2015
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.29.1_supplement.571.3
Subject(s) - chrysene , pyrene , chemistry , anthracene , benzo(a)pyrene , immunoglobulin light chain , microbiology and biotechnology , biochemistry , antibody , organic chemistry , biology , genetics
Polycyclic aromatic hydrocarbons (PAHs) are one of the most widespread organic pollutants. In order to select anti‐PAHs antibodies, the conjugate of a benzo[a]pyrene (BP) with BSA (BP‐BSA) was used to screened 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 from seven scFvs amino acid residues sequences without stop codons in frame were selected for proteomic analysis with each other. Four scFvs encoded unique nonstructural proteins with low sequence identity among them. The proteomic analysis of all CDRs and the boundaries in the CDR3 formation were carried out. Two of scFvs (T68 and T72) were expressed in E.coli and purified using a nickel resin. The affinity of T68 and T72 was determined by surface plasmon resonance. The KDs values of T68 and T72 to BP‐BSA were 2.93x10 ‐7 and 1.37x10 ‐6 , respectively, which were close to the previously obtained monoclonal antibodies against BP. The KDs values of T68 to PAHs (BP‐BSA, chrysene‐BSA, pyrene‐BSA, anthracene‐BSA, and benzo[a]anthracene‐BSA) were similar to 10 ‐7 . T72 KDs values were 1.37x10 ‐6 , 2.63x10 ‐7 , 8.22x10 ‐6 , 3.74x10 ‐6 , and 9.42x10 ‐8 to BP‐BSA, chrysene‐BSA, pyrene‐BSA, anthracene‐BSA, and benzo[a]anthracene‐BSA, respectively. The active centers of T68 and T72 computer built models were different from each other which explained difference in KDs values.