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.

New human single chain anti-idiotypic antibody against benzo[a]pyrene

“…Earlier, high affinity murine scFv Ab1 against BP with K d = 0.7 × 10 –8 M [ 25 , 31 ] was obtained and monoclonal antibodies with K d = 0.68 × 10 –10 M [ 32 ], as well as human scFv against BP with K d = 2.93 × 10 –7 M [ 26 ]. The high affinity of human anti-idiotype scFv against BP was described for the first time in the current article.…”

“…The amino acid sequences of pSh and T72 were presented by Ustinov et al . [ 25 , 26 ], respectively. The identity between pSh and T72 was low, because of different amino acid sequences: Max score 185, Query cover 92%, Ident 47% (data not shown).…”

“…T72 was human idiotype scFvs against BP. Both scFvs purifications were processed as indicated in [ 25 ] and [ 26 ], respectively. pSh and T72 have CBD in each molecule.…”

“…The purification of His-tagged A4 was performed using Ni 2+ resin as described in [ 26 ]. The resulting DNA from A4 of M13 phage encoding scFv against pSh was transformed into E. coli HB2151 strain for protein expression.…”

“…pSh was used for phage library screening. However also A4 showed a positive reaction with the T72 in the assay used, despite the fact that the active sites of pSh and T72 were different [ 25 , 26 ]. A4 binding had a dose-dependent manner in both cases and was very similar.…”