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.
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.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.