title: [Preparation of monoclonal antibody against 4-amylphenol and homology modeling of its Fv fragment].
abstract: Objective To prepare and characterize a monoclonal antibody (mAb) against 4-amylphenol (4-AP), clone its cDNA sequence and make homology modeling for its Fv fragment. Methods A high-affinity anti-4-AP mAb was generated from a hybridoma cell line F10 using electrofusion between splenocytes from APA-BSA-immunized mouse and Sp2/0 myeloma cells. Then we extracted the mRNA of F10 cells and cloned the cDNA of mAb. The homology modeling and molecular docking of its Fv fragment was conducted with biological software. Results Under the optimum conditions, the ic-ELISA equation was y=A2+(A1-A2)/(1+(x/x0)p) (A1=1.28; A2=-0.066; x0=12560.75; p=0.74) with a correlation coefficient (R2) of 0.997. The lowest detectable limit was 0.65 μg/mL. The heavy and light chains of mAb respectively belonged to IgG1 and Kappa. The homology modeling and molecular docking studies revealed that the binding of 4-Ap and mAb was attributed to the hydrogen bond and hydrophobic interactions. Conclusion The study successfully established a stable 4-AP mAb-secreting hybridoma cell line. The study on spatial structure of Fv fragment using homology modeling provided a reference for the development and design of single chain variable fragments.
authors.affiliation: State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, China.
authors.forename: Lei
authors.initials: L
authors.lastname: Cheng
authors.affiliation: State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, China. *Corresponding author, E-mail: wuhzh@ecust.edu.cn.
authors.forename: Haizhen
authors.initials: H
authors.lastname: Wu
authors.affiliation: Industrial Products and Raw Materials Testing Center, Shanghai Entry-exit Inspection and Quarantine Bureau, Shanghai 200237, China.
authors.forename: Jing
authors.initials: J
authors.lastname: Fei
authors.affiliation: State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, China.
authors.forename: Lujia
authors.initials: L
authors.lastname: Zhang
authors.affiliation: State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, China.
authors.forename: Jiang
authors.initials: J
authors.lastname: Ye
authors.affiliation: State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, China.
authors.forename: Huizhan
authors.initials: H
authors.lastname: Zhang
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D007114:Immunization; D007074:Immunoglobulin G / Q000032:analysis; D007135:Immunoglobulin Variable Region / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D062105:Molecular Docking Simulation; D008969:Molecular Sequence Data; D019800:Phenol / Q000737:chemistry / Q000276:immunology*
chemical_list: D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G; D007135:Immunoglobulin Variable Region; D019800:Phenol