title: In vitro affinity maturation and characterization of anti-P24 antibody for HIV diagnostic assay.
abstract: P24 antigen is the main structural protein of HIV-1, its detection provide a means to aid the early diagnosis of HIV-1 infection. The aim of this study was to improve the selectivity and sensitivity of the HIV P24 diagnostic assay by developing a cohort of 9E8 affinity-matured antibodies through in vitro phage affinity maturation which was performed by complementarity determining region (CDR)-hot spot mutagenesis strategy. Antibody 9E8-491 had an affinity constant of 5.64 × 10(-11) M, which was 5.7-fold higher than that of the parent antibody (9E8). Furthermore, the affinity, sensitivity and specificity of 9E8-491 were higher than those of 9E8, which indicate that 9E8-491 is a good candidate detection antibody for HIV P24 assay. Structure analysis of matured variants revealed that most hydrogen bonds resided in HCDR3. Among the antibody-antigen predicted binding residues, Tyr(100A/100B) was the original conserved residue that was commonly present in HCDR3 of 9E8 and variants. Arg(100)/Asp(100C) was the major variant substitution that most likely influenced the binding differences among variants and 9E8 monoclonal antibody. Both efficient library panning and predicted structural data were in agreement that the binding residues were mostly located in HCDR3 and enabled identification of key residues that influence antibody affinity.
authors.affiliation: State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, National Institute of Diagnostics and Vaccine Development in Infectious Diseases, School of Life Science, Xiamen University, Xiamen 361105, China and.
authors.forename: Lin
authors.initials: L
authors.lastname: Xia
authors.affiliation: State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, National Institute of Diagnostics and Vaccine Development in Infectious Diseases, School of Life Science, Xiamen University, Xiamen 361105, China and.
authors.forename: Juan
authors.initials: J
authors.lastname: Zhang
authors.affiliation: State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, National Institute of Diagnostics and Vaccine Development in Infectious Diseases, School of Life Science, Xiamen University, Xiamen 361105, China and.
authors.forename: Chuanjia
authors.initials: C
authors.lastname: Cui
authors.affiliation: State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, National Institute of Diagnostics and Vaccine Development in Infectious Diseases, School of Life Science, Xiamen University, Xiamen 361105, China and.
authors.forename: Xingjian
authors.initials: X
authors.lastname: Bi
authors.affiliation: State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, National Institute of Diagnostics and Vaccine Development in Infectious Diseases, School of Life Science, Xiamen University, Xiamen 361105, China and.
authors.forename: Junhui
authors.initials: J
authors.lastname: Xiong
authors.affiliation: State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, National Institute of Diagnostics and Vaccine Development in Infectious Diseases, School of Life Science, Xiamen University, Xiamen 361105, China and.
authors.forename: Hai
authors.initials: H
authors.lastname: Yu
authors.affiliation: State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, National Institute of Diagnostics and Vaccine Development in Infectious Diseases, School of Life Science, Xiamen University, Xiamen 361105, China and Texas Therapeutics Institute, The Brown Foundation of Molecular Medicine, University of Texas Health Science Center at Houston, Houston TX 77030, USA.
authors.forename: Zhiqiang
authors.initials: Z
authors.lastname: An
authors.affiliation: State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, National Institute of Diagnostics and Vaccine Development in Infectious Diseases, School of Life Science, Xiamen University, Xiamen 361105, China and.
authors.forename: Wenxin
authors.initials: W
authors.lastname: Luo
authors.affiliation: State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, National Institute of Diagnostics and Vaccine Development in Infectious Diseases, School of Life Science, Xiamen University, Xiamen 361105, China and.
authors.forename: Ningshao
authors.initials: N
authors.lastname: Xia
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology* / Q000378:metabolism; D000915:Antibody Affinity; D001483:Base Sequence; D016466:CHO Cells; D022801:Complementarity Determining Regions / Q000235:genetics; D003412:Cricetulus; D004797:Enzyme-Linked Immunosorbent Assay; D015483:HIV Antibodies / Q000235:genetics / Q000276:immunology*; D016655:HIV Core Protein p24 / Q000276:immunology*; D015658:HIV Infections / Q000175:diagnosis*; D015497:HIV-1 / Q000276:immunology* / Q000378:metabolism; D006801:Humans; D006860:Hydrogen Bonding; D008969:Molecular Sequence Data; D016296:Mutagenesis; D019151:Peptide Library; D012680:Sensitivity and Specificity
chemical_list: D000911:Antibodies, Monoclonal; D022801:Complementarity Determining Regions; D015483:HIV Antibodies; D016655:HIV Core Protein p24; D019151:Peptide Library
keywords: HIV P24; affinity maturation; diagnostic assay; sensitivity; specificity