title: Generation of high-affinity antibody against T cell-dependent antigen in the Ganp gene-transgenic mouse.
abstract: Generation of high-affinity Ab is impaired in mice lacking germinal center-associated DNA primase (GANP) in B cells. In this study, we examined the effect of its overexpression in ganp transgenic C57BL/6 mice (Ganp(Tg)). Ganp(Tg) displayed normal phenotype in B cell development, serum Ig levels, and responses against T cell-independent Ag; however, it generated the Ab with much higher affinity against nitrophenyl-chicken gammaglobulin in comparison with C57BL/6. To further examine the affinity increase, we established hybridomas producing high-affinity mAbs and compared their affinities using BIAcore. C57BL/6 generated high-affinity anti-nitrophenyl mAbs (K(D) approximately 2.50 x 10(-7) M) of IgG1/lambda1 and contained the V(H)186.2 region with W33L mutation. Ganp(Tg) generated much higher affinity (K(D) > 1.57 x 10(-9) M) by usage of V(H)186.2 as well as noncanonical V(H)7183 regions. Ganp(Tg) also generated exceptionally high-affinity anti-HIV-1 (V3 peptide) mAbs (K(D) > 9.90 x 10(-11) M) with neutralizing activity. These results demonstrated that GANP is involved in V region alteration generating high-affinity Ab.
authors.affiliation: Department of Immunology, Graduate School of Medical Sciences, Kumamoto University, Honjo, Kumamoto, Japan. nobusaka@kaiju.medic.kumamoto-u.ac.jp
authors.forename: Nobuo
authors.initials: N
authors.lastname: Sakaguchi
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Kimura
authors.forename: Shuzo
authors.initials: S
authors.lastname: Matsushita
authors.forename: Satoru
authors.initials: S
authors.lastname: Fujimura
authors.forename: Junji
authors.initials: J
authors.lastname: Shibata
authors.forename: Masatake
authors.initials: M
authors.lastname: Araki
authors.forename: Tamami
authors.initials: T
authors.lastname: Sakamoto
authors.forename: Chiemi
authors.initials: C
authors.lastname: Minoda
authors.forename: Kazuhiko
authors.initials: K
authors.lastname: Kuwahara
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000235:genetics; D000915:Antibody Affinity; D001402:B-Lymphocytes / Q000166:cytology / Q000276:immunology; D001483:Base Sequence; D002645:Chickens; D018076:DNA, Complementary / Q000235:genetics; D015483:HIV Antibodies / Q000096:biosynthesis; D015497:HIV-1 / Q000276:immunology; D006801:Humans; D006825:Hybridomas / Q000276:immunology; D007135:Immunoglobulin Variable Region / Q000235:genetics; D066298:In Vitro Techniques; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D009500:Neutralization Tests; D009687:Nuclear Proteins / Q000235:genetics* / Q000276:immunology*; D010750:Phosphoproteins / Q000235:genetics* / Q000276:immunology*; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid; D013601:T-Lymphocytes / Q000276:immunology*; D014301:Trinitrobenzenes / Q000276:immunology
chemical_list: D000911:Antibodies, Monoclonal; D018076:DNA, Complementary; C481510:GANP protein, mouse; D015483:HIV Antibodies; D007135:Immunoglobulin Variable Region; D009687:Nuclear Proteins; D010750:Phosphoproteins; D014301:Trinitrobenzenes