Generation of high-affinity antibody against T cell-dependent antigen in the Ganp gene-transgenic mouse.
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.
Sakaguchi
Nobuo
N
Department of Immunology, Graduate School of Medical Sciences, Kumamoto University, Honjo, Kumamoto, Japan. nobusaka@kaiju.medic.kumamoto-u.ac.jp
Kimura
Tetsuya
T
Matsushita
Shuzo
S
Fujimura
Satoru
S
Shibata
Junji
J
Araki
Masatake
M
Sakamoto
Tamami
T
Minoda
Chiemi
C
Kuwahara
Kazuhiko
K
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
D000911:Antibodies, Monoclonal; D018076:DNA, Complementary; C481510:GANP protein, mouse; D015483:HIV Antibodies; D007135:Immunoglobulin Variable Region; D009687:Nuclear Proteins; D010750:Phosphoproteins; D014301:Trinitrobenzenes