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Generation of monoclonal autoantibodies from Babesia rodhaini-infected mice.

Chiou SP, Kitoh K, Igarashi I, Takashima Y - J. Vet. Med. Sci. (2014)

Bottom Line: The presence of anti-erythrocyte autoantibodies in animals infected with various Babesia species is well reported.Five clones were generated.By Western blotting analysis using whole erythrocyte antigens, one clone reacted with a broad band around 90-150 kDa, and the 2 clones reacted with a band larger than 150 kDa.

View Article: PubMed Central - PubMed

Affiliation: United Graduate School of Veterinary Science, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan.

ABSTRACT
The presence of anti-erythrocyte autoantibodies in animals infected with various Babesia species is well reported. However, the pathogenesis of autoantibodies in babesiosis is poorly understood. Here, we demonstrated that anti-erythrocyte immunoglobulin (Ig) M and IgG were present in B. rodhaini-infected mice at 6 and 8 days after infection, respectively. Furthermore, we generated monoclonal antibodies against erythrocyte antigen from B. rodhaini-infected mice. Five clones were generated. By Western blotting analysis using whole erythrocyte antigens, one clone reacted with a broad band around 90-150 kDa, and the 2 clones reacted with a band larger than 150 kDa. B. rodhaini-infected mice and/or autoreactive monoclonal antibodies established in this study might be a powerful tool for in vivo pathogenesis studies of autoantibody development in infectious diseases.

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Related in: MedlinePlus

Anti-erythrocyte autoantibodies generated by B. rodhaini infection. A.Development of anti-erythrocyte IgG (●) and IgM (■) after B. rodhainiinfection (n=15 at 2, 4, 6, 8 and 10 days and n=14 at 11 days post-infection,***P<0.0001: compared with day 0, by Student’s ttest). B. Western blotting analysis using B. rodhaini-infected serum.As a negative control, mock-infected serum was used. (Open arrowhead: Band 3-like area,right panel: long exposed). C. Western blotting analysis using monoclonal antibodiesproduced by 5 hybridoma clones. As a negative control, supernatant of myeloma cellcultures was used (lane (−)). Whole erythrocyte lysate was used as an antigen.
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fig_001: Anti-erythrocyte autoantibodies generated by B. rodhaini infection. A.Development of anti-erythrocyte IgG (●) and IgM (■) after B. rodhainiinfection (n=15 at 2, 4, 6, 8 and 10 days and n=14 at 11 days post-infection,***P<0.0001: compared with day 0, by Student’s ttest). B. Western blotting analysis using B. rodhaini-infected serum.As a negative control, mock-infected serum was used. (Open arrowhead: Band 3-like area,right panel: long exposed). C. Western blotting analysis using monoclonal antibodiesproduced by 5 hybridoma clones. As a negative control, supernatant of myeloma cellcultures was used (lane (−)). Whole erythrocyte lysate was used as an antigen.

Mentions: Infection of C57BL/6 mice (CLEA Japan Inc., Tokyo, Japan) with B. rodhainiand detection of autoantibodies were performed as reported previously with minor modifications[5]. Briefly, 106 parasitized erythrocyteswere intravenously injected into 8-week-old female C57BL/6 mice, and serum samples werecollected every other day. The experiments were performed in accordance with the GifuUniversity Animal Care and Use Committee guidelines. Autoantibodies reacting with erythrocyteswere detected by enzyme-linked immunosorbent assay (ELISA) using erythrocyte membrane lysateas an antigen. To detect antigen-specific immunoglobulin (Ig) G and IgM, horseradishperoxidase (HRP)-conjugated rabbit-anti-mouse IgG or HRP-conjugated rabbit-anti-mouse IgM wasused. After B. rodhaini infection, IgM reacting with erythrocytes wasdetected 6 days after infection (Fig. 1AFig. 1.


Generation of monoclonal autoantibodies from Babesia rodhaini-infected mice.

Chiou SP, Kitoh K, Igarashi I, Takashima Y - J. Vet. Med. Sci. (2014)

Anti-erythrocyte autoantibodies generated by B. rodhaini infection. A.Development of anti-erythrocyte IgG (●) and IgM (■) after B. rodhainiinfection (n=15 at 2, 4, 6, 8 and 10 days and n=14 at 11 days post-infection,***P<0.0001: compared with day 0, by Student’s ttest). B. Western blotting analysis using B. rodhaini-infected serum.As a negative control, mock-infected serum was used. (Open arrowhead: Band 3-like area,right panel: long exposed). C. Western blotting analysis using monoclonal antibodiesproduced by 5 hybridoma clones. As a negative control, supernatant of myeloma cellcultures was used (lane (−)). Whole erythrocyte lysate was used as an antigen.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4197159&req=5

fig_001: Anti-erythrocyte autoantibodies generated by B. rodhaini infection. A.Development of anti-erythrocyte IgG (●) and IgM (■) after B. rodhainiinfection (n=15 at 2, 4, 6, 8 and 10 days and n=14 at 11 days post-infection,***P<0.0001: compared with day 0, by Student’s ttest). B. Western blotting analysis using B. rodhaini-infected serum.As a negative control, mock-infected serum was used. (Open arrowhead: Band 3-like area,right panel: long exposed). C. Western blotting analysis using monoclonal antibodiesproduced by 5 hybridoma clones. As a negative control, supernatant of myeloma cellcultures was used (lane (−)). Whole erythrocyte lysate was used as an antigen.
Mentions: Infection of C57BL/6 mice (CLEA Japan Inc., Tokyo, Japan) with B. rodhainiand detection of autoantibodies were performed as reported previously with minor modifications[5]. Briefly, 106 parasitized erythrocyteswere intravenously injected into 8-week-old female C57BL/6 mice, and serum samples werecollected every other day. The experiments were performed in accordance with the GifuUniversity Animal Care and Use Committee guidelines. Autoantibodies reacting with erythrocyteswere detected by enzyme-linked immunosorbent assay (ELISA) using erythrocyte membrane lysateas an antigen. To detect antigen-specific immunoglobulin (Ig) G and IgM, horseradishperoxidase (HRP)-conjugated rabbit-anti-mouse IgG or HRP-conjugated rabbit-anti-mouse IgM wasused. After B. rodhaini infection, IgM reacting with erythrocytes wasdetected 6 days after infection (Fig. 1AFig. 1.

Bottom Line: The presence of anti-erythrocyte autoantibodies in animals infected with various Babesia species is well reported.Five clones were generated.By Western blotting analysis using whole erythrocyte antigens, one clone reacted with a broad band around 90-150 kDa, and the 2 clones reacted with a band larger than 150 kDa.

View Article: PubMed Central - PubMed

Affiliation: United Graduate School of Veterinary Science, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan.

ABSTRACT
The presence of anti-erythrocyte autoantibodies in animals infected with various Babesia species is well reported. However, the pathogenesis of autoantibodies in babesiosis is poorly understood. Here, we demonstrated that anti-erythrocyte immunoglobulin (Ig) M and IgG were present in B. rodhaini-infected mice at 6 and 8 days after infection, respectively. Furthermore, we generated monoclonal antibodies against erythrocyte antigen from B. rodhaini-infected mice. Five clones were generated. By Western blotting analysis using whole erythrocyte antigens, one clone reacted with a broad band around 90-150 kDa, and the 2 clones reacted with a band larger than 150 kDa. B. rodhaini-infected mice and/or autoreactive monoclonal antibodies established in this study might be a powerful tool for in vivo pathogenesis studies of autoantibody development in infectious diseases.

Show MeSH
Related in: MedlinePlus