| Budget Amount *help |
¥3,000,000 (Direct Cost: ¥3,000,000)
Fiscal Year 2002: ¥1,200,000 (Direct Cost: ¥1,200,000)
Fiscal Year 2001: ¥1,800,000 (Direct Cost: ¥1,800,000)
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| Research Abstract |
We are addressing to produce recombinant human antibodies. The aim of this study is the production of human anti-HLA antibodies. In HLA-typing analysis performed in organ transplantation, paternity test, and analysis of diseases, human anti-HLA serum are used, but the cross reaction occurs in some percentage because human sera are not monoclonal. In addition, since the most sera are obtained from multigravida, it is difficult to prepare all sets of sera which show high specificity and affinity characteristics, and also identify rare case of HLA.
In this study, we aimed to produce recombinant anti-HLA class I B51 and class II DR9 antibodies using PCR primers and expression vector constructed for phage display system.
Memods were as follows: Firstly, B cells isolated from peripheral blood which were donated from B51 and DR9 antibody positive individuals were immortalized by EBV infection. Secondly, in the case of B51, immortalized cells were fused with hetero-myeloma cells and established anti-B51 antibody producing cell lines. In the case of DR9, antibody producing cells were isolated from immortalized cells by EBV infection. RNA was extracted from antibody producing cella and Fab regions of antibodies were PCR amplified. Amplified DNA fragments were cloned to expression vector. In these manners, antibody genes were preserved. Finally, antibodies were expressed and specificities of antibodies were analyzed.
DNA sequencing analysis of L and H chains and Western blot analysis of expressed Fab antibody revealed that anti B51antibody was IgG/λα and DR9 was IgG/κ. Recombinant anti B51 Fab antibody was expressed and purified, and used for FACScan and AHG-LCT analyses. B51 antibody reacted specifically for B51 positive cells, but not for negative cells. The cloning, expression and purification of anti DR9 antibody were completed and analysis of its specificities was under investigation.
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