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KMID : 0378020080510110038
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New Medical Journal
2008 Volume.51 No. 11 p.38 ~ p.46
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Reformed Ant-CD1a Monoclonal Antibody Synthesis by Molecular Genetic Method
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Oh Seung-Hwan
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Abstract
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Purpose: CD1a is known to present lipid antigens and observed in various diseases. Anti-CD1a monoclonal antibody can be used to treat and diagnose for a lot of disorders. In this study, reformed anti-CD1a monoclonal antibody was synthesized with modification of pre-defined procedures to find more useful and productive methods.
Methods: The cDNA of anti-CD1a monoclonal antibody was constructed with RT-PCR using NA1/34.HLK cells. Single chain variable fragment (scFv) antibody was expressed on transformed Eschericia coli and secreted into bacterial culture media at 25?C and 30?C with various sucrose concentration by phage display method. The presence of scFv was
screened with flow cytometry by binding on thymocytes and specificity of it was clarified by blocking of its reaction with anti-CD1a NA1/34.HLK antibody.
Results: There were 17 clones which secreted scFv on the media binding to thymocytes but only two clones(22A, 34B) showed specificity. The 22 A clone medias which cultured at both 25?C and 30?C showed the binding (D value =0.88, 0.80). But the 34B had it only at 25?C (D values = 0.47) and not at 30?C (D value =0.27). After blocking thymocytes, all the three above mentioned scFv media had significantly decreased the reaction. All three D values which compared the scFv binding histograms between before and after blocking were over 0.3. The difference of the binding activity of scFv among each sucrose concentration on media (absence, 0.2 M, 0.4 M) was not significant (D values <0.2)
Conclusion: Lowering incubation temperature can be effective to product anti-CD1a scFv antibody, but sucrose would not. This finding could be helpful to synthesis and study for anti-CD1a scFv.
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KEYWORD
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Single-Chain Fv, Flow Cytometry, Anti-CD1a monoclonal antibody
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