Fc受体结合型与非Fc受体结合型CD3单抗的制备
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摘要
目的研究Fc受体结合型与非Fc受体结合型CD3单抗的制备及单抗的特性。方法利用杂交瘤细胞株扩增,使用体外培养法制备Fc受体结合型CD3单抗,利用动物体内诱生法制备非Fc受体结合型CD3单抗。使用亲和层析法纯化抗体并与市售的CD3单抗比较,行淋巴细胞培养、流式细胞术等了解Fc受体结合型与非Fc受体结合型CD3单抗的特性。结果使用体外培养法制备蛋白G琼脂糖纯化了Fc受体结合型CD3单抗;使用动物体内诱生法制备A蛋白亲和层析法纯化了非Fc受体结合型CD3单抗;CD3单抗与市售的CD3单抗一致;Fc受体结合型与非Fc受体结合型CD3单抗的增殖及T细胞受体内化效应和文献报道一致。结论利用杂交瘤细胞株扩增并成功制备了Fc受体结合型与非Fc受体结合型CD3单抗,并为后续工作的进行奠定了良好的基础。
Objective To purify Fc receptor binding and Fc receptor non-binding anti-CD3 monoclonal antibody.Methods Hybridoma strains were amplified.In vitro culture was applied to produce Fc receptor binding anti-CD3.Ascites were applied to produce Fc receptor non-binding anti-CD3.Affinity chromatography was used for the purification of antibodies.The products were compared to the commercial CD3 monoclonal antibody.Also,lymphocyte culture and flow cytometry were used to understand the characteristics of Fc receptor binding and Fc receptor non-binding CD3 monoclonal antibody.Results Fc receptor binding anti-CD3 was produced by protein G-agarose purification.Fc receptor non-binding anti-CD3 was produced by Protein A ascites purification.The products were not different with the commercial CD3 monoclonal antibody and consist with previous papers.Conclusion By purification of Fc receptor binding and Fc receptor non-binding anti-CD3 monoclonal antibody,agood foundation is established for the follow-up work.
Objective To purify Fc receptor binding and Fc receptor non-binding anti-CD3 monoclonal antibody.Methods Hybridoma strains were amplified.In vitro culture was applied to produce Fc receptor binding anti-CD3.Ascites were applied to produce Fc receptor non-binding anti-CD3.Affinity chromatography was used for the purification of antibodies.The products were compared to the commercial CD3 monoclonal antibody.Also,lymphocyte culture and flow cytometry were used to understand the characteristics of Fc receptor binding and Fc receptor non-binding CD3 monoclonal antibody.Results Fc receptor binding anti-CD3 was produced by protein G-agarose purification.Fc receptor non-binding anti-CD3 was produced by Protein A ascites purification.The products were not different with the commercial CD3 monoclonal antibody and consist with previous papers.Conclusion By purification of Fc receptor binding and Fc receptor non-binding anti-CD3 monoclonal antibody,agood foundation is established for the follow-up work.
引文
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[2]Harris A C,Ferrara J L,Levine J E.Advances in predicting acute GVHD[J].Br J Haematol,2013,160(3):288-302.
[3]Blazar B R,Murphy W J,Abedi M.Advances in graft-versushost disease biology and therapy[J].Nat Rev Immunol,2012,12(6):443-458.
[4]Inamoto Y,Flowers M E,Lee S J,et al.Influence of immunosuppressive treatment on risk of recurrent malignancy after allogeneic hematopoietic cell transplantation[J].Blood,2011,118(2):456-463.
[5]Carpenter P A,Lowder J,Johnston L,et al.A phase II multicenter study of visilizumab,humanized anti-CD3antibody,to treat steroid-refractory acute graft-versus-host disease[J].Biol Blood Marrow Transplant,2005,11(6):465-471.
[6]Li X,Deng R,He W,et al.Loss of B7-H1expression by recipient parenchymal cells leads to expansion of infiltrating donor CD8+T cells and persistence of graft-versus-host disease[J].J Immunol,2012,188(2):724-734.
[7]He W,Racine J J,Johnston H F,et al,Depletion of host CCR7(+)dendritic cells prevented donor T cell tissue tropism in anti-CD3-conditioned recipients[J].Biol Blood Marrow Transplant,2014,20(7):920-928.
[8]Li N,Chen Y,He W,et al.Anti-CD3preconditioning separates GVL from GVHD via modulating host dendritic cell and donor T-cell migration in recipients conditioned with TBI[J].Blood,2009,113(4):953-962.
[9]Li N,Zhao D,Kirschbaum M,et al.HDAC inhibitor reduces cytokine storm and facilitates induction of chimerism that reverses lupus in anti-CD3conditioning regimen[J].Proc Natl Acad Sci USA,2008,105(12):4796-4801.
[10]黄晓军.构建有中国特色的造血干细胞移植体系[J].中华血液学杂志,2012,33(8):321-324.
[11]Zhang C,Lou J,Li N,et al.Donor CD8+T cells mediate graft-versus-leukemia activity without clinical signs of graft-versus-host disease in recipients conditioned with anti-CD3 monoclonal antibody[J].J Immunol,2007,178(2):838-850.
[12]Yi T,Li X,Yao S,et al.Host APCs augment in vivo expansion of donor natural regulatory T cells via B7H1/B7.1in allogeneic recipients[J].J Immunol,2011,186(5):2739-2749.
[13]Jayashree B S,Nigam S,Pai A,et al.Targets in anticancer research-A review[J].Indian J Exp Biol,2015,53(8):489-507.
[14]卢克友.粪便隐血试验单克隆抗体法与联苯胺法的比较[J].福建医药杂志,2008,30(2):109-110.