鸡新城疫病毒中和单克隆抗体的制备及鉴定

Production and Characterization of Neutralizing Monoclonal Antibodies against Newcastle Disease Virus

采用差速离心法纯化鸡新城疫病毒(NDV)标准毒株F48E8,免疫Balb/c小鼠,应用杂交瘤细胞技术制备抗NDV单克隆抗体,旨在为NDV中和抗原表位分析奠定基础。将NDV感染BHK-21细胞,建立异源免疫过氧化物酶单层细胞试验(IPMA)的单克隆抗体检测方法,筛选获得14株分泌抗NDV单克隆抗体杂交瘤细胞株,其单克隆抗体腹水IPMA效价均在0.50×10-2~2.56×10-5(F48E8株和La Sota株)。血凝抑制试验(HI)结果表明,单克隆抗体1G6、2C1、4D2、5F2和13A5具有血凝抑制活性,其HI效价在(6~12)log2(F48E8株)和(9~11)log2(La Sota株)。病毒中和试验结果表明,单克隆抗体5F2和13A5对F48E8株和La Sota株均有明显的病毒中和活性,中和效价分别为1∶400~1∶800和1∶25。夹心ELISA结果表明,单克隆抗体5F2识别NDV HN蛋白,13A5识别F蛋白。综上,成功制备了具有中和活性的NDV单克隆抗体(5F2和13A5)。

Balb/c mice were immunized by the Newcastle disease virus （ NDY ） reference strain F48E 8 purified by differential centrifugation, and used for monoclonal antibodies （ mAbs ） production using hybri- doma technology. After screened by a heterologous immunoperoxidase monolayer assay （IPMA ） based on the NDY-infected BHK -21 cells, fourteen hybridoma cell lines secreting mAbs specific for NDY were ob-tained, whose antibody titers of ascites were determined to be 0. 50 ×10^-2-2.56 ×10^-5 to F48E 8 and to LaSotain by IPMA respectively. In hemagglutination inhibition （ HI） assay , mAbs 1G6 ,2C1 ,4D2,5F2 and 13 A5 showed hemagglutination-inhibitory activity with the HI titers of 6-12log2 （ F48E8 ） and 9-11 log2 （LaSota） , of which mAbs 5F2 and 13 A5 showed significant neutralizing activity to both NDY virulent and vaccine strains in the virus neutralization （ YN） test with the titers of 1：400- 1：800 and 1： 25 respective-ly. Furthermore, sandwich ELISA using the recombinant proteins showed that mAb 5F2 reacted with HN protein of NDY, while mAb 13 A5 recognized the F protein. In conclusion, the NDY mAbs of 5F2 and 13A5 with neutralization activity were obtained successfully.