鸭肝炎病毒现场分离株单克隆抗体的研制

Preparation and identification of monoclonal antibodies against locale isolated strain of duck hepatitis virus

为有效防治鸭肝炎,建立特异性强、敏感性高、简便快速的DHV检测方法,对分离的DHV进行纯化,并制备单克隆抗体。鸭胚尿囊液中的DHV经冻融、氯仿反复处理、PEG浓缩和超速离心纯化后,免疫BALB/c小鼠,用杂交瘤技术,通过间接ELISA法筛选及多次亚克隆,成功获得8株阳性杂交瘤细胞,分别命名为DHV-1、DHV-6、DHV-7、DHV-8、DHV-9、DHV-10、DHV-11和DHV-12,并制备出腹水。8株单抗腹水经ELISA法检测,DHV-7、DHV-8的效价为1∶2 000;DHV-6、DHV-10的效价为1∶8 000;DHV-1、DHV-9、DHV-11和DHV-12的效价为(1∶32 000)^(1∶512 000)。抗体亚类鉴定,DHV-6、DHV-7、DHV-8、DHV-10为IgG1;DHV-1、DHV-12为IgG2a;DHV-2、DHV-9、DHV-11为IgG2b。上述抗体均为k链。特异性鉴定表明,上述单克隆抗体只能与从尿囊液中纯化的DHV病毒包板反应,与非DHV抗原无反应。鸭胚中和试验及雏鸭保护试验结果表明,DHV-6、DHV-7、DHV-9、DHV-10具有较好的中和活性,并对雏鸭有不同程度的保护作用。

The BALB/c mice were immunized with DHV of allantoid liquid in duck embryo which were purified by freezing and melting repeatedly,dealt with by chloroform,concentrated by PEG and overspeed membrane filtrating.Eight hybridoma cell lines were obtained after detecting and 3 sub-cloning by I-ELISA.They were named DHV-1,DHV-6,DHV-7,DHV-8,DHV-9,DHV-10,DHV-11 and DHV-12 respectively.The ascites titres of these MAbs were between 1∶2 000 and 1∶512 000.The ascites had no cross reaction with other antigen and the normal allantoid liquid in duck embryo.MAbs belonged to IgG with κ-chain.neutralization test and young duck pretection test indicated that neutralization characters of DHV-6,DHV-7,DHV-9,DHV-10 are stronger.