血清Ⅲ型马立克病病毒单克隆抗体的制备及在疫苗蚀斑计数中的应用
Preparation of serotype-Ⅲ specific monoclonal antibody against MDV and its pre-liminary applications in plaque counting of vaccines
李文超 1刘丹 2杨承槐 2李启红 2李欣 1张莹 1毛娅卿 2李俊平2
作者信息
- 1. 沈阳工学院生命工程学院,辽宁 抚顺 113122
- 2. 中国兽医药品监察所,北京 100081
- 折叠
摘要
通过制备马立克病病毒(Marek's disease virus,MDV)血清Ⅲ型特异性单克隆抗体,并对其特性进行鉴定,为建立鸡马立克病(Marek's disease,MD)二价活疫苗间接免疫荧光蚀斑计数方法奠定基础.以纯化的MDV血清Ⅲ型HVT Fc-126株免疫BALB/c小鼠,采用常规单克隆抗体技术,经间接免疫荧光方法筛选杂交瘤细胞株;选取1株制备MDV Ⅲ型单克隆抗体,建立MD二价活疫苗的间接免疫荧光蚀斑计数方法.结果筛选出2株稳定分泌抗MDV血清Ⅲ型单克隆抗体的杂交瘤细胞株,并选择1D2株单抗建立了检测MD二价活疫苗的间接免疫荧光蚀斑计数方法,可快速准确地计数二价活疫苗中血清Ⅲ型病毒的蚀斑形成单位数(Plaque forming unit,PFU),与琼脂覆盖法相比,敏感性和特异性大大提高.因此,制备的MDV单克隆抗体具有良好的血清型特异性,可用于MD二价活疫苗的间接免疫荧光蚀斑计数.
Abstract
In order to prepare and characterize serotype-Ⅲ specific monoclonal antibody(McAb)a-gainst Marek's disease virus(MDV),and then utilized it for establishing plaque counting method by indirect immunofluorescent staining.Purified MDV serotype-Ⅲ HVT Fc-126 strains were used to immunize BALB/c mice.The hybridoma cultured supernatants were screened by indirect immu-nofluorescent assay with HVT Fc-126 infected chicken embryo fibroblast cells(CEF).The hybri-doma clones were selected and employed for preparation of McAb.Plaque counting method by indi-rect immunofluorescent staining was established with the McAb to detect the MDV serotype-Ⅲplaque forming units(PFU)in Marek's Disease bivalent live vaccines.Two hybridoma clones that constitutively secrete McAb against MDV serotype-Ⅲ were screened.High purified McAb was pre-pared with 1D2 hybridoma clone.Compared with agarose plaque assay,the indirect immunofluores-cent staining method characterized higher sensitivity,specificity,and more rapidity.Significantly,the indirect immunofluorescent staining method could accurately count PFU of serotype-Ⅲ viruses in bivalent vaccine containing two serotype viruses.Hence,serotype-Ⅲ specific McAb against MDV has been prepared successfully,and could be applied in plaque counting of bivalent vaccines by in-direct immunofluorescent staining method.
关键词
马立克病病毒/血清型/单克隆抗体/二价疫苗/蚀斑计数Key words
Marek's disease virus/serotype/monoclonal antibody/bivalent vaccine/plaque counting引用本文复制引用
基金项目
辽宁省教育厅科学研究经费面上基金(2021)(LJKZ1339)
兽药行业公益性重点专项(GY202105)
出版年
2024
NETL
NSTL
万方数据