血清1型马立克氏病病毒单克隆抗体的制备及其特异性鉴定

Preparation and Specificity identification of Monoclonal Antibody against Serotype 1 Marek’s Disease Virus

关键词:  马立克氏病病毒  单克隆抗体  鉴定。

Key words:  Marek’s disease virus  monoclonal antibody  identification.

摘要:

为制备血清1型马立克氏病病毒（Marek’s disease virus，MDV）单克隆抗体，以超速离心浓缩的血清1型MDV CVI988/Rispens毒株免疫BALB/c小鼠，应用杂交瘤技术制备单克隆抗体。通过亚克隆、间接ELISA和间接免疫荧光（Indirect immunofluorescence assay, IFA）筛选，获得3株分泌抗血清1型MDV单克隆抗体的杂交瘤细胞系，分别命名为4D9、1C5和1F10，并对其进行特异性分析。间接ELISA结果显示，4D9、1C5和1F10制备的腹水ELISA效价分别达5.1x104、8.2x105、4.1x105，可用于后续建立ELISA检测方法；IFA结果显示，4D9、1C5和1F10制备的腹水与不同血清1型MDV毒株具有良好的反应性，效价可分别达1: 12800、1: 25600和1: 12800，与血清3型MDV毒株和其他常见禽源病毒均没有交叉反应，特异性良好；亚型检测结果显示，4D9抗体为IgG2a/κ型，1C5抗体为IgG2b/κ型、1F10抗体为IgG1/κ型；应用1C5建立的IFA方法能检出至少5PFU CVI988/Rispens毒株感染，适用于血清1型MDV的检测。综上，制备的单克隆抗体可用于血清1型MDV的检测，为MDV致病机制研究及诊断试剂研发提供基础。

Abstract:

To prepare the monoclonal antibody (McAb) of serotype 1 Marek’s disease virus, BALB/c mice were immunized with the serotype 1 MDV CVI988/Rispens strain concentrated by ultracentrifugation, and McAbs were prepared by hybridoma technique. By subcloning, indirect ELISA and indirect immunofluorescence assay (IFA) identification, three positive McAb cell lines were obtained and named as 4D9, 1C5 and 1F10, respectively. Then, the specificity of the obtained McAbs was analyzed. The ELISA titers of 4D9, 1C5 and 1F10 reached 5.1x104, 8.2x105 and 4.1x105, indicating that all of the three could be used to establish ELISA detection methods; IFA results showed that the ascites prepared by 4D9, 1C5 and 1F10 had good reactivity with different serotype 1 MDV strains with the titers of 1:12800, 1:25600 and 1:12800, respectively, and all of the three have no cross-reactivity with serotype 3 MDV strains and other common avian-derived viruses; The subtype test of the three above McAbs indicated that 4D9 was IgG2a/к subtype, 1C5 was IgG2b/κ subtype and 1F10 was IgG1/κ subtype. The IFA method established with 1C5 could detect more than 5 PFU CVI988/Rispens, confirming that this method is suitable for the detection of serotype 1 MDV. In summary, the McAbs obtained in this study can be used for the detection of serotype 1 MDV and provide the basis for further researches on MDV pathogenicity and the development of diagnostic reagents.