小鼠呼肠孤病毒Ⅲ型标准化血清制备及ELISA检测方法的建立

目的制备标准化小鼠呼肠孤病毒III型（reovirus 3,Reo-3）免疫血清,建立小鼠Reo-3抗体ELISA检测方法。方法使用动物来源的Reo-3病毒感染BALB/c小鼠,获得高效价免疫血清;以BHK-21细胞制备Reo-3病毒抗原,同时制备做正常对照抗原。滴定抗原和标准化小鼠Reo-3血清的最佳工作浓度,进行特异性、敏感性、重复性、稳定性等实验。结果制备18 mL抗Reo-3血清,经检测,IFA效价达1∶640,IEA效价达1∶160;Reo-3抗原和标准化小鼠Reo-3免疫血清最佳工作浓度分别为5μg/mL和1∶2400;该ELISA检测体系Reo-3特异抗原批内变异系数为3.8%,批间变异系数为4.0%;检测灵敏度〉1∶4400;与仙台病毒（SV）、小鼠肝炎病毒（MHV）、小鼠腺病毒（Mad）、小鼠脑脊髓炎病毒（TMEV）、小鼠多瘤病毒（POLY）均无交叉反应。经37℃破坏性试验,2 d稳定性试验相对偏差〈27%。结论本研究制备的Reo-3抗血清达到同批次大量高滴度的水平,可作为检测实验小鼠Reo-3病原的标准化质控血清。本研究建立的ELISA方法重复性、稳定性好,特异性、敏感性强。该体系可用于大、小鼠等实验动物Reo-3抗体的检测。

Preparation of Antisera and Establishment of an ELISA Detection Technique for Reovirus III in Laboratory Mice

Objective To prepare a high-titer standard mouse serum against reovirus 3 for quality control of viral infection in laboratory mice and to establish an ELISA method for detecting Reo-3 antibody.Methods BALB/c Mice were infected with Reo-3.Sera were harvested and identified by IFA,IEA.The Reo-3 specific antigen was prepared by raising BHK-21 cells.Results The titers of mouse anti-Reo-3 sera were 1∶ 640 by IFA and 1∶ 160 by IEA,respectively.The best working density of Reo-3 antigen and the mouse anti-Reo-3 serum were 5 μg/mL and 1∶ 2400,respectively.The inter-assay coefficient of variation of normal antigen and specicic antigen was 3.8%.The intra-assay average coefficient of variation was 4.0%.The detection sensitivity was 1∶ 4400.There was no cross-reactivity with Sendai virus（SV）,mouse hepatitisvirus（MHV）,mouse adenovirus（MAd）,vaccinia virus and mouse polyoma virus（POLY）.The stability test showed that the relative deviation was below 27% within 2 days at 37℃.Conclusion An anti-Reo-3 serum from immunized mice has been prepared.It is with a high titer,sensitive and specific to Reo-3 detection.It can be widely used as a standard control serum to detect Reo-3 serologically.The ELISA method can be used in detecting the Reo-3 antibody in rodent colony with good reproducibility,duplication,stability,specificity,and sensitivity,