【作者】 张玲妮；

【导师】 姚火春；

【作者基本信息】 南京农业大学 ， 预防兽医学， 2004， 硕士

【摘要】 应用0.8％甲醛灭活的猪链球菌2型9801全菌作为抗原，免疫BABL／C小鼠，待抗体水平达到1：100000时，取脾细胞并与对数期生长良好的SP2／0进行融合。应用亲和层析纯化的原核表达的溶菌酶释放蛋白(MRP)为抗原建立了间接ELISA筛选阳性克隆，将阳性克隆多次有限稀释(每孔约0.5～1个细胞)得到MRP阳性单克隆细胞8株。将10~5～10~7个细胞注射到BABL／C小鼠腹腔制备腹水，效价约1：10000，并用Westen-blot鉴定此单抗。应用兔源全菌抗体和MRP单抗建立双夹心间接ELISA，用于检测猪链球菌2型，试验结果证实可以区分猪链球菌2型与A群、B群、C群、D群链球菌、大肠杆菌、金黄色葡萄球菌等菌株。 应用甲醛灭活的猪链球菌2型9801菌株作为标准菌株建立了检测猪链球菌2型抗体的菌体包被间接ELISA，并应用该菌的荚膜多糖提取物建立了荚膜多糖包被间接ELISA。并对两种间接ELISA方法进行了比较。试验结果显示，利用荚膜多糖包被的间接ELISA检测猪链球菌抗体虽然非特异性低，但试验结果阳性血清OD值偏小，试验方法的灵敏度降低，荚膜多糖作为包被抗原对ELISA板的要求很高，国产普通ELISA板包被效果很差；而应用菌体包被间接ELISA进行猪链球菌抗体检测，非特异性比用荚膜多糖包被间接ELISA高，但比超声波抗原的非特异性低，非特异性随着抗体稀释度的增加而逐渐减小，试验可以找到一个灵敏度与特异性均较高的稀释度，因而适用于猪链球菌2型抗体的检测。与毒力因子作为包被抗原的间接ELISA相比，菌体包被间接ELISA进行猪链球菌抗体检测还具有全面准确的特点。菌体作为包被抗原时对ELISA板的要求不高，可以降低检测的成本，在-20℃可保存一年半以上。菌体包被间接ELISA优于荚膜多糖包被间接ELISA，可用于猪链球菌的抗体监测，便于推广应用。 应用猪链球菌2型9801株菌体包被间接ELISA分别对70份阳性血清和70份阴性血清进行1：100、1：200、1：400、1：800稀释检测，制作了不同稀释度下阳性血清和阴性血清的OD值与出现频率的正态分布图，以及血清不同稀释倍数的检测结果的ROC曲线，利用血清流行病学原理对检测结果进行分析，确定了检测方法的最佳单一稀释度1：400，以及判定阳性血清和阴性血清的OD值：以OD值≥0.6为检测阳性，以OD值≤0.3为检测阴性，0.3＜OD值＜0.6为检测可疑值。并应用建立的方法及阳性摘要血清和阴性的判定标准，对1 997年采集的江苏地区的猪血清进行了检测.阳性率高达48%，尤其是双甸马塘地区，阳性率高达7 2.4%.而杭体阳性率高的地区也正是1998年至1 999年间江苏省首先幕发猪健球菌2型的地区，推测在98年猪链球菌大爆发的前期，有的猪场已经存在猪链球菌病感染，可能由于自身抵抗力和杭生素的作用，没有在当时就一下基发，因而及时对杭体监控是预防控制该病的重要措施.更多还原

【Abstract】 The BALB/C mice were immunized with the inactivated streptococcus suis type 2 by 0.8% formaldehyde.When the serum anti-MRP-antibody titre came to 1:104, the spleen cells were fused with the SP2/0 by PEG-1500. Indirect ELISA was founded to detect the hybridoma surprunt by purified expressed MRP with the His-Bind Resin . Eight hybridoma strains secreting McAb against MRP were obtained by limited diluting several times and the ascites was identified with Westen-blot. A double antibody sandwich ELISA was established to detect streptococcus suis type 2.A Formalin-killed whole bacteria cells of HA9801 antigen-based enzyme-linked immunosorbent assay(WCA-ELISA) to detect antibodies against Streptococcus suis type 2 was developed and compared with a purified capsular polysaccharide antigen-based indirect ELISA (CPS-ELISA). Although the standardized gave satisfactory results that most cross-reactions decreased significantly, the result of positive sera of animals associated with Streptococcus suis type 2 and the result of negtive sero by CPS-ELISA did not present liters significantly different. According to the results, it could be concluded that the CPS-ELISA developed in this study used as a diagnostic tool to identify infected animals was discredited. Specificity is improved by diluting the sera with WCA-ELISA. A appropriate dilution can be found out to make the result of detection accuratest. It is proved that the result was more reliable with WCA-ELISA than with CPS-ELISA.70 shares of positive sera and 70 shares of negtive sera diluted to100, 200, 400 , 800, 1600 were tested with a whole bacteria cells of HA9801 antigen-based enzyme-linked immunosorbent assay. The result was analysed to determine the appropriate dilution and the criterion to makesure the negtive sera and the positive sera by epidemiology. It proved that: Beforel998, there was the infection ofStreptococcus suis type 2 in Jangsu province by detecting the sera collected in 1997.更多还原