猪伪狂犬重组抗原间接ELISA诊断方法的建立

本研究应用重组抗原,成功建立了检测猪伪狂犬病病毒血清抗体的间接ELISA诊断方法。通过方阵滴定确定了抗原最适包被量为1.45μg/孔,血清最适稀释度为1∶80,其作用时间为60 min,酶标抗体最适作用时间为60 min。其阳性临界值为OD≥0.123。此外,该抗原不与猪其他疾病的阳性血清反应,具有良好的特异性;批内重复试验,变异系数小于10%。该方法与进口诊断试剂盒的符合率为96.3%。本研究为现地免疫猪群抗体检测和进行PRV流行病学调查提供了一种简便的血清学诊断方法。     

抗猪附红细胞体单克隆抗体的制备

以纯化的猪附红细胞体免疫BALB/c小鼠,运用淋巴细胞杂交瘤技术进行细胞融合,并用间接ELISA方法进行筛选,经过间接ELISA方法、免疫印迹和间接免疫荧光试验进行鉴定,共获得5株分泌抗猪附红细胞体单克隆抗体的杂交瘤细胞,分别命名为1H1、1H2、3A5、5B1和7E11,其单抗亚类鉴定分别属于IgG2b、IgG1、IgG2b、IgG2b和IgG2b。这5株McAb均能与猪附红细胞体全菌蛋白发生特异性反应,而不与猪肺炎支原体、猪链球菌、大肠杆菌和猪繁殖与呼吸综合征病毒发生反应,并且1H1、3A5和5B1能识别同一抗原位点,1H2和7E11识别另一抗原位点。     

蜂蜜中氯霉素残留量检测方法研究概况

综述了近几年来国内外蜂蜜中氯霉素残留检测方法.着重介绍了高效液相色谱法、毛细管电泳法、酶联免疫法、色/质联用分析法以及生物传感器法,并分别综述了各种方法的优势与不足,最后展望了蜂蜜中氯霉素残留检测的发展趋势.     

猪流感抗体间接ELISA检测方法的建立

猪流感病毒A／Swine／Fujian／668／2001（H3N2）株感染的鸡胚尿囊液，经差速离心后，再经蔗糖密度梯度离心，提纯、纯化的猪流感病毒经NP-40处理并反复冻融，作为猪流感间接ELISA抗原，确立了间接ELISA检测方法。对29份HI试验猪流感为阴性的血清进行了检测，经统计学分析，确定间接ELISA判定标准，被检血清OD490nm值≥0．20判定为阳性。该方法对猪瘟等11种猪疫病阳性血清无交叉反应，批内和批间重复试验的吸收变异系数分别在3．34％～8．12％和6．2％～9．04％之间。与HI的符合率达到92．8％，经卡方检验（P〈0．01）比HI试验敏感。为猪流感抗体检测提供了快速、准确、简便的方法。     

BSA对牛结核抗体ELISA检测的影响

分别研究了封闭液、血清稀释液及酶标二抗稀释液中BSA成分对牛结核抗体ELISA检测的影响.结果显示,酶标二抗稀释液中BSA的存在使ELISA检测值普遍偏低,且BSA的含量越高,检测值越低;为了提高ELISA检测值,使其达到理想检测效果,必须提高酶标二抗的使用浓度.BSA中可能存在的牛血清抗体杂质对酶标二抗起到了"中和"作用,降低了酶标二抗与目标抗体结合的实际浓度,使检测结果普遍偏低.     

人乳铁蛋白转基因小鼠杂交选育的研究

根据孟德尔遗传原理,对表达人乳铁蛋白转基因小鼠进行近交和远交,获得了纯合子小鼠(P品系1♀、Q品系1♀1♂)和双重杂合子小鼠(PQ杂合3♀3♂).催乳获得小鼠乳汁,ELISA检测乳汁中人乳铁蛋白的含量.通过比较发现,纯合子小鼠和单纯杂合子小鼠在表达量上没有明显差异,而双重杂合子小鼠的表达量则较前两者有明显提高.     

Kinetics of Interferon-Gamma Production and its Comparison with Anti-Listeriolysin O Detection in Experimental Bovine Listeriosis

The kinetics of the production of interferon gamma (IFN-) in whole blood culture and its comparison with anti-listeriolysin O (ALLO) detection by ELISA were studied during oral infection of calves with Listeria monocytogenes. Culture filtrate antigen (CFA), listeriolysin O (LLO), and sonicated antigen (SA) were used to prime the peripheral blood mononuclear cells (PBMCs) and the plasma from orally infected calves. IFN- and ALLO appeared as early as day 7 of an oral infection. IFN- was detected earlier with LLO than with SA. The Max50 interleukin (IL-2) activity and IFN- estimated in the culture supernatant from PBMCs primed in vitro with different antigens of L. monocytogenes revealed high induction of IL-2 and IFN- by CFA, LLO and live antigen. IFN- assay and ALLO detection were used for testing cases of repeat breeding in dairy cattle. It appeared that detection of IFN- employing LLO can be used to diagnose listerial infections.     

Induced and spontaneous IgE antibodies to Dermatophagoides farinae in dogs and cats: evidence of functional heterogeneity of IgE

Enzyme-linked immunosorbent assays (ELISAs) were developed to measure IgE antibodies specific for Dermatophagoides farinae in dogs and cats. Although higher levels were detected in atopic dogs and cats than in normal animals without skin disease, the differences were not statistically significant. On the other hand, levels in dogs and cats that were reared under laboratory conditions, and thus presumably not exposed to house dust mites, were either very low or undetectable. IgE antibodies were induced in 10 laboratory-reared cats using low-dose antigenic stimulation in aluminium hydroxide. All cats developed detectable IgE, but not all developed positive skin tests. However, serum from those cats with positive skin tests were able to give positive Prausnitz–Küstner (PK) tests. The canine data, together with previous work on basophil histamine release, suggests that the distinction between atopic and normal dogs may result from a heterogeneity of either IgE or of the high-affinity mast cell receptor. The feline data can only be explained by the existence of a heterogeneity of IgE.     

抗猪流行性腹泻病毒单克隆抗体的研制及其特性鉴定

以新生乳猪增殖猪流行性腹泻病毒（ＰＥＤＶ）,通过蔗糖密度梯度离心将其纯化,获得较为完整的病毒颗粒。病毒ＥＬＩＳＡ效价为１∶３×１０５,蛋白含量为３．９６ｇ／Ｌ。将提纯的ＰＥＤＶ免疫ＢＡＬＢ／ｃ小鼠,取免疫鼠脾细胞与ＳＰ２／０骨髓瘤细胞进行融合,融合率为９５．６％。用间接ＥＬＩＳＡ进行筛选,共检出３８个阳性孔,阳性率为２８．８％。对其中１５个阳性值较高的孔进行３次再克隆,阳性率达１００％,最后获得１５株稳定分泌特异性抗ＰＥＤＶ单抗（ＭｃＡｂ）的杂交瘤细胞。ＥＬＩＳＡ阻断试验与交叉试验证明,其分泌的抗体具有高度特异性。腹水和细胞培养上清的ＥＬＩＳＡ效价分别为１∶１０３～１∶１０６和１∶１２８～１∶５１２。经鉴定,１５株ＭｃＡｂ的分子类型均为ＩｇＧ,均无免疫沉淀特性。杂交瘤细胞的染色体数目为９０～１１０,平均９５。尤为重要的是,这些杂交瘤细胞所分泌的抗体,有的对ＰＥＤＶ具有中和作用,能够阻断病毒对猪只的侵害作用。     

用Dot—ELISA检测鸡新城疫病毒的研究

本文建立了检测鸡新城疫病毒的Ｄｏｔ－ＥＬＩＳＡ法。用该法检测自然发病鸡群的粪便样品和脾组织匀浆各８２份,鸡新城疫病毒的检出率（５１．２％,７４．４％）高于血球凝集试验（３６．６％,６１．０％）,且两种方法具有良好的平行关系。该法操作简单,结果明显,直观,适用于基层检测鸡新城疫病毒。