FAVN与RFFIT在动物和人狂犬病中和抗体检测中的比较

目的比较荧光抗体病毒中和试验(FAVN)和快速荧光灶抑制试验(RFFIT)两项技术用于动物和人血清狂犬病中和抗体检测的差异。方法以FAVN和RFFIT两种方法分别对60份动物及人血清进行狂犬病中和抗体的平行定量检测,应用配对定量资料的t检验对所得数据进行统计学分析。结果以0.5IU/mL的国际推荐标准,对同一样品经FAVN和RFFIT两种方法测定的中和抗体效价进行定性判定,二者对全部60份样品的检测符合率为96.67%(58/60)。统计学分析显示,两种检测方法对总体样品及不同动物样品的定量检测结果差异均无统计学意义(P0.05)。结论 FAVN和RFFIT可通用于动物和人血清的狂犬病中和抗体检测。     

双重平行检测批量样品对两个实验室RFFIT检测效能的评价

快速荧光灶抑制试验(RFFIT)是世界卫生组织(World Health Organisation,WHO)推荐的狂犬病病毒中和抗体检测标准方法之一,国内进行RFFIT检测的不同实验室之间互评可促进该方法推广时的标准化。病毒病预防控制所和中国药品生物制品检定所对200例人血清进行了双重平行RFFIT检测。结果显示双方检测中和抗体效价几何均值(genomic means titration,GMT)为1.89、1.84I U/mL,差别无统计学意义(t=-0.94,P>0.05);血清中和抗体阳性率为95.00%、93.00%,差别无统计学意义(χ2=0.71,P>0.05);双方结果定性一致率为89.00%,发生结果定性不一致的22例(11.00%)血清双方检测结果均<1I U/mL;67例双方检测结果均<1I U/mL的血清样品中有22例(32.84%)发生结果定性不一致。     

采用快速荧光灶抑制试验对一种狂犬病病毒抗体竞争性ELISA检测法的评价

目的以快速荧光灶抑制试验(RFFIT)检测结果为标准,确定竞争性酶联免疫吸附试验(ELISA)检测人血清样品中狂犬病病毒抗体的能效。方法对100份人血清样品采用RFFIT检测狂犬病病毒中和抗体,采用竞争性ELISA检测狂犬病病毒抗体,分析两种方法检测结果的相关性和一致性。结果经回归分析,两方法检测结果之间有线性关系,回归方程Y=2.320+0.333X,相关系数r=0.504;经χ2检验,两种方法阳性检出率差异无统计学意义(χ2=3.70,P>0.05),两种方法有相关性(χ2=16.50,P<0.05);经Scheff啨可信区间法分析,RFFIT不同检测值范围内ELISA阳性检出率之间差异无统计学意义(P>0.05)。结论本组评价的竞争性ELISA与RFFIT检测结果之间有相关性,但一致性较差,还需要进一步改进。     

狂犬病病毒中和抗体检测试剂盒的效果评价

目的应用快速荧光灶抑制试验(RFFIT)对狂犬病病毒中和抗体检测试剂盒的检测效果进行评价。方法用RFFIT和狂犬病病毒中和抗体检测试剂盒分别对135份人血清和164份动物血清进行狂犬病病毒中和抗体(RVNA)效价检测,应用SPSS22.0分析两种检测方法所得结果的相关性和一致性。结果两种方法所得结果的阳性检出符合率为99.33%(297/299);两种方法对动物血清和人血清的检测结果之间有较好的直线相关关系,相关系数分别为0.897和0.941;同一份标本的重复性检验显示,试剂盒检测方法具有较好的稳定性。结论狂犬病病毒中和抗体检测试剂盒可以用于人和动物血清狂犬病病毒中和抗体的检测。     

狂犬病疫苗接种者抗狂犬病病毒中和抗体影响因素分析

目的探讨狂犬病疫苗接种后体内抗狂犬病病毒中和抗体产生的影响因素。方法采集2018年1-8月山东、江苏、河南、河北、湖南和安徽6省疑似狂犬病Ⅱ/Ⅲ级暴露后接种狂犬病疫苗的暴露者血清标本,快速荧光灶抑制实验(RFFIT)检测抗狂犬病病毒中和抗体(RVNA)水平,采用SPSS 19.0软件进行数据统计分析。结果全程接种狂犬病疫苗的血清样本为498份,RFFIT检测结果显示,RVNA几何平均滴度(GMT)为4.94 IU/ml,血清阳性率为96.18%(479/498)。不同组别抗体阳性率分析结果显示,免疫时间各组阳性率之间差异有统计学意义(χ^2=7.888,P=0.039),30~90 d组抗体阳性率高于91~180 d组,其他各组比较差异均无统计学意义。多因素分析结果显示年龄、免疫时间是RVNA水平的重要影响因素,其中年龄与RVNA水平呈正相关,免疫时间与RVNA水平呈负相关。免疫时间对RVNA水平的影响最大,影响重要性为0.62。结论接种狂犬病疫苗后能产生可靠的免疫效果,年龄、免疫时间等因素对抗体阳性率及RVNA水平有一定影响。暴露后在及时进行规范完整的暴露后处置外,应对经常暴露于狂犬病的高危人群、免疫力低下者或患有免疫抑制性疾病的人群定期检测血清抗体,对于RVNA滴度小于0.5 IU/ml的接种者应及时进行加强免疫,补接种疫苗,从而有效预防狂犬病。     

用斑点免疫结合测定对狂犬病作血清学诊断

大多数实验室喜欢用在免疫荧光抗体染色法使用活病毒的组织培养中和试验(快速荧光焦点抑制试验〔RFFIT〕)诊断狂犬病.斑点免疫结合测定法(DIA)常规检测各种病毒的抗体的成功,提示其可用于测定狂犬病病毒抗体.作者使用了33份人血清(17份源自阴性未免疫接种个人,16份源自阳性免疫接种个人)和22份狗血清(3份源自阳性接种狗,19份对照),比较了DIA和标准方法(RFFIT)的结果.     

人狂犬病毒IgG抗体定量测定试剂盒(酶联免疫法)的评价

目前,被国际兽疫局（OIE）和世界卫生组织（WHO）狂犬病专家委员会认可的检测狂犬病病毒血清／血浆抗体的方法有小鼠中和试验（MNT）、快速荧光灶抑制试验（RFFIT）、荧光抗体病毒中和（FAVN）试验口,但均存在耗时长、操作繁琐、需要昂贵荧光显微镜以及专业人员培训等缺点,只能在有条件的实验室进行。而抗狂犬病毒抗体的ELISA测定方法因其特异、     

狂犬病毒核蛋白单抗和荧光抗体中和试验的建立与应用

目的建立狂犬病毒血清中和抗体效价的快速检测方法，以实现狂犬疫苗免疫效果的定量评价，指导高危人群及动物种群的免疫预防。方法采用常规方法建立杂交瘤细胞株并制备抗狂犬病毒核蛋白单抗，以亲和层析法进行纯化，异硫氰酸荧光黄标记制备荧光抗体；以BHK-21细胞系培养狂犬病毒细胞适应株CVS-11，以染毒细胞测定荧光抗体的工作浓度后，通过荧光抗体染色法测定CVS-11细胞半数感染量（TCID50）；利用狂犬病毒灭活疫苗免疫犬制备抗狂犬病毒血清，并以国际兽疫局标准品进行标定；参考世界卫生组织及国际兽疫局推荐方法，确定中和试验程序，对51份样品血清进行测定，并与国际狂犬病参考实验室的检测结果进行比较，评价其精确及可靠程度。以Kaber法公式和Microsoft Excel软件为基础，设计被测样品中和效价计算方法。结果制备的抗狂犬病毒核蛋白单抗的亚型为IgG2a；纯化、标记后的荧光抗体工作浓度为1：400；CVS-11株的使用量为每孔100 TCID50；本研究建立的检测程序对51份人及犬血清测得的中和效价结果与国际参考实验室的测定结果一致；自行设计的计算方法操作简便，计算快捷。结论成功建立了与国际标准相当的狂犬病毒中和抗体效价测定和计算方法。     

狂犬病毒核蛋白抗原ELISA定量法的建立及初步验证

目的建立检测狂犬病疫苗各工序产品中核蛋白抗原含量的双抗体夹心ELISA法。方法以兔抗狂犬病毒多抗为包被抗体,辣根过氧化物酶标记的抗狂犬病毒核蛋白单抗作为酶标记抗体,对狂犬病毒核蛋白抗原含量进行定量测定,并对该方法进行初步验证。结果此方法线性相关系数大于0.97;最佳线性范围为0.000625～0.01IU/ml,定量限度为0.000625IU/ml;准确性为102%～109%;变异系数(CV)为7.2%～9.4%;与小牛血清、牛血清清蛋白(BSA)、卵清蛋白(OVA)、流感疫苗纯化液、乙脑疫苗纯化液、甲肝疫苗纯化液等均无交叉反应。结论该方法特异性强,灵敏度高,准确性、重复性和稳定性好,可用于狂犬病疫苗各工序产品中核蛋白抗原的定量检测。     

Antigenic variants of rabies virus

Antigenic variants of CVS-11 strain of rabies virus were selected after treatment of virus populations with monoclonal antibodies directed against the glycoprotein antigen of the virus. These variants resisted neutralization by the hybridoma antibody used for their selection. Two independently mutating antigenic sites could be distinguished when five variants were tested with nine hybridoma antibodies. The frequency of single epitope variants in a cloned rabies virus seed was approximately 1:10,000. Animals were not or only partially protected when challenged with the parent virus or with another variant, but were fully protected against challenge with the virus used for immunization. Variants were also detected among seven street viruses obtained from fatal cases of human rabies. Animals immunized with standard rabies vaccine were protected against challenge with some but not all street rabies variants. A comparative antigenic analysis between vaccine strain and challenge virus by means of monoclonal antiglycoprotein antibodies showed a slightly closer degree of antigenic relatedness between vaccine and challenge strain in the combinations where vaccination resulted in protection. It remains unknown, however, whether these apparently minor antigenic differences in the glycoproteins account for the varying degrees of protection. The results of this study clearly indicate that the selection of vaccine strains and the methods used to evaluate the potency of rabies vaccines need to be revised.     

Is rinderpest virus the archevirus of the Morbillivirus genus?

Groups of 6-39 monoclonal antibodies identifying 3-18 distinct epitopes on the nucleoprotein (NP), polymerase (P), hemagglutinin (H; equivalent in canine distemper and rinderpest viruses), and fusion (F) components of measles and canine distemper viruses were characterized in immunofluorescence tests with fixed Vero cell cultures infected with measles, canine distemper and rinderpest viruses. The majority of NP-specific monoclonal antibodies reacted with all three viruses, but one-third of the antibodies only reacted with the homologous virus. A few antibodies detected epitopes uniquely shared between either measles and rinderpest viruses or canine distemper and rinderpest viruses. Of the P-specific antibodies, two-thirds only reacted with the homologous virus, one antibody detected an epitope shared between canine distemper and rinderpest viruses, and the rest reacted with all three viruses. Also, the majority of antibodies against the H component were type-specific, but four antibodies reacted both with measles and rinderpest viruses. In contrast, the F component was antigenically highly conserved. 17 of 21 antibodies against this component reacted with all three viruses; one antibody reacted only with measles and rinderpest virus F components, and three antibodies reacted only with the homologous virus. No monoclonal antibody of any specificity selectively reacted with only measles and canine distemper viruses. Furthermore, the measles virus H component appeared to be more closely related to the equivalent rinderpest virus component than to the canine distemper virus component. Thus, it is proposed that rinderpest virus is the archevirus of the morbillivirus group from which canine distemper virus was first to evolve and, more recently (perhaps about 5,000 years ago), measles virus.     

P选择素在原发性高血压伴腔隙性脑梗死患者发病机制中的作用

目的观察血小板活化的标志———P-选择素在原发性高血压伴腔隙性脑梗死患者发病机制中的作用。方法利用FITC标记的抗CD42a单抗(鼠IgG1),PE标记的抗CD62P单抗(鼠IgG1),CY标记的抗CD45单抗(鼠IgG1),FITC标记的鼠IgG1同种型对照免疫球蛋白,PE标记的鼠IgG1同种型对照免疫球蛋白,上述单抗分别标记全血中血小板及白细胞的相关抗原;同时利用流式细胞仪,采取全血三色法流式细胞术方法,检测全血血小板上P-选择素阳性表达:全血标本中分别加入3种荧光标记的单克隆抗体FITC-CD42a,PE-62P及CY-CD45,室温避光反应20min,10g/L多聚甲醛固定10min,用PBS洗2次,上机分析。结果原发性高血压伴腔隙性脑梗死患者血小板P-选择素阳性表达犤(23.0±4.0)%犦明显高于原发性高血压无腔隙性脑梗死患者犤(16.9±1.8%)犦及正常人组犤(5.2±0.7)%犦,三者之间相比差异有显著性意义(t=137.4,28.4,P<0.05)。结论P-选择素在原发性高血压伴腔隙性脑梗死发病中起重要作用P-选择素与血管渐进损伤有关。     

Validation of the rapid fluorescent focus inhibition test for rabies virus-neutralizing antibodies in clinical samples

Monoclonal antibodies are successful biologics in treating a variety of diseases, including the prevention or treatment of viral infections. CL184 is a 1:1 combination of two human monoclonal IgG1 antibodies (CR57 and CR4098) against rabies virus, produced in the PER.C6 human cell line. The two antibodies are developed as replacements of human rabies immune globulin (HRIG) and equine rabies immune globulin (ERIG) in postexposure prophylaxis (PEP). The rapid fluorescent focus inhibition test (RFFIT) is a cell-based virus neutralization assay which is usually performed to determine the biological potency of a vaccine and to measure the levels of protection against rabies in humans and animals. In order to confirm the suitability of this assay as a pharmacodynamic assay, we conducted a validation using both HRIG- and CL184-spiked serum samples and sera from vaccinated donors. The validation results met all analytical acceptance criteria and showed that HRIG and CL184 serum concentrations can be compared. Stability experiments showed that serum samples were stable in various suboptimal conditions but that rabies virus should be handled swiftly once thawed. We concluded that the assay is suitable for the measurement of polyclonal and monoclonal rabies neutralizing antibodies in clinical serum samples.     

Detection of rabies virus antigen or antibody using flow cytometry

BACKGROUND: Rabies is invariably a fatal encephalomyelitis that is considered to be a serious public health problem. Rabies diagnosis must be rapid and conclusive. Detection and quantification of antirabies antibodies is used for assessment of the effectiveness of rabies vaccines. Hence, computer-automated detection of fluorescence using flow cytometry was attempted to reduce the work time required to undertake the conventional rapid fluorescent focus inhibition test (RFFIT). METHODS: Pasteur virus (PV)-infected mouse neuroblastoma (MNA) cells were stained with rabies virus antinucleocapsid antibody, fluorescein isothiocyanate (FITC) conjugate, and the percentage of infected cells at 24, 48, and 72 h postinfection (PI) was determined using flow cytometry. Serum samples containing known antibody titres estimated by RFFIT in terms of IU/ml were used to neutralize 50 FFD50 dose of PV. The percentage of MNA cells infected by the un-neutralized virus was estimated by flow cytometry. Using the value of the percentage of cells infected in the presence of known negative serum as 100%, the infection inhibition caused by antibodies at each dilution of positive reference serum was calculated and a regression equation generated for the prediction of rabies virus antibody titres in test sera samples as equivalent units per ml (EU/ml). RESULTS: There was a significant increase in the percentage of infected cells between 24 and 48 h PI from 26.45 to 75.28%. The percentage of cells having high side scatter was also highest at 72 h PI (11.11%). Antibody titres predicted by flow cytometry and those estimated by RFFIT as IU/ml showed a correlation coefficient of 0.74. CONCLUSIONS: Thus, flow cytometry could be used to detect rabies virus antigen in infected cells and to predict serum antibody titres from a single dilution of serum tested with the potential advantages of automation, rapidity, and lack of subjectivity. It has the potential to replace the time-tested RFFIT in rabies serology in the years to come.     

浙江省台州市两株狂犬病病毒流行株的基因序列分析

目的分析浙江省台州市2株狂犬病病毒核蛋白及糖蛋白的基因序列,了解狂犬病病毒野毒株与人用及兽用狂犬病疫苗株间的差异。方法以免疫荧光法检测2008年采自台州市的144只犬脑标本,以RT-(nested)PCR法扩增病毒核蛋白及糖蛋白全基因,克隆测序后以生物信息学软件进行遗传特征分析。结果检测到2株狂犬病病毒阳性样品,序列分析表明两样品所携病毒均为基因1型狂犬病病毒,所携病毒的N基因核苷酸及氨基酸同源性均为100%,G基因核苷酸和氨基酸同源性分别为99.8%和99.4%。所携病毒与CTN疫苗株核苷酸同源性较高,N基因与G基因分别为88.8%和85.9%~86.1%,两样品所携病毒与Hep-Flury疫苗株的核蛋白氨基酸同源性最高,为97.6%,CTN次之,为97.1%,与CTN糖蛋白氨基酸同源性较高,为92.3%~92.5%。与诸基因1型狂犬病病毒参考株相比,两样品所携病毒与浙江温州Wz0(H)、衢州株ZJ-QZ及印度尼西亚株病毒同源性最高。系统发育分析表明XY20及JJ22与浙江温州Wz0(H)、衢州株ZJ-QZ以及印度尼西亚株、疫苗株CTN,以及泰国与马来西亚株进化关系最近,而与疫苗株aG、PV、ERA,及标准攻击毒CVS株等进化关系较远。结论两份阳性犬脑所携狂犬病病毒是基因1型狂犬病病毒,但无论是N基因还是G基因的核苷酸序列,以及推导出来的氨基酸序列与已知的1型狂犬病病毒株及疫苗株存在差异。     

流式微球分析技术检测人心肌钙蛋白T方法的建立

目的建立流式微球分析技术（cytoometric bead assay,CBA）检测人肌钙蛋白T（cTnT）方法。方法用鼠抗人的cTnT的单克隆抗体包被在已激活的羧基化聚苯乙烯微球上,然后再用包被好的微球与检测标本进行抗原抗体免疫反应,并加入羊抗人cTnT的多克隆抗体和异硫氰酸荧光素（FITC）标记的驴抗羊的IgG,室温避光免疫反应一定时间后洗涤一次,上流式细胞仪检测FITC的荧光强度,以此测定标本中cTnT含量。结果通过正交试验,在100μl反应体系中,微球含量大约为1.25×106,选择10μg的鼠抗人cTnT单克隆抗体为最佳加入量,加入标本和抗体后,选择避光反应时间为120 min,洗涤选择一次,选择8μg/ml羊抗人cTnT多克隆抗体和20μg/ml FITC标记驴抗羊IgG为最佳工作浓度;方法检测灵敏度为16.0 pg/mL,线性范围是16-5 000 pg/mL,批内重复性变异系数为5.2-11.3%,回收率是94.7-111.5%,高浓度的胆红素、胆固醇和甘油三脂无明显干扰,与Roche Elecsys的电化学发光法有较好的相关性。结论自建CBA检测cTnT技术性能较好,可在临床工作中推广使用。