轮状病毒LLR株在两种不同细胞中病毒滴度的比较

目的:通过轮状病毒LLR株在牛肾细胞和Vero细胞中培养效果的比较,为轮状疫苗的生产筛选最佳的细胞基质和培养条件。方法:将轮状病毒LLR株按MOI 0.02分别接种牛肾细胞和Vero细胞,在相同培养条件下进行培养,每天观察两种细胞病变的情况,同时抽样检测病毒滴度,分析两种细胞对轮状病毒LLR株的敏感性。结果:牛肾细胞在感染轮状病毒LLR株后d 3病毒滴度达到最高,为6.8 lgCCID50.mL-1;而Vero细胞在感染轮状病毒LLR株后d 8病毒滴度达到最高,为7.3 lgCCID50.mL-1。轮状病毒LLR株在牛肾细胞和Vero细胞上均能达到满意的病毒表达量。结论:轮状病毒LLR株在Vero细胞中培养能够达到理想的病毒表达量,利用Vero细胞培养轮状病毒LLR株,有利于提高疫苗的产量和质量。     

瑞士青少年中VZV IgG抗体的血清阳性率及血清阴性的危险因素分析

作者对瑞士青少年中的水痘 -带状病毒(VZV) Ig G抗体进行了血清阳性率调查。　　血清样本来自瑞士八个城市及农村地区的 13～ 15岁学生 ,共 1788份。用 EL ISA检测 VZV Ig G,对于 EL ISA不能确定的及阴性或弱阳性的样本 ,则加用 VZV感染细胞的膜抗原荧光抗体染色法 (FAMA)。 1788份样本中 ,用 EL ISA确定具有 VZV Ig G抗体的有170 7份 ,占 95 .5 % ;2 0份 (1.1% )不能肯定 ;6 1份 (3.4 % )为阴性。 13份弱阳性的和 15份不能肯定的样本 ,用 FAMA法测定均为阳性。 EL ISA法检测血清抗体阴性者 ,2 0人中有 17人 (85 % ) FA…     

成人T细胞白血病病毒引起的脑干炎

作者利用明胶颗粒凝集试验、间接免疫荧光抗体试验对35份各种神经系统疾病患者血清进行检测,在1份患重症脑干炎患者血清中发现成人T细胞白血病病毒抗体强阳性,在国内尚无报道。     

乙型脑炎减毒活疫苗检定用BHK21工作细胞库的质量控制

目的  对乙型脑炎减毒活疫苗（乙脑疫苗）检定用BHK21工作细胞库进行全面质量控制以确保其质量。方法  按照《中华人民共和国药典》2010年版三部收录的细胞鉴别试验、无菌检查、支原体检查、外源病毒污染检查和病毒敏感性检查方法,对检定用BHK21工作细胞库进行全面质量检定。结果  待检BHK21细胞的同工酶电泳图谱与BHK21细胞标准图谱一致。细菌、真菌、支原体、外源病毒检测结果均符合规定要求。病毒敏感性检测显示,待检BHK21细胞培养的乙脑疫苗病毒滴度为6.93 IgPDF/ml,与合格BHK21细胞培养的乙脑疫苗（病毒滴度为6.89 IgPDF/ml）相似,均达到质量标准要求（6.48~7.12 IgPDF/ml）。结论 合格的BHK21工作细胞库可用于乙脑疫苗的质量检定来保证乙脑疫苗质量。     

肠道病毒71型假病毒荧光定量检测方法在疫苗中和抗体检测中的应用

目的假病毒荧光定量方法（PVLA）应用于EV71疫苗动物样本和人体样本检测的适用性研究。方法分别采用细胞病变法（CPE）和PVLA方法检测82份EV71试验小鼠血清以及27份疫苗临床试验人体血清样本,比较两者检测的一致性。结果应用两种方法检测82份疫苗免疫小鼠血清,EV71中和抗体滴度的相关系数为0．842,Bland—Altman分析显示高度一致性;检测27份EV71临床试验人体血清样本的中和抗体,两种方法具有高度一致性。结论PVLA与CPE方法具有高度一致性,适用于疫苗免疫动物样本和临床评价人体样本EV71中和抗体的检测。     

两种双抗原夹心法检测抗人类免疫缺陷病毒抗体的方法比较

目的 探讨酶联免疫吸附试验(ELISA)与胶体金法检测抗人类免疫缺陷病毒抗体(抗HIV抗体)的灵敏度及特异度.方法(1)灵敏度试验:将15份经免疫印迹法确诊为获得性免疫缺陷综合征(AIDS)患者的血清进行倍比稀释,应用ELISA法和胶体金法检测所有患者血清原液、稀释血清的抗HIV抗体,记录两种方法抗HIV抗体检测结果为阳性的最大稀释倍数.(2)特异度试验:应用上述两种方法同时检测200份非AIDS患者的血清,记录两种方法的阴性率.结果(1)灵敏度试验:ELISA法的最大稀释倍数显著高于胶体金法(P＜0.05).(2)特异度试验:两种方法的阴性率均为100％,差异无统计学意义(P＞0.05).结论 ELISA法抗HIV抗体的灵敏度优于胶体金法,但是两种方法检测血清原液的灵敏度、特异度结果均一致,因此胶体金法可以代替ELISA法用于AIDS的初筛试验.     

MDCK细胞基质与鸡胚基质单价流感疫苗在小鼠中诱导的免疫反应的比较研究

目的:比较鸡胚基质和细胞基质的H1N1单价流感疫苗在小鼠体内诱导的免疫反应的差异。方法:分别采用鸡胚基质和MDCK细胞基质H1N1流感疫苗注射C57BL/6小鼠,通过体外中和实验,胞内细胞因子染色及多细胞因子检测比较2种基质生产的单价流感疫苗诱导的体液及细胞免疫反应的差异。结果:体液免疫方面,细胞基质疫苗组血清滴度与鸡胚基质疫苗组血清滴度基本一致。在细胞免疫方面,细胞基质疫苗组的细胞免疫相关细胞因子分泌水平更加显著。结论:由于病毒培养基质的不同,2种流感单价疫苗在诱导免疫反应时间存在一定差异。细胞疫苗具有诱导多种免疫反应的优势,综合细胞工艺的其他优势,说明利用细胞工艺生产流感疫苗将会成为趋势。本实验有助于评价不同基质制备的单价流感疫苗在小鼠中诱导的免疫水平。     

假病毒在中和抗体检测中的应用研究进展

目前成功的病毒性疫苗大多通过诱导广谱有效的中和抗体来保护机体免受感染,因此检测血清中和抗体水平对疫苗的免疫效果评价具有重要意义.中和抗体检测的传统方法包括微量细胞病变效应抑制法、蚀斑减少试验等,均需进行活病毒操作.近年来,随着逆转录病毒载体和重组病毒表达技术的发展,假病毒得到越来越多的研究,并被广泛应用在新型疫苗研发、中和抗原表位鉴定、细胞嗜性研究、抗病毒药物筛选、中和抗体检测、基因治疗等方面.此文主要对假病毒在中和抗体检测中的应用进行综述,并总结相关领域的研究进展.     

人二倍体细胞2BS株和KMB17株在疫苗研发中的应用

人二倍体细胞是WHO认可的更安全的疫苗生产用细胞基质,已成为全世界疫苗生产首选的细胞基质。我国自主研制的二倍体细胞KMB17株和2BS株来源于人胚肺细胞,因其无致癌性、无外源因子污染,且细胞性状比较稳定,越来越多地在人用疫苗研发与生产中使用。由于KMB17细胞和2BS细胞对多种病毒具有敏感性,因此也作为细胞基质被应用于许多病毒性疫苗的研究开发。目前,用这两种人二倍体细胞生产的疫苗,包括甲型肝炎疫苗、脊髓灰质炎疫苗和肠道病毒71型灭活疫苗等,已在我国大量上市。     

酶免疫试验——一种检测麻疹免疫水平的血清学方法

在对人群进行麻疹免疫的流行病学调查中,传统的方法为血凝抑制试验(HI),但因它需使用猴血球而受到一定的限制。近年来,酶免疫试验(EIA)作为一种简便、特异和敏感的方法,在麻疹抗体的检测中也得到了应用。作者用苏联医学科学院病毒制品研究所生产的麻疹抗体EIA试剂盒,同时应用HI方法对曾在5、6年前接种过麻疹疫苗的492名6～8岁儿童进行了检测分析。每次试验中均选用下列血清作对照:1份高及中等滴度麻疹抗体,1份低滴度(1:10)麻疹抗体,1份为阴性。在EIA试验中血清以1:10稀释     

Specific protection against bovine leukemia virus infection conferred on cattle by the Romanian inactivated vaccine BL-VACC-RO

The Romanian BL-VACC-RO vaccine against bovine leukemia virus (BLV) infection was prepared with ethylenimine-inactivated BLV obtained from persistently infected cell lines. Intramuscular administration of two vaccine doses (each containing 0.40 mg virus glycoprotein with adjuvant), 2 weeks apart, conferred protection against challenge infection with BLV on 18 of the 20 vaccinated calves. Two calves were not protected, in spite of their positive serologic response to vaccination. The results demonstrate the efficiency of the BL-VACC-RO vaccine in preventing BLV infection.     

二代测序技术检测生物制品中外源病毒的技术方法和应用进展

生物制品中外源病毒污染一直是监管机构对其安全性评价的重要内容,目前主要采用的技术包括体外细胞培养、接种动物、种属特异性检查等传统方法,但是传统方法普遍存在灵敏度低、检测范围窄等缺点,因此有些病毒可能无法检出。二代测序法(Next Generation Sequencing,NGS)曾被用于发现已获批上市的疫苗中存在外源病毒污染,说明该法具有全面检测病毒的潜力。使用NGS法检测外源病毒,能从源头上保障生物制品的安全。然而,生物制品中的病毒含量可能很低,甚至还可能和已知病毒的基因序列存在较大差异,NGS法能否全面准确地检出病毒尚不清楚;而且,NGS是一项复杂的技术, 在操作流程和数据解读方法等方面尚无任何指导原则。本文首先回顾了世界范围内的主要药品生产企业和监管机构对NGS技术检测生物制品中外源病毒的观点,接着详细讨论了该方法的技术流程和将方法标准化时面临的挑战,最后陈述了部分NGS技术检测生物制品中外源病毒的应用实例,为利用NGS技术检测生物制品中外源病毒方法的建立、标准化和验证提供启示。     

泉州地区5岁以下住院患儿呼吸道感染非细菌病原体检测结果分析

目的:检测儿内科5岁以下呼吸道感染住院患儿的非细菌病原体IgM抗体,为临床诊疗提供依据。方法:收集11 976例5岁以下儿内科呼吸道感染住院患儿的血清标本,采用间接免疫荧光法检测其中8种常见非细菌病原体的IgM抗体。结果:检测11 976例标本非细菌病原体IgM抗体,其中流感病毒B型(FluB)、肺炎支原体(MP)阳性率较高,其次为呼吸道合胞病毒(RSV)、流感病毒A型(FluA)、嗜肺军团菌(LP)。MP在2013-2014 年5、6、7、8月份的阳性率均较高,FluA在1、2月份和11、12月份感染率高,FluB、副流感病毒在6、12月份的感染率高于其他月份。MP阳性率在春夏季显著高于秋冬季。MP感染率2岁以上儿童高于2岁以下婴幼儿,RSV主要感染2岁以下的婴幼儿。结论: 2012年11月至2015年8月泉州地区引起5岁以下儿童呼吸道感染的非细菌病原体主要是FluB、MP,不同病原体在不同的月份感染率有差别。MP在春夏季的感染率较高。2岁以上儿童MP感染率较高,而RSV主要感染2岁以下的婴幼儿。     

Development of a microplate duplex immunoassay to simplify detection of growth hormone doping: Proof of concept

The World Anti-Doping Agency (WADA) prohibits athletes from using recombinant growth hormone (GH). The validated method used in antidoping laboratories for the direct detection of exogenous GH in serum requires two immunoluminometric assays (ILMAs): The first mainly measures the concentration of the full-length (22 kDa) form of GH (recGH), and the second measures concentrations of multiple GH fragments produced by the pituitary gland (22 kDa, 20 kDa and other forms) (pitGH). The tube-by-tube analysis is laborious. A recent development opened new possibilities to simplify the detection of recGH in serum: multiplexed immunoassays that detect multiple targets in a single well of a 96-well plate using an ELISA-like procedure with high sensitivity. Our aim was to evaluate this technology by developing a customized assay for GH detection. One pair of antibodies with specificities similar to those of the recGH assay and one pair of antibodies compatible with pitGH detection were selected for a single duplex assay. Forty-eight serum samples (negative athlete samples and positive samples following GH administration) were analyzed using the two methods. The microplate duplex assay discriminated between the negative athlete samples and the positive controls, although the rec/pit ratios from the duplex assay were lower than those obtained with the ILMAs. This new assay would offer a modern alternative to ILMAs, with fewer analytical steps and a smaller sample volume. However, an adaptation of the decision limits seems mandatory.     

An ELISA method for detection of human antibodies to an immunotoxin

INTRODUCTION: The use of biological molecules, such as immunotoxins, as pharmaceuticals is limited by the presence and development of human antibodies to these agents. This immune response can cause significant inflammatory-related toxicities and can interfere with the efficacy of the biological agent. Therefore, a clinically applicable method to detect these human antibodies is needed for screening patients prior to enrollment and for monitoring patients during treatment. The SS1(dsFv)-PE38 immunotoxin currently in clinical trials is a hybrid molecule targeted against mesothelin-expressing cancer cells via the Fv portion of a murine antibody linked to the Pseudomonas exotoxin (PE), which can inhibit protein synthesis leading to cell death. The objective of this study was to determine if an enzyme-linked immunosorbent assay (ELISA)-based method could be used to detect human anti-SS1(dsFv)-PE38 antibodies in patient serum. METHODS: Human antibodies to the immunotoxin in serially diluted serum specimens were captured on immunotoxin-coated ELISA plates, and detected using a secondary goat antihuman antibody linked to biotin in combination with horseradish peroxidase linked to avidin D (HRP-Avidin). The color was developed with tetramethyl benzidine (TMB). Curves of optical density (OD(630)) versus dilution for 44 serum specimens were compared with positive and negative control serum specimens to classify the serum as positive or negative for anti-immunotoxin antibodies. RESULTS: Ten out of the 40 patients screened were positive for anti-immunotoxin antibodies. Repeated testing of seven samples produced the same results in two independent experiments. The first two patients treated with the immunotoxin developed anti-immunotoxin antibodies during treatment. The results were in perfect concordance with a tissue culture-based neutralization assay performed by an independent laboratory. DISCUSSION: An ELISA-based strategy using an immunotoxin to capture human anti-immunotoxin antibodies provides a consistently accurate technology for screening and monitoring patient serum specimens in clinical trials.     

Immunogenic comparison of two coupling methods of marine polysaccharide to bovine serum albumin

Two conjugates of marine polysaccharide (MPS) and bovine serum albumin (BSA) were prepared using two methods, periodate oxidation and reductive amination, with the intent of enhancing its immunogenicity. Sera samples from Balb/c mice immunized with the products named MPS-BSAp and MPS-BSAr respectively were evaluated by enzyme-linked-immunosorbent assay (ELISA). The results showed that mice immunized with MPS-BSAp produced antibodies not against MPS but rather against MPS-BSAp, while the mice immunized with MPS-BSAr produced high titer antibodies only specific for MPS. The difference was attributed to the fact that the epitopes of MPS had been changed in the coupling process by periodate oxidation. A mouse immunized with MPS-BSAr was chosen to prepare monoclonal antibodies (mAbs) specific for polysaccharide MPS. A hybridoma cell line that secreted monoclonal antibody recognizing specifically polysaccharide MPS was established.     

国产与进口牛血清促进Vero细胞生长的比较

 目的  通过对国产与进口牛血清促进Vero细胞生长的比较,筛选可替代进口血清用于轮状病毒疫苗生产中Vero细胞培养的国产牛血清。方法  以国产的3批胎牛血清和1批新生牛血清为待评血清,以进口胎牛血清为对照血清,制备细胞培养液。在T175细胞培养瓶（T瓶）和2层细胞工厂中连续传代培养Vero细胞各3代,观察每一代次的培养上清液、细胞形态和细胞汇合度,计算细胞收获量和与对照血清相比较的相对增长率。采用单样本t检验对细胞收获量与生产要求的细胞产量（T瓶：>1.50×10⁷ 个/ml;细胞工厂：>3.00×10⁸ 个/ml）进行比较。根据细胞相对增长率判断国产血清与进口对照血清促细胞生长活性的相近程度。结果  国产血清培养的Vero细胞生长状态均良好。T瓶培养时,国产血清组的细胞收获量为（1.56～9.30）×10⁷ 个/ml,与生产要求细胞产量的差异有统计学意义（t=2.44～3.76,P<0.05）,且t值均>0,说明符合生产要求。细胞相对增长率接近或超过100%。细胞工厂培养时,国产血清组的细胞收获量为（1.80～4.92）×10⁸ 个/ml,与生产要求产量的差异无统计学意义（t=－0.23～1.16,P>0.05）,但是只有1批胎牛血清和新生牛血清的细胞收获量均值>3.00×10⁸ 个/ml,且细胞相对增长率>90%。 结论  经与进口牛血清比较,国产的1批胎牛血清和1批新生牛血清可替代进口血清,用于轮状病毒疫苗生产中的Vero细胞培养。     

Detection of biologically active botulinum neurotoxin--A in serum using high-throughput FRET-assay

IntroductionThe goals of this project were to compare fluorescent resonance energy transfer (FRET) assays using a customized FRET substrate (substrate–substrate-A, SSA) with a commercially available FRET substrate (SNAPtide); optimize the assay conditions for SSA for lowest level of detection; and apply SSA to detect botulinum neurotoxin-A (BoNTA) in serum samples.MethodsBiological activity of BoNTA and light-chain-A (LCA) was verified by murine phrenic nerve-hemidiaphragm bioassay and western blot before use in both FRET assays. The reaction conditions were optimized to determine the smallest amount of toxin that could be detected. A range of serum samples was investigated for interference in the SSA-based FRET assay. Detection of BoNTA from rat serum samples was performed over time.ResultsWe found that BoNTA and LCA were able to cleave the substrates whereas mutated LCA and a different serotype of BoNT, BoNTB, could not. SSA had significantly more arbitrary fluorescing units compared to the FRET substrate SNAPTide, and the SSA assay could detect 0.1 nM of BoNTA or LCA comfortably (p = < 0.05) in a 20-μl reaction. No significant interference was observed when serum was present in the reaction buffer. Due to negligible background noise, the SSA FRET assay could detect BoNTA from spiked rat serum even after 256 min.DiscussionThe greatest advantage of the FRET assay is its extreme rapidity, its cost effectiveness, and unlike ELISA, its ability to detect biologically active toxin. SSA is a better FRET substrate for detecting BoNTA toxin (detected 0.1 nM concentration). Because serum present in the assay reaction did not cause any appreciable interference, the assay can be used to detect BoNTA in serum samples. Therefore, the SSA FRET assay can be used for pharmacokinetic and pharmacodynamic studies, screening inhibitors, and detecting BoNTA in serum samples.     

Intestinal immunoglobulin a response to naturally acquired enterotoxigenic Escherichia coli in US travelers to an endemic area of Mexico

BACKGROUND: Simple methods for detecting secretory immunoglobulin A (sIgA) immune responses following natural enteric infection and oral immunization are needed. METHODS: Fourteen students from the United States acquiring enterotoxigenic Escherichia coli (ETEC) diarrhea in Mexico were studied for fecal immunoglobulin A (IgA) response to their homologous infecting ETEC and to heat-labile (LT) toxin of ETEC using Dot-Blot microfiltration and enzyme-linked immunosorbent assay (ELISA) methods. Paired stool samples were collected on the day of presentation and 5 days later. RESULTS: Twelve of 14 (86%) patients with ETEC diarrhea (5 heat-stable [ST]/LT positive, 4 LT-only, and 5 ST-only) developed sIgA antibodies directed against their homologous ETEC and 6 (66%) of the 9 patients harboring ST/LT or LT-only strains developed sIgA LT-antibody responses. Single fecal samples from 9 healthy controls were negative for ETEC specific antibodies. CONCLUSIONS: Patients with diarrhea due to noninvasive ST/LT ETEC and LT ETEC commonly produce a specific sIgA antibody response early in the illness. We feel that the methods employed will be useful to detect antibodies during natural infection by enteric pathogens and following oral enteric vaccine administration.     

荧光灶试验和TCID50-ELISA方法在Ⅲ价轮状病毒重配疫苗感染滴度测定中的对比研究

目的:对比荧光灶试验和TCID50-ELISA方法测定III价轮状病毒基因重配疫苗感染滴度的精确性,重复性和可行性。方法:同时采用TCID50-ELISA方法和荧光灶试验方法测定Ⅲ价轮状病毒基因重配疫苗的感染滴度,对测定结果进行比较分析。结果与结论:在Ⅲ价轮状病毒基因重配疫苗的感染滴度测定中,荧光灶试验方法和TCID50-ELISA方法各有其特点,荧光灶试验方法更加精确和快速,相对于TCID50-ELISA方法,具有更低的变异性,即其重复性较高;而TCID50-ELISA方法受主观因素的影响较低。