双抗体夹心ELISA与NIH法在狂犬病疫苗抗原检测中的比较

通过用单克隆抗体制备双抗体夹心ELISA,快速检测狂犬病疫苗中狂犬病毒糖蛋白(G)的含量,将狂犬病疫苗的检定时间由28天缩短至2个工作日,以此缩短疫苗库存待检时间,提高疫苗的生产和销售效率,并最终替代NIH动物法.将待检的狂犬病疫苗样品在同一性别12～14g昆明鼠体内作效力检定试验(NIH),同时用双抗体夹心ELISA检测狂犬病疫苗中狂犬病毒G蛋白含量,用Microsoft Office Excel做出标准品和各样品疫苗的线性关系图并计算出疫苗效力值E-NIH.结果用双抗体夹心ELISA所得的E-NIH与对应的小鼠效力试验所得结果M-NIH之间呈正相关性;同一批狂犬病疫苗分次测得E-NIH值在2.41～5.85之间,而相应的M-NIH值在5.11～10.19之间.从而得出E-NIH与相应的M-NIH之间存在明显的线性关系;与NIH法比较,ELISA具有重复性好、成本低、快速等优点;用双抗体夹心ELISA替代小鼠效力试验是可能并可行的.     

狂犬病病毒抗体胶体金检测试纸的制备

通过胶体金免疫层析技术建立一种特异、便捷、快速的狂犬病病毒抗体检测方法,对犬等动物免疫狂犬病疫苗后的抗体水平监测提供参考。用醋酸锌沉淀法沉淀狂犬病病毒CVS11,Sepharose 4FF进行层析纯化。用柠檬酸三钠还原法制备的胶体金,标记纯化的狂犬病病毒,喷于试纸的结合释放垫 (金标垫),将SPA (葡萄球菌表面A蛋白) 和纯化的兔抗狂犬病病毒IgG分别喷于试纸的T (检测线) 处和C (对照线) 处,组装试纸条。用制备的试纸条对261份犬血清进行检测,与快速荧光灶抑制试验 (RFFIT) 检测的结果一致;对已知效价的犬狂犬病毒中和抗体 (VNA) 大于0.5 IU,结果为阳性;对狂犬病病毒中和抗体 (VNA) 小于0.5 IU/mL的血清,结果为阴性。制备的狂犬病病毒抗体胶体金检测试纸检测犬血清抗体,具有特异、便捷、快速的特点,能够检测出狂犬病病毒中和抗体大于0.5 IU的血清,适用于临床犬血清抗体水平监测,具有良好的应用前景。     

狂犬病毒糖蛋白的克隆表达及其在中和抗体检测中的应用

目的:制备基因工程表达的狂犬病毒糖蛋白优势表位抗原,并评价其在疫苗免疫后中和抗体检测中的应用价值。方法:TRIzol法从狂犬病疫苗中提取总RNA,经RT-PCR获得糖蛋白目的基因片段,构建相应的原核表达质粒,转化大肠杆菌HB101,诱导表达获得纯化重组蛋白,以重组蛋白作为包被抗原,初步建立检测糖蛋白中和抗体的ELISA方法。结果:获得狂犬病毒糖蛋白优势表位区段抗原,建立了糖蛋白中和抗体ELISA检测方法。该检测方法对59例健康献血员血浆样本检测特异性为98.31%(58/59),接种狂犬疫苗免疫个体血浆样本抗体阳性率为98.95%(94/95)。结论:基因工程表达的狂犬病毒糖蛋白优势表位抗原可用于人接种狂犬疫苗后疫苗免疫效果评价。     

双抗体夹心ELISA与NIH法在狂犬病疫苗抗原检测中的比较

通过用单克隆抗体制备双抗体夹心ELISA,快速检测狂犬病疫苗中狂犬病毒糖蛋白(G)的含量,将狂犬病疫 苗的检定时间由28天缩短至2个工作日,以此缩短疫苗库存待检时间,提高疫苗的生产和销售效率,并最终替代 NIH动物法。将待检的狂犬病疫苗样品在同一性别12～14g昆明鼠体内作效力检定试验(NIH),同时用双抗体夹 心ELISA检测狂犬病疫苗中狂犬病毒G蛋白含量,用Microsoft Office Excel做出标准品和各样品疫苗的线性关 系图并计算出疫苗效力值E-NIH。结果用双抗体夹心ELISA所得的E-NIH与对应的小鼠效力试验所得结果M- NIH之间呈正相关性;同一批狂犬病疫苗分次测得E-NIH值在2．41～5．85之间,而相应的M-NIH值在5．11～ 10．19之间。从而得出E-NIH与相应的M-NIH之间存在明显的线性关系;与NIH法比较,ELISA具有重复性好、 成本低、快速等优点;用双抗体夹心ELISA替代小鼠效力试验是可能并可行的。     

狂犬病毒抗体ELISA检测试剂盒的改进研究

为了提高狂犬病毒抗体检测的灵敏度和特异性,采用狂犬病毒单克隆抗体包被酶标板,再分别加入重组的狂犬病毒糖蛋白或细胞培养抗原做固相层的方法(抗体捕捉法),用传统的间接ELISA法做对照,按常规方法检测抗狂犬病毒抗体。结果显示,抗体捕捉法的非特异性反应低于间接法,而灵敏度达到0．51U水平,高于间接法。在800份临床标本检测中,检出率明显高于间接法。用15份阳性血清作小鼠中和试验,并和抗体捕捉ELISA法比较具有高度的一致性。试验结果充分表明,该方法优于传统的ELISA间接法。因此可作为临床注射狂犬疫苗后检测血清中狂犬病毒抗体的常规方法。     

不同狂犬病毒株抗原反应性的比较研究

应用ELISA法对285份免疫前及健康人血清和742份以不同毒株制备的狂犬病疫苗(aG株、CaG株、CTN株,PM株)全程免疫后人群的血清标本进行抗狂犬病毒抗体的检测。结果显示CTN株病毒抗原对不同毒株狂犬病疫苗免疫后血清的抗体阳性检出率均能达到90％以上,且与SNT效价的相关性较好;而aG株抗原对除aG株以外其它毒株狂犬病疫苗免疫后血清的阳性检出率仅为50％左右。因此不同毒株狂犬病毒抗原的反应性存在明显差异,选用纯度高、活性好的CTN株病毒或两株病毒以不同比例混合作为ELISA检测用抗原,测定疫苗免疫后人群的抗体水平,可获得满意的结果。     

重组人源抗狂犬病毒单克隆抗体SO57、SOJB对狂犬病毒G蛋白亲和力的比较研究

一直以来,狂犬病是一种严重的人类致死性传染病。对狂犬病暴露后的预防主要是采用抗狂犬病毒免疫球蛋白（RIG）结合狂犬疫苗注射的方法。目前使用的两类RIG为人RIG（HRIG）和马RIG（ERIG）,它们都是从免疫血清中分离出来的。由于HRIG成本高且产量少,质量难以控制,有潜在病毒污染的风险;而ERIG存在引起过敏反应等问题,因此,人们期望抗狂犬病毒人单克隆抗体能够取代RIG,用于狂犬病的暴露后预防。在狂犬病毒的多种免疫原物质中,狂犬病毒糖蛋白（以下简称G蛋白）是诱导产生抗病毒免疫保护的一种主要抗原,同时也是诱导产生病毒中和抗体并与之反应的唯一抗原。针对G蛋白的抗狂犬病毒抗体中和细胞外的狂犬病毒,并介导感染细胞的裂解及抗体依赖性的细胞毒性。在Dietzschold等发现的几株针对G蛋白的人单克隆中和抗体中,中和毒株的范围最广、抗体效价最高的为SO57,除此之外,SOJB也是目前研究较多的针对G蛋白的人单克隆中和抗体。     

抗狂犬病毒中和抗体研究进展

狂犬病是一种人兽共患传染病,人和动物一旦发病后死亡率几乎百分之百,而有效的暴露后预防措施可以将死亡风险降至零。根据WHO推荐的狂犬病暴露后预防方案,一般狂犬病暴露者需要进行疫苗注射,严重者则需在进行疫苗注射的同时注射抗狂犬病毒中和抗体。常用的中和抗体有马抗狂犬病毒免疫球蛋白和人抗狂犬病毒免疫球蛋白,然而两者都存在引起过敏反应或血液疾病的风险。人源抗狂犬病毒中和抗体则因为具有安全性高、成本低、可量产等优点有望代替免疫球蛋白用于暴露后预防。基因工程抗体技术的发展加速了抗体人源化的进程。就抗狂犬病毒中和抗体的发展历程,不同类型中和抗体的优缺点以及中和抗体的未来研究方向作了综述及展望,以期为新一代狂犬疫苗的研发提供参考。     

狂犬病毒蚀斑方法的建立

我们用CTN-1狂犬病毒二倍作细胞适应株[1]经蚀斑纯化而获得的CTN-2病毒株在BHK-21单层细胞培养建立了狂犬病毒的简易蚀斑技术。用连续10倍稀释的病毒悬液接种BHK-21细胞而产生的蚀斑数呈10倍规律的减少。该蚀斑技术用于蚀斑减少中和试验测定不同国际单位(IU)效价的抗狂犬病血清与原抗血清(IU)效价均高低成正比。用蚀斑中和试验测定了七批我国生产的人用精制抗狂犬病血清的IU效价与原抗血清IU效价的高低是相符的。     

应用生物反应器建立人用冻干狂犬病疫苗(Vero细胞)生产工艺的研究

应用15L生物反应器,采用片状载体对Vero细胞进行高密度培养、制备高毒力滴度的狂犬病毒收获液,经纯化后生产人用冻干狂犬病疫苗。采用15L生物反应器对培养方法(批次培养和连续灌流培养)进行试验,收获高毒力滴度的狂犬病毒收获液并制备人用冻干狂犬病疫苗。结果表明:Vero细胞在接种狂犬病毒后可以连续收获病毒液达到25d以上,冻干狂犬病疫苗的效价可以达到5.54IU/剂。本工艺可以用于进行大规模的人用冻干狂犬疫苗的生产。     

A semi-quantitative serological method to assess the potency of inactivated rabies vaccine for veterinary use

Potency is one of the most important indexes of inactivated vaccines. A number of methods have been established to assay the potency, of which the NIH test and single-dose mouse protection test are the “prescribed methods”. Here, we report a method to semi-quantitatively assay the potency of an inactivated rabies vaccine, which uses fewer animals and takes less time to complete. Depending on the quality requirements of a vaccine (e.g. minimum potency), a rabies reference vaccine is, for example, diluted to the minimum potency, and 50 μL of the dilution is taken to inoculate 10 mice. The same amount of the test rabies vaccine is inoculated into another 10 mice. After two weeks, all mice are bled and serum samples are assayed for viral neutralizing antibody by the fluorescent antibody virus neutralization (FAVN) test. By comparing the median and interquartile range of antibody titers of the reference vaccine with those of the test vaccine, the test vaccine potency can be semi-quantitatively judged as to whether it is in accord with the required quality. The reliability of this method was also confirmed in dogs. The procedure can be recommended for batch potency testing during inactivated rabies vaccine production.     

Use of a monoclonal antibody for quantitation of rabies vaccine glycoprotein by enzyme immunoassay

The use of a monoclonal antibody specific for the envelope glycoprotein of rabies virus has been used in a direct enzyme immunoassay to quantify the glycoprotein content of rabies vaccines produced in two kinds of cell culture. The results of this direct enzyme immunoassay are well correlated with the in vivo NIH potency test. This test may be a useful tool to rapidly and accurately control the antigenic value of a vaccine. It could also be used for the "in process' control of vaccine production before deciding whether a vaccine batch may reasonably be subjected to the NIH potency test.     

Standardization of an enzyme immunoassay for the in vitro potency assay of inactivated tissue culture rabies vaccines: determination of the rabies virus glycoprotein with polyclonal antisera

A non-competitive enzyme-linked immunoassay (ELISA) has been standardized to supplement the in vivo potency test used for the quality control of inactivated tissue culture vaccines against rabies. The essentials of the ELISA were: fixation of the virus in different dilutions of vaccine on the surface of microtitre plates; testing of the reference and up to six test vaccines on one plate; incubation with polyclonal antisera to rabies virus glycoprotein containing an excess of antibody; further incubation with a species-specific anti-IgG coupled to peroxidase; a final incubation with a substrate. The incubation periods were 1 h, 1 h and 30 min both at +37 degrees C. The relative potency determinations were made graphically or by a computer using a parallel line bioassay in which the potencies of the vaccines of unknown potency were tested against the reference preparation on a single microtitre plate. Under these conditions inactivated rabies vaccines of different types (virus strains, cell substrates, inactivation and concentration procedures) were tested for potency. Furthermore, it was possible with this in vitro method to assay adjuvanted vaccines, in process samples such as tissue culture supernatants with live or inactivated rabies virus, concentrates, and vaccines undergoing thermal stability tests. The rabies glycoprotein antigen-antibody reaction was highly specific according to the results and the glycoprotein content was measured quantitatively. The potency determined by the in vitro ELISA correlated with the in vivo NIH protection potency test. The lower limit of detection of the ELISA was 0.015 IU/ml. Quantitative antigen determination was possible with both homologous and heterologous antisera to rabies virus glycoprotein when vaccines of the same virus strain were tested. When the potencies of vaccines of different virus strain specificity were calculated, it was necessary to take into account the strain-specific antigenicity. Even so vaccines of high potency were found to give a stronger reaction with a heterologous serum than did weak vaccines with a homologous antiserum. Stability tests made on inactivated tissue culture vaccines such as vaccine from the human diploid cell strain (HDCS), from purified chicken embryo cell (PCEC) or from purified Vero cell rabies vaccine (PVRV), showed high stability of the glycoprotein antigen even after four months of storage at +37 degrees C or 24 h at +56 degrees C, provided that the vaccines were stored in a lyophilized state. The antigenicity of liquid vaccines was inactivated after a few hours at +56 degrees C. For tropical areas, therefore, only lyophilized vaccines should be considered.     

A simple immuno-capture ELISA to estimate rabies viral glycoprotein antigen in vaccine manufacture.

Rabies is an endemic, fatal zoonotic disease in the developing countries. Prevention and post-exposure therapy require safe and efficacious vaccines. The vaccine potency depends on the amount of immunogenic rabies viral glycoprotein antigen in the vaccine preparation. In order to estimate the rabies viral glycoprotein antigen, a specific monoclonal antibody was developed and used in an immuno-capture ELISA (IC-ELISA). The monoclonal antibody binds a conformational epitope on the natively folded rabies viral glycoprotein as indicated by specific, membrane fluorescence on unfixed, rabies virus infected murine neuroblastoma (MNA) cells and glycoprotein gene encoding plasmid transfected COS cells. In addition, the monoclonal antibody competes with and blocks a glycoprotein antigen site III binding monoclonal antibody (mAb-D1, Institut Pasteur, Paris, France). The monoclonal antibody was used in an IC-ELISA using an in-house standard to quantify the rabies viral glycoprotein antigen in 12 vaccine preparations with potency values ranging from 4 to 18 IU. The results indicated a good correlation with the NIH mouse potency assay (r=0.83). The immuno-capture ELISA described in this study can be used to quantify the immunogenic rabies viral glycoprotein antigen in the inactivated rabies viral antigen preparation in a simple and rapid format, which enables better vaccine formulation.     

Evaluation of the single radial-immunodiffusion assay for measuring the glycoprotein content of rabies vaccines

The glycoprotein content of rabies vaccines containing the Pitman-Moore strain of rabies virus was measured by the single radial immunodiffusion assay and correlated with vaccine potency. The variability of this assay was 6.3% for a single vaccine lot tested over a one-year period. Using sera prepared against rabies virus glycoprotein from different strains of virus, the assay gave different values. These differences could be eliminated by using a homologous vaccine strain as an internal reference. Single radial-immunodiffusion values for Pitman-Moore vaccines correlated with the manufacturers' NIH potency assay, but required a mathematical transformation to convert values from one assay to the other. Single radial-immunodiffusion values for Street Alabama Dufferin and Flury-LEP vaccines did not correlate with NIH values. Modification of the single radial immunodiffusion technique and the feasibility of using this assay for the determination of rabies vaccine potency are discussed.     

Immune Response in Primates Vaccinated with Duck Embryo Cell Culture Rabies Vaccine

Adult rhesus monkeys (Macaca mulata) were vaccinated with four inactivated rabies vaccines, including two cell culture vaccines, one zonal purified cell culture vaccine, and a 10% extracted duck embryo vaccine. The vaccines were potency tested by both National Institutes of Health (NIH) and Habel methods and passed one or both tests. However, a vaccine having acceptable potency by one method frequently failed or was marginal by the other procedure. Groups of three monkeys were inoculated with each vaccine by one of two schedules. The first consisted of four weekly 1-ml doses followed by a 1-ml booster dose at 6 months, and the second consisted of seven daily 1-ml doses of vaccine with no booster. Both zonal purified and extracted duck embryo vaccines induced detectable neutralizing antibody by day 7 with either schedule, and antibody titers elicited by the cell culture vaccine remained high through 210 days. However, antibody titers produced by the 10% duck embryo vaccine dropped sharply after their 28-day peak. Duck embryo cell culture vaccines with low or marginal potency as measured by Habel or NIH tests still produced rapid, high levels of serum-neutralizing antibody in primates. LD(50) or NIH and Habel tests as measured in mice were not necessarily good indices of antibody response in the primate host. The need for a cell culture potency test that will yield a more predictable correlation with the definitive host's antibody response is discussed.     

应用细胞工厂建立人用狂犬病疫苗连续培养多次收获工艺

目的为提高生产效率、增加原代地鼠肾细胞单产量及狂犬病病毒产量,建立人用狂犬病疫苗（地鼠肾细胞）连续培养多次收获工艺。方法选用12-14日龄SPF地鼠,无菌取肾经消化,制备成细胞悬液,分装到40层细胞工厂并培养细胞成单层;接种狂犬病病毒固定毒aG株,连续培养病毒并多次收获。分别对同一细胞批制备的多个单次病毒收获液的免疫原性、病毒滴度和地鼠肾细胞蛋白质含量进行检测。结果用40层细胞工厂培养原代地鼠肾细胞和狂犬病病毒,细胞接种浓度为1．0×10。～1．5x10。cells／mL,（36±1）℃培养72h成致密单层;按0．1MOI病毒接种,可进行6次收获病毒;多个单次病毒收获液病毒滴度均不低于6．0lgLD50／mL;免疫原性检查保护指数不低于100;地鼠肾细胞蛋白质残留量随着收获次数的增加而不断降低。结论用细胞工厂建立了人用狂犬病疫苗连续培养多次收获工艺,能显著提高地鼠。肾单产量,增加产能。     

高效价冻干人用狂犬病疫苗暴露后免疫程序的研究

初步确定高效价冻干人用狂犬病疫苗（6．0IU／剂）暴露后免疫程序。制备高效价的冻干人用狂犬病疫苗（6．0IU／剂）,以狂犬病街毒CNX8601和BD06分别攻击小鼠和比格犬的咬肌,接种不同效价的狂犬病疫苗,以RFFIT法检测中和抗体,根据动物死亡情况,计算暴露后疫苗保护率,对不同效价的疫苗进行中和抗体测定和保护率统计分析。在以小鼠为实验动物的疫苗保护率研究中,冻干人用狂犬病疫苗（3．1IU／剂）0／3／7／14／28免疫程序的保护率为40．6％,高效价的冻干人用狂犬病疫苗（6．0IU／剂）0／3／14免疫程序的保护率为56．2％,中和抗体比较,P〈0．05,2组间有显著性差异;在以比格犬为实验动物的保护效果研究中,冻干人用狂犬病疫苗的保护率（3．1IU／剂）为70％;高效价的冻干人用狂犬病疫苗（6．0IU／剂）的保护率为80％,中和抗体的比较,P〉0．05,没有显著性差异。高效价冻干人用狂犬病疫苗暴露后免疫程序可初步确定为0、3、14d免疫。     

In vitro rabies vaccine potency appraisal by ELISA: advantages of the immunocapture method with a neutralizing anti-glycoprotein monoclonal antibody

The replacement of the in vivo potency test (NIH test) for rabies vaccine evaluation by in vitro methods is at present discussed in many reports and also by WHO expert working groups. For this purpose, in vitro glycoprotein titration has been proposed. Among the different glycoprotein assays, we have studied two ELISA methods (immunocapture and direct plate coating with the antigen to be tested) using neutralizing mono- and polyclonal antibodies. In our view, the immunocapture method based on the use of a neutralizing monoclonal anti-glycoprotein antibody seems to be a convenient tool for the determination of the in vitro potency of rabies vaccine and of the products corresponding to the different steps of their production process.