双抗夹心ELISA法快速检测人用狂犬病疫苗的效价

目的：用单克隆抗体制备的双抗夹心ELISA法，快速检测人用狂犬病疫苗中的狂犬病毒糖蛋白G的含量，并探讨其替代NIH法的可行性。方法：将疫苗样品用NIH法检定，同时以国家标准品为参考疫苗，用双抗夹心ELISA法检测。结果：用双抗夹心ELISA法测得的IgED50为2．60～2．78；用NIH法测得的IgED50为2．55～2．85。对两种检测方法进行F检验，两种检测方法的灵敏度有显著差异（F=5．76，P〈0．05）；用t检验法对这两种检测方法进行分析，表明两组测定值的差异无统计学意义（t=0．187，P〉0．05）。结论：双抗夹心ELISA法与NIH法测得的IgED50呈正相关性。与NIH法相比，双抗夹心ELISA法具有重复性好、成本低、快速等优点。用双抗夹心ELISA法取代NIH法测狂犬病疫苗效价是可行的。     

检测乙型脑炎病毒中和抗体的简化试验

在亚洲乙型脑炎 ( JE)病毒是病毒性脑炎的常见原因 ,估计每年有 4 50 0 0例病例 ,并且发病率和病死率较高。　　由于登革热也在 JE流行区流行 ,因此为了与登革热区分 ,所有 JE阳性血清必须检测 JE特异性中和抗体才能确诊。蚀斑减少中和试验 ( PRNT)是定量检测 JE中和抗体的金标准。然而该方法需在 6孔或 2 4孔板上进行 ,需时 1周 ,这样就限制了其在大规模筛选 ,尤其是在疫苗效力研究和血清流行病学调查中的应用。因此 ,目前已建立了几种改进的定量检测 JE中和抗体的方法。　　微灶减少中和试验和荧光灶抑制试验 ,应用过氧物酶结合抗…     

韩国儿童初免和加强免疫乙型脑炎病毒SA14-14-2株疫苗后的免疫应答

乙型脑炎病毒（ＪＥＶ）ＳＡ１４－１４－２株减毒活疫苗由中国研制，为评价其在中国以外地区的使用效果，作者对韩国儿童初次和加强免疫该疫苗后的免疫应答进行了研究。研究对象为乙型脑炎低流行区的１～３岁儿童，对８４名儿童接种由中国成都生物制品研究所生产的ＳＡ１４－１４－２疫苗，滴度为６．８ｌｏｇ＿（１０）ｐｆｕ／０．５ｍｌ，其中６８名儿童为初免，１６名儿童有ＪＥＶ灭活疫苗接种史或ＪＥＶ接触史。另外，以２５名接种过２剂灭活疫苗（中山株）的儿童作为对照。采集所选对象免疫前和免疫后４周的静脉血，用蚀斑减少中和试…     

抗HPV11病毒样颗粒单克隆抗体的性质鉴定及初步应用

目的分析和鉴定抗HPV11病毒样颗粒(virus-like particle,VLP)鼠源单克隆抗体的性质,筛选性质和生物学活性较优的抗体,并初步应用于抗原或疫苗的质量分析。方法分别利用间接ELISA法和Western blot对HPV11的22株单克隆抗体的亚类、与HPV11 VLP的结合能力和构象敏感性进行检测;采用血凝抑制实验对单克隆抗体的血凝抑制活性进行分析;运用基于假病毒的抗体中和实验对单克隆抗体的中和活性进行鉴定,选出中和活性高的单抗进行两两配对,采用双抗夹心ELISA法捕获单抗并筛选合适的配对双抗。结果对22株单抗的性质进行了详细和完整的鉴定,并根据构象敏感性进行排序,筛选出6株型别特异、结合活性强且中和活性高的单抗(2A2、4A1-3、16G7、14A6、9C12和19C7);成功建立了基于单抗的双抗夹心(14A6∶Ag∶9C12-HRP)ELISA定量分析方法。结论获得了较全面的HPV11 VLP单抗性质信息,建立了重组HPV11抗原质量分析的双抗夹心ELISA法,为HPV11抗原的生命周期管理或尖锐湿疣疫苗的研发、工艺优化、产品放行和稳定性研究等提供了技术支持。     

毒素中和试验和ELISA法测定小鼠和豚鼠免疫血清中破伤风抗毒素滴度的比较

作者应用ELISA法测定了小鼠和豚鼠免疫后不同时间的血清破伤风抗体的滴度,并与毒素中和（TN）试验的结果进行了相关性分析,结果如下: 8只豚鼠每只注射7.5絮状反应单位（Lf）破伤风类毒素（TT）,免疫后9天血清中无中和抗体,而ELISA法测得IgG滴度为0.18～0.71ELISA抗毒素单位（EAU）/ml。注射后4周用ELISA法测得IgG滴度比TN法高6～10倍。但注射后6周用ELISA和TN法测定豚鼠血清,抗体滴度非常相似〔相关系数（r）=0.88〕。     

不同类型麻疹、腮腺炎、风疹疫苗及初免年龄对抗体水平的影响

作者以加拿大魁北克省作为试验基地,用MMRⅡ和Trivirix两种不同配方的麻疹、腮腺炎、风疹（MMR）疫苗对468名12～24月龄儿童进行接种。其中241人接种MMRⅡ疫苗,其余接种Trivirix疫苗。此两种疫苗中,风疹疫苗均用RA27/3株制备,且含量相同〔1000半数组织培养感染量（TCID_(50)）〕,但在麻疹和腮腺炎病毒和含量上有所差别,MMRⅡ疫苗含Enders Edmonston麻疹病毒1000 TCID_(50),Teryl Lynn腮腺炎病毒5000TCID_(50),Trivirix疫苗含Schwarz麻疹病毒1000 TCID_(50),Urabe Am 9腮腺炎病毒20000TCID_(50)。初免后5～6年,取静脉血,分别用蚀斑减少中和（PRN）试验、酶免疫试验和血凝抑制（HI）试验检测麻疹、腮腺炎和风疹抗体。     

乙型脑炎瘦苗IC51

澳大利亚IntercellAG公司开发的乙型脑炎病毒（Japaneseencephalitisvirus，JEV）疫苗IC51，由培养于Vero细胞的SA圹14-2毒株经纯化和甲醛灭活制得，该疫苗于2009年上半年分别获得澳大利亚治疗产品管理局（TherapeuticGoodsAdministration，TGA）、美国FDA和欧洲药品管理局（EuropeanMedivinesAgency，EMEA）批准，用于成人预防乙型脑炎。     

双抗体夹心ELISA法检测肾综合征出血热汉滩病毒糖膜蛋白

用双抗体夹心ELISA法检测肾综合征出血热（HFRS）汉滩病毒LR1株糖膜蛋白（GP）,并用SDS－PAGE电泳对ELISA检测结果进行验证,实验结果显示,双抗体夹心ELISA法特异性强,重复性好,简便易行,是HFRS汉滩病毒糖膜蛋白较为理想的检测方法,对HFRS疫苗的生产和质量控制有重要意义。     

酶联免疫吸附试验检测乙型肝炎病毒影响因素分析

目的：探讨酶联免疫吸附试验检测乙型肝炎病毒（HBsAg）的影响因素。方法：参照《全国临床检验操作规程》ELISA双抗夹心法,按照说明书进行操作。结果：影响酶联免疫吸附试验检测HBsAg的因素主要有边缘效应、ED-TA、肝素、RF、AFP、补体、自身抗体等。结论：ELISA检测检测时要严格按照规程操作,提高检测人员的素质,把好质量关,提高检测报告的准确率。     

Vero细胞乙型脑炎灭活疫苗的研制

目的 以Vero细胞为基质,研制安全有效的乙型脑炎灭活疫苗.方法 将经Vero细胞适应的乙型脑炎病毒P3株接种Vero细胞,培养后收获病毒液.采用β-丙内酯灭活,通过超滤浓缩、硫酸鱼精蛋白沉淀、蔗糖密度梯度离心等方法纯化病毒,最终配制成预防乙型脑炎病毒感染的疫苗.通过临床试验考察疫苗的安全性及免疫原性.结果 该疫苗的主要考核指标,如病毒滴度、总蛋白含量、Vero细胞DNA残留量、效价等均符合国家标准.临床观察结果显示,接种本疫苗后局部及全身反应轻微;各年龄组乙型脑炎病毒中和抗体阳转率均>90%.结论 采用本法制备的疫苗安全有效.     

非典型肺炎灭活疫苗免疫动物效果评价

目的应用空斑减少中和试验对非典型肺炎灭活疫苗动物免疫血清进行中和抗体测定,以评价其免疫效果。方法用Vero-E6细胞接种6孔塑料细胞培养板,加两层含琼脂糖培养基,以中性红为染色剂建立空斑试验,以能减少50%的空斑为标准测定抗SARS中和抗体。结果三批非典型肺炎灭活疫苗免疫小鼠和家兔均产生较高的中和抗体。结论非典型肺炎灭活疫苗对两种动物具有良好的免疫效果。     

Comparison of poliovirus antigen measurements in vitro and in vivo. Preliminary work towards the standardisation of inactivated poliovirus vaccine potency

The potency of various batches of inactivated poliovirus vaccines have been determined in vitro using an enzyme-linked immunosorbent assay (ELISA), and the results compared with in vivo potency values obtained in guinea pigs and expressed as antibody titres and antigen extinction limits, revealing three possible expressions of vaccine quality. The purpose of the study was to collect information needed to regulate the vaccine dose within a standard potency range using all three values obtained. The results expressed in D antigen units as determined by ELISA suggest that this technique is more sensitive in measuring differences in antigen concentration than either of the methods chosen to evaluate vaccine potency in vivo.     

副黏病毒Tianjin株重组血凝素-神经氨酸酶单克隆抗体的制备及在临床检测中的初步应用

目的:建立双抗体夹心ELISA法,调查副黏病毒Tianjin株引起婴幼儿下呼吸道感染情况。方法:用纯化的重组HN片段免疫BALB/c小鼠,按常规方法制备单克隆抗体。以兔抗Tianjin株多克隆抗体为捕获抗体,单抗G7G7E7为检测抗体,建立双抗体夹心ELISA法,检测104例下呼吸道感染患儿支气管肺泡灌洗液(BALF)标本。结果:获得3株能稳定分泌单克隆抗体的杂交瘤细胞株G7H4D3、G7D9G3和G7G7E7。3株单克隆抗体与副黏病毒Tianjin株有较高结合活性,与甲、乙型流感病毒,新城疫病毒(NDV),人副流感病毒(hPIV)1型、3型、肺炎支原体均无交叉反应性。ELISA相加试验和阻断试验表明,3株单抗识别相同或相近表位区域,识别的表位在抗病毒免疫应答中处于免疫优势地位。双抗体夹心ELISA法检测阳性率为1.92%(2/104)。结论:与RT-PCR和间接ELISA法比较,双抗体夹心ELISA法,具有敏感性高、特异性强的优点,适于临床检测使用。副黏病毒Tianjin株是婴幼儿下呼吸道感染的重要致病因子之一。     

无明胶保护剂冻干乙型脑炎灭活疫苗（Vero细胞）稳定性研究

目的  观察不含明胶的不同保护剂配方冻干乙型脑炎灭活疫苗（Vero细胞）的稳定性。方法  在现行冻干乙型脑炎灭活疫苗制备工艺的基础上,以乳糖替代明胶,并适当增加人血白蛋白用量至5、10和20 g/L,制成A、B、C三个保护剂配方疫苗。对疫苗进行25 ℃加速试验、37 ℃热稳定性试验和2～8 ℃长期稳定性试验,检测疫苗的有效抗原含量和效力（T值）,并与现行疫苗（对照）进行比较。结果  三个保护剂配方疫苗于25 ℃放置24周后,A、B疫苗有效抗原含量仅分别降低2.2%和5.9%,效力稳定;而C疫苗有效抗原含量降低13.6%,效力降低5.0%。37 ℃放置8周后,A、B、C疫苗有效抗原含量分别降低11.2%、13.2%、15.2%,效力分别降低5.8%、9.0%、7.2%。2～8 ℃放置48个月后,A、B、C疫苗有效抗原含量分别降低17.2%、18.4%、21.9%,效力均符合《冻干乙型脑炎灭活疫苗（Vero细胞）注册标准》。在上述试验中,A、B疫苗均优于或等于对照疫苗;C疫苗与对照疫苗差异较大,但均符合相关标准。结论  含A配方保护剂的冻干乙型脑炎灭活疫苗稳定性高且白蛋白用量少,因此,建议首选A配方保护剂。     

抗H7N9流感病毒中和抗体快速检测方法的建立及应用

目的：对建立的抗H7N9流感病毒中和抗体快速检测方法进行方法学验证及初步应用。方法：分别采用不同代次细胞对高、中、低不同滴度的阳性血清进行多次平行检测,考察细胞代次对检验结果的影响;采用NIBSC提供的参考品对方法学的特异性进行验证;同时应用抗H7N9的阳性血清检测进一步评估方法的准确性和精密性;采用ELISA-MNT法和血凝抑制（hemagglutination inhibition,HI）试验分别接种H7N9灭活流感疫苗免疫的小鼠血清样本20份,分析两种方法检测结果的相关性。结果：采用ELISA-MNT中和法,使用不同代次MDCK细胞（25、30和35代）检测相同的血清样本的中和抗体滴度结果相同;该方法只对羊抗H7N9的血清具有较高保护力,对其他血清基本没有交叉反应;该方法准确性良好;该方法组间、组内精密性的平均变异系数分别为4%和11%。该方法测定H7N9型流感疫苗免疫后的小鼠血清抗体效价,其结果与HI抗体的相关系数为0.61,表明两种方法的检测结果之间呈良好的正相关性。结论：建立的微量病毒中和法能够满足H7N9流感病毒中和抗体效价检测的要求,可用于H7N9新型大流行流感疫苗的免疫评价。     

Collaborative study for validation of a serological potency assay for rabies vaccine (inactivated) for veterinary use

European Pharmacopoeia (Ph. Eur.) monograph 0451 on Rabies vaccine (inactivated) for veterinary use describes an in vivo batch potency test that is based on the NIH test. This assay uses a large number of mice and results in a significant degree of suffering. In the interest of replacement, reduction and refinement of animal tests (3R) a serological potency assay for Rabies vaccine (inactivated) for animal use, developed and validated at the Paul-Ehrlich-Institut, has been assessed in a collaborative study organised by the EDQM (European Directorate for the Quality of Medicines & HealthCare). The goal was to demonstrate the wider transferability of the proposed assay and confirm its suitability. The study involved 13 laboratories and assessed 4 different vaccines from the EU market. Results of the study confirm that a limit test using a relatively small number of animals in a serological assay is possible, reproducible and reliable. The optimal number of animals per vaccine is product specific but may roughly be indicated to be between 8 and 10 for the products included in this study. Non-responders should be included in the analysis because they may reflect sub-potent vaccines. However, there may be a need to impose a maximum on the number of non-responders allowed for the reference vaccine as a monitor for assay validity. This assay provides a significant 3R improvement in terms of both the number of animals used and the amount of suffering entailed and provides a more reliable and reproducible assay format than the vaccination challenge assay. It also reduces the time required as compared to the vaccination challenge assay. It has been recommended to the Ph. Eur. group of experts 15V that this assay be included as an alternative to the batch potency assay in the Ph. Eur. monograph 0451.     

Establishment of batch 4 of the Biological Reference Preparation (BRP) for rabies vaccine (inactivated) for veterinary use

9 laboratories from 7 countries including both laboratories from the public and private sector participated in a collaborative study organised under the aegis of the European Directorate for the Quality of Medicines Biological Standardisation Programme in order to establish batch 4 of the European Pharmacopoeia (Ph. Eur.) Biological Reference Preparation (BRP) for rabies vaccine (inactivated) for veterinary use. Establishment of Ph. Eur. BRP batch 4 was necessary in order to replace Ph. Eur. BRP batch 3, the stocks of which were dwindling. 8 laboratories provided results. Ph. Eur. BRP batch 4 was calibrated against the 5th International Standard for inactivated rabies vaccine in International Units (IU) using the vaccination challenge method of the Ph. Eur. monograph 0451. The International Standard (IS), Ph. Eur. BRP batch 4 and batch 3 are all freeze-dried vaccines prepared by beta-propiolactone inactivation of the Pitman Moore strain of rabies. Based on the results of the study, a potency of 11 IU/vial was assigned to Ph. Eur. BRP batch 4 for rabies vaccine (inactivated) for veterinary use. Nevertheless, it was noted that the vaccination challenge assay used as the "golden standard" for potency determination of inactivated rabies vaccines for veterinary use is a crude assay requiring the use of a large number of animals. Evidence from this study and from the collaborative study to establish Ph. Eur. BRP batch 3 suggests that the assay is difficult to perform and provides highly variable results. The validation of a suitable in vitro alternative is therefore highly recommended, as is the possible improvement of the in vivo assay, which will most likely remain the "golden" standard.     

Feasibility study to evaluate the correlation between results of a candidate in vitro assay and established in vivo assays for potency determination of Newcastle disease vaccines

A Newcastle disease virus antigen quantification assay has been developed at CIDC-Lelystad as a candidate in vitro potency test for inactivated Newcastle disease vaccines. In studies performed at CIDC-Lelystad, a high correlation was demonstrated between the results of this candidate in vitro potency assay and the results of the serological potency assay (European Pharmacopoeia monograph 0870; test A). Furthermore, a high correlation between the serological data (Haemagglutination Inhibition-antibody titres) and clinical protection after challenge was demonstrated. The aim of the feasibility study was to confirm the correlation between the results obtained using the candidate in vitro potency assay and the results from both the in vivo potency assays currently prescribed in Ph Eur monograph 0870, in different laboratories and to determine whether a large-scale validation study of the in vitro method should ensue. In the feasibility study three Official Medicines Control Laboratories tested the potency of 5 different inactivated Newcastle disease vaccines and one experimental vaccine, using both of the in vivo methods described in the European Pharmacopoeia and the candidate in vitro method. The 6 vaccine batches represented a quantitative range of Newcastle disease virus antigen content and were produced by different manufacturers. Statistical evaluation of all results indicated that a satisfactory correlation was found in all laboratories between the two types of in vivo tests currently in place, and the candidate in vitro test. An excellent reproducibility of the proposed in vitro method was observed with respect to the ranking of the vaccines included in this study. It is concluded that the results of this feasibility study indicate that a large-scale collaborative study can be organised to validate the in vitro method and the suitability of the reference preparation.