目的:比较麻腮风类减毒活疫苗的两种取样方式测定疫苗各组分滴度是否存在差异.方法:采用多支疫苗混合滴定和单支疫苗滴定两种方法对国内上市的麻腮风类疫苗进行检测,对两种方法测定的结果平行比对.结果:单支疫苗滴定后的均值与多支疫苗混合后的检测结果基本一致;腮腺炎疫苗的支间滴度差异略高,麻疹、风疹疫苗的支间滴度差异较小.结论:目前使用的多支混合滴定的检测方法能够满足对麻腮风类疫苗质控的要求,但单支滴定法对工艺改进更有指导意义.

目的:对水痘减毒活疫苗病毒滴度测定用样品的不同取样方法进行优化研究.方法:采用国外药典测方法,对我国上市水痘减毒活疫苗进行单支测定后计算平均滴度,并与我国多支混合后测定的结果进行比较.结果:采用单支检测后计算的均值与多支混合后测定结果基本一致,多数企业制品支间均一性良好.结论:《中国药典》中对水痘减毒活疫苗病毒滴度测定方法能满足确保疫苗有效性的需要,但在反映支间均一性方面可借鉴国外药典方法.

Objective:To develop a new method based on neuraminidase (NA) activity for detec-ting virus titers of cell culture-based influenza vaccines and preliminarily analyze its application.Methods:Reaction conditions including the substrate concentration for enzymatic reaction, stop solution, the number of initially infected target cells and cell lysis buffer were optimized. The titers of cell culture-based influenza vaccine strains were detected by the established method and the results were compared with those by the traditional viral titration test.Results:The optimal substrate concentration for enzymatic reaction was 25 μmol/L, and the optimal stop solution was 0.2 mol/L Na 2CO 3. In the detection of NA activity in infected cells, the maximum relative fluorescence value was obtained by infecting 4×10 4 cells/well with influenza virus for 48 h and using 0.5% TritonX-100 for lysis. The developed method showed no significant differences with the traditional virus titration test in detecting the titers of four batches of influenza vaccine virus strains ( P>0.05), indicating that the two methods had a good consistency. Conclusions:This study established a new method based on NA activity to detect virus titers of cell culture-based influenza vaccines. The method could be used for the detection of virus strains used in the production of cell cultured-based influenza vaccines.

目的 建立人类乳头瘤病毒(human papillomaviruses,HPV)特异性抗体单一稀释度ELISA检测法,并进行方法验证及初步应用.方法 以HPV16及HPV18 L1病毒样颗粒蛋白作为包被抗原,血清样品采用单一稀释度稀释(未免疫血清及免疫后血清分别进行100及500倍稀释),建立HPV特异性抗体的单一稀释度ELISA检测法,并验证方法的特异性、准确性、线性范围、检测限度及精密性.采用建立的方法检测自然感染HPV及接种疫苗后的血清样本(各28份),并与假病毒中和试验(pseudovirus-based neutralization assay,PBNA)检测结果进行比较.结果 100份阴性血清样本HPV16及HPV 18 IgG抗体均为阴性;HPV16及HPV18抗体滴度的平均回收率分别为74％和73％;HPV16及HPV18抗体的定量检测范围分别为0.049～0.370 IU/mL及0.030～0.231 IU/mL;该方法重复性CV在2.9％～6.6％之间,日间精密性CV在7.7％～ 11.9％之间.接种疫苗后的血清样本检测值显著高于自然感染样本(P＜0.05),接种疫苗后的血清样本检测值与PBNA法检测结果具有显著相关性(HPV16和HPV18抗体检测结果的r值分别为0.727和0.800).结论 成功建立了HPV特异性抗体的单一稀释度ELISA检测法,且具有较好的特异性、准确性、精密性,可用于HPV疫苗免疫效果评价及疫苗上市后抗体水平的监测.

目的 优化流感减毒活疫苗诱发黏膜IgA抗体的ELISA检测方法,并制备IgA抗体实验室内控品.方法 采用ELISA检测法对流感病毒抗原(全病毒及其相应HA抗原)及包被浓度(0.5、1、2、4 μg/mL)、封闭液(1％ BSA、10％ FBS、5％脱脂奶粉)及封闭时间(1、1.5、2h)、样本稀释液(含1％ BSA的PBST、含2％脱脂奶粉的PBST、样本保存液)及样本作用时间(45、60、75 min)、酶标抗体稀释度(1:1 000、1∶2000、1∶4000、1∶8000)进行优化.采用优化的方法制备针对H1N1、H3N2和B型3个型别流感病毒的IgA抗体检测用实验内控品,并用该方法对接种三价流感减毒活疫苗的20名志愿者的鼻咽拭子样本进行检测.结果 最适间接ELISA法检测条件为:以全病毒作为包被抗原,包被浓度为1μg/mL;以含1％牛血清白蛋白为封闭液封闭2h;样本稀释液为2％脱脂奶粉的PBST,作用时间为60 min;酶标抗体稀释度为1∶1 000.采用优化方法制备的H1N1、H3N2、B型IgA抗体阳性实验室内控品几何平均滴度分别为23、45、35,可接受范围分别为16～32、32～64、32～64.接种疫苗后20名志愿者中3种型别IgA抗体滴度较接种前均显著增高(P＜0.001),40％～50％志愿者接种后IgA抗体滴度比接种前至少升高2倍.结论 成功优化了流感减毒活疫苗诱发黏膜IgA抗体的ELISA检测方法,制备的IgA抗体的实验室内控品可用于流感减毒活疫苗接种后鼻咽拭子抗体的检测.

Objective To develop an enzyme-linked lectin assay ( ELLA ) for measuring neuraminidase inhibition (NI) antibody titers in subjects vaccinated with H7N9 influenza vaccine. Methods Neuraminidase substrate, the dilution and incubation time of enzyme-labeled antibody, the concentration of influenza antigen for coating and pH value of the dilution buffer were optimized. Based on that, ELLA was established and used to detect anti-influenza neuraminidase antibody titers in serum samples of 34 subjects before and after vaccination with H7N9 influenza vaccine. Results The optimal neuraminidase substrate was fetuin at a coating concentration of 7. 5 μg/ml. The optimal dilution of enzyme-labeled antibody was 1 : 500. The virus strain of influenza H7N9 vaccine was used as antigen at a concentrations of 4. 5lgCCID50/ml in solution with a pH of 6. 5. Influenza-specific NI titers detected after immunization with vaccine were significantly higher than those before vaccination (P<0. 001). In the 34 subjects receiving H7N9 vaccine, the seroconversion rate of NI antibody was 47％ (≥40 in NI titer ), which was lower than that of HI antibody (P<0. 05). Conclusions An ELLA with natural substrate for measurement of anti-in-fluenza NI antibody was developed. It is simple and practical and might be used in the establishment of im-mune evaluation system for influenza vaccines and NI antibody.

目的:对建立的检测流感病毒特异性IgA抗体检测方法进行验证,并且用于评价流感减毒活疫苗(LAIV)粘膜免疫反应.方法:使用来自英国生物制品所(NIBSC)的流感抗原国际参考品作为包被抗原,检测鼻咽拭子阳性样品,确定流感病毒型别特异性的IgA抗体滴度,考察该方法的特异性;通过对高低不同抗体滴度的阳性样本多次平行检测来评价该方法的重复性;在此基础上,对526份鼻咽拭子样本进行IgA抗体滴度检测.结果:只针对包被抗原阳性的样品检测到高滴度的IgA抗体,其他型别的抗体未检测到,方法特异性显著.鼻咽拭子IgA抗体检测方法的组间重复性、组内重复性、方法重复性良好.检测接种LAIV疫苗免疫前后的鼻咽拭子样本,疫苗组H1N1、H3N2以及B型IgA抗体几何平均滴度(GMT)接种前后的比值分别为1.15、1.45和1.18,均高于安慰剂组;疫苗接种组IgA抗体滴度高于免疫前2倍的比率,也高于安慰剂对照组(P＜0.05).鼻咽拭子IgA抗体检测结果与血清样本HI抗体结果缺乏相关性(p＜0.001).结论:建立的流感病毒特异性IgA抗体的检测方法能够满足LAIV粘膜免疫评价的需要.

Objective: To carry out methodological validation and preliminary application of the rapid detection method for neutralizing antibody against influenza A (H7N9) virus. Methods: Different generations of positive cells were used to perform multiple parallel tests on high, medium and low titers of positive serums, and the effects of cell generation on the test results were investigated. Specificity of the method was verified by the reference material provide by NIBSC. Accuracy and precision of the method were evaluated by anti-H7N9 virus positive serums. ELISA-MNT and hemagglutination inhibition (HI) assay were separately used to test the antibody titers from mouse serums immunized with inactivated influenza H7N9 vaccine and the correlation of the two assays was evaluated. Results: The neutralizing antibody titers of the same serum samples were identical by using the ELISA-MNT neutralization method and different generations of MDCK cells (the 25 th, 30 th and 35 th generation). The method only had high protection against the serum of sheep anti-H7N9 and had no cross reaction with other serums. The method had good accuracy and average coefficient variations of intra-assay and inter-assay were 4 ％ and 11.2％, respectively. The correlation coefficient between the antibody titers from mice immunized with H7N9 influenza vaccine measured by the ELISA-MNT assay that obtained from the HI assay was 0.61, indicating that there was a good positive correlation between the detection results of the two methods. Conclusion: The established micro-virus neutralization method can meet the requirement of H7N9 influenza virus neutralizing antibody titer detection, and can be used for immunological evaluation of vaccine for H7N9 new pandemic influenza.

目的 对人乳头瘤病毒(human papilloma virus,HPV)假病毒中和试验起始稀释倍数进行研究,确定该系统中合适的起始稀释倍数.方法 为了排除血清中HPV抗体以及母传抗体的干扰,选取50份15～ 18月龄的儿童血清作为验证样本.血清以不同的稀释倍数,与适量的假病毒中和.以光学显微镜观察接种细胞的形态,来判定不同稀释度的血清对细胞生长状况的影响;观察感染细胞表达的绿色荧光蛋白的强度及数量,以此考察血清对假病毒感染过程的影响.结果 血清稀释度1∶40对细胞生长状况影响较小;该稀释倍数的病毒进入细胞表达的荧光强度与不加血清的阳性对照类似;假病毒感染细胞导致的荧光斑点数与真实病毒对照相比减少了10％,远低于中和试验判定阈值(相对抑制率50％).结论 在该实验系统中,HPV假病毒中和试验的血清起始稀释度以1∶40较为合适.

对于大多数的生物制品而言,特别是减毒类活疫苗产品,冻干是提升疫苗稳定性极为重要的制剂手段.为提高冻干产品的稳定性和复水性,制品中加入保护剂是常用的方法.使用保护剂可改变疫苗在冻干过程中由于环境改变造成的损害,能较好地保持生物样品原有的结构和生物活性,同时可提升产品贮藏期内蛋白质的稳定性[1].

目的:建立联合酶联免疫的微量病毒中和法(ELISA-MVN法),用于大流行流感疫苗流感病毒中和抗体效价的检测,并进行方法学验证.方法:采用4种羊抗流感病毒血清参考品,与人感染禽流感H5N1病毒疫苗株(A/Vietnam/1194/2004-NIBRG-14)病毒液,分别于37℃孵育18 h后,固定细胞,ELISA检测细胞内流感病毒核蛋白表达,确定其中和抗体效价,考察该方法的特异性;通过对高中低不同抗体滴度的血清样本的多次平行检测来评价该方法的重复性;通过ELISA-MVN法和血凝抑制试验(HI试验)分别检测大流行流感疫苗接种者血清样本,比较2种方法的灵敏性和相关性.结果:ELISA-MVN结果显示羊抗H5N1的血清中和抗体检测滴度为5 120,其他3种血清的中和抗体滴度小于10,该方法特异性良好;采用ELISA-MVN法对3种不同滴度的血清进行重复检测,组内重复性试验的6次平行试验结果的RSD为3.5％.组间重复性5次结果,RSD为4.8％～8.6％.2种方法测定疫苗接种者血清样本抗体效价的结果具有显著相关性(P＜0.001),相关系数r=0.932.ELISA-MVN法检测结果的几何平均滴度高于HI试验检测结果,该方法较HI试验在检测大流行流感疫苗血清样本灵敏度高.结论:ELISA-MVN可满足大流行流感疫苗血清学检测的要求,并可用于评价大流行流感疫苗的免疫效果.

目的 建立检测流感病毒中和抗体效价的微量病毒中和法(microneutralization tests,MNT),并进行验证.方法 建立改进的微孔板病毒中和法,并对该方法的关键影响因素(不同代次MDCK细胞和细胞培养板边缘效应)以及方法的准确性和精密性进行验证;采用改进的微孔板病毒中和法和血凝抑制(hemagglutination inhibition,HI)试验分别检测甲型H1N1流感疫苗免疫的血清样本85人份,分析两种方法检测结果的相关性.结果 采用改进的微孔板病毒中和法,用不同代次MDCK细胞(25、30、35代)以及在细胞培养板不同位置(边缘孔、非边缘孔)检测相同的血清样本的中和抗体滴度结果相同;该方法准确性和精密性良好;改进的微孔板病毒中和法和HI法测定甲型H1N1流感疫苗免疫后血清抗体效价结果的相关系数为0.81,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可满足流感病毒中和抗体效价检测的要求,可用于评价疫苗的免疫效果.

目的:制备腮腺炎减毒活疫苗国家参考品,并进行标定.方法:选用国内腮腺炎疫苗生产毒株S79制备腮腺炎疫苗参考品.在生产过程中严格控制精密性、水分含量,对候选参考品进行鉴别试验、病毒滴度、水分含量、牛血清蛋白残留量及无菌检查等试验.检验合格后组织4个实验室对候选参考品病毒滴度进行协作标定,计算实验室内变异及实验室间变异系数;对候选国家参考品进行稳定分析.结果:制备的候选参考品鉴别试验、无菌检查结果均符合规定,水分含量为1.6％.经协作标定,腮腺炎减毒活疫苗病毒滴定候选国家参考品的病毒滴度为(4.96±0.38) lgCCID50·mL-1,实验室内变异系数在2.0％ ～4.1％之间;稳定性考核数据显示制备的参考品具有较好的稳定性.结论:制备的参考品符合作为腮腺炎减毒活疫苗国家参考品的要求.

目的 纯化B型流感病毒血凝素(hemagglutinin,HA).方法 使用不同浓度的菠萝蛋白酶酶切B型流感病毒HA,超速离心获得含HA的上清后,经蔗糖密度梯度离心进行纯化,HPLC分子筛分析纯化效果.纯化后的蛋白经糖苷酶PNGaseF处理后,切取丰度较高的蛋白条带,进行质谱鉴定.结果 病毒蛋白与菠萝蛋白酶蛋白含量的最佳比例为40:1;5％ ～ 30％梯度浓度的蔗糖能很好地去除低相对分子质量的蛋白,流感病毒内丰度较高的NP及M蛋白得到很好的去除;纯化的HA蛋白的纯度大于90％,且HPLC分析显示仅有1个主峰,均一性良好.结论 优化了B型流感病毒HA蔗糖密度梯度离心纯化方法,获得了高纯度的HA蛋白,为制备HA标准抗血清奠定了基础.

Objective To develop and validate a pseudovirion-based neutralization assay(PBNS)in determination the titers of serum neutralizing antibodies,including HPV 16 and HPV 18 antibodies.Methods PBNS assay was repeatedly used in detection the titers of both antibodies of HPV 16 and HPV 18 in three different serum samples for observing the effects of different batches of pseudovirion and different passages of cells on the experiment results.The developed method was also evaluated for accuracy,specificity and reproducibility based on the detection antibodies of HPV 16 and HPV 18 by positive sera in both positive(HPV 16 and HPV 18) valent or single positive(either HPV16 or HPV18) valent.Results The effects of different conditions including batches of pseudovirion and passages of cells on the antibody titers varied within a quadruple range.Furthermore,the accuracy,specificity and reproducibility of the developed method were acceptable.Conclusion The established PBNS can meet the detection requirements for neutralizing antibodies of HPV 16 and HPV 18,which could be used in evaluating the immune effect of the related vaccine.

Objective To evaluate the immune-persistence of inactivated poliomyelitis vaccine(IPV) in infants in China.Methods A total of 372 healthy infants from 11 towns of Pingle County,Guangxi Zhuang Autonomous Region were randomly into two groups.The 176 children in trial group were immunized with 3 doses of IPV,while the 196 children in control group with 3 doses of oral poliovirus vaccine(OPV),at ages of 2,3 and 4 months,respectively.The sera were collected one month after primary immunization and at 18 months of age respectively,and determined for neutralizing antibody titer against poliomyelitis virus of types Ⅰ,Ⅱ and Ⅲ by micro-neutralization test.Results The GMTs of neutralizing antibodies against poliomyelitis virus of types Ⅰ,Ⅱ and Ⅲ in sera of infants in two groups at 18 months of age were significantly lower than those one month after primary immunization(P < 0.001).The serum antibody positive rates against poliomyelitis virus of types Ⅰ,Ⅱ and Ⅲ at 18 months of age were significantly lower than those one month after primary immunization in trial group(P < 0.001),while showed no significant difference in control group(P = 0.111).However,the serum neutralizing titers against poliomyelitis virus of types Ⅰ,Ⅱ and Ⅲ were less than 1︰8 in 11.4%,17.1% and 18.2% of infants in trial group,respectively.Conclusion The protective level of antibody against poliomyelitis virus in infants after primary immunization with IPV decreased significantly at 18 months of age,indicating a necessity of booster immunization.

Single Radial Immunodiffusion(SRID) is used for the determination of Hemagglutinin(HA) content in influenza vaccines.The standard antisera for SRID test are routinely provided by WHO collaborative laboratories.The traditional preparation method involves purification and cleavage of virus,further separating of HA fragments for immunization of animals.This whole process could take more than 3 months for completion,which delay the vaccine development in due course.In this study,the HA gene fragment was obtained by RT-PCR and cloned into bacterial expression vector.The recombinant proteins were purified and identified by SDS-PAGE and Western blot.After immunization of rabbits with re-naturation proteins,the antiserum was proved to be suitable for SRID test.The new method is helpful to a fast preparation of antiserum to be used for influenza vaccine SRID test.

The single radial immunodiffusion (SRID) method currently used to determine the hemagglutinin (HA) content of the inactivated influenza vaccines depends on the availability of reference HA antigen and corresponding anti-serum, updated and provided annually by World Health Organization (WHO) collaborative centers. Particularly early in a pandemic outbreak, reference reagents could be the bottleneck in vaccine development and release. Therefore, other reliable tests capable of quantifying HA content could substantially shorten the time needed for vaccine formulation. Here electrophoretic separation of deglycosylated samples in conjunction with densitometry was used to quantify HA contents of H1N1 vaccine at multiple manufacturing sites. We found the overall consistency between the alternative method and traditional SRID was 88–122% in seven lots of vaccine bulks from four subtypes (types) of influenza vaccine, confirming its suitability to quantify HA content. Moreover, we used the alternative method to prepare a national HA antigen reference in China for quality control of 2009 pandemic influenza A (H1N1) vaccines prior to the arrival of the WHO SRID reference standards, subsequently confirming good agreement between both methods. The alternative method for vaccine quantification enabled the Chinese health authority to approve H1N1 vaccine 1 month earlier than otherwise possible.

To investigate the BSA testing kit interfering factors,twelve categories,totally 28 samples from 19 manufactures,including freeze-dry vaccines and liquid vaccines were chosen to test BSA content.15ng/ml BSA sample,two-diluted vaccine samples and diluted vaccine samples piked with 15ng/ml BSA were detected respectively.The value of the vaccine samples added with 15ng/ml BSA subtract the value of the samples should be within the 95% internal confidence of the 15ng/ml BSA sample average value.The results showed that most samples' recovery rates are between 85% and 115%.Very few samples' recovery rates are between 82% and 83%.The vaccine samples assayed in the study do not contain interfering factors,the residual BSA testing kit currently in use is reliable.

Objective To establish an alternative method for determination of haemagglutinin(HA)content in influenza vaccine so as to solve the problem of quantitative determination of HA in vaccine bulk at early stage of outbreak of pandemic influenza.Methods Influenza vaccine bulk was treated with PNGase F under an optimized condition,and separated by reduced SDS-PAGE,in which the percentage of HA protein was determined by densitometric analysis.The HA content was calculated based on the percentage of HA and total protein content in test sample.The HA contents in seven batches of vaccine bulks were determined by the alternative method and traditional SRID method respectively,and the results were compared.Results The optimal total protein content of bulk for treatment with PNGase F was 400 μg /ml,and the optimal ratio of PNGase F to bulk was 1:50(v /v).After treatment,various protein bands in test samples were differentiated clearly by reduced SDS-PAGE,of which two HA subgroups were identified by sequencing,with the identical relative molecular masses to those expected.The coincidence rates of determination results of HA contents in seven batches of vaccine bulks by the alternative method and traditional SRID method were 87.90% ~ 122.20%.Conclusion An alternative method for determination of HA content in influenza vaccine is preliminarily developed,which may be used for the quantitative determination of HA in vaccine bulk when the WHO reference materials are not available in outbreak of pandemic influenza.