米酵菌酸多克隆抗体的制备

米酵菌酸是椰毒假单胞菌及其酵米菌亚种的毒性代谢产物之一，是引起酵米面和变质银耳等多种食品中毒致死的主要病因。作者成功地制备了米酵菌酸牛血清白蛋白结合物（ＢＡＢＳＡ）、卵清蛋白结合物（ＢＡ－ＥＡ），并在国内首次获得了抗ＢＡ半抗原的多克隆抗体，血清效价可达１：１２×１０４和１：８×１０４。建立了检测ＢＡ的直接和间接竞争ＥＬＩＳＡ法。为进一步研制抗ＢＡ的单克隆抗体、探寻特异性的免疫治疗手段提供了有价值的科学基础。     

抗河豚毒素单克隆抗体的制备及其特性的初步研究

将用合成的匙孔形虫戚血蓝蛋白－甲醛－河豚毒素（ＫＬＨ－ＨＣＨＯ－ＴＴＸ）连接物免疫的ＢＡＬＢ／ｃ小鼠的脾细胞与小鼠骨髓瘤细胞系Ｓｐ２／０融合，经３～４次亚克隆，建立了３个稳定分泌抗河豚毒素的杂交瘤细胞株，分别命名为１Ｇ３、４Ｇ３、６Ｄ９。其中１Ｇ３和４Ｇ３分属ＩｇＧ２ｂ亚类，６Ｄ９属ＩｇＧ１亚类。腹水的抗体稀释度为１∶１０５～１：５×１０６。用竞争抑制性酶联免疫吸附试验（ＥＬＩＳＡ）测定三种抗体的最低反应浓度为１×１０－３ｍｇ／Ｌ。     

人类白细胞抗原DRB1等位基因与幽门螺杆菌感染的关系

目的 研究人类白细胞抗原（ＨＬＡ）ＤＲＢ１等位基因与幽门螺杆菌（Ｈｐ）感染的关系。方法 用Ｂｉｏｓｅｅｄ Ｈｐ－ＩｇＧ定量酶联免疫试剂盒检测４６例胃癌、７５例食道癌和１００例人群对照的Ｈｐ－ＩｇＧ抗体，用Ｂｉｏｔｅｓｔ低解析水平ＨＬＡ－ＤＲＢ酶联免疫探针杂交测定试剂盒检测ＨＬＡ－ＤＲＢ１等位基因。结果 （１）Ｈｐ－ＩｇＧ阳性组ＤＲＢ１＊０８基因频率显著高于Ｈｐ－ＩｇＧ阴性组（１３．１％ｖｓ４．４％     

甲肝减毒活疫苗(LA-1株)两针法的免疫应答

为探讨国产规范化甲肝减毒活疫苗（ＬＡ－１株）免疫两针的抗体水平和适宜程序,将６￣１０岁的抗ＨＡＶ－ＩｇＧ阴性儿童分为Ａ、Ｂ两组。分别按０,６程序和０,１２程序进行接种（疫苗滴度为１０＾６．７５ＴＣＩＤ５０）。于第２针免后１个月要血,用Ａｂｂｏｔｔ公司的ＩＭｘｍＥＩＡ试剂及相应的自动检测仪检测血清抗ＨＡＶ－ＩｇＧ和滴度。结果,两组的抗体阳转率均为１００％,０,１２程序组的ＧＭＴ（３１４５．４ｍＩＵ／     

视黄醇对人淋巴细胞抗体生成能力的促进作用

将新生儿脐带血和成人静脉血中淋巴细胞与不同浓度的视黄醇进行体外培养试验，通过对培养液中免疫球蛋白含量的测定观察维生素Ａ对淋巴细胞抗体生成能力的影响。结果表明，对于自脐血中分离的淋巴细胞，视黄醇的最佳效应浓度为１×１０￣（－６）ｍｏｌ／Ｌ，该浓度恰是人体血清的生理水平，在此条件下淋巴细胞产生的ＩｇＭ水平高于无视黄醇培养对照纽约５．９倍。相反，自成人血中分离的淋巴细胞在视黄醇浓度低为１×１０（－１０）至１０（－１４）ｍｏｌ／Ｌ时表现出ＩｇＧ的产量增加，提示正常血清水平的维生素Ａ对维持和促进小儿淋巴细胞对外来性抗原的反应有非常重要的影响。     

肾综合征出血热患病病后特异性抗体测定

用ＥＬＩＳＡ法测定了４７例病后肾综合征出血热患者特异性ＩｇＡ，ＩｇＥ，ＩｇＧ，ＩｇＭ抗体水平，同时用血凝抑制试验作血清学分型。病后１－６年的病例４种特异性抗体检出率分别为７６．５８％，６８．０９％，９７．８７％，３６．１７％；抗体几何平均滴度分别为１８．９９，１８．３５，１０７．００，１０．８３；ＨＦＲＳ－ＩｇＡ在病后第４年，ＨＦＲＳ－ＩｇＥ及ＩｇＭ在第３年后检出率及滴度水平均明显下降，ＨＲＦＳ－     

婴儿母传抗－HAVIgG的动态研究

本文通过对宁海县随机抽取的５０名未经甲肝疫苗免疫预防的健康产妇及其所生婴儿抗－ＨＡＶＩｇＧ的动态观察。结果表明，健康产妇及其刚生婴儿抗体阳性率均达９８．００％；且４９名抗－ＨＡＶＩｇＧ阳性婴儿其抗体阳性率及平均滴度均随月龄增大而呈阶梯式下降，至９月龄时，婴儿母传抗－ＨＡＶＩｇＧ阳性率已降为２４．４９％，ＧＭＴ为１∶２．０２，这一研究结果，提示在甲肝高发区选择１０～１２月龄婴儿可作为甲肝疫苗初免的最佳月龄。     

ELISA法检测风疹疫苗免疫效果的应用

目前，国内文献报道的风疹疫苗免疫学效果观察采用的检测方法多为血凝抑制试验（ＨＩ）〔１，２〕，由于检测的ＨＩ抗体包含ＩｇＧ和ＩｇＭ抗体，而在机体抗微生物免疫中起主要作用的血清球蛋白是ＩｇＧ。因此，检测其免后特异性ＩｇＧ抗体滴度，对疫苗的免疫原性考核或效果观察更具有实际意义。中国预防医学科学院病毒学研究所研制的ＥＬＩＳＡ检测风疹ＩｇＧ抗体试剂盒具有敏感性高、特异性强等优点。为此，我们试用该法检测风疹疫苗的血清免疫应答效果，结果报告如下：１　材料和方法１．１　疫苗　ＢＲＤ－Ⅱ株冻干风疹活疫苗　系北京生…     

麻疹野病毒的分离及血清学的初步分析

从１９９３年山东省和湖南省１０例麻疹急性期患者的咽拭子中，用Ｂ９５ａ细胞分离到７株麻疹野病毒，同时用 Ｖｅｒｏ细胞分离未获成功。将此 ７株病毒以 １０４ＴＣＩＤ５０／ｍｌ剂量分别接种到 Ａ－Ｖｅｒｏ细胞，只有１株出现融合病变，其它毒株未见增殖。抗麻疹血凝素（Ｈ蛋白）单克隆抗体（１２Ｅ）对分离株的中和滴度低于Ｌ４株６０倍以上。在同期收集的１３例麻疹患者血清，经ＥＬＩＳＡ法检测，３ 例ＩｇＧ抗体阴性，３例抗体滴度为１：２００～１：８００，但７例滴度在１：３２００～１：１０２　４００之间，占５４％．１３例血清中１２例ＩｇＭ抗体阳性。     

杂色曲霉素单克隆抗体的研制与特性鉴定

杂色曲霉素（ＳＴ）衍生后偶联到牛血清白蛋白（ＢＳＡ）或血蓝蛋白（Ｈ）上，制得复合抗原ＢＳＡ－ＳＴ和Ｈ－ＳＴ。将ＢＳＡ－ＳＴ免疫Ｂａｌｂ／ｃ小鼠，取脾细胞与ＳＰ２／０骨髓瘤细胞融合。筛选到一株稳定分泌抗ＳＴ抗体的单克隆杂交瘤细胞株，该株细胞诱导的小鼠腹水及血清中均富含抗ＳＴ单克隆抗体（ＭｃＡｂ－ＳＴ）。经鉴定，ＭｃＡｂ－ＳＴ属于ＩｇＧ＿１，与Ｈ－ＳＴ反应之亲和力常数为１．２９ｘ１０￣９Ｌ／ｍｏｌ，其分子量为１６８０００（重链５３０００，轻链３１０００）。ＭｃＡｂ－ＳＴ的结构类似物的相对交叉反应下超过２％。应用ＭｃＡｂ－ＳＴ及Ｈ－ＳＴ建立的ＩＣ一ＥＬＩＳＡ法测定ＳＴ的线性范围是０．１～１０ｎｇ／ｍｌ，最低检测量是０．５ｐｇ／　２５μｌ，为快速检测粮食中ＳＴ提供厂重要手段。     

抗A型产气荚膜梭菌α毒素单克隆抗体杂交瘤细胞系的建立与中和效应

用提取的Ａ型产气荚膜梭菌α毒素包涵体免疫ＢＡＬＢ／ｃ小鼠，取小鼠脾细胞与ＳＰ２／０骨髓瘤细胞进行融合和克隆化，经ＥＬＩＳＡ筛选，共获得７株稳定分泌单克隆抗体（ＭｃＡｂ）的杂交瘤细胞株，分别命名为１Ａ８、１Ｃ３、１Ｄ５、１Ｄ８、１Ｆ１、１Ｈ１和２Ｅ３。经鉴定，７株ＭｃＡｂ的Ｉｇ亚类有ＩｇＧ１（１Ｄ８）、ＩｇＧ３（１Ａ８、１Ｃ３和２Ｅ３）和ＩｇＭ（１Ｄ５、１Ｆ１和１Ｈ１）。细胞培养上清和腹水抗体效价分别为：１∶５１２～１∶１０２４和１∶１０６～１∶１０８。尤为重要的是，２Ｅ３杂交瘤细胞株分泌的ＭｃＡｂ不仅能够中和α毒素的磷脂酶Ｃ活性和溶血活性，而且能够对致死性腹腔感染小鼠产生良好的被动保护作用     

抗黄曲霉毒素M1的单克隆抗体细胞株的建立

本研究建立了抗黄曲霉素素ＡＦＭ１的单克隆抗体细胞株,用ＡＦＭ１－肟＝朱血清白蛋白（ＡＦＭ１－ｏｘｉｍｅ－ＢＳＡ）ＷＪＭ ＢＵ ＴＲ ＱＫＵＭ本研究建立     

具备中和中东呼吸综合征冠状病毒活性的单克隆抗体的制备

目的制备并鉴定对中东呼吸综合征冠状病毒(MERS-CoV)具有中和活性的单克隆抗体,为进一步开展MERS-CoV感染与免疫保护机制研究,以及发展诊断与治疗手段奠定基础。方法 MERS-CoV S蛋白受体结合区与人IgG Fc片段融合表达的重组蛋白免疫BALB/c小鼠,将免疫后小鼠脾细胞与SP2/0骨髓瘤细胞融合,通过ELISA筛选出阳性克隆,随后通过多次亚克隆筛选出稳定分泌特异性抗体的杂交瘤细胞株,制备单抗腹水并进行抗体效价测定及亚类鉴定;通过MERS-CoV假病毒及活病毒中和试验筛选出中和单抗。结果获得了10株能够稳定分泌抗原特异性单抗的杂交瘤细胞株。制备的腹水单抗亚类均为IgG1;其中7株单抗抗体ELISA效价达到10 000以上,并有1株单抗对MERS-CoV具有良好的中和活性。结论本研究获得了1株对MERSCoV具有良好中和活性的单克隆抗体。     

Complement-independent dengue virus type 1 infection-enhancing antibody reduces complement-dependent and -independent neutralizing antibody activity

Dengue fever and dengue hemorrhagic fever are globally important mosquito-transmitted viral diseases. However, the only licensed vaccine is not highly protective. Viremia is related to disease severity in infected humans, and it is thought to be reduced by neutralizing antibodies but increased by infection-enhancing antibodies. We established an assay system to measure the balance between neutralizing and enhancing antibodies and found that most dengue-immune individuals in endemic areas carry complement-independent enhancing antibodies (CiEAb). Studying CiEAb is important for dengue vaccine development because the enhancing activity of CiEAb does not decrease in the presence of complement, which can reduce the enhancing activity of other antibodies in vitro. Here, we investigated the effects of CiEAb on the activity of neutralizing antibodies (mainly, complement-dependent neutralizing antibodies; CdNAb) using cocktails of mouse monoclonal antibodies (MAbs) against dengue virus type 1 (DENV-1). These cocktails included MAbs with enhancing activity only (represented by D1-V-3H12 [3H12]) or neutralizing activity only (represented by D1-IV-7F4 [7F4]). Because 3H12, an IgG1 subclass antibody, is complement-independent and cross-reacted with all dengue serotypes, it is a suitable model of CiEAb. An approximately equal amount of 3H12 abolished the neutralizing activity of 7F4. The complement-dependent neutralizing activities of the IgG2a and IgG2b variants of 7F4 were also completely inhibited by ⩾3-fold concentrations of the IgG1 variant. The complement-dependent antibody activities of other anti-DENV-1 MAbs and those of MAbs directed against other serotypes were inhibited 50% by 3H12 at various mixing ratios, ranging from one-hundredth to 10-fold. The complement-dependent neutralizing activities of dengue-immune mouse ascites fluids were also effectively inhibited by 3H12. This suggests that concomitantly induced CiEAb exerts an unwanted effect on the protective capacity of a vaccine. Thus, the effective inhibition of the neutralizing activity of CdNAb by CiEAb has implications for dengue pathogenesis and vaccine development.     

甲型肝炎病毒H2株单克隆抗体的制备及鉴定

目的 为甲型肝炎病毒(HAV)的诊断和相关研究提供高效价的抗体来源. 方法 用细胞培养获得的H_2株HAV免疫BALB/C小鼠,经细胞融合技术,ELISA筛选和有限稀释克隆化培养. 结果 获得3株能稳定分泌抗HAV单克隆抗体(McAb)的杂交瘤细胞株,命名为6-A8、6-F2和1-H3.经鉴定,其上清和腹水的抗体效价分别为1∶(2048～16 824)和1∶(65 536～524 288).6-A8和6-F2属IgG2a亚型,1-H3属IgG1亚型.染色体数分布在92～98之间.3株细胞株McAb分别能与HAV不同抗原位点结合. 结论 3株能稳定分泌抗HAV McAb的杂交瘤细胞株成功建立,并获得高效价的抗HAV McAb.     

Human Antibody Responses to Avian Influenza A(H7N9) Virus, 2013

Understanding host antibody response is crucial for predicting disease severity and for vaccine development. We investigated antibody responses against influenza A(H7N9) virus in 48 serum samples from 21 patients, including paired samples from 15 patients. IgG against subtype H7 and neutralizing antibodies (NAbs) were not detected in acute-phase samples, but ELISA geometric mean titers increased in convalescent-phase samples; NAb titers were 20–80 (geometric mean titer 40). Avidity to IgG against subtype H7 was significantly lower than that against H1 and H3. IgG against H3 was boosted after infection with influenza A(H7N9) virus, and its level in acute-phase samples correlated with that against H7 in convalescent-phase samples. A correlation was also found between hemagglutinin inhibition and NAb titers and between hemagglutinin inhibition and IgG titers against H7. Because of the relatively weak protective antibody response to influenza A(H7N9), multiple vaccinations might be needed to achieve protective immunity.     

抗乙型副伤寒沙门菌鞭毛抗原McAb的特性分析与初步应用

目的：通过对抗乙型副伤寒沙门菌鞭毛抗原单克隆抗体的特性分析，研制检测乙型副伤寒沙门菌的ELISA法，对副伤寒传染病进行早期诊断。方法：用杂交瘤技术制备单克隆抗体，并进行鉴定，用双抗体夹心ELISA建立检测乙型副伤寒沙门菌鞭毛抗原ELISA方法，对临床标本进行检测。结果：7株抗乙型副伤寒沙门菌鞭毛抗原的单克隆抗体IgG15株、IgG2a1株、IgG2b1株，均不与相关沙门菌、肠道菌反应，用菌体免疫制备的MeAb 3B5、3G12、7H1、2B5，与乙型副伤寒沙门菌、乙型副伤寒沙门菌鞭毛抗原反应，而用纯化的鞭毛抗原免疫制备的单克隆抗体1F4、5D3、3H7，只与乙型副伤寒沙门菌鞭毛抗原，385与7H1识别同一抗原表位，3B5、7H1与3G12、2B5相互识别不同抗原表位，1F4、5D3、3H7相互识别不同抗原表位。3B5包被、过氧化物酶（HRP）标记3G12建立双抗体夹心ELISA，鞭毛抗原、菌体的最低检出量分别为5ng／ml、10^5个／ml。检测159份献血员血清及70份发热待查血清为阴性，1份发热待查血清、9份血培养阳性患者血清为阳性。结论：用纯化的鞭毛抗原免疫制备的单克隆抗体不适于检测乙型副伤寒沙门菌，菌体免疫制备的单克隆抗体特异性强，建立的双抗体夹心ELISA法，可对乙型副伤寒进行早期诊断。     

抗黄曲霉毒素B1单抗制备及免疫学定量方法建立

目的本研究合成并鉴定了AFB1人工抗原,制备AFB1的单克隆抗体(AFB1mAb)。方法采用NHS法将AFB1分别偶联于载体蛋白BSA和OVA上,分别合成W人工抗原AFB1-BSA和AFB1-OVA,紫外分光光度法和SDS-PAGE进行鉴定;AFB1-BSA免疫BALB/C小鼠,通过间接ELISA和阻断ELISA法选择细胞融合备用鼠;用杂交瘤技术制备AFB1mAb,并对其效价、亲和力、敏感性、特异性、亚型进行鉴定;体内诱生腹水法大量制备单抗。结果 UV图谱和SDS-PAGE图表明结半抗原AFB1和载体BSA及OVA偶联成功;筛选出2H5-F6、2H5-C9、2H9-C3三株杂交瘤细胞;鉴定单抗亚型均为IgG1;细胞上清效价1∶2.0×102～1∶1.28×103,2H5-F6的腹水效价1∶1.28×106,AFB1mAb亲和常数Ka为2.65×1010 L/moL,对AFB1的IC50为2.58 ng/mL;与AFB2的交叉反应率为1.61%,与其他类药物无交叉反应。结论通过试验获得高效价、敏感、特异的AFB1mAb,可用于各种食品中AFB1残留的快速免疫学检测试验。     

H7N9禽流感病毒血凝素线性B细胞抗原表位单克隆抗体的制备和鉴定

目的研制用于H7N9禽流感病毒诊断的血凝素线性B细胞抗原表位单克隆抗体。方法采用H7N9禽流感病毒血凝素特异性线性B细胞抗原表位免疫BALB/c小鼠,通过杂交瘤细胞技术和间接ELISA法筛选、鉴定获得稳定分泌抗H7N9禽流感病毒血凝素单克隆抗体的杂交瘤细胞株;通过SDS-PAGE、间接ELISA和斑点免疫印迹技术鉴定其亚型、效价、纯度等生物学特征。结果共获得2株单克隆抗体(7C8H8和8E9E2),其中7C8H8抗体亚类为IgG2a,腹水抗体效价>1∶512000,且可以特异性结合H7N9禽流感病毒血凝素。8E9E2仅具有较低的H7N9禽流感病毒血凝素蛋白结合力。结论获得1株可分泌高效价、高特异性的抗H7N9禽流感病毒血凝素单克隆抗体的杂交瘤细胞株7C8H8。     

Pneumococcal conjugate vaccine primes mucosal immune responses to pneumococcal polysaccharide vaccine booster in Papua New Guinean children

IntroductionInvasive pneumococcal disease remains a major cause of hospitalization and death in Papua New Guinean (PNG) children. We assessed mucosal IgA and IgG responses in PNG infants vaccinated with pneumococcal conjugate vaccine (PCV) followed by a pneumococcal polysaccharide vaccine (PPV) booster.MethodsInfants received 7-valent PCV (7vPCV) in a 0–1–2 (neonatal) or 1–2-3-month (infant) schedule, or no 7vPCV (control). At age 9 months all children received 23-valent PPV (23vPPV). IgA and IgG to 7vPCV and non-7vPCV (1, 5, 7F, 19A) serotypes were measured in saliva collected at ages 1, 2, 3, 4, 9, 10 and 18 months (131 children, 917 samples). Correlations were studied between salivary and serum IgG at 4, 10 and 18 months.ResultsSalivary IgA and IgG responses overall declined in the first 9 months. Compared to non-7vPCV recipients, salivary IgA remained higher in 7vPCV recipients for serotypes 4 at 3 months, 6B at 3 months (neonatal), and 14 at 3 (neonatal), 4 and 9 months (infant); and for salivary IgG for serotypes 4 at 3, 4 and 9 months, 6B at 9 months, 14 at 4 (neonatal) and 9 months, 18C at 3, 4, and 9 (infant) months, and 23F at 4 months. Following 23vPPV, salivary 7vPCV-specific IgA and IgG increased in 7vPCV-vaccinated children but not in controls; and salivary IgA against non-PCV serotypes 5 and 7F increased in 7vPCV recipients and non-recipients. Salivary and serum IgG against 7vPCV-serotypes correlated in 7vPCV-vaccinated children at 4 and 10 months of age.ConclusionsPCV may protect high-risk children against pneumococcal colonization and mucosal disease by inducing mucosal antibody responses and priming for mucosal immune memory that results in mucosal immune responses after booster PPV. Saliva can be a convenient alternative sample to serum to study PCV-induced systemic IgG responses.