14株抗乙型肝炎表面抗原单克隆抗体与变异表面抗原的反应模式及应用

目的制备抗乙型肝炎表面抗原(hepatitis B surface antigen,HBsAg)的单克隆抗体,检测单克隆抗体与15种变异HBsAg的反应模式。用筛选出的单抗建立快速检测变异HBsAg的ELISA实验方法,并做初步评价。方法用中国乙型肝炎病毒感染者血清中分离的HBsAg免疫BALB/c小鼠,通过杂交瘤细胞融合技术制备抗-HBs单克隆抗体。检测不同单克隆抗体与野生及变异HBsAg的反应性。筛选出两种可以较好识别变异HBsAg的单克隆抗体McAb2和McAb3,建立两种抗体ELISA检测HBsAg的方法。结果制备了14株抗-HBs单抗。经过初筛,有4种可以较好识别包括G145R在内的大多数变异HBsAg。优化了McAb2和McAb3检测HBsAg的条件,检测HBsAg的灵敏度较好,检测变异HBsAg的能力优于2种现行国产HBsAg检测试剂盒。结论用本实验制备的单抗可以很好地识别包括G145R在内的大多数变异HBsAg。     

抗HBs mAb的研制及其对野生与免疫逃逸变异HBsAg的交叉反应特征

目的:抗HBs G6 mAb制备及其对重组野生与免疫逃逸变异HBsAg结合能力与特点的评价.方法:常规制备并纯化抗HBs mAb,以纯化抗HBs mAb IgG包被,采用ELISA对17种野生及"a"决定簇替代性变异全基因重组表达HBsAg进行检测,并与几种市售HBsAg检测试剂进行比较.结果:该杂交瘤细胞生长与分泌特性稳定,其培养上清与腹水效价分别为2048及4096×103;对野生HBsAg检测(ELISA)敏感性不低于0.125 μg/L.该抗体可与15种变异抗原中12种发生反应(P/N≥2.5),反应信号强度分别为低反应组(2份,与野生株A值比较,下同)平均7.55%;中反应组(1例)为59.40%;高反应组(9种)分别为野生株的92.1%～109.4%,低反应或无反应表达产物的免疫逃逸变异部位集中在HBV"a"决定簇I环的起始部即120～124序列之间.部分表达产物采用市售试剂作同步检测,平均显色强度高于或明显高于几种国内应用最为普遍的HBsAg ELISA(P<0.05).结论:抗HBs G6 mAb对多数免疫逃逸变异HBsAg有很强的结合能力,所针对的变异类型亦表现出某种特殊的规律.     

ELISA差减分析——一种简便实用的免疫逃逸变异HBsAg检测新方法

目的:立足现有实验技术与试剂,建立一项血清免疫逃逸变异HBsAg检测的新方法。方法:以市售ELISA试剂筛选其检测信号在灰区范围的HBV感染者;采用2种不同免疫逃逸变异HBsAg检测能力的ELISA试剂对选留血清进行检测,以"ELISA差减分析"方式确认免疫逃逸变异HBsAg阳性者;以中和实验及雅培化学发光试剂验证检测结果的特异性;并对部分免疫逃逸变异HBsAg阳性者以PCR及直接测序做HBV DNA分析。结果:自10231例受检者中筛选HBsAgELISA检测信号灰区标本244例;复核检测显示G6ELISA、市售ELISA两者对野生HBsAg检测敏感性分别为0.0625ng/ml,与0.125ng/ml;检测阳性率分别为16.80%(G6ELISA)及5.73%(市售ELISA)。ELISA差减分析确定免疫逃逸变异HBsAg阳性者27例,阳性率为11.07%。对部分免疫逃逸变异HBsAg阳性血清做进一步考核,抗HBs中和强度为(84.07%±6.32%),中和率100%(13/13);雅培化学发光试剂复核阳性率为94.4%(17/18,阳性者中包括两份单项G6-ELISA检测最低阳性信号标本);HBV DNA阳性率36.9%(7/19),略低于同期HBsAg低信号阳性者(6/14,42.9%,P0.05);直接测序发现其中"a"区变异3例,分别为P142L、T126A及M133T/K160T;S蛋白亲水区非"a"决定簇变异1例,变异类型为L109Q。结论:ELISA差减分析法能有效地用于部分免疫逃逸变异HBsAg的临床诊断,其诊断能力视所依托试剂免疫逃逸变异检测能力不同而有显著差别。     

抗肌红蛋白单克隆抗体的制备与鉴定及其初步应用

目的 制备和鉴定抗肌红蛋白(Mb)单克隆抗体,为人肌红蛋白检测试剂的研制奠定基础.方法 用人肌红蛋白抗原免疫BALB/c小鼠,采用杂交瘤技术进行细胞融合,用ELISA方法筛选,有限稀释法进行克隆化培养阳性杂交瘤细胞株.ELISA法测定小鼠腹水滴度,CLIA法对抗体进行配对实验与特异性鉴定.结果 筛选培养出9株分泌抗肌红蛋白单克隆抗体的杂交瘤细胞株,腹水滴度为1∶5×104 ～ 1∶2×105.通过配对实验,有5株抗体6种组合可以成功配对.这5株抗体的亲和常数为6.46×108 ～ 3.68×109 L/mol.用5株抗体所建立的6种化学免疫分析方法与人Hb交叉反应率为7.434×103％ ～2.904×10-2％,与人ALB交叉反应率为4.980×10-7％ ～3.830×10-5％.用Mb17B6与Mb21E8两株抗体建立的CLIA标准曲线的线性系数为0.997,灵敏度为6.02ng/mL.结论 成功制备了可以应用于免疫分析的抗肌红蛋白单克隆抗体.     

特异性HER2单克隆抗体的制备及初步应用

目的:制备特异性识别天然构象的人表皮生长因子受体-2(HER2)的单克隆抗体(mAb),用以指导Herceptin的临床应用.方法:利用纯化的原核表达的HER2融合蛋白作为抗原免疫BALB/c小鼠,取脾细胞与NS1骨髓瘤细胞按常规方法融合,ELISA法进行杂交瘤的初步筛选.构建HER2酵母展示系统,用酵母cell base ELISA进行第2次筛选.将筛选到的阳性克隆株进行亚克隆获得稳定分泌细胞株,用免疫组织化学的方法鉴定其与SK-Br-3细胞和293a细胞的反应性.结果:通过ELISA筛选得到168株阳性杂交瘤,用酵母cell base ELISA最终得到8株阳性杂交瘤,此8株免疫组化检测均与SK-Br-3细胞反应,而与293a细胞无反应.结论:成功制备了8株识别天然构象的HER2的mAb,能够用于免疫组化,为临床上使用Herceptin进行个性化治疗乳腺癌打下了基础.     

抗人甘露聚糖结合凝集素(MBL)单克隆抗体的制备、鉴定与检测方法的建立

目的 制备抗甘露聚糖结合凝集素(MBL)的单克隆抗体(McAb),建立免疫学检测方法.方法 以纯化MBL为抗原,免疫Ualb/c小鼠,制备单克隆抗体,鉴定其特性,建立并验证夹心ELISA定量检测法.结果 筛选出2株稳定分泌抗MBL的单抗杂交瘤细胞株,免疫球蛋白亚类均为IgG1.两单抗间抑制率＜50%,结合位点不同两单抗特异识别MBL.选择包被抗体B10浓度为8μg/mL,酶标抗体B3稀释度为1:500,建立夹心EIJSA定量检测法.该法检出范围为1～100 μg/mL,与其他抗原无交叉反应.重复性试验的批内批间变异均小于10%,回收率在101%～103%之间,cv小于7%.检测结果与国外试剂盒比较无显著差异.结论 制备的抗MBL单克隆抗体可用于MBL的免疫学检测.     

基于抗原表位制备抗人GP73单克隆抗体

目的:制备抗GP73蛋白的单克隆抗体(mAb)。方法:将GP73蛋白N末端的肽段(AAAERGAVELK)展示于T7噬菌体表面,扩增重组噬菌体作为抗原免疫小鼠,制备抗体。通过ELISA法检测小鼠血清抗体的效价,筛选分泌抗GP73蛋白抗体的杂交瘤细胞株;通过ELISA、Westernblot等方法检测mAb的特异性。结果:利用表面展示GP73抗原表位的重组噬菌体为抗原免疫小鼠,通过细胞融合和筛选得到了能识别GP73蛋白的mAb,且特异性较好。结论:将蛋白质抗原的合适抗原表位展示在T7噬菌体表面,用这种重组噬菌体作为替代抗原可以制备识别全蛋白的mAb。     

乙型脑炎病毒prM蛋白单克隆抗体的制备

目的 克隆日本乙型脑炎病毒(Japanese encephalitis virus,JEV)前膜蛋白(prM)编码基因,原核表达、纯化prM蛋白,以纯化产物为免疫原制备单克隆抗体(mAb);方法从感染JEV病毒的鼠脑中克隆编码JEV prM蛋白的基因并将其克隆入原核表达载体pET32a,转化大肠埃希菌BL21(DE3)LvsS后以IPTG诱导表达.表达产物经Ni-NTA纯化后进行SDS-PAGE分析.用纯化的蛋白免疫BALB/c小鼠,经细胞融合和亚克隆后筛选出能分泌识别prM蛋白的mAb的杂交瘤细胞株.用Western Blot和免疫组化方法检测单克隆抗体的特异性.结果 从鼠脑中克隆出约500 bp的JEV prM基因,将其克隆入原核表达载体中,在大肠埃希菌中获得了较好表达.纯化的prM蛋白免疫BALB/c小鼠,经传统细胞融合及筛选方法制备出单克隆抗体,抗体滴度为105.ELISA、Western Blot和免疫组化检测结果证实该株单抗具有较好的特异性.结论 成功的表达和纯化了日本乙型脑炎病毒的prM蛋白,并完成了单克隆抗体的制备,为乙型脑炎病毒感染的早期诊断及预防的研究奠定了良好的基础.     

单个B细胞制备CRP单克隆抗体及其ELISA检测体系的初步建立

目的:用单个B细胞抗体制备技术结合Exip293真核表达系统制备hs-CRP单克隆抗体。方法:用重组CRP蛋白免疫小鼠,荧光标记探针和流式分选技术分离出表达CRP单克隆抗体的单个B细胞,通过PCR方法扩增出编码CRP单克隆抗体蛋白的轻重链核酸序列并构建重组质粒,质粒转染到Expi 293细胞中制备出单克隆抗体蛋白。结果:用间接ELISA法筛选出两个具有良好特异性的单克隆抗体20#,22#,将这两个抗体配对并建立ELISA双抗体夹心检测方法,CRP浓度在15～250 ng/ml范围内有较好的检测线性(y=0. 003x-0. 035 8,R2=0. 997 4),精密度分析的批内变异系数CV为6. 7%～9. 37%,批间变异系数为8. 52%～9. 10%,平均回收率为98%,与临床血清检测值具有良好的相关性,达到CRP检测要求。结论:本研究所用的单个B细胞分离分选方法结合Expi 293真核表达系统在制备单克隆抗体方面具有省时、高效、特异性好、灵敏度高的特点,具有很好的应用价值。     

淋巴细胞脉络丛脑膜炎病毒GP2单克隆抗体的制备和鉴定

目的制备淋巴细胞脉络丛脑膜炎病毒(LCMV)单克隆抗体(m Ab)。方法采用杂交技术获得并筛选出杂交瘤细胞,收集含单克隆抗体的培养上清行免疫荧光(IFA)、dot-blot、ELISA法鉴定抗体重链类型和测定单克隆抗体的免疫效价。用蛋白G Sepharose亲和层析填料纯化抗体,纯化后的抗体采用SDS-PAGE检测纯化效果,并进一步用Western blot检查抗体对LCMV的作用部位。结果成功获得能稳定分泌抗LCMV的特异性杂交瘤细胞株,顺利获得纯化的单克隆抗体,命名为anti-GP2。免疫荧光、ELISA结果均证实获得的单克隆抗体能识别LCMV抗原。用ELISA法鉴定出该单克隆抗体重链类型为Ig G2b,培养液免疫效价大约为1.35×10~3。蛋白G Sepharose亲和层析填料纯化抗体后经SDS-PAGE检测证实纯化抗体成功。Western blot提示该单克隆抗体对LCMV的作用部位为病毒颗粒表面糖蛋白抗原GP2。结论成功制备出了针对LCMV病毒颗粒表面糖蛋白抗原GP2的单克隆抗体(anti-GP2 m Ab)。     

Detection of hepatitis B surface antigen (HBsAg) in peripheral blood mononuclear cells of patients with acute and chronic active hepatitis B

Seventy five patients with acute and chronic active hepatitis (CAH) were studied by indirect immunofluorescence with monoclonal antibodies for the presence of hepatitis B surface antigen (HBsAg) on peripheral blood mononuclear cells (PBMC). The viral surface antigen was detected in the PBMC of all the patients with hepatitis B virus (HBV)-induced CAH and in acute patients with more than 2 months of evolution. No HBsAg was detected in the samples obtained from 12 normal controls or from 14 non-A, non-B CAH patients. Analysis of PBMC subsets revealed that HBsAg was present in non-T cells; dual fluorescence studies showed HBsAg on surface Ig-positive lymphocytes. The binding of anti-HBs monoclonal antibodies was higher than that of a goat anti-HBs serum, and the highest reactivity was observed with an antibody against the pre-S(2)-region sequence. Both HBsAg and hepatitis B core antigen (HBcAg) were also detected in lysates of PBMC by dot blot analysis.     

Qualitative analysis of the humoral immune response to the "a" determinant of HBs antigen after inoculation with plasma-derived or recombinant vaccine

Competitive inhibition assays using monoclonal antibodies reacting with defined sequence of the "a" determinant of hepatitis B surface antigen (HBsAg) indicate that the humoral response to plasma-derived and recombinant HBsAg vaccine is similar in quantitative and qualitative terms. Both vaccines evoke an antibody response to the RFHBs 1 epitope, which is known in animal experiments to be protective.     

Mutant hepatitis B virus surface antigens (HBsAg) are immunogenic but may have a changed specificity

Mutant hepatitis B virus with substitutions within the coding region for HBV surface antigen (HBsAg) has been found naturally in chronic carriers. It is therefore important to clarify whether the identified substitutions within the HBsAg have impact on the antigenicity and immunogenicity of HBsAg. A total of nine mutated HBV s-genes with single representative mutations were generated by site-directed mutagenesis and subcloned into an expression vector. The binding of polyclonal and monoclonal antibodies to these mutant HBsAg (mtHBsAg) was tested by immunofluorescence (IF) staining of cells transfected with the expression vectors. The amino acid (aa) substitutions like G145R, F134S, and C147W affected the binding of anti-HBs antibodies to corresponding mtHBsAg to different extents. The impact of aa substitutions G145R and F134S on the immunogenicity was accessed by genetic immunization of mice with vectors expressing middle HBsAg with the corresponding mutations. The immunized mice developed antibodies to recombinant HBsAg containing the HBV preS region and HBsAg-specific cytotoxic T-cell. However, the development of antibody response to wild-type small HBsAg was significantly impaired by the aa substitutions in HBsAg. Based on this fact, we further investigated whether the mtHBsAg with the aa substitution G145R is able to induce mutant-specific antibody responses. Strikingly, serum samples from mice immunized with mtHBsAg with G145R recognized plasma-derived mtHBsAg. Two mouse MAbs specific to mtHBsAg were generated. One MAb recognized mtHBsAg with G145R but not wild type and other mtHBsAg. We conclude that HBsAg with aa substitutions are immunogenic but may have a changed fine specificity.     

Structural characterization of viral neutralizing monoclonal antibodies to hepatitis B surface antigen

In this study we describe the viral neutralizing activity of murine monoclonal antibodies (MAb) specific for hepatitis B surface antigen (HBsAg). This viral neutralizing activity was assessed in vitro by employing Hepatitis Delta Virus (HDV) and human hepatocytes as target cells. To further characterize these viral neutralizing antibodies we generated a panel of anti-idiotypic (anti-Id) reagents and serologically characterized these antibodies for epitope specificity, Id specificity, and Id heterogeneity. Direct binding and competitive inhibition solid phase enzyme immunoassay have demonstrated that two murine MAb specific for HBsAg (anti-HBs), designated A1.2 and A3.1, recognize similar or overlapping epitopes on HBsAg, while monoclonal anti-HBs, designated A2.1 recognizes a unique HBsAg epitope. Further, Id analysis using monoclonal and polyclonal anti-Id reagents have identified both a private and a cross-reactive Id, respectively, on the anti-HBs, A1.2 preparation. The source of the idiotypic cross-reactivity between A1.2 and A3.1 has been identified, using Western blot analysis, to conformational determinants expressed by the heavy (H) and light (L) chains of these monoclonal anti-HBs. Lastly, the intrastrain antibody repertoire induced following HBsAg immunization was found to be relatively restricted in heterogeneity by clonotype analysis using isoelectric focusing and affinity immunoblot analysis. Interspecies variability in the anti-HBs response was observed based on epitope recognition using purified anti-HBs from a variety of species.     

乙型肝炎病毒表面抗原突变体稳定表达细胞系的建立和抗原性鉴定

目的 探讨乙型肝炎(乙肝)病毒(HBV)表面抗原(HBsAg)阴性HBV感染的分子机制。方法 在对46例HBsAg阴性但血清HBVDNA阳性感染者的S基因序列进行分析的基础上，构建了6株新的HBsAg变异株(4株联合点突变株，2株插入变异株)的EBO-plpp真核表达载体，并转染了COS7细胞，建立了这6株HBsAg变异株的稳定表达细胞系。分别用酶联免疫法(ELISA)和放射免疫法(RIA)对细胞表达的HBsAg抗原性进行检测。结果 除1例插入变异株可检测到较弱的阳性外，其余5株均检测不到HBsAg的存在。结论 新发现的HBsAg联合点突变和氨基酸插入变异，均对HBsAg的抗原性有负面的影响，是造成HBsAg阴性HBV感染的直接原因。     

Non-productive infection of human newborn blood mononuclear cells with herpes simplex virus: effect on T cell activation, IL-2 production and proliferation.

IgG subclasses of antibodies to the hepatitis B surface antigen (HBsAg) in sera from 40 healthy infants immunized with the vaccine against hepatitis B virus (HBV) were detected using an enzyme-linked immunosorbent assay (ELISA) with monoclonal antibodies. The infants were born to asymptomatic HBsAg-positive mothers. Total serum IgG subclasses were also tested to exclude a deficiency of certain subclasses in these infants but their distribution was the one expected according to age. In contrast, IgG subclass antibodies to HBsAg were predominantly IgG1 and IgG4. The collected data indicate that infants produce significantly higher levels of IgG1 and IgG4 than IgG2 and IgG3 in response to the vaccine for HBV. The IgG4 response to anti-viral vaccinations is uncommon. The role of that IgG4 subclass is not yet clear: even if an anaphylactic role was suggested, no adverse reactions were observed in vaccinated children.     

Impact of amino acid sequence variation of a determinant(s) on the antigenic properties of hepatitis B virus HBsAg

Impact of amino acid sequence variation on the antigenic properties of the surface hepatitis B virus antigen HBsAg was studied. Eleven recombinant HBsAg variants of wild (adr, ayw2, adw2, adw4, aywl, adw2) and vaccine escape (adw2 T126S, adw2 Q129L, adw2 Q129R, adw2 T143K, adw2 Q145R, aywl Q145A) were obtained. All the recombinant antigens were tested on a panel of 43 monoclonal antibodies (MAb) specific to different HBsAg determinants. Amino acid sequence variation of the a-determinant of HBsAg was shown to significantly affect its immunological responsiveness and antigenic properties. Amino acid substitution in different positions or in the same position, but for various amino acids may differently affect these properties.     

血清乙型肝炎病毒前S1抗原检测及其与病毒复制的关系

用抗Ｓ和抗前Ｓ１单抗的双抗体夹心ＥＬＩＳＡ法检测１５０例慢性乙型肝炎患者、乙型肝炎病毒表面抗原（ＨＢｓＡｇ）携带者和健康人血清中的ＨＢＶ前Ｓ１抗原，其结果和ＨＢＶＤＮＡ聚合酶链反应（ＰＣＲ）、乙型肝炎血清标志的检测结果进行比较。结果表明：前Ｓ１抗原在乙型肝炎病毒ｅ抗原（ＨＢｅＡｇ）阳性组中的检出率和相对滴度显著高于ＨＢｅＡｇ阴性组（Ｐ＜０．０１）；在ＨＢｅＡｇ阴性组中，抗－ＨＢｅ阴性人群前Ｓ１抗原的检出率和相对滴度也显著高于抗－ＨＢｅ阳性人群（Ｐ＜０．０１）。前Ｓ１抗原和ＨＢＶＤＮＡ检测结果的符合率达８０％，两者检出率的相关系数ｒ＝０．９８２６（Ｐ＜０．０１）。结论：血清前Ｓ１抗原和乙型肝炎病毒的存在关系密切。     

乙型肝炎表面抗原124aa分子在大肠杆菌中的高效表达

利用聚合酶链反应特异性扩增编码乙型肝炎表面抗原羧基末端１２４个氨基酸的基因片段,并将其大肠杆菌中获得了高效表达。表达产物氨基末端有１个含６组氨酸肽段的２０氨基酸融合部分,分子量１６１７８Ｄａ,以包含体形式存在,约占细菌总蛋白２２％。