A novel diagnostic monoclonal antibody specific for Helicobacter pylori CagA of East Asian type

Yasuda A, Uchida T, Nguyen LT, Kawazato H, Tanigawa M, Murakami K, Kishida T, Fujioka T, Moriyama M. A novel diagnostic monoclonal antibody specific for Helicobacter pylori CagA of East Asian type. APMIS 2009; 117: 893–9. Molecular biological and epidemiological studies have suggested that Helicobacter pylori producing East Asian CagA protein variant is more virulent than that producing Western CagA. In the present study, we developed and validated an enzyme‐linked immunosorbent assay (ELISA) using a monoclonal antibody specifically recognizing East Asian CagA‐positive H. pylori. A total of 32 H. pylori strains were tested and the data were subjected to receiver‐operator characteristic (ROC) curve analysis. The accuracy of the test, determined by calculating the area under the curve, was 0.96, which indicated a high level of accuracy. At the ROC optimized cutoff, the sensitivity and specificity of our ELISA method were 88.0% and 100%, respectively. The validated ELISA showed good performance in terms of sensitivity and specificity. These results suggest that this test is suitable for the diagnostic detection of East Asian CagA carrying strains. We also analyzed the localization of the CagA protein in H. pylori‐infected gastric mucosa with fluorescence immunohistochemistry, and found that CagA protein expression was up‐regulated by adhesion to epithelial cells.     

Production of a monoclonal antibody against SARS-CoV spike protein with single intrasplenic immunization of plasmid DNA

Severe acute respiratory syndrome (SARS) is a highly infectious disease caused by a novel coronavirus (SARS-CoV). Specific monoclonal antibodies (mAbs) against the SARS-CoV are vital for early diagnosis and pathological studies of SARS. Direct intrasplenic inoculation of plasmid DNA encoding antigen is an effective and fast approach to generate specific mAb when the protein antigen is difficult to prepare or dangerous in use. In this study, we selected one fragment of SARS-CoV spike protein (S1-₃) as antigenic determinant by immunoinformatics. Single intrasplenic immunization of plasmid DNA encoding S1-₃ induced anti-spike protein antibodies. We established one hybridoma cell line secreting specific mAb and evaluated this mAb with murine leukemia virus pseudotyped with SARS-CoV spike protein (MLV/SARS-CoV). The mAb could recognize the spike protein on the MLV/SARS-CoV-infected Vero E6 cells albeit with no neutralizing effect on the infectivity of the pseudotype virus. Our results show that a single-shot intrasplenic DNA immunization is efficient for the production of specific mAb against SARS spike protein, and a linear epitope of the spike protein is recognized in this study.     

Identification of a B-cell antigenic epitope at the N-terminus of SARS-CoV M protein and characterization of monoclonal antibody against the protein

To identify the potential B-cell antigenic epitopes within the N-terminus of SARS-CoV (SARS-associated coronavirus, SARS-CoV) M protein and characterize monoclonal antibody (MAb) against the protein as well as its recognizing region, we expressed and purified a portion of SARS-CoV M protein (amino acid 1–43) in Escherichia coli (E. coli). By using Western blot and enzyme-linked immunosorbent assay (ELISA), we showed that the purified recombinant M protein could be recognized by four SARS-CoV-positive human sera even when those sera were 12,800-fold diluted. Furthermore, we characterized one representative IgG2 MAb, 3H9, which exhibited a strong immunoreaction to both recombinant M protein and native viral protein of SARS-CoV. We found a B-cell antigenic epitope located between amino acid 1–15 and defined the MAb recognizing region within amino acid 16–28 of M. These findings not only suggest that both recombinant M protein and its specific MAbs may be used as the diagnostic reagents for SARS, but also provide a potential target site for the design of an epitope-based vaccine against SARS. Chao Qian and Di Qin contributed equally to this work.     

重组灵芝免疫调节蛋白(rLZ-8)单克隆抗体的制备及鉴定

目的:制备重组灵芝免疫调节蛋白(rLZ-8)单克隆抗体,为rLZ-8的药效学及药代动力学研究奠定基础.方法:以纯度99%的rLZ-8免疫BALB/c小鼠;应用常规的细胞融合技术建立一种能够稳定分泌抗rLZ-8单克隆抗体的杂交瘤细胞株;采用小鼠腹腔接种法制备腹水;Protein A Sepharose 亲和层析柱在AKTA纯化仪上对抗体进行纯化;间接ELISA方法测定单克隆抗体的效价;Western blot胶片曝光方法对rLZ-8单克隆抗体特异性进行分析.结果:筛选出2株可特异性分泌抗rLZ-8单克隆抗体的杂交瘤细胞株.分别命名为clone13-2-3和clone11-4-4.其抗体亚型分别为IgG1亚型和IgG2a亚型.腹水抗体效价分别为1:12 000和1:7 500,细胞培养上清效价分别为1:3 000和1:2 800.Western blot检测证明这两株杂交瘤细胞所分泌的单克隆抗体均具有良好的特异性.结论:成功制备出较高效价和较高特异性的鼠抗rLZ-8的单克隆抗体.     

金葡菌肠毒素C2的单克隆抗体制备、鉴定及初步应用

目的：制备抗金葡菌肠毒素C2（SEC2）的单克隆抗体（MeAb），并建立SEC2检测方法。方法：以金葡菌培养液中纯化的SEC2为抗原，免疫BALB／c小鼠，制备单克隆抗体；并对单克隆抗体的特性进行鉴定；利用纯化后的抗体建立了夹心ELISA定量检测SEC2方法，并进行了验证和初步应用。结果：筛选出4株稳定分泌抗SEC2抗体的杂交瘤细胞株，其免疫球蛋白亚类均为IgG1，除两株单抗的SEC2识别位点相同外，其余单抗均特异性识别SEC2的不同结合位点。建立的夹心ELISA定量检测法，特异性、灵敏度、重复性均较好，检测SEC2范围为0．5—20ng,／ml，回收率在97．8％-101％，变异系数为2％-5％。结论：本研究制备的抗SEC2的单克隆抗体，可用于建立了SEC2定量检测方法，为控制金葡素制品质量和金葡菌肠毒素研究提供了较实用的方法。     

Induction of a protective response with an IgA monoclonal antibody against Mycobacterium tuberculosis 16 kDa protein in a model of progressive pulmonary infection

Mycobacterium tuberculosis is a facultative intracellular pathogen for which cell-mediated immunity is considered the major component of the immune response. For many decades, the prevailing scientific view has been the antibodies have little or no role in modifying the course of M. tuberculosis infection. In recent years, several studies have challenged this dogma, and there is a body of evidence that supports a role of antibodies against M. tuberculosis. In the present work, we evaluated the protective activity of two monoclonal antibodies (TBA61 and TBA84). Here, we chose the intratracheal model of pulmonary infection to evaluate bacterial load and morphometric and histological changes in the lungs of treated mice. Data obtained revealed the reduction of bacterial load and milder morphometric and histopathological changes in mice treated with TBA61 at 21 days post-infection with M. tuberculosis H37Rv compared to those treated with TBA84 and control mice. These results allow continuing exploring the potential use of monoclonal antibodies as prophylactic and therapeutic agents against intracellular pathogens such as M. tuberculosis.     

人卵泡刺激素β亚基单克隆抗体的制备及鉴定

目的　建立分泌抗人卵泡刺激素 (FSH) β亚基 5 1～ 6 0片段单克隆抗体细胞株 ,并对单抗进行免疫学鉴定。 方法　以人工合成的FSHβ蛋白抗原决定簇多肽为免疫原 ,采用脾内包埋法免疫Balb c小鼠 ,取其脾细胞与SP2 0小鼠骨髓瘤细胞融合 ,杂交瘤细胞用人工合成的人FSHβ亚基 5 1～ 6 0片段包被、间接ELISA法筛选。结果　获得稳定分泌抗人FSHβ片段单抗细胞 1株 (F1C)。ELISA检测腹水效价为 1∶1.6× 10 4 ,与黄体生成素 (LH)、促甲状腺素 (TSH)、人绒毛膜促性腺激素 (hCG)等无交叉反应 ,免疫印迹 (Westernblot)显示该单抗特异性强。结论　本实验所制备的单抗具有良好的灵敏性、特异性和实用性 ,可用于酶免疫检测方法的建立 ,为检测试剂盒的研制及其临床应用提供了有力的依据     

Immunohistological characterization of a monoclonal antibody (OV632) against epithelial ovarian carcinomas

Summary A hybridoma cell line (OV632) producing monoclonal antibody against ovarian carcinomas was developed from the spleen cells of a mouse immunized with cystic fluid from a serous cystadenocarcinoma. Immunohistological studies in frozen sections showed that 22 out of 28 nonmucinous ovarian carcinomas, which included serous, endometrioid, clear cell, and undifferentiated tumours, reacted with this antibody. Three out of 7 mucinous ovarian carcinomas were positive, whereas only 7 out of 122 extra-genital malignant lesions, predominantly adenocarcinomas, were positive. The negative cases included 38 breast carcinomas and 24 colon carcinomas, tumours which are responsible for most of metastatic disease in the ovary. On the basis of these findings, the antibody OV632 is considered appropriate for histodiagnostic purposes as an aid in the distinction between primary and secondary ovarian cancer.     

抗人巨细胞病毒单克隆抗体的建立及初步临床应用

应用人巨细胞病毒AD169株(HcMV-AD169)免疫BALB/c小鼠,取脾细胞与Sp2/0小鼠骨髓瘤细胞融合,获得两株(1F9 2H10)分泌抗HCMV单克隆抗体(McAb)的杂交瘤细胞系。经鉴定,两株McAb的Ig亚类为IgG1,腹水效价间接ELISA法为10~(-5)和10~(-6)。两株McAb仅与HCMV反应,而与其他疱疹病毒无反应,2H10有中和病毒作用而1F9则无。用HCMV-McAb建立抗体捕获ELISA法测定150例孕妇血清中HCMV-IgM抗体,阴阳性总符合率与间接ELISA法相比较,为99.3％(149/150)。文中尚对HCMV-McAb用途作了讨论。     

抗肾综合征出血热病毒人单克隆抗体杂交瘤细胞株的建立及初步鉴定

以亲和层析技术纯化的肾综合征出血热病毒（ＨＦＲＳＶ）结构蛋白（５５０００，６７０００）为特异性抗原，体外免疫正常人外周血淋巴细胞（ＰＢＬ），然后将体外免疫的淋巴细胞与人－鼠种间杂交瘤细胞（Ｋ６Ｈ６／Ｂ５）融合，经ＥＬＩＳＡ间接法筛选出２株（２Ｄ５，１Ｂ７）分泌抗－ＨＦＲＳＶ人单克隆抗体（Ｈ－ＭｃＡｂ）杂交瘤细胞株，４次克隆化后，１００％阳性。对２Ｄ５株初步鉴定表明，其抗体类型为ＩｇＧ１；培养上清中抗体浓度为２０～３０μｇ／ｍｌ；特异性免疫荧光反应证明２Ｄ５株Ｈ－ＭｃＡｂ是ＨＦＲＳＶ特异性；中和效价为１∶１６０；体外连续传代６个月仍稳定分泌抗体。     

The use of human antigen-specific monoclonal antibodies in an enzyme-linked immunosorbent assay for the determination of anti-HBsAg antibody subclasses

A human monoclonal anti-HBsAg antibody (IgGl, λ) was used as a reference for an ELISA determination of the serum levels of anti-HBsAg antibodies in human volunteers after vaccination with H-B vax. The IgG subclass distribution of specific antibodies showed a marked dominance of IgGl antibodies. In addition, small amounts of specific IgG4 antibodies were occasionally found, suggesting a different pattern from that found after natural disease. The novel use of a human monoclonal antibody to measure specific antibodies may give a more accurate determination of specific IgG subclass levels than previously available methods.     

抗重组幽门螺杆菌VacA单克隆抗体的制备

目的 制备抗重组幽门螺杆菌致细胞空泡毒素抗原(VacA)的单克隆抗体(mAb).方法 用基因工程菌pQr30-v-DH5α大最表达重组蛋白VaeA,经Ni2+-NTA树脂纯化后,Western blot鉴定抗原性,免疫家兔后ELISA法检测血清VacA抗体鉴定其免疫原性.用重组vacA免疫Balb/c小鼠.取免疫鼠脾细胞与骨髓瘤SP2/O细胞融合,HAT选择性培养和间接EIJSA进行筛选,并检测所分泌抗体的效价和分析Ig类别.结果 获得4株能稳定分泌VacA mAb的杂交瘤细胞,能分泌IgG2b、lgM和IgG1 3类抗体,轻链均为k型.其中,IgG1 mAb经Western blot鉴定能与重组VacA发牛特异性反应.结论 应用纯化的重组VacA,成功获得了能稳定分泌幽门螺杆菌VacA单克隆抗体的杂交瘤细胞,并制备了单克隆抗体.为进一步研制检测VacA的试剂盒及探讨VacA的致病机制奠定了基础.     

抗抗CD3 ScFv单克隆抗体的制备及鉴定

目的：制备抗抗CD3 ScFv单克隆抗体并研究其生物活性.方法：分离纯化后的抗CD3 ScFv蛋白免疫BALB/c小鼠,采用传统杂交瘤技术制备抗抗CD3 ScFv单克隆抗体;采用ELISA和Western blot鉴定其亚类和抗原结合特异性;采用FACS测定抗抗CD3 ScFv单克隆抗体对Jurkat细胞特异活性.结果：成功筛选出一株能稳定分泌抗抗CD3 ScFv单克隆抗体的杂交瘤细胞株（10B7）,其分泌的抗体亚类为IgG1.该抗抗CD3 ScFv单克隆抗体直标后可特异性结合抗CD3 ScFv蛋白和抗CD3抗体.结论：文中所研制的抗抗CD3 ScFv单克隆抗体具有与抗CD3抗体、抗CD3 ScFv蛋白特异结合的活性,在肿瘤导向治疗中抗CD3/抗肿瘤双特异抗体的亲和层析纯化、药代动力学监测等方面具有广泛的应用前景.     

Pfs25蛋白单克隆抗体的制备及双抗体夹心ELISA的建立

目的:制备恶性疟原虫子孢子囊表面膜蛋白Pfs25的单克隆抗体( mAb),建立检测Pfs25蛋白的双抗体夹心ELISA方法.方法:纯化毕赤酵母表达的重组Pfs25蛋白,并免疫BALB/c小鼠,采用骨髓瘤细胞Sp2/0与免疫BALB/c鼠脾细胞杂交的细胞融合技术,通过间接ELISA检测获得分泌抗Pfs25抗体的阳性杂交瘤细胞株,通过免疫F1鼠诱生腹水,纯化腹水,并进行mAb的各项生物学鉴定.辣根过氧化物酶(HRP)标记纯化后的抗体,以4B7为包被抗体,1B4为酶标抗体,建立了双抗体夹心ELISA法.结果:获得3株抗Pfs25的杂交瘤细胞株,其中2株有良好的稳定性和特异性.并建立了双抗体夹心ELISA检测法,检测有效范围在0.07～1 mg/mL,其检测灵敏度为41.6 ng/mL.结论:成功制备抗Pfs25蛋白的单克隆抗体,并建立了一种可用于Pfs25蛋白检测的双抗体夹心ELISA法,为Pfs25蛋白制备传播阻断型疟疾疫苗奠定了基础.     

幽门螺杆菌尿素酶B单克隆抗体的制备、鉴定及应用

目的：制备幽门螺杆菌（Hp）尿素酶B（ureB）单克隆抗体并鉴定。 方法： 以Hp重组纯化蛋白ureB为抗原，运用杂交瘤技术制备ureB单克隆抗体，用间接ELISA法检测抗体免疫学活性，用双抗夹心的IRMA法检测不同单抗是否识别相同的表位，并用于检测Hp感染。 结果： 获得两株识别不同表位的ureB单抗杂交瘤细胞，可测得Hp 感染的相关抗原。 结论： 抗ureB单抗可特异性与Hp结合，可用于建立幽门螺杆菌现症感染的检测方法。     

HCV NS3蛋白单克隆抗体的制备及其识别区域的分析

目的 制备针对丙型肝炎病毒（HCV）非结构区NS3全长蛋白的单克隆抗体（MAb）,并分析获得的单抗识别表位所在区域,为建立以NS3蛋白为靶位的抗HCV研究提供抗体工具。方法 用原核表达的HCV非结构区NS3全长蛋白作为免疫原,采用小鼠腹股沟皮下NC膜包埋法免疫小鼠,按常规杂交瘤细胞的制备方法,经细胞融合、克隆化制备抗NS3蛋白的MAb。用间接免疫荧光法和Westem blot鉴定其特异性。分别构建NS3丝氨酸蛋白酶（NS3蛋白的N末端1／3）编码基因的真核表达质粒pcDNA3．1（-）-ns3p、NS3解旋酶（NS3蛋白的C末端2/3）编码基因的真核表达质粒pcDNA3．1（-）-ns3A,将其瞬时转染COS-7细胞后,以获得的单克隆抗体作为一抗,通过免疫荧光分析获得单抗识别表位所在的区域。结果 获得了2株抗NS3蛋白的单克隆抗体,这2株MAbs均特异识别NS3蛋白,并确定了它们的结合区域。结论 获得了针对NS3蛋白的单克隆抗体,并对其单抗识别表位所在的区域进行了分析,为下一步进行以NS3蛋白为靶位的抗HCV研究奠定了良好的基础。     

Identification by monoclonal antibody of a major (28 kDa) surface membrane antigen of Schistosoma mansoni

Monoclonal antibody M.1 was generated from mice immunized with membrane enriched extracts of mechanically transformed schistosomula. M.1 bound to the surface membranes of cercariae and young (0-24 h post-transformation) schistosomula but did not bind to older schistosomula or cultured worms. M.1 immunoprecipitated an antigen of approximate molecular weight 27-28 kDa from schistosomula. Experiments using metabolic labeling showed that the antigen was actively synthesized by developing schistosomula. Further M.1 immunoprecipitated a similar 27-28 kDa antigen from membrane-enriched extracts of miracidia, lung and adult worms as well as from schistosomula.     

沙眼衣原体pORF5质粒蛋白单克隆抗体2H4的纯化及其免疫学特性研究

目的 纯化抗沙眼衣原体pORF5单克隆抗体2H4并鉴定其免疫学特性.方法 大量培养pORF5单克隆抗体阳性杂交瘤细胞株并收集培养上清,采用G蛋白免疫亲和层析法纯化2H4单克隆抗体;酶联免疫吸附试验(ELISA)测定2H4效价及抗体亚类;Western blot鉴定其特异性;免疫荧光试验(IFA)检测2H4单克隆抗体的衣原体种属特异性.结果 纯化后2H4抗体的纯度高达93％;效价为1:1024,免疫球蛋白类型为IgG2a;2H4抗体不仅能特异性识别pORF5融合蛋白,而且能特异性识别Ct血清型A、D、L2、鼠农原体(MoPn)、鹦鹉热嗜农原体(6BC)质粒所编码的内源性pORF5蛋白,但不识别衣原体其他质粒蛋白和肺炎嗜衣原体(Cpn).结论 获得了高纯度的能特异识别pORF5质粒蛋白的单克隆抗体,为进一步研究pORF5蛋白结构和功能以及沙眼衣原体诊断试剂盒的研制奠定了良好的基础.     

抗重组人白细胞介素18单克隆抗体的制备与特性研究

目的 :获得有生物活性的小鼠抗重组人白细胞介素 18(rhIL 18)单克隆抗体。方法 :采用重组hIL 18免疫BALB C小鼠 ,应用杂交瘤技术 ,ELISA法筛选阳性杂交瘤细胞株并经多次克隆化。结果 :建立了 2株小鼠抗hIL 18单克隆抗体杂交瘤细胞 1C7和 1F5 ,染色体数目分别为 92条和 90条 ,所分泌的抗体分别为IgG2a和IgG1,轻链均为κ型 ,腹水IgG抗体经亲和层析法纯化后纯度达 95 %以上 ,效价为 1× 10 - 4和 1× 10 - 5,Westernblot显示 2株单抗均能特异性识别 18 3kD处的rhIL 18蛋白。 1C7单抗的亲和常数Ka=1 7× 10 5,1F5的亲和常数Ka=1 3× 10 5。 2株单抗识别不同抗原表位。结论 :制备的 2株单抗为进一步研究IL 18的分子结构、生物学功能及其与免疫相关性疾病的关系提供了实验材料。