人血清OSM-IRMA的建立及初步应用

本文利用富含肿瘤相关糖抗原sTn的羊颌下腺粘蛋白(OSM)作为抗原,建立了两株sTn表位的mAb(7F4,2F6)和双位点夹心OSM-IRMA,以探讨其对肿瘤早期诊断的可能性.结果表明,该方法的灵敏度为0.1U/mL;批内和批问CV分别为4.13%和9.7%;平均回收率为106.9%;含高浓度sTn的血清样品系列倍比稀释后测定,实测值和计算值呈线性相关,相关系数大于0.99,30名健康成人空腹血清OSM均值为4.9士1.16U/mL,以7.2U/mL为正常值上限,在胃癌、结肠癌和肺癌的阳性率分别为46.7%、40%和20%,表明该OSM-IRMA在胃癌诊断中具有较好的应用价值.     

肝癌相关的单克隆抗体的制备及其抗原表位分析

目的:制备肝癌相关的单克隆抗体(mAb)用于肝癌的诊断。方法:用高转移肝癌细胞系HCCLM-6细胞免疫BALB/c小鼠后,取小鼠脾细胞与Sp2/0融合建立杂交瘤细胞系。通过ELISA法筛选HCCLM-6细胞蛋白的特异性mAb,用免疫组化染色法筛查对肝癌组织阳性率高的mAb,并用免疫荧光方法鉴定其在不同肿瘤细胞系中的表达,最后通过筛选Uni-ZAP XR人肝细胞cDNA表达文库初步鉴定mAb识别的抗原及其表位。结果:建立了28株杂交瘤细胞株,用ELISA法和免疫组化染色法筛选出肝癌阳性率较高的mAbQGA062。通过人肝细胞cDNA表达文库筛选初步鉴定表明mAb QGA062识别的抗原是纤维连接蛋白(FN),经分析其抗原表位位于肽段YTVSLVAIKGNQESPK。结论:获得与肝癌相关的mAb QGA062,并鉴定了其识别的抗原和表位,为肝癌的诊断和FN参与肝癌转移的机制研究提供了重要的制剂。     

抗人c-erbB2单克隆抗体的制备及其特异性鉴定

目的:制备小鼠抗人c-erbB2mAb,并进行特异性鉴定。方法:应用计算机软件分析人源c-erbB2抗原表位,人工合成羧基端含优势表位的13肽,与钥孔戚血蓝蛋白(KLH)偶联后,免疫BALB/c小鼠。取免疫小鼠脾细胞与Sp2/0骨髓瘤细胞常规融合,依次经HAT选择培养、间接ELISA法、克隆化和免疫组化染色法筛选出稳定分泌抗天然人源c-erbB2mAb的杂交瘤细胞株。用交叉反应试验和阻断试验检测mAb的特异性。结果:获得1株可稳定分泌抗天然人源c-erbB2抗体的杂交瘤细胞株。该mAb与已知的c erbB2抗原阳性的乳腺癌标本起反应;与其他不表达c-erbB2分子的细胞不起反应。用合成的13肽阻断后,失去与c-erbB2抗原的反应性。结论:用合成的13肽作为免疫原成功地制备出1株抗c-erbB2的mAb。     

抗人硫氧还蛋白-1单克隆抗体的制备与鉴定及初步应用

目的:制备抗硫氧还蛋白-1(TRX-1)的单克隆抗体(mAb)并进行特性鉴定.方法:以原核表达的TRX-1为抗原免疫BALB/c小鼠,按常规方法进行细胞融合.采用有限稀释法和间接ELISA法克隆和筛选阳性杂交瘤细胞株,用ELISA检测mAb腹水的效价、相对亲和力和进行表位分析,用Western blot法对mAb的特异性进行鉴定.结果:得到筛选出3株能稳定分泌抗TRX-1的杂交瘤细胞株B6、D5和E3,免疫球蛋白亚类均为IgG>(к),腹水mAb效价均在106以上,3株抗体分别针对不同抗原表位.用mAb建立的竞争抑制ELISA灵敏度达1.22 μg/L.结论:获得3株特异性好、亲和力高、能稳定分泌抗TRX-1的杂交瘤细胞株,为研究TRX-1在人类细胞、血液、组织中的表达及定位提供了可能,并为探索相关炎症、癌症发病机制及新治疗方法提供了强有力的工具.     

人成纤维细胞生长因子-21单克隆抗体的制备及抗原表位的确定

目的 制备小鼠抗人成纤维细胞生长因子(hFGF)-21单克隆抗体(mAb),通过细菌展示确定该mAb的抗原表位.方法 用hFGF-21作为检测和免疫抗原,间接ELISA筛选分泌抗人hFGF-21 mAb的杂交瘤细胞株；用FITC标记该mAb,并克隆hFGF-21的不同片段到展示载体Apex上,通过流式细胞仪筛选其抗原表位.结果 成功筛选出1株抗hFGF-21抗体的细胞株,其分泌抗体重链的亚型为IgG 2b,轻链为Kappa链；该杂交瘤细胞株腹水的效价为1:4.096×106；传30代及液氮中保存3个月,该细胞株能稳定分泌抗hFGF-21 mAb,且效价稳定；Western blot法检测证明该抗体与人FGF-21有很好的特异性；该mAb与小鼠FGF-21有交叉反应；通过流式细胞仪对抗原表位的筛选,该mAb可与hFGF-21下游的第107～121个氨基酸反应.结论 成功的制备出特异性、高稳定性的小鼠抗hFGF-21 mAb,确定了该mAb的抗原表位在第107～121位氨基酸.     

鼠抗人CD20单克隆抗体的制备与鉴定

目的:制备CD20融合蛋白及制备CD20的单克隆抗体(mAb),并对其进行特性鉴定.方法:以人单核细胞cDNA文库为模板,构建重组表达质粒pGEX- 4T-1-CD20 (即CD20-GST) 和pET-32a-CD20(即CD20-His).以融合蛋白CD20-GST为免疫源免疫BALB/c雌性小鼠制备(mAb),用间接ELISA法测定抗体的效价,Western blot、免疫荧光鉴定其特异性及免疫组化染色法对mAb相应的抗原进行组织定位.结果:CD20-GST 和CD20-His蛋白在大肠杆菌中可高效表达,经纯化后可获得高纯度的CD20蛋白,经细胞融合得到1株稳定分泌CD20抗体的杂交瘤细胞株,Western blot和免疫组化显示抗CD20 mAb与CD20蛋白具有良好的反应性.结论:成功制备高纯度CD20蛋白并以此为抗原制备了1株能够稳定分泌抗人CD20的鼠mAb的杂交瘤细胞株BD11F4,为进一步研究CD20对非霍奇金淋巴瘤的诊断治疗提供基础.     

抗丁型肝炎病毒mAb的制备及其初步应用

目的 :以基因工程表达丁型肝炎病毒抗原蛋白 (HDAg)为抗原 ,建立分泌单克隆抗体 (mAb)的杂交瘤细胞系 ,并对其分泌的mAb进行鉴定。方法 :用Ni NTA技术纯化HDAg免疫BALB/c小鼠 ,取脾细胞及骨髓瘤细胞按常规方法融合、筛选、克隆化及腹水注射制备mAb。采用ELISA及免疫组化技术进行鉴定。结果 :筛选出 2株能稳定分泌特异性抗 HD的mAb杂交瘤细胞株 (HD1,HD2 ) ,经多次复苏传代仍能稳定分泌抗体 ,特异性强 ,效价高。应用于ELISA测定患者血清HDAg均呈特异性阳性反应。免疫组化检测肝组织显示 ,针对该mAb的抗原主要分布于细胞核或浆内。结论 :此两株丁肝杂交瘤细胞株分泌的抗体对丁肝抗原具有特异亲和性 ,为丁型肝炎病毒的临床诊断及病理检测提供技术基础     

抗GRP78单克隆抗体的制备及初步鉴定

目的:原核表达GRP78并制备抗GRP78的特异性单克隆抗体(mAb),并初步鉴定相应mAb的特性。方法:从肺癌患者组织中抽提总RNA,RT-PCR获得grp78全长基因,并通过原核重组表达GRP78蛋白;以纯化的GRP78蛋白免疫BALB/c小鼠,成功免疫的小鼠脾细胞与骨髓瘤Sp2/0细胞融合,筛选得到分泌特异性抗GRP78的mAb的杂交瘤细胞株;并通过Western blot及免疫组化初步鉴定其特异性。结果:成功构建重组表达质粒PET-28a-grp78并由大肠杆菌表达获得GRP78蛋白,被该蛋白免疫的BALB/c小鼠与Sp2/0细胞融合、筛选,获得3株稳定分泌抗GRP78抗体的杂交瘤细胞株,分别命名为2A1、4B8和4A12。采用其中1株4A12 mAb对3个肺癌患者组织样本进行Western blot检测以及免疫组化分析,结果均显示4A12 mAb能够特异识别肺癌组织表达的GRP78。结论:成功制备了抗GRP78的mAb,并能够特异识别肺癌组织表达的GRP78,为进一步研究GRP78在肿瘤治疗中的作用提供基础。     

抗人RANTES分子单克隆抗体的制备及特性鉴定

目的:制备抗人RANTES分子单克隆抗体(mAb)并进行初步鉴定,为研究RANTES分子的组织分布和功能提供实验手段。方法:应用淋巴细胞杂交瘤技术,制备小鼠源性抗人RANTESmAb。用ELISA法鉴定腹水mAb的效价。用Q FastFlow阴离子交换柱纯化mAb。用Westernblot鉴定mAb的抗原结合活性。用免疫组化染色法,对RANTES分子在进行小肠移植术的大鼠移植肠组织中的分布进行鉴定。结果:获得4株分泌抗RANTESmAb的杂交瘤细胞株。间接ELISA法测定腹水mAb的效价均达1×10-6,3株mAb为IgG1亚类(κ),1株为IgG2b(κ)。Westernblot的结果显示,3株mAb与人RANTES均有良好的结合活性。用3株mAb进行免疫组化染色的结果显示,RANTES分子在进行小肠移植术的大鼠小肠腺上皮细胞胞质中呈高表达。结论:获得4株能特异性识别天然RANTES分子的mAb。大鼠小肠移植后其肠上皮细胞中可高水平地表达RANTES。     

抗血型M、N及抗血型糖蛋白A/B单克隆抗体的制备及其特性鉴定

目的:制备抗血型M、N及抗血型糖蛋白A/B(GPA/GPB)的单克隆抗体(mAb),并进行特性鉴定。方法:用人“O”型血红细胞作为免疫源,免疫BALB/c小鼠。采用淋巴细胞杂交瘤技术制备mAb,用谱红细胞筛选阳性克隆;采用直接、间接血凝试验检测杂交瘤细胞培养上清及腹水中mAb的效价。分别用快速定性试纸和酶处理红细胞检测mAb的Ig亚类及抗原表位。用Western blot鉴定抗GPA/GPB mAb的特异性。结果:获得4株分泌抗M、1株抗N及3株抗GPA/GPB mAb的杂交瘤细胞株。杂交瘤细胞培养上清mAb的效价介于1×2-4~1×2-8之间,腹水mAb的效价在1×2-7~1×2-12之间。除1株mAb 1C1C9C4为IgM外,其他7株mAb均为IgG。通过杂交瘤细胞培养上清与谱红细胞的反应格局,结合mAb抗原表位的检测,确定4株和1株mAb可分别特异性结合于GPA的M、N抗原表位;另3株mAb 6D7C9、7C9H4和7C9G11与“O”型血红细胞膜的Western blot结果显示,均可结合GPA、GPB蛋白。结论:成功地建立了4株分泌抗M、1株分泌抗N及3株分泌抗GPA/GPB mAb的杂交瘤细胞株,可用于MNSs血型系统的研究及鉴定,并为制备双功能抗体用于病毒、肿瘤疾病的诊断和治疗打下了坚实的基础。     

Carcinoembryonic Antigen and Nonspecific Cross-reacting Antigen in Medullary Carcinoma of the Thyroid

Carcinoembryonic antigen (CEA) and nonspecific cross-reacting antigen (NCA) were studied immunohisto-chemically in formalin fixed, paraffin embedded tissues of 73 cases of medullary carcinoma of the thyroid (MTC) using 2 polyclonal antibodies (CEA antisera cross-reactive with or without NCA), 3 monoclonal antibodies recognizing epitopes only on CEA, and one monoclonal antibody against NCA. The staining patterns of the 5 antibodies against CEA in MTCs were not different, and they reacted with 86.3% of all cases. With regard to the effects of fixatives on the staining patterns, samples fixed with formalin or 4% paraformaldehyde demonstrated CEA immunoreactivity in both the cell membrane and cytoplasm. In Bouin fixed tissue, the immunoreactivity was predominant on the cell membrane, whereas cytoplasmic positivity predominated in alcohol fixed specimens. Thus the difference in fixatives used in previous studies does not appear to be a major reason for the difference in the reported incidence of CEA positive MTCs. It is concluded that CEA is still a useful tumor marker for MTC and that it is detectable only in thyroid tumors originating from C cells, as seen in our series. The epitope defined by monoclonal antibody F106 88, present only on NCA, was found in 42.5% of all cases (49.2% of CEA positive MTCs). The NCA immunoreactivity was located in the tumor cell cytoplasm as globular aggregates, which were also labeled for CEA.     

Monoclonal antibodies against surface antigens of Bacteroides gingivalis.

Monoclonal antibodies against the various surface antigens of Bacteroides gingivalis were obtained by the fusion of murine myeloma cells (SP2/0-Ag14) with spleen cells of BALB/c mice immunized with the whole cells. Two monoclonal antibodies reacted with lipopolysaccharide, and the other two reacted strongly with capsule antigen. One showed reactivity with the hemagglutinin of the cells. The five monoclonal antibodies reacted with sonicated antigen from all B. gingivalis strains tested. No cross-reactivity of the monoclonal antibodies with antigens from nine species of other black-pigmented Bacteroids strains was observed. An immunoblotting test involving the use of these monoclonal antibodies indicated that the epitope of B. gingivalis lipopolysaccharide was polysaccharide with a high molecular weight of 40,000 to 60,000. The immunoblotting test also demonstrated that the epitopes of capsule antigen and of hemagglutinin were 27,000- and 40,000-molecular-weight proteins, respectively.     

Production of a monoclonal antibody as immunohistochemical marker on paraffin embedded tissues using a new immunization method

This report describes a method for the production of murine monoclonal antibodies (MAbs) against cellular antigens preserved during formol fixation and paraffin embedding of human tissues in an attempt to select markers that would be useful in immunopathology. Hybridomas were prepared using spleen cells from mice immunized with cell suspensions obtained from formalin-fixed paraffin block sections of a human breast carcinoma. A monoclonal antibody 83 D4 was selected, which was reactive with paraffin embedded breast carcinoma tissues, but not with normal breast. The reactive antigen has a high molecular weight (400-1000 kD) and was detected on the cell surface of live human breast cancer cell lines and on frozen tissues sections. These results demonstrate that the MAb 83 D4 identifies a native breast tumor associated epitope conserved during tissue fixation and embedding and could be used as an immunohistochemical marker.     

Production and characterization of monoclonal antibodies against serotype strains of Pseudomonas aeruginosa

Monoclonal antibodies against 12 of the 17 IATS serotype strains of Pseudomonas aeruginosa were produced. Eighty-seven hybridoma clones were isolated, and the antibodies secreted were found to be reactive with both Formalin-fixed whole cells and purified lipopolysaccharide of homologous strains in enzyme-linked immunosorbent assays. Among these monoclonal antibodies, the predominant antibody class was immunoglobulin M (IgM) (76%), although antibodies of the IgG2a and IgG3 isotypes were also produced. The monoclonal antibodies could further be divided into two groups based on their ability to agglutinate whole cells of homologous strains. The agglutinating monoclonal antibodies were found to immunoblot with the O side chains of homologous lipopolysaccharide, while the nonagglutinating monoclonal antibodies were found to be reactive with outer membrane protein-associated lipopolysaccharide. The applicability of monoclonal antibodies for serotyping was examined, and several antibodies were found to agglutinate whole cells and immunoblot with the O antigen of corresponding serotypes of clinical isolates from cystic fibrosis patients. In conclusion, a set of monoclonal antibodies against the IATS serotype strains of P. aeruginosa have been produced. These antibodies represent a bank of invaluable immunological reagents which may have application in serotyping, epitope mapping, lipopolysaccharide structural determination, and studies of protection against P. aeruginosa.     

Enhancement of metastatic ability by ectopic expression of ST6GalNAcI on a gastric cancer cell line in a mouse model

ST6GalNAcI is a sialyltransferase responsible for the synthesis of sialyl Tn (sTn) antigen which is expressed in a variety of adenocarcinomas including gastric cancer especially in advanced cases, but the roles of ST6GalNAcI and sTn in cancer progression are largely unknown. We generated sTn-expressing human gastric cancer cells by ectopic expression of ST6GalNAcI to evaluate metastatic ability of these cells and prognostic effect of ST6GalNAcI and sTn in a mouse model, and identified sTn carrier proteins to gain insight into the function of ST6GalNAcI and sTn in gastric cancer progression. A green fluorescent protein-tagged human gastric cancer cell line was transfected with ST6GalNAcI to produce sTn-expressing cells, which were transplanted into nude mice. STn-positive gastric cancer cells showed higher intraperitoneal metastatic ability in comparison with sTn-negative control, resulting in shortened survival time of the mice, which was mitigated by anti-sTn antibody administration. Then, sTn-carrying proteins were immunoprecipitated from culture supernatants and lysates of these cells, and identified MUC1 and CD44 as major sTn carriers. It was confirmed that MUC1 carries sTn also in human advanced gastric cancer tissues. Identification of sTn carrier proteins will help understand mechanisms of metastatic phenotype acquisition of gastric cancer cells by ST6GalNAcI and sTn.     

胃癌组织匀浆中一组新肿瘤相关抗原MG-Ags的测定及其价值

作者应用5种抗胃癌单克隆抗体(MG5,MG7 MG9 MGb2和MGd1)的等量混合物,以间接ELISA测定了14例胃癌组织及癌旁组织匀浆中胃癌相关抗原MG-Ags、发现14例癌组织中MG-Ags的平均含量是癌分组织的5.2倍,其中有12例(85.7％)癌组织中MG-Ags高于癌旁组织,说明MG-Ags与胃癌组织具有明显关系。     

Human Trophoblast-Specific Surface Antigens Identified Using Monoclonal Antibodies*

ABSTRACT: Mouse monoclonal antibodies have been produced against syncytiotrophoblast plasma membrane preparations isolated from term human placentas. Of 20 positive clones, two antibodies (H309, H318) were directed against the C_γ2 domain of IgG, one (H312) was directed against albumin, and another (H303) was directed against a further normal human serum component (not transferrin). One monoclonal antibody (H310) recognized an antigenic epitope restricted to trophoblast and lymphocytes: this antibody did not inhibit mitogenic or allogeneic stimulation of lymphocytes. Two monoclonal antibodies (H315 and H317) reacted with trophoblast-specific antigenic determinants. The H315 antigen was present on first trimester syncytiotrophoblast, unlike the H317 antigen.     

Monoclonal antibodies specific for Shigella flexneri lipopolysaccharides: clones binding to type IV, V, and VI antigens, group 3,4 antigen, and an epitope common to all Shigella flexneri and Shigella dysenteriae type 1 stains.

Monoclonal antibodies reactive with Shigella flexneri O antigens were generated in both mouse and rat systems. Antibody-producing hybridomas were screened in an enzyme-linked immunosorbent assay using chemically defined lipopolysaccharides as antigens, and the epitope specificities were determined with a panel of lipopolysaccharides and synthetic O-antigen-specific glycoconjugates as antigens. To verify the specificity seen in the enzyme-linked immunosorbent assay, the antibodies were used in agglutination against a large number of S. flexneri strains. Monoclonal antibodies with the following specificities were identified: type, antigen IV (reactive with serotype 4a and 4b bacteria); type antigen V (reactive with serotype 5a and 5b bacteria); type antigen VI (reactive with serotype 6 bacteria); group antigen 3,4(reactive with serotype 1a, 2a, 3b, 4a, 5a, and Y bacteria); and group antigen 1 (reactive with an epitope present on all S. flexneri and Shigella dysenteriae type 1 bacteria). Furthermore, a monoclonal antibody defining a new O-antigenic epitope present on some S. flexneri strains of serotypes 4a, X, and Y was characterized (4X). The monoclonal antibodies analyzed in this study define epitopes described by polyclonal antisera (type antigens IV, V, and VI), define a hitherto uncharacterized epitope (group antigen 1), and finally identify new epitopes in what has previously been considered as one epitope (group antigen 3,4 and type antigen IV). These immunochemically characterized monoclonal antibodies may have a powerful potential in studies of the importance of humoral immunity in shigellosis.     

Monoclonal antibodies produced in BALB.B10 mice define new antigenic determinants in culture filtrate preparations of Mycobacterium tuberculosis.

A panel of monoclonal antibodies were derived from BALB.B10 mice immunized with a culture filtrate from Mycobacterium tuberculosis H37Rv. Of these antibodies, 10 were examined more closely for antigen specificity and interspecies reactivity. Six antibodies were used as immunosorbents for affinity purification of their corresponding antigens. Two monoclonal antibodies (HBT 2 and HBT 11) reacted with a 17-kilodalton antigen, and a competition assay showed that these antibodies are directed against the same epitope or against epitopes that are sterically very close to each other. Monoclonal antibody HBT 12 reacted with the same molecule with which a previously described 38-kilodalton reactive antibody reacted but was directed against a different epitope. Antibody HBT 10 reacted with a culture filtrate of M. tuberculosis but not of Mycobacterium bovis BCG. This latter finding was further studied by testing different preparations of M. tuberculosis H37Rv antigens and, additionally, culture filtrates of four M. tuberculosis and two BCG strains. Interspecies reactivity was assayed by immunoblotting and revealed that the majority of the monoclonal antibodies were specific to M. tuberculosis complex.     

Expression of tumor-associated Thomsen-Friedenreich antigen (T Ag) in Helicobacter pylori and modulation of T Ag specific immune response in infected individuals

We tested the hypothesis that the gastric cancer associated bacteria, Helicobacter pylori (H. pylori) express the cancer-related Thomsen-Friedenreich (T) antigen. We also analysed whether infection with H. pylori alters the amount of natural anti-T antibodies in the patients' sera. Cell surface membrane extracts of H. pylori NCTC 11637 strain and clinical isolates of H. pylori (n = 13) were analysed by immunoblotting and cell-ELISA with five different T antigen-specific monoclonal antibodies (MAbs). Two major protein bands of approximately 68 kDa and 58 kDa were immunostained on blots of H. pylori extracts with T specific MAbs but not immunostained with unrelated MAb. The specificity was shown in that immunostaining was blocked with peanut agglutinin (PNA) and rabbit antiserum to T antigen. The binding of T specific MAb to the 58 kDa protein band was also blocked by rabbit antiserum against heat shock proteins of H. pylori. The relative expression of T antigen-related proteins differed among H. pylori strains, with 68 kD associated T antigen expression higher in patients with more severe pathology. The level of IgG antibody to T epitope in patients with gastric cancer (n = 66) and normal blood donors (n = 62) were compared and the level of anti-T Ab in gastric cancer patients was significantly lower than that in normal blood donors. A significant positive correlation between T specific antibody in serum and H. pylori IgG antibody level was found in H. pylori-infected normal blood donors (P < 0.001), but this correlation was not found in H. pylori-infected cancer patients. In summary, the cancer related T epitope is expressed in H. pylori and modulation of T antigen-specific immune response in H. pylori-infected individuals suggests that H. pylori infection may alter natural immune mechanisms against cancer.