抗人肝癌单克隆抗体HL2的制备及鉴定

用肝癌细胞株QGY-7703、BEL-7402、SMMC-7721以贯序法免疫BALB/c小鼠,获得一株分泌肝癌单抗的杂交窟株HL_2,其染色体数目为97—105。单抗HL_2系IgG_2b亚类。吸收实验及阻断实验证明HL_2抗原与CEA、AFP、HBsAg、HBcAg及HBeAg无免疫同源性。ABC法和ELISA法说明单抗HL_2与四株肝癌细胞、六例肝癌组织均呈明确阳性反应,除与SGC-7901、H_(128)、K_(562)、Hela细胞株及部分胚胎肝脏,胚胎结肠有较弱性反应外,与肝癌旁组织、正常肝脏、其它肿瘤组织和细胞株、二种正常人细胞及其它胚胎组织无反应。显示了单抗HL_2的特异性较好、阳性率较高,是一个有应用前景的单抗。     

抗人C4结合蛋白单克隆抗体的制备及某些方法的改进

用易于获得的C4BP/C4混合抗原免疫小鼠,EAC1q4C4BP血球间接血凝法作为筛选手段,获得了分泌抗C4BP抗体的杂交瘤细胞株。用单克隆抗体制成的亲和柱分离的C4BP,有C4BP的电泳特性和抑制EAC14与C2形成C3转化酶的活性。本文同时报告单克隆抗体制备方案的某些改进:1.小鼠免疫过程中用尾尖近似定量微量采血法对血中抗体水平定期监测,选择合适的小鼠供脾;2.用戊二醛固定的检测血球筛选杂交瘤,方法简便迅速,改变了杂交瘤筛选工作繁重的局面;3.用单细胞分离法达到单克隆化,缩短工作周期。     

抗人CD3单链抗体基因的构建及序列分析

本文在已克隆抗人ＣＤ３抗体ＶＨ和ＶＫ基因的基础上，设计并合成了ＰＣＲ引物。两个外侧引物分别含有ＥｃｏＲＩ和ＳａｌＩ酶切位点及起始码和终止码序列，４个内侧引物各含部分连肽基因序列，回收后混合退火。     

Generation of the Single Chain Antibody Fragment Conserves the Idiotypic Profile of the Anti-CD30 Monoclonal Antibody HRS3

Recombinant single chain antibody fragments (scFv) derived by combining immunoglobulin VL and VH regions provide valuable antibody-like reagents. A number of them are shown to have retained the antigen specificity of the parental monoclonal antibody (MoAb). Little is known about the idiotypic profile of scFv fragments compared with that of the parental MoAb. To address this question we analysed the idiotypic profile of a scFv that was derived by phage-display techniques from the anti-CD30 MoAb HRS3.We assayed (i) binding of HRS3-scFv to recombinant CD30-Fc antigen and to four different anti-idiotypic MoAbs defining at least three different idiotopes on HRS3, and (ii) cross-competition with the parental MoAb HRS3 and the closely related anti-CD30 MoAb HRS4. The assays revealed that the HRS3-scFv fragment exhibits the same specificity for both CD30 antigen and the tested anti-idiotypic MoAbs compared with the parental MoAb demonstrating that the recombinant scFv fragment has retained the complete idiotope of the parental MoAb.     

抗人结肠癌单克隆抗体SC3A在荷瘤裸鼠体内的放射免疫定位研究

本文用~(131)I标记自制的抗人结肠癌单克隆抗体SC3A,进行荷人结肠癌裸鼠体内生物学分布和肿瘤的放射免疫显像研究。结果在注射~(131)I饲—SC3A后24～120h,肿瘤部位的放射性均显示出选择性浓聚,以72～120h的影像最为清晰;而注射~(131)I—鼠IgG则呈全身均匀性分布,无肿瘤部位选择性波聚影像。在72h,13种器官的T/NT均大于2,肿瘤LI为6.94,与扫描显像结果吻合。这些都表明SC3A在生物体内对结肠癌具有良好的选择性和导向作用,抗体可对其供临床体内进一步应用提供了依据。     

MCAb、PcAb不同标记ELISA技术对HBeAg／抗－HBe检测的评估

用单克隆抗体（ＭｃＡｂ）和多克隆抗体（ＰｃＡｂ）进行辣根过氧化物酶标记或生物素标记作酶联免疫吸附试验（ＥＬＩＳＡ），检测乙型肝炎病毒ＨＢｅＡｇ／抗－ＨＢｅ标志物。结果显示，ＭｃＡｂ的灵敏性高于ＰｃＡｂ，且标记用的抗体量较少，一孔一期法同时测ＨＢｅＡｇ／抗－ＨＢｅ只能使用ＭｃＡｂ。利用生物素与亲和素系统与ＭｃＡｂ建立的Ｍｃ－ＡＢＣ－ＥＬＩＳＡ技术，其灵敏性与放射免疫法（ＲＩＡ）相同，特异性好，稳定性优于ＲＩＡ。生物素标记抗体至少１年保持效价不变，是一种新的较理想的检测ｅ系统的方法之一。     

鼠抗人CD28功能性单克隆抗体的研制及其生物学特性的鉴定

以天然表达CD28分子的人多发性骨髓瘤(multiple myeloma,MM)细胞:XG-1为免疫原,常规免疫BALB/c小鼠,采用B淋巴细胞杂交瘤技术进行细胞融合,CD28转基因细胞株(T-28)及XG-1为抗体筛选阳性细胞株,经免疫荧光标记分析反复筛选和多次的克隆化培养,最终获得1株持续、稳定分泌鼠抗人CD28单克隆抗体的杂交瘤细胞株,命名为10F3。对这株单抗生物学功能的研究结果表明,这株抗体能特异性地识别人CD28分子和介导有效的共刺激信号,激发T细胞的活化和增殖。     

抗HSPC238单克隆抗体的制备和初步鉴定

目的制备抗HSPC238的单克隆抗体，为HSPC238的功能研究奠定基础。方法以纯化的重组蛋白HSPC238为抗原，免疫BALB／c小鼠，运用杂交瘤技术制备HSPC238单克隆抗体，并用间接ELISA法和Western—blot法对单克隆抗体的特性进行鉴定。结果成功建立两株稳定分泌抗HSPC238的单克隆抗体杂交瘤细胞株，分别命名为E001和E002。ELISA检测抗HSPC238单克隆抗体的腹水效价为1：12800和1：25600。两株单克隆抗体的免疫球蛋白亚类均为IgG1。通过Western—blot实验证实，两株单克隆抗体均能特异性结合真核细胞内源性HSPC238蛋白。结论成功制备了两株效价高、特异性好的抗HSPC238单克隆抗体，制备的抗HSPC238单克隆抗体可用于HSPC238蛋白的鉴定，为HSPC238蛋白的生物学功能研究奠定了基础。     

抗激活小鼠T细胞抗原单克隆抗体（2H3）识别的靶抗原分析

2H3为一株抗激活小鼠T细胞表面抗原的IgG_1型单克隆抗体。间接ELISA实验表明:它能与ConA 激活的小鼠脾细胞及IL—2依赖的CTLL—2细胞发生特异性结合。靶细胞免疫荧光染色法表明:上述两种靶细胞可与荧光标记的抗IL—2R单抗结合并可被事先与2H3作用所阻断,但该两种靶细胞与荧光标记抗Ia抗体的结合不能被2H3所阻断。对2H3所结合的靶细胞膜蛋白提取物进行的Western-Blotting实验表明:2H3识别的靶抗原分子量约为50～60KD。结果提示:2H3识别的靶抗原与小鼠细胞膜表面的IL—2R(P_(55)蛋白)是一致的,其免疫生物学活性正在进行分析中。     

Development of a Monoclonal Antibody to a Ureaplasma urealyticum Serotype 9 Antigen

We produced a monoclonal antibody (MAb) to Ureaplasma urealyticum Vancouver, the serotype 9 standard strain. By immunoblotting, this MAb showed a single, 85-kDa band with the homologous serotype and a minor, 100-kDa band with serotype 2 but did not react with any other serotype standard strain. Clinical isolates of U. urealyticum were tested with this MAb and with two sets of polyclonal antisera against the 14 serotype standard strains. The use of MAb 9-2H9 correctly identified certain serotype 9 strains but did not react with wild-type strains lacking the serotype 9 determinant.     

Expression and Characterization of a Divalent Chimeric Anti-Human CD3 Single Chain Antibody

Murine anti-CD3 monoclonal antibodies (MoAbs) are used in clinical practice for immunosuppression. However, there are two major drawbacks to this treatment: the associated cytokine release syndrome and human anti-mouse antibody response. To overcome these side-effects, the authors generated a chimeric anti-human CD3 single chain antibody, scUCHT1. It is an IgM variant of UCHT1, a mouse IgG1 MoAb directed against human CD3. scUCHT1 consists of the light and heavy variable chain binding domains of UCHT1 and a human IgM Fc region (CH₂ to CH₄). scUCHT1 was produced by COS-7 and SP2/0 transfectants, and mainly assembled in a dimeric form. It retained the binding specificity and affinity of the parental MoAb UCHT1. In contrast to UCHT1, scUCHT1 did not induce T-cell proliferation and cytokine release (TNF-α and IFN-γ) in in vitro assays. These results suggest that the engineered chimeric anti-CD3 single chain antibody (scUCHT1) may be useful in clinical immunosuppressive treatment.     

Monoclonal Antibody against Babesia equi: Characterization and Potential Application of Antigen for Serodiagnosis

Monoclonal antibody (MAb) BEG3 was produced against Babesia equi parasites to define a species-specific antigen for diagnostic use. The MAb reacted with single, paired, and Maltese cross forms of B. equi, and no reaction was observed with this MAb on acetone-fixed Babesia caballi, Babesia ovata, or Babesia microti parasites in the indirect immunofluorescent antibody test. Confocal laser and immunoelectron microscopic studies showed that the antigen which was recognized by this MAb was located on the surface of B. equi parasites. This MAb recognized a 19-kDa protein of B. equi antigen and did not react with B. caballi antigen or normal horse erythrocytes in immunoblot analysis. This MAb also significantly inhibited the in vitro growth of the B. equi parasite. Preliminary studies using partially purified antigen, which was separated by high-pressure liquid chromatography and recognized by the MAb, suggested that it is a suitable antigen for enzyme-linked immunosorbent assay detection of anti-B. equi antibodies in naturally infected horse sera.     

Presence of an Endothelial Antigen on the Syncytiotrophoblast of Human Chorionic Villi: Detection by a Monoclonal Antibody

ABSTRACT: We describe a monoclonal antibody, B721. This antibody reacts with an antigen present on vascular endothelium and on the syncytiotrophoblast of term chorionic villi. The antigen is absent from the trophoblast of the chorion, from the amniotic epithelium, and from normal peripheral blood or lymph node lymphocytes. We discuss the possible functional roles of the antigen. We propose that the syncytiotrophoblast, by expressing endothelial antigens, mimics endothelium and may perform endothelial functions.     

Characterization of the Immune Response to an Epitope on Mycobacterium leprae Antigen 7 Defined by a Monoclonal Antibody

A mouse monoclonal antibody (038D-C6) was shown by crossed immunoelectrophoresis and radioimmunoassay to react with an epitope on the Mycobacterium leprae antigen 7. This epitope was highly crossreactive with BCG/M. tuberculosis and of a non-arabinogalactan-arabinomannan nature. A solid-phase radioimmunoassay (SPRIA) was applied, based on competitive inhibition by human sera of antigen binding by this anti-M. leprae monoclonal antibody. Inhibitory activity determined by this assay decreased markedly upon treatment in both lepromatous and tuberculoid leprosy patients. A correlation was found between the bacterial load of the patient and the inhibitory activity measured in the SPRIA assay. Serum-inhibitory activity could therefore perhaps be used as a follow-up test for patients on treatment or as a screening method to detect infective cases. A dot enzyme-linked immunosorbent assay based, like the SPRIA assay, on competitive inhibition by human sera, was explored as an inexpensive and technically simple alternative also applicable under field conditions.     

Characterization of an Early Activation-Dependent Antigen on Lymphocytes Defined by the Monoclonal Antibody BL-Ac(F2)

A novel activation-dependent lymphocyte cell-surface antigen which is recognized by a MoAb, BL Ac(F2), is described. Although not found on resting lymphocytes the antigen is induced rapidly wiihin 2–4 h following stimulation of the cells using mitogens or antibodies against the T-eell CD3 antigen and sIgM on B cells, respectively. Immunopreeipitation and Western Blotting indicated that the MoAb recognizes a molecule in a range of 78 85 kDa. Beyond its activalion-dependent expression on lymphocytes the antigen was detected also on myelo-monocytic cells. Expression kinetics and cellular distribution of this molecule suggest that it is distinct from previously described activation-dependent cell-surface antigens sueh as CD69, CD25 and 4F2.     

Characterization of Monoclonal Antihuman-B-Cell Antibody BL13 as an Anti-C3d-Receptor (CR2) Antibody

BL13, a mouse monoclonal IgG1 antibody raised against human B cells, blocked the function of the C3d receptor (CR2) and bound with high affinity (5 X 10(8) L M-1) to CR2 on B lymphoma cells. Following capping with the second antibody, BL13 inhibited C3d-dependent rosette formation of Daudi and Raji cells and C3b-dependent CR2-mediated rosette formation with B lymphoma cells, but did not inhibit CR1-mediated rosettes between C3b-bearing cells and peripheral blood lymphocytes. Competitive binding experiments between biotinylated BL13 or anti-CR2 antibody HB-5 and unlabelled antibodies demonstrated that BL13 bound to an epitope that is distinct from that recognized by HB-5, and closely associated with that recognized by monoclonal antibody anti-B2. BL13 only reacted with some B cells and follicular dendritic cells in germinal centres in human lymph nodes, whereas HB-5 strongly reacted with circulating B cells and bound to most cells in the follicles. These results demonstrate the heterogeneity of antigenically defined CR2.     

抗人结肠癌单克隆抗体MC_6的制备及其相应抗原的免疫组化定位研究

本研究利用手术中取得的新鲜结肠癌组织免疫Balb/c小鼠。制得5株能持续、稳定分泌抗体的杂交瘤细胞系。其中HC6株杂交瘤细胞经过180天连续培养，单克隆抗体MC6分泌稳定，选择性强，效价高。免疫组化检测发现，MC6相应抗原在结肠癌组织及结肠癌细胞系高度表达（90％～100％），显著高于其它癌组织，癌旁和正常粘膜及癌细胞系（0％～44．4％）；中度以上染色者占阳性者总数80％以上，主要分布于细胞膜上，且除癌细胞膜和胞浆呈阳性反应外，腺腔内粘液亦可见不同程度着色。表明MC6相应抗原是一种在结肠癌细胞膜上占优势的抗原，并可脱落至腺腔。此单克隆抗体的成功研制为发现新的结肠癌生化标志，为结肠癌早期诊断及术后病情监测提供了可能性。     

基因免疫诱导9.1C3分子内影像类抗独特型抗体的产生

本文分别采用真核表达载体ｐＥＦ－ＢＯＳ及ｐＣＭＶ４构建含起始码和终止码的抗９．１Ｃ３分子单克隆抗体重链可变区基因重组表达质粒９．１Ｃ３ＶＨｐＥＦ－ＢＯＳ及９．１Ｃ３ＶＨｐＣＭＶ４，并将其分别免疫ＢＡＬＢ／ｃ小鼠。取免疫小鼠血清对Ｋ５６２细胞进行间接免疫染色和流式细胞仪分析表明，基因免疫小鼠体内可诱导９．１Ｃ３分子内影像类抗独特型抗体的产生。