Production and characterization of monoclonal antibodies with diagnostic potential against Shigella flexneri

Highly specific monoclonal antibodies (McAbs), 1aM3, 2aM1 and 2bM2 were produced and characterized against Sh. flexneri 1a, 2a and 2b respectively. IaM3 is an IgG3 (lambda) type antibody reactive against whole bacteria or the lipopolysaccharide (LPS) only; 2aM1 is an IgG1 (kappa) type antibody which reacts with the polysaccharide component of LPS; 2bM2 is an IgG3 (lambda) type antibody which binds with both the polysaccharide and protein/peptide components of the LPS. The results indicate that specific McAbs with diagnostic potential can be produced using acetone-killed and dried cells as antigen. Ascitic fluid produced in mice using the anti-Sh. flexneri hybridomas was found not to contain higher titres of McAbs against the bacteria. A "nutritionally-deprived" medium was, therefore, constructed which produced greater than five times the concentration of the McAbs than that could be obtained using normal culture fluid.     

Production and characterization of monoclonal antibodies with therapeutic potential against Shiga toxin

Four monoclonal antibodies (T2D1, T3B1, T7B2 and T4C4) were produced and characterized against Sh. dysenteriae type 1. These monoclonal antibodies (McAbs) were shown to be directed against the same or adjacent determinants on the A-subunit of the Shiga toxin. All the anti-Shiga toxin McAbs are IgG1 (kappa) antibodies and have the capacity to neutralize the cytotoxic and neurotoxic effects of the Shiga toxin: the data strongly suggest that both of the activities, associated with the toxin, are due to the same molecule. In addition, the McAbs were found to be highly protective, at low dose, when administered therapeutically to lethally-treated animals.     

Production and characterization of monoclonal antibodies against Trypanosoma cruzi-associated antigens

Nine monoclonal antibodies (mAb) recognizing characteristic antigens in Trypanosoma (T.) cruzi were obtained from hybridomas which had been established by a fusion between mouse myeloma cells and the spleen cells of a mouse immune to the epimastigote form of T. cruzi (Tulahuen strain). Antigen specificities of these mAb were assessed by an indirect immunofluorescence (IIF) method using T. cruzi in different life cycles (amastigote and trypomastigote) and other members of the Trypanosomatidae. The mAb were classified into 3 groups from their reaction patterns to different parasites: 1) The strain specific mAb that reacted only with T. cruzi Tulahuen epimastigote, 2) the species specific mAb that reacted with all T. cruzi strains but not with other species of parasites, and 3) the mAb that were cross-reactive with other species of Trypanosomatidae. Most mAb were specific to epimastigote form of T. cruzi, but some reacted weakly with trypomastigote and amastigote form of the parasites. Immunoblotting and glycolipid analyses of the membrane fraction of homogenized parasites using the mAb identified at least 3 distinct antigenic molecules; those of protein nature having Mr. 43,000 and 58,000 and Mr. 43,000 and 62,000 and molecule(s) of glycolipid nature.     

Bifunctional antibodies and their potential clinical applications

Summary Bifunctional antibodies are monovalent, bispecific, antibody-derived molecules. They have been produced by both chemical and biological means. They are thought to have several advantages over monoclonal antibodies in both immunotherapy and immunodiagnosis. Bifunctional antibodies have been shown to be efficient in the targeting of drugs, toxins, radiolabelled haptens and effector cells on to diseased tissues, primarily cancer cells. In addition, bifunctional antibodies have been used to develop novel immunoassays. The full potential of bifunctional antibodies has yet to be realised.     

Evaluation of the optimal incubation temperature for detecting certain IgG antibodies with potential clinical significance

It is commonly believed that IgG antibodies react optimally at 37 degrees C, but there are few published data supporting this. In this study, 140 antibodies from the Rh, Kell, Duffy, Kidd, and SsU blood groups were studied by a low-ionic-strength solution indirect antiglobulin technique at four different temperatures of incubation: 10, 22, 30, and 37 degrees C. Only titration score differences of greater than 10 were considered significant. None of the 140 antibodies sensitized red cells (RBC) significantly better at 10, 22, or 30 than at 37 degrees C. All antibodies, except one example of anti-c, sensitized RBCs as well at 30 as at 37 degrees C. At 22 degrees C, 100 percent of Kidd and SsU, 94 percent of Kell, and 82 percent of Duffy, but only 49 percent of Rh antibodies sensitized RBCs as well as they did at 37 degrees C. It is possible that these differences reflect the influence of antigenic structures and/or topography on the thermal dynamics of the antigen-antibody bond.     

Bifunctional antibodies and their potential clinical applications.

Bifunctional antibodies are monovalent, bispecific, antibody-derived molecules. They have been produced by both chemical and biological means. They are thought to have several advantages over monoclonal antibodies in both immunotherapy and immunodiagnosis. Bifunctional antibodies have been shown to be efficient in the targeting of drugs, toxins, radiolabelled haptens and effector cells on to diseased tissues, primarily cancer cells. In addition, bifunctional antibodies have been used to develop novel immunoassays. The full potential of bifunctional antibodies has yet to be realised.     

Production and characterization of a cytotoxic monoclonal antibody reacting with rat islet cells.

We have produced a murine monoclonal antibody (F43 A1D2) that binds to the cell surface of both rat islet tumor cells (RINm clone 5F and RINm clone 14B) and normal rat islet cells. This antibody is cytotoxic in the presence of complement for RIN tumor cells as well as A, B, and D pancreatic polypeptide rat islet cells. Antibody A1D2 does not bind to rat thymus cells, pancreatic acinar cells, or fibroblasts. Antigen A1D2 (termed ICSAn-1 [islet cell surface antigen 1]) is a trypsin-sensitive glycoprotein with a molecular mass of approximately 24,000 daltons. In addition to purification of its islet cell antigen, antibody A1D2 can be used to identify and isolate living islet cells from pancreatic digests.     

多种肿瘤标志物在肺癌细胞上的表达及临床应用

目的　研究CD4 0、主要组织相容性抗原 I(MHC Ⅰ )、Fas、细胞粘附分子 1(ICAM 1)、上皮细胞生长因子受体 (EGFR)、P糖蛋白 (PGP)、肺相关蛋白 (LRP)、bcl2、突变型P53 和细胞倍体分析等多个指标在实体肺癌细胞上的表达及其临床意义 ,旨在寻找对肺癌诊断、预后判断及评价发生发展的新指标。方法　通过流式细胞术检测 6 8例实体组织标本上各种指标的表达情况 ,进而分析这些参数与肺癌的临床关系。结果　肺癌组的EGFR、PGP和P53 的表达明显高于对照组 ;CD4 0、EGFR和CD5 4在已转移的原发性肺癌组织中的表达明显高于未发生转移者 (P <0 0 0 5 ) ;这些肿瘤标志物与肺癌组织类型之间无明显相关性 (P >0 0 5 ) ;所有多倍体均为原发性肺癌组织 ,原发性肺癌组织中 86 1%为多倍体 ,良性肺占位和正常肺组织均为二倍体。在生存期超过 5年者 (7例 )中 ,以CD4 0低表达者 (表达率 <80 % )和PGP不表达者 (表达率 <10 % )为主 ;在生存期不足 5年者 (8例 )中 ,以CD4 0高表达者和PGP表达者为主。结论　多倍体的出现是肺癌的特异性指标 ;EGFR、PGP和突变型P53 有望成为诊断肺癌的监测指标 ;CD4 0、EGFR和CD5 4有可能成为判断肿瘤发生发展的有用指标 ;CD4 0和PGP有可能成为判断预后的指标。     

Recombinant forms of glycophorin C as a tool for characterization of epitopes for new murine monoclonal antibodies with anti-glycophorin C specificity

Glycophorin C (GPC) and glycophorin D (GPD) are minor but important components of human RBC membranes. They carry the high-frequency antigens Ge2, Ge3 and Ge4 of the Gerbich blood group system. The epitopes for five new monoclonal antibodies (MoAbs) with anti-GPC specificity were characterized. Two antibodies (4G11 and 5B11) reacted with glycosylated N-terminal epitopes, and three reacted with internal epitopes of GPC. Pepscan analysis showed that the MoAb RB11 required for binding the EPDP sequence, occurring twice in GPC polypeptide chain. The MoAb 7F11 recognized the sequence 13PLSLEPDP20, and the MoAb RB8 did not react with synthetic peptides. Further characterization of the internal epitopes was performed in fluorescence-activated cell sorter (FACS) with the use of recombinant GPC and its variant forms transiently expressed on COS-7 cells. The results indicated that the MoAb RB11 recognized distinctly its target sequence EPDP only in a normal GPC molecule. The reactivity of the MoAb 7F11 with the PLSLEPDP sequence was confirmed and found to be enhanced by the O-glycan at the Ser15 residue. The MoAb RB8 recognized the glycopeptidic epitope in proximity to the Ser15 residue, requiring the presence of O-glycan. The combination of immunochemical techniques with the use of the recombinant forms of GPC has made it possible to define the role of sugar chains in the recognition of peptidic epitopes in glycosylated antigen and sheds new light on the Gerbich system antigens.     

Production and characterization of murine monoclonal antibodies specific for serogroups E1 and E4 Salmonella.

Two anti-Salmonella serogroup E-specific monoclonal antibodies (MAbs) are described. Neither antibody reacted with any of the 58 strains of serogroups A-D Salmonella tested by enzyme immunoassays nor did they react with any of the 21 other species of enterobacteria, 15 species of other Gram-negative bacteria, and 6 species of Gram-positive bacteria. In contrast, all 14 strains of serogroups E1 and E4 Salmonella reacted with both antibodies. Ascitic fluids of these two antibodies agglutinated all 42 strains of serogroups E1 and E4 Salmonella tested by slide agglutination method but did not agglutinate any of the 107 strains of other serogroups of Salmonella. Lysogenic conversion of serogroup E1 Salmonella strains by phages epsilon 15 and epsilon 34 resulted in loss of reactivities of these strains with the MAbs.     

Clinical assessment of monoclonal antibodies for the treatment and diagnosis of colorectal cancers

The clinically useful monoclonal antibodies(Mabs) for colorectal cancers were reviewed. Since 1980's, immunoscintigraphy has been performed for the detection of occult colorectal cancers. However, it may be substituted with the development of positron emission tomography. As for the treatment, some Mabs have been shown to be effective for the adjuvant therapy of postoperative colorectal cancers. Some Mabs to epidermal growth factor receptors(EGFr) are quite promising since they block the functions necessary for the tumor growth and enhance the cytotoxicity of chemotherapy. Recent advances for the development of humanized Mabs will improve the chance of Mabs to be used as an effective adjuvant.     

Characterization and application of a monoclonal antibody with dual specificity for hemoglobins S and C

In an effort to develop a rapid screening immunoassay for the presence of hemoglobin S (Hb S) in cord blood, we have produced a hybridoma (beta s-1) secreting a monoclonal antibody (MAb) with strict specificity for Hb S over hemoglobin (Hb A). A reactivity was observed for hemoglobin C (Hb C) that was weaker than that for Hb S but still greater than 10(3) times greater than that seen for Hb A. Application of this antibody in a dot blot assay provided for a rapid (50-minute) single-step confirmatory test for Hb S, Hb C, or both in cord blood hemolysates. The sensitivity of the test would allow for mass screening of cord blood hemolysates (40 mg total hemoglobin per milliliter) and detection of Hb S, Hb C, or both at concentrations greater than or equal to 1%.     

人血清白蛋白单克隆抗体的制备、鉴定与初步应用

目的制备人血清白蛋白(HSA)单克隆抗体(单抗)并以之制备亲和层析柱去除甘露聚糖结合凝集素(MBL)制剂中污染的HSA。方法采用杂交瘤技术制备单抗,通过ELISA、Western-blotting鉴定其特性;制备亲和层析柱去除MBL制剂中的污染HSA。结果筛选出4株稳定分泌HSA单抗的杂交瘤细胞株1C11、2E9、3A5和4H1,单抗亚类均为IgG1,亲和常数分别为7.19×109、1.46×109、6.94×109和2.28×1010;1C11与3A5的识别位点相似或相同,其他单抗识别位点均不同;4H1可与兔血清发生交叉反应,余者与其他种属血清均无交叉反应。结论所制备的HSA单抗1C11、2E9、3A5能特异结合HSA且与其他种属血清无交叉反应,抗HSA单抗亲和层析柱可用于去除人血清蛋白制剂中的污染HSA。     

心肌钙蛋白T单克隆抗体制备及应用

目的　人心肌肌钙蛋白T单克隆抗体制备及在心肌梗死诊断中的初步应用。方法　在研究心肌特异性肌钙蛋白T(cTnT)提纯的基础上成功地制备出 3株特异性抗cTnT单克隆抗体 ,用其中的 2株Mcab建立了检测cTnT双单克隆抗体的夹心ELISA法。结果　对 30名健康献血员和 32例心肌梗死患者血中cTnT水平进行临床检测 ,正常人血清cTnT浓度平均为 (0 .12 7± 0 .0 84) μg L ,最低为 0 ,最高为 0 .42 μg L ,以血清cTnT >0 .40 μg L为诊断心肌梗死的临界值。 32例患者血清cTnT值平均为 (2 .10 8± 1.974) μg L ,患者血清cTnT分别用BM试剂盒及ELISA法检测 ,经统计学处理 ,二者相关系数为 0 98,符合临床检测要求。结论　该法灵敏度、准确度和精确度较高 ,具有推广价值