Production and properties of a monoclonal antibody against human epidermal growth factor

A monoclonal antibody against human epidermal growth factor (hEGF) was obtained from a mouse hybridoma cell line. The purified monoclonal antibody from the ascites fluid of a mouse injected with one of the cell lines was specific for hEGF and did not cross-react with mouse EGF (mEGF). Its Kd value for hEGF was 1.4 X 10(-9) M. This monoclonal antibody inhibited the biological activities of hEGF, including its binding to the receptor of BALB/3T3 cells and its stimulation of DNA synthesis in the cells, but did not affect the activities of mEGF. The monoclonal antibody completely inhibited DNA synthesis stimulated by human urine from a patient without a tumor, but only partially inhibited the stimulatory activity in urine from a tumor-bearing patient.     

抗人B7-H1单克隆抗体的制备和鉴定

目的:采用杂交瘤技术制备抗人B7-H1单克隆抗体,并对其进行鉴定。方法:经抗原免疫的小鼠脾细胞与小鼠骨髓瘤细胞以常规方法融合;用间接ELISA法筛选分泌抗体的杂交瘤细胞株;阳性克隆用有限稀释法获得稳定分泌抗人B7-H1单克隆抗体的杂交瘤细胞株;扩增杂交瘤细胞注射进小鼠腹腔后制备腹水;纯化腹水中的单克隆抗体并对其亚型进行鉴定;用间接ELISA法测抗体效价;将肺癌组织制成石蜡切片,用抗人B7-H1抗体进行免疫组化染色。结果:获得1株稳定分泌抗人B7-H1单克隆抗体的杂交瘤细胞株,所分泌的单抗类型为IgG1;抗体效价为1×108,纯化后的抗体含量为6.76g/L;免疫组化实验中,单抗可与肺癌组织表面的B7-H1蛋白特异地结合。结论:制备了人B7-H1单克隆抗体,为B7-H1检测试剂盒的研制奠定了基础。     

Development of a mouse antiperoxidase secreting hybridoma for use in the production of a mouse PAP complex for immunocytochemistry and as a parent cell line in the development of hybrid hybridomas

Mouse antibodies are increasingly used as primary antibodies for immunocytochemistry as more mouse monoclonal antibodies are being produced. The localisation of these antibodies by the PAP technique requires mouse antiperoxidase antibody. A monoclonal antiperoxidase would obviate the limitations of production of a polyclonal mouse antiperoxidase. This paper describes the development of a mouse hybridoma producing such an antibody (MAP A6-2) and the use of this antibody to localise a number of mouse primary antibodies by the PAP technique for both light and electron microscopy. The antibodies localised include monoclonal antienkephalin and antityrosine hydroxylase. MAP A6-2 had a higher affinity in immuno-diffusion experiments and gives slightly better staining with an horse radish peroxidase of a different type from that used for immunisation. Staining was optimum with horse radish peroxidase type X whereas horse radish peroxidase type VI was used for immunisation. Also described is the production of a HAT sensitive variant cell line allowing the possibility of using this hybridoma as a parent cell line for the production of hybrid hybridomas secreting bi-specific antibodies.     

人IL17-RD胞外区真核表达及其单克隆抗体的制备

为了进一步研究白介素17受体D (IL-17RD) 在IL-17信号的调节作用,探索是否可以通过单克隆抗体阻断IL-17RD介导的IL-17信号通路而缓解自身免疫疾病,利用昆虫表达载体从Sf9细胞中表达纯化人IL-17RD-ECD蛋白,免疫Balb/C小鼠30 d,取小鼠脾脏细胞并与小鼠骨髓瘤细胞SP2/0进行融合,应用有限稀释法进行筛选,经过克隆化后筛选到一株能稳定分泌抗IL-17RD-ECD的杂交瘤细胞株1F8。经过初步鉴定,该细胞株分泌的抗体类型为IgG1+kappa类,经过Western blot     

Development of a mouse antiperoxidase secreting hybridoma for use in the production of a mouse PAP complex for immunocytochemistry and as a parent cell line in the development of hybrid hybridomas

Summary Mouse antibodies are increasingly used as primary antibodies for immunocytochemistry as more mouse monoclonal antibodies are being produced. The localisation of these antibodies by the PAP technique requires mouse antiperoxidase antibody. A monoclonal antiperoxidase would obviate the limitations of production of a polyclonal mouse antiperoxidase. This paper describes the development of a mouse hybridoma producing such an antibody (MAP A6-2) and the use of this antibody to localise a number of mouse primary antibodies by the PAP technique for both light and electron microscopy. The antibodies localised include monoclonal antienkephalin and antityrosine hydroxylase. MAP A6-2 had a higher affinity in immuno-diffusion experiments and gives slightly better staining with an horse radish peroxidase of a different type from that used for immunisation. Staining was optimum with horse radish peroxidase type X whereas horse radish peroxidase type VI was used for immunisation. Also described is the production of a HAT sensitive variant cell line allowing the possibility of using this hybridoma as a parent cell line for the production of hybrid hybridomas secreting bi-specifie antibodies.     

Macrophage antigens associated with adhesion: identification by a monoclonal antibody specific for Lewis lung carcinoma cells

A monoclonal antibody specific for Lewis lung carcinoma (3LL) cells (Mab 5B5) was found to recognize antigens expressed on murine macrophages and on a macrophage hybridoma line upon cell adhesion on plastic surfaces. These antigens were also present on the surface of murine macrophage tumor M5076 cells which develop solid tumors and metastases. The M5076 tumor cells freshly isolated from the primary tumor and from hepatic metastases strongly bound Mab 5B5 but lost this capacity after adhesion. Freshly isolated thioglycolate-elicited peritoneal mouse macrophages were not labeled by Mab 5B5; however, after 1 h of adhesion, 50% of the adherent macrophages were directly incubated with Mab 5B5 prior to harvesting by scraping. Permeabilization of peritoneal macrophages by saponin showed that the antigens recognized by Mab 5B5 were present inside the cells before adhesion. Similar results were obtained with the 2C11-12 macrophage hybridoma cells. P388D1 cells (a weakly adherent macrophage tumor cell line), HL60 cells (a human promyelocytic cell line), and human monocytes were poorly labeled without permeabilization but were strongly labeled by Mab 5B5 upon permeabilization. The specificity of the monoclonal antibody in relation to the adherence capacity of these cells is discussed.     

抗胶质瘤细胞单克隆抗体SZ-38及其识别抗原的生化特征

本文以人脑胶质瘤细胞系SHG-44为抗原,利用杂交瘤技术建立了一株恒定地分泌抗胶质瘤细胞单克隆抗体(McAb)的杂交瘤细胞株SZ-38。McAb SZ-38与9/10胶质瘤细胞系发生强结合反应。而与淋巴细胞、ABO型红细胞等所有正常血液细胞及绝大多数被检测肿瘤细胞系无反应。经免疫转移电泳及免疫沉淀法鉴定,该McAb识别抗原为胶质瘤细胞膜上Mw47,000糖蛋白。应用SPA-Sepharose 4B提纯MeAb SZ-38,再把纯化抗体交联于Sepharose 4B,以McAb亲和层析法提取SZ-38抗原,经SDS-PAGE证实其有较高的纯度。     

Immunohistochemical and immunoelectron microscopic analyses of alpha-amylase isozymes in human intrahepatic biliary epithelium and hepatocytes.

The expression and localization of the pancreatic and salivary isozymes of alpha-amylase in the intrahepatic biliary epithelium and hepatocytes were examined by the immunohistochemical method with polyclonal and monoclonal antibodies in 45 normal autopsied human livers. Immunoelectron microscopic studies with the protein A-gold method were performed with the monoclonal antibodies (MAb) on seven of the livers. The intrahepatic biliary system was divided into large ducts, septal ducts, interlobular ducts, bile ductules, and peribiliary glands. Immunohistochemically, pancreatic isozyme was observed in the supranuclear cytoplasm of the epithelium of large ducts, septal ducts, and peribiliary glands in almost all livers. Interlobular ducts expressed pancreatic isozyme in only four (9%) livers. Bile ductules and hepatocytes were negative for pancreatic isozyme in all cases. Expression of salivary isozyme was observed in the supranuclear cytoplasm of the epithelium of large ducts, septal ducts, interlobular ducts, bile ductules, and peribiliary glands in almost all livers, although the expression in interlobular ducts and bile ductules was weak. Hepatocytes were weakly positive for salivary isozyme. Immunoelectron microscopy revealed that both pancreatic and salivary isozymes were located in the supranuclear cytoplasm of the epithelium of large ducts, septal ducts, and peribiliary glands, and that hepatocytes had no pancreatic isozyme but contained salivary isozyme. These data suggest that pancreatic and salivary isozymes of alpha-amylase are produced by the intrahepatic biliary epithelium and secreted into intrahepatic biliary lumens, and that they may play an important role in the physiology of the intrahepatic biliary tree and hepatic bile. It is also suggested that hepatocytes produce a small amount of salivary alpha-amylase that may be secreted into the biliary tree.     

Anti-major histocompatibility complex immunity detected prior to intentional alloimmunization. II. Monoclonal H-2-specific antibodies obtained from an unimmunized C57BL/KaLwRij (H-2b) mouse

A monoclonal 'natural' anti-H-2 IgM antibody produced by a hybridoma cell line OL-3.17 (H-2 m. 209) is described. The OL-3.17 monoclonal antibody was obtained by hybridization of spleen B cells from an unimmunized C57BL/Ka (H-2b) mouse in the serum of which simultaneously an IgM kappa paraprotein of high concentration and a natural H-2-specific antibody of high titer was detected. The monoclonal antibody OL-3.17 reacted strongly with H-2d and H-2s and weakly with H-2k,q,r lymphocytes, thereby detecting a hitherto unknown H-2 public determinant. The target molecules for OL-3.17 cocapped with class-I H-2 antigens, but immunoprecipitation of H-2 antigens was not achieved. This is the first monoclonal H-2-specific antibody obtained from a mouse without intentional immunization and, with high probability, was derived from a B-cell clone which produced natural H-2-specific antibodies detectable in the serum of the original mouse.     

Isolation of a monoclonal antibody viral polypeptide VP2 of simian virus 40

A stable hybridoma cell line, IIG8-203-2, that secretes a monoclonal antibody of the immunoglobulin subclass M was obtained by fusion of mouse myeloma cells with spleen cells of mice that had been immunized with the viral polypeptide VP2 of simian virus 40. The monoclonal antibody recognizes viral polypeptides that migrate with VP2 polypeptides in a sodium dodecyl sulfate-polyacrylamide gel. It also recognizes two intracellular polypeptides (29,000 and 37,000 daltons) in a detergent-insoluble fraction extracted 30 h after virus infection of TC7 cells.     

Monoclonal antibodies against Mycoplasma hyorhinis: A secondary effect of immunization with cultured cells

Monoclonal antibodies were prepared against two different human tumour cell lines, the melanoma cell line SK-Mel-25 and the acute lymphoblastic leukemia T cell line CCRF-CEM. Presence of antibodies against human tumour cells in the supernatants of hybridoma cultures was tested by binding of ¹²⁵I-F(ab′)₂ anti-mouse IgG. On two occasions a hybridoma culture, initially selected for subsequent cloning as it seemingly produced antibodies against tumour cells, was later found to produce monoclonal antibodies specific for Mycoplasma hyorhinis. In immunofluorescent staining patchy structures were visible which seemed to be attached to the cell surface. By combined staining with FITC-conjugated anti-mouse immunoglobulin for monoclonal antibody, Evans blue for cytoplasm and Hoechst compound no. 33258 for DNA, the reaction against mycoplasma could be recognized. These results demonstrate that if cultured cells are used for preparation of monoclonal antibodies, there is a good chance that the selected hybridomas may produce antibodies against ‘culture artifacts’ such as mycoplasmas, in addition to the target antigens. Thus mycoplasma contamination of cell cultures poses a serious problem in the hybridoma research and the testing system for antibody specificity should be carefully monitored.     

A human monoclonal antibody to cytomegalovirus (CMV)

We report the development of a human monoclonal antibody to cytomegalovirus (CMV) produced from a human X human hybridoma. This hybrid was developed by fusion of an EBV-transformed cell line making antibody to CMV and a human lymphoblastoid cell line WI-L2. The antibody is directed to a CMV-specific antigen primarily in the nucleus of CMV-infected human fibroblasts. It cross-reacts with at least 10 different strains of CMV and may provide a method for the rapid in vitro diagnosis of CMV infections. The production of CMV-specific human man monoclonal antibodies from human-human hybridomas for future therapeutic use is now technically feasible with this specific method of production.     

大鼠脑红蛋白(NGB)可溶性原核表达和单克隆抗体制备及鉴定

脑红蛋白 (NGB)是新发现的脑特异的携氧蛋白 .为了对其功能进行深入的研究 ,应用已构建大鼠NGB基因编码区原核表达载体pGEX 4T 2 NGB转化的大肠杆菌BL2 1 ,获得可溶性表达产物 .用谷胱甘肽S 转移酶 (GST)亲和层析柱纯化后作为抗原 ,免疫Balb c小鼠 .通过传统的细胞融合方法制备了抗大鼠NGB的单克隆抗体 ,并对全部 4株单抗进行了亚类和亚型、效价和特异性鉴定 .为NGB结构与功能的深入研究提供了重要工具     

Nematospiroides dubius: passive transfer of protective immunity to mice with monoclonal antibodies

Nine hybridoma cell lines secreting monoclonal antibodies specific for Nematospiroides dubius were produced by fusion of the mouse myeloma cell line NS-1 to either spleen cells or mesenteric lymph node cells from mice repeatedly infected with N. dubius. Seven of the antibodies were identified as IgM and two as IgG1. Each monoclonal antibody bound to polypeptide epitopes on both infective larvae (L3) and adult worms. However, five antibodies bound preferentially to L3 and three to adult worms. All nine antibodies reacted with high molecular weight protein antigens. Passive protective immunity in Balb/c mice was demonstrated with monoclonal antibodies Nd2 and Nd3 in ascites fluid which stunted both male and female worms and reduced parasite fecundity.     

A New Monoclonal Antibody that Recognizes 180 kDa Polypeptide Expressed on Early Mouse Embryos and Mouse Embryonal Carcinoma Cells

A hybridoma clone 1F7 producing a monoclonal antibody against OTF9-63 mouse embryonal carcinoma cell line was isolated with the aid of the intrasplenic primary immunization technique. This antibody was capable of recognizing embryonal carcinoma cell lines originated from certain mouse strains, including 129/Sv, but not those which were established from other strains as well as human and other murine cell lines except FM3A, a mouse mammary carcinoma cell line. Indirect immunofluorescence study revealed that 1F7 antigen was expressed on early mouse embryos in a stage specific manner. In order to characterize the 1F7 antigenic molecules, we analyzed both glycoshingolipids and glycoproteins recovered from OTF9-63 by means of immunostaining after thin layer chromatography and SDS-polyacrylamide gel electrophoresis followed by Western blotting, respectively. It was concluded that 1F7 antigenic determinants were carried by 180 kDa polypeptides. One of the most interesting characteristics of 1F7 antigen is complete failure to express on the embryonic ectoderm of 5.5d and 6.5d embryos, one of cell types most closely related to embryonal carcinoma cells. 1F7 antibody may help analyse the process of teratocarcinogenesis in 129/Sv mice.     

Identification of a glycoprotein, gp21, of adult T cell leukemia virus by monoclonal antibody

A mouse hybridoma cell line producing monoclonal antibody, F10, was established from mice hyperimmunized with cells bearing adult T cell leukemia (ATL) virus (ATLV). F10 antibody reacted with an ATLV structural polypeptide ( gp21 ) with a m.w. of 21,000 that was glycosylated on cell surfaces. The gp21 was expressed on cell surfaces of all ATL-associated antigen (ATLA)-positive human cell lines but not on ATLA-negative cell lines nor peripheral blood leukocytes stimulated with mitogens. The gp21 was also detected with anti-ATLA-positive human serum, and the binding of F10 antibody to ATLA-positive cell surfaces was significantly blocked by pretreatment with anti-ATLA-positive human sera. Double immunofluorescence staining with F10 antibody and anti-ATLA-positive human serum caused co-capping on cell surfaces, which suggests that gp21 co-exists with other ATLV antigens expressed on cell surfaces. Immunoprecipitation studies also suggested that the gp21 is a minor component of the ATLV envelope.     

Establishment of hybridoma secreting human monoclonal antibody against hepatitis B virus surface antigen

The HAT (hypoxanthine, aminopterin, thymidine) sensitive and ouabain resistant human B lymphoblastoid cell line TAW-925 was obtained from 6-thioguanine resistant B lymphoblastoid cell line WI-L2. Hybridomas were obtained at a high frequency (10(-4)-10(-5) when TAW-925 was hybridized with cells transformed with Epstein-Barr virus. Using TAW-925 as a parental cell line, we have obtained a hybridoma which stably secretes human monoclonal antibody against hepatitis B virus surface antigen.     

A monoclonal antibody that precipitates the glycoprotein receptor for epidermal growth factor is directed against the human blood group H type 1 antigen

Monoclonal antibody 101 produced by a hybridoma obtained by fusion with NS-1 myeloma cells of spleen cells from a mouse immunized with the human epidermoid carcinoma cell line, A431, specifically precipitates epidermal growth factor receptor, a glycoprotein of 170,000 Mr solubilized from A431 cell membranes (Richert, N. D., Willingham, M. C., and Pastan, I. H. (1983) J. Biol. Chem. 258, 8902-8907). The antibody also binds to neutral glycolipids extracted from A431 cells as evidenced by solid phase radioimmunoassay and by autoradiography. Binding of antibody to its target is inhibited by lacto-N-fucopentaose I but not by 2'-fucosyllactose or related oligosaccharides. Thus, antibody 101 is probably directed against the human blood group H type 1 sugar sequence Fuc alpha 1-2Gal beta 1-3GlcNAc . . .. This sequence presumably occurs on the epidermal growth factor receptor. Monoclonal antibody 102 produced by another hybridoma from the same fusion has the same cell specificity as antibody 101 and also binds to neutral glycolipids. However, binding of antibody 102 to its target is inhibited by 2'-fucosyllactose and not by lacto-N-fucopentaose I or related oligosaccharides. Thus, antibody 102 is probably directed against the human blood group H type 2 sugar sequence Fuc alpha 1-2Gal beta 1-4GlcNAc . . .. Antibody 102 does not precipitate solubilized epidermal growth factor receptor. Both antibodies bind to neutral glycolipids extracted from human erythrocytes belonging to blood group O but not to neutral glycolipids extracted from human erythrocytes with the "Bombay" phenotype.     

Chemical structure of carcinoma ganglioside antigens defined by monoclonal antibody C-50 and some allied gangliosides of human pancreatic adenocarcinoma

A hybridoma, C-50, obtained by fusion of mouse myeloma cells with spleen cells from a mouse immunized with cells from the colorectal carcinoma cell line COLO 205, produced antibodies that detected ganglioside antigen in human adenocarcinomas in many organs. The major ganglioside antigen fraction isolated from liver metastases of a pancreatic adenocarcinoma, behaving as a homogenous band on thin-layer chromatography, consisted of three different gangliosides. One of them, A (25%), had the same carbohydrate structure as the ganglioside antigen defined by monoclonal antibody 19-9, NeuAc alpha 2-3Gal beta 1-3(Fuc alpha 1-4)GlcNAc beta 1-3Gal beta 1-4Glc-Cer(Fuc-3'-isoLM1) Magnani, J.L., Nilsson, B., Brockhaus, M., Zopf, D., Steplewski, Z., Koprowski, H. and Ginsburg, V. (1982) J. Biol. Chem. 257, 14365-14369). The major ganglioside, B (60%), was the isomeric hexasaccharide ganglioside (NeuAc alpha 2-3Gal beta 1-4(Fuc alpha 1-3)GlcNAc beta 1-3-Gal beta 1-4Glc-Cer(Fuc-3'-LM1) and the third ganglioside, C, was 6'-LM1, NeuAc alpha 2-6Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc-Cer (15%). Ganglioside B, isolated from human kidney, did not react with the C-50 MAb. Based on this result and on studies of COLO 205 cell induced tumours where the ganglioside antigen fraction only consisted of A, it is suggested that the C-50 MAb defines an antigen determinant present in A.     

茶尺蠖核型多角体病毒多角体蛋白单克隆抗体的制备

为了建立一种基于免疫反应检测茶尺蠖核型多角体病毒的方法,以纯化后的茶尺蠖核型多角体病毒作为抗原,免疫BALB/c小鼠,将小鼠脾脏细胞与小鼠骨髓瘤细胞Sp2/0融合,经间接ELISA筛选及克隆得到了一株稳定分泌单克隆抗体的杂交瘤细胞株,命名为7D3。同时克隆并在大肠杆菌中表达了EoNPV多角体蛋白基因,获得重组多角体蛋白。经Western blotting鉴定,该抗体可与EoNPV的多角体蛋白特异性结合。利用制备EoNPV多角体蛋白的单克隆抗体,建立了间接ELISA测定EoNPV的方法。