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1.
Ricin-resistant human T-cell hybridomas producing interferon gamma   总被引:1,自引:0,他引:1  
Ricin-resistant variants of the SH9 T-cell line were selected after growth of this line in medium containing toxic amounts of ricin, a lectin derived from Ricinus communis. The ricin-resistant SH9 lines, SH9.R0 and SH9.R1, were demonstrated to be deficient in cell surface ricin-binding sites, but otherwise had the cellular phenotype of SH9 cells. Ricin-resistant T-cell hybridomas were prepared by fusion of SH9.R0 and SH9.R1 with activated T-lymphocytes. The presence of ricin in the selection medium rapidly killed unfused T-lymphocytes and prevented cell transformation by human T-cell leukaemia virus type 1 (HTLV-1) which is shed by the SH9.R0 and SH9.R1 cells. This ensured that the cells growing out were indeed hybridomas. Ricin-resistant T-cell hybridomas were characterised and also shown to lack cell surface receptors for ricin. Analysis of T-cell surface markers indicated that the T-cell hybridomas could be the result of fusions between SH9.R1 cells and T-helper lymphocytes or T-suppressor lymphocytes. All of the T-cell hybridomas prepared in this study spontaneously produced interferon gamma (IFN gamma).  相似文献   

2.
A B Lang  U Schuerch  S J Cryz 《Hybridoma》1991,10(3):401-409
To facilitate the production and purification of human monoclonal antibodies, we evaluated the ability of human hybridomas to adapt to chemically defined-serum free media. From a panel of human hybridomas secreting antibody against serotype specific lipopolysaccharide determinants of gram-negative bacteria, the growth and secretion properties of the two hybridomas producing antibodies against two strains of Pseudomonas aeruginosa, 4-8KH15 and 4-10KH139, were analysed. Both clones did not grow in protein-free medium. However, it was possible to adapt them to serum-free media consisting of a basal medium supplemented with insulin, transferrin, ethanolamine, and selenite. Antibody secretion rates were equal (4-8KH15: 26-31 micrograms IgM/10(6) cells/day) or higher (4-10KH139: 58-90 micrograms IgM/10(6) cells/day) in serum-free media as compared to conventional serum-supplemented medium. Our studies suggest that adaptation of the described hybridomas to selected serum-free media results in an antibody production which is very high as compared with reports in comparable systems. The establishment of these conditions will significantly facilitate the production of large amounts of human monoclonal antibodies which is a prerequisite for a therapeutical application.  相似文献   

3.
A collection of hybridomas from C57BL/6 mice producing antibodies to dextran B512 was analysed and found to reflect the immune response in vivo with regard to immunoglobulin class expression, T cell dependency and antibody affinity. IgM-, IgG-3, and IgG-2b, and IgA-producing hybridomas were found. IgG3-producing hybridomas were obtained from nude mice, indicating T cell independent IgG3 synthesis. All monoclonal antibodies were of kappa light chain. A major anti-dextran idiotype was expressed in many monoclonals. Secondary immune responses to dextran were also suppressed at the hybridoma cell level. However, hybridomas from secondary responses produced antibodies expressing the major idiotype, suggesting that anti-idiotype mediated suppression was not responsible for the reduced secondary response. Most monoclonals belonged to the VHJ558 family, but the IgG3-producing hybridomas showed a preferential use of genes from the VHX24 family. All monoclonals were directed against internal structures of the dextran molecule. The affinity for dextran of the IgG antibodies produced in secondary immune responses was drastically increased, even when the mice were immunized with thymus-independent forms of dextran, indicating that T helper cells need not be involved in affinity maturation of the immune response.  相似文献   

4.
Hybridomas were derived from lipopolysaccharide-reactive splenic B cells of adult germ-free BALB/c mice fed a chemically defined ultrafiltered "antigen-free" diet (GF-CD) and from splenic B cells of 5-day-old conventional (CV-NEO) BALB/c mice. The monoclonal antibodies (mAb) from both collections of hybridomas were tested for reactivity against a large panel of antigens of exogenous and endogenous origin. As a source of natural exogenous antigens 36 different bacteria and 9 different viruses were used, while as endogenous antigens frozen tissue sections of stomach, liver and kidney, the Hep-2 cell line and the anti-idiotopic mAb Ac38 and Ac146 were used. In both collections of mAb approximately 70% reacted with one or more bacterial antigens, while no reactivity could be detected against the viral antigens. Of the GF-CD and CV-NEO hybridomas, 16% and 19%, respectively, reacted with one or more frozen tissue sections. Overall 56% and 68% of the GF-CD and CV-NEO hybridomas, respectively, were producing multireactive antibodies reactive to several exogenous and/or endogenous antigens. Among the GF-CD hybridomas a correlation was found between multireactivity and the usage of the VH gene family PC7183. In CV-NEO hybridomas, however, the preferential utilization of the VH gene family PC7183 was found among both mono- and multireactive hybridomas. The results suggest (a) that the actual B cell repertoire of neonatal mice consists of a large proportion of multireactive B cells which are reactive with autoantigens and bacterial antigens, but not viral antigens and (b) that in antigen-deprived mice the neonatal repertoire is largely preserved during maturation of the mice.  相似文献   

5.
Epithelial cells of the respiratory and gastrointestinal tracts are extremely vulnerable to the cytotoxic effects of ricin, a Shiga-like toxin with ribosome-inactivating properties. While mucosal immunity to ricin correlates with secretory immunoglobulin A (IgA) antibody levels in vivo, the potential of IgA to protect epithelial cells from ricin in vitro has not been examined due to the unavailability of well-defined antitoxin IgA antibodies. Here we report the characterization of four monoclonal IgA antibodies (IgA MAbs) produced from the Peyer's patches and mesenteric lymph nodes of BALB/c mice immunized intragastrically with ricin toxoid. Two IgA MAbs (33G2 and 35H6) were active against ricin's lectin subunit (RTB), and two (23D7 and 25A4) reacted with the toxin's enzymatic subunit (RTA). All four IgA MAbs neutralized ricin in a Vero cell cytotoxicity assay, blocked toxin-induced interleukin-8 release by the human monocyte/macrophage cell line 28SC, and protected polarized epithelial cell monolayers from ricin-mediated protein synthesis inhibition. 33G2 and 35H6 reduced ricin binding to the luminal surfaces of human intestinal epithelial cells to undetectable levels in tissue section overlay assays, whereas 23D7 had no effect on toxin attachment. 23D7 and 25A4 did, however, reduce ricin transcytosis across MDCK II cell monolayers, possibly by interfering with intracellular toxin transport. We conclude that IgA antibodies against RTA and RTB can protect mucosal epithelial cells from ricin intoxication.  相似文献   

6.
Ricin is a highly toxic, dichain ribosome-inactivating protein present in the seeds of Ricinus communis (castor), grown principally as a source of high quality industrial lubricant and as an ornamental. Because of its presence in industrial byproducts and its documented use for intentional poisoning, there is a need for analytical methodology to quantify ricin in both castor extracts and food matrices. We developed a panel of monoclonal antibodies to ricin, with most having strong cross-reactivity with RCA-1, a homologous but less toxic castor agglutinin. Some of the IgM-producing hybridomas appeared to produce a second, IgG isotype and were further analysed by fluorescence-activated cell sorting. The antibodies were effective in various ELISA formats, many with IC50's in the range of 0.1–10 ng/mL and minimal matrix effects in skim milk. Assay specificity can be adjusted for analytical needs by varying the combination of antibodies in a sandwich ELISA format.  相似文献   

7.
Eleven mouse monoclonal antibodies directed against epitopes on CNBr peptides of the major sialoglycoconjugate of the human red blood cell, glycophorin A, have been produced by hybridomas derived from P3-X63-Ag8.653 myeloma cells and spleen cells from BALB/c mice immunized with purified glycophorin. The monoclonal antibodies could be divided into four groups according to their reactivities with CNBr peptides in a direct ELISA assay: one antibody (6B5) that binds solely to the aminoterminal octapeptide (CNBr3); two antibodies (8F10 and 9C3) that bind to CNBrl (residues 9-81); two antibodies (3D2 and 4C6) that are reactive with CNBr2, The C-terminal portion of the molecule (residues 82-131); six antibodies (1B4, 4C3, 4E7, 7B10, 7C11 and 9D6) which are cross-reactive with an epitope on both CNBr1 and CNBR3 glycopeptides. This cross-reactive epitope(s) appears to involve both carbohydrate and protein residues.  相似文献   

8.
9.
人酸性成纤维细胞生长因子单克隆抗体的制备   总被引:2,自引:0,他引:2  
王芳  徐秀英 《免疫学杂志》1993,9(4):267-270
以纯化的重组人酸性成纤维细胞生长因子(haFGF)为抗原,免疫BALB/C小鼠,取脾细胞与SP2/0细胞融合,获得一株稳定分泌抗haFGF的单克隆抗体的杂交瘤细胞株,基DNA含量为脾细胞与SP2/0细胞DNA含量之和,所分泌抗体为小鼠IgG1亚类,免疫印迹显示:此单抗只与大肠杆菌中的haFGF有结合,与牛aFGF则没有结合反应。  相似文献   

10.
In an attempt to develop highly efficient antibody—drug conjugates for passive immunotherapy of cancer the A-chain of the potent toxin, ricin, was coupled to antibodies in order to render them specifically cytotoxic for target cells without the participation of complement. The antibody—toxin conjugates (immunotoxins) showed no loss of antibody or A-chain activity. In vitro, highly purified A-chain was about 5000 times less toxic on HeLa cells than whole ricin. Unconjugated A-chain made no difference between TNP-HeLa and HeLa cells but when coupled to anti-DPN antibodies it became about 500 times more cytotoxic to TNP-HeLa cells than to HeLa cells. In vivo A-chain (LD50: 20 mg/kg) was about 3000 times less toxic than whole ricin and treatment with immunotoxin significantly inhibited tumor take and tumor growth of TNP-HeLa cells in nude mice.  相似文献   

11.
12.
Secretory immunoglobulin A (IgA) antibodies directed against cholera toxin (CT) are thought to be important in resistance to oral challenge with virulent Vibrio cholerae, although alternative mechanisms for protection of intestinal epithelia against CT-induced fluid secretion have been proposed. The ability of anti-CT IgA to block the effects of CT on human enterocytes has not been directly tested because of the lack of a well-defined in vitro intestinal epithelial cell system to directly measure toxin action and the limited availability of purified anti-CT IgA antibodies. We have generated hybridomas that produce monoclonal IgA and IgG antibodies directed against CT by fusion of Peyer's patch cells with mouse myeloma cells after oral-systemic immunization of mice with CT and CT B-subunit protein. All of the anti-CT antibodies recognized the B subunit. Three clones (designated anti-CTB IgA-1, IgA-2, and IgA-3) which produced IgA antibodies in dimeric and polymeric forms were selected. Checkerboard immunoblotting demonstrated that IgA-1 recognized an epitope distinct from that recognized by IgA-2 and IgA-3 and that none of the antibodies were directed against the binding site of GM1, the intestinal cell membrane toxin receptor. The protective capacity of these IgAs was tested in vitro with human T84 colon carcinoma cells grown on permeable supports as confluent monolayers of polarized enterocytes. When each anti-CTB IgA was mixed with 10 nM CT and applied to the apical surfaces of T84 cell monolayers, all three IgAs blocked CT-induced Cl- secretion in a dose-dependent manner and completely inhibited binding of rhodamine-labelled CT to apical cell membranes. Thus, monoclonal anti-CTB IgA antibodies are sufficient to protect human enterocytes in vitro against CT binding and action.  相似文献   

13.
Pathogenic mechanisms of the demyelinating encephalopathy featuring the nervous phase of human African trypanosomiasis (HAT) are largely unknown. They might include autoimmune disorders. A variety of autoantibodies is detected during the disease and we have previously evidenced anti-galactocerebroside (GalC) antibodies in the serum and cerebrospinal fluid (CSF) from patients in the nervous stage (stage II) of HAT. We now show that anti-GalC antibodies recognize an antigen located on the parasite membrane and common to different strains of trypanosomes. By using affinity chromatography with a rabbit anti-GalC antiserum, a 52-kD proteolipid was isolated from the membrane of Trypanosoma brucei (T. b.) brucei AnTat 1.9, AnTat 1. 1E, and T. b. rhodesiense Etat 1.2/R and Etat 1.2/S. Antibodies directed against this antigen were found in the CSF from patients with nervous stage HAT. These CSF also contained anti-GalC antibodies and adsorption with the proteolipid decreased anti-GalC reactivity. Immunization of mice with this antigen induced the production of antibodies which cross-reacted with GalC but no protection from experimental infection with T. b. brucei. These data support the hypothesis that anti-GalC antibodies detected in the CSF from HAT patients might be induced by molecular mimicry with a parasite antigen.  相似文献   

14.
Monoclonal antibodies against ricin toxin were produced and some of their properties investigated. Antibodies 196 C12 and 197 C7 raised against A-chain reacted with a CnBr fragment probably comprised between amino acid 254 and 262. Antibodies 193 A9, 196 A3, and 191 B7 recognized a 6-7 kD CnBr peptide. A second set of antibodies was raised against whole inactivated ricin. Most of them bound in a solid phase radioimmunobinding assay only to ricin and few had a low activity against purified A-chain. Different effects were noted on toxin action in cultured leukemic cells. If cells were preincubated with ricin followed by antibodies, MAb 207 E5 and 216 B3 had a strong enhancing effect on toxin action. If antibodies and toxin were mixed and then added to sensitive cells, antibody 207 E5 gave a strong protection while 216 B3 maintained its enhancing activity. The effect of antibody 216 B3 was further investigated by quantitative cloning experiments which showed that toxin had a fivefold enhancement in its activity by a preincubation with this antibody. Binding of fluoresceinated ricin to leukemic target cells was inhibited by a preincubation with antibody 207 E5 while antibody 216 B3 had no effect.  相似文献   

15.
Hybridomas producing antibodies against soluble antigens have in most cases been difficult to establish. After fusion of myeloma cells with spleen cells obtained from mice immunized with a soluble protein, hybridomas secreting specific antibodies have been observed to occur very rarely among non-specific hybridomas. We found that the frequency of specific hybridomas correlates directly with the increase over background of the frequency of blast and/or plasma cells in the spleen (measured by cell size analysis) after antigenic stimulation. High yields of specific hybridomas were obtained simply by following a novel immunization technique consisting of several conventional preimmunization courses followed by 4 very high doses of antigen in saline on each of the last 4 days before fusion.  相似文献   

16.
To prepare hybridomas secreting monoclonal antibodies (MoAb) against human alpha-interferon (alpha-IFN), BALB/c mice were immunized with IFN produced in Namalwa cells. Native alpha-IFN, as well as partially purified or on cellulose adsorbed alpha-IFN preparations were used for immunization. Seven hybridomas continuously secreting IgG against human alpha-IFN were prepared by fusion of splenocytes from immunized donors with the mouse myeloma cells. MoAb reacted in ELISA as well as in neutralization test with human lymphoblastoid, leukocytic and recombinant alpha-IFN.  相似文献   

17.
目的 :制备鼠抗人白细胞介素 15(hIL 15)单克隆抗体 (mAb) ,并鉴定其特性。方法 :自重组人白细胞介素 15(rhIL 15)基因工程菌中 ,提取融合蛋白GST IL 15,以 12 0g/LSDS PAGE分离鉴定 ,切取含有目的条带的凝胶 ,免疫BALB/c小鼠。取免疫小鼠的脾细胞与Sp2 / 0骨髓瘤细胞常规融合 ,依次进行HAT选择培养 ,间接ELISA法筛选抗体阳性的杂交瘤细胞及克隆化。对杂交瘤细胞株的稳定性及其分泌的mAb的特性进行鉴定。另外 ,以rhIL 15包涵体蛋白 (rhIL 15IBP)免疫新西兰白兔 ,制备抗hIL 15的多克隆抗体 (多抗 )。用抗hIL 15的mAb与多抗建立双抗体夹心间接ELISA。结果 :获得 1株可稳定分泌特异性抗hIL 15mAb的杂交瘤细胞。建立了双抗体夹心间接ELISA ,检测rhIL 15蛋白的敏感性达 10 μg/L。结论 :成功地制备抗hIL 15mAb ,并建立了一种可用于hIL 15检测的双抗体夹心间接ELISA。  相似文献   

18.
Hybridomas producing monoclonal antibodies against human alpha interferon (hu-IFN alpha) were constructed by fusion of NSO myeloma cells with spleen cells of BALB/c mice immunized with purified hu-IFN alpha. Altogether, 527 hybridomas were prepared in two separate experiments. From this cohort of hybridomas, 51 produced monoclonal antibodies against hu-IFN alpha. Seventeen out of fifty one hybridomas produced antibodies with neutralizing capacity for IFN while 34 hybridomas produced monoclonal antibodies with binding ability not accompanied with the neutralization of biological activity of IFN. The specificity of antibodies was determined with 3 types of tests: ELISA, ELISAN (modified ELISA) and neutralization test. Using isotype analysis, it has been found that 23 monoclonal antibodies were of IgM class, 20 were of IgG1 subclass, 4 were of IgG2b and 4 of IgG3 subclass. The average number of chromosomes in hybridomas was between 61.35 and 78.55. Their average doubling time was between 13.95 and 25.76 hrs.  相似文献   

19.
Monoclonal antibodies against the biologically active N-terminal fragment of human parathyroid hormone, hPTH (1-34), were produced. The procedure included the use of novel secondary immunization in vitro of mouse spleen cell cultures. Dissociated spleen cells from primary immunized Balb/c mice, were cultured for five days in the presence of thymocyte conditioned media (TCM) and synthetic hPTH (1-34). Contrary to previous findings by other workers, in our hands Balb/c mice responded well. Following immunization the spleen cells were fused with NSl myeloma cells and cultured for eleven days before screening for antibody. Using an enzyme linked immunosorbent assay (ELISA) a number of positive clones were detected. Positive cells were cloned by limiting dilution and fifteen specific monoclonal hybridomas were produced. The immunoglobulin class of the different monoclonal antibodies was found to be IgGl. The immunocytochemical reaction was tested with chief cell carcinoma tissue and found to be clearly positive.  相似文献   

20.
Sera from Brucella abortus-infected and -vaccinated bovines recognized four lipopolysaccharide (LPS) determinants: two in the O-polysaccharide (A and C), one in the core oligosaccharide from rough Brucella LPS (R), and one in lipid A (LA). From 46 different hybridomas secreting monoclonal antibodies (MAbs) against various LPS moieties, 9 different specificities were identified. Two epitopes, A and C/Y, were present in the O-polysaccharide. Two epitopes were found in the core oligosaccharide (R1 and R2) of rough Brucella LPS. MAbs against R1 and R2 epitopes reacted against LPS from different rough Brucella species; however, MAbs directed to the R2 epitope also reacted against enterobacterial LPS from deep rough mutants. Three epitopes (LA1, LA2, and LA3) were located in the lipid A backbone. Different sets of MAbs recognized two epitopes in the lipid A-associated outer membrane protein (LAOmp3-1 and LAOmp3-2). LPS preparations from smooth brucellae had small amounts of rough-type LPS. Although LPS from rough brucellae did not show smooth-type LPS in western blots (immunoblots), two hybridomas generated from mice immunized with rough B. abortus produced antibodies against smooth B. abortus LPS. Results are discussed in relation to the structure and function of B. abortus LPS and to previous findings on the epitopic density of the molecule.  相似文献   

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