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1.
Elongation factor-1alpha (EF-1alpha) is an enzyme that is essential for protein synthesis. Although EF-1alpha offers an excellent target for the disruption of insect metabolism, agents known to interfere with EF-1alpha activity are toxic to humans. In this article, we describe the development of monoclonal antibodies (MAbs) that can disrupt the activity of insect EF-1alpha without cross-reacting with the human enzyme. MAbs were generated to EF-1alpha from Sf21 cells derived from the fall armyworm, Spodoptera frugiperda, by immunizing mice with EF-1alpha eluted from SDS-PAGE gels. The MAbs reacted with EF-1alpha in eggs and first through fifth instars of the fall armyworm in immunoblots of SDS-PAGE gels, but did not recognize EF-1alpha in human carcinoma cells and normal tissues. MAbs with the ability to recognize EF-1alpha in its native conformation, identified through immunoprecipitation experiments, were added to Sf21 cell lysates to determine whether the antibodies could inhibit incorporation of [(35)S]methionine into newly synthesized in vitro translation products. Of the four EF-1alpha-specific MAbs tested, three significantly inhibited protein synthesis when compared to the negative control antibody (P < 0.001, one-way ANOVA; followed by Dunnett's test, P < 0.05). 相似文献
2.
V. P. Baklaushev K. A. Pavlov V. P. Chekhonin 《Biochemistry (Moscow) Supplemental Series B: Biomedical Chemistry》2009,3(2):105-115
The review considers the modern methods of radioimmune diagnostics of high-grade gliomas using monoclonal antibodies and also other approaches of multi-component radioimmunolocalization with pretargeted bispecific antibodies. High-grade tumor-related proteins have been analyzed as potential targets for radiolabeled antibodies. Recent experimental and clinical data on immunolocalization of brain tumors and the most promising immunochemical approaches for diagnostic and targeted therapy of gliomas with tumor-specific antibodies are discussed. 相似文献
3.
Hybridomas were made by fusing mouse myeloma cells with spleen cells from mice immunized with bovine red cells. Sixteen cloned lines which secreted haemolytic monoclonal antibodies reacting with antigens in the A, B, F, Z and S blood group systems were established; one of the antibodies identified a new factor in the B system. Extensive tests on red cells from 1000 animals indicated that several of the antibodies are suitable for use in routine blood typing; others are of potential use for genetic studies of the bovine blood group systems. 相似文献
4.
Isolation and characterization of monoclonal antibodies reactive with endothelial cells 总被引:2,自引:0,他引:2
Monoclonal antibodies were generated to antigens on cultured human umbilical vein endothelial cells. Spleen cells from BALB/c mice, immunized with low passage cultures of human umbilical vein endothelial cells, were fused with the non-secretory myeloma line, P3 x 63Ag 8.653. Hybridoma supernatants were screened for the desired immunological reactivity using ELISA binding assays. Hybridomas secreting antibodies reacting with the immunizing endothelial cells, but not with peripheral blood mononuclear cells, were cloned by limiting dilution and three stable clones were chosen for study. Further testing by ELISA revealed that each antibody displayed a unique pattern of reactivity. One antibody, 14E5, reacted with the macrophage-like cell line DHL-2, cultured macrophages derived from peripheral blood monocytes, and macrophages derived from malignant effusions. The antibody failed to react with fibroblasts or bovine endothelial cells. The second antibody, 12C6, reacted with human and primate fibroblasts and endothelial cells derived from bovine arteries, but not with mature macrophages. The third clone, 10B9, reacted only with the immunizing endothelial cells and the immature-macrophage line U-937. All three antibodies failed to react with long-term human B or T lymphoblastoid cell lines, leukemic cell lines, or murine macrophage lines. None of the antibodies reacted with a battery of human epithelial derived cell lines or primary cultures of human epithelial cells. Indirect immunofluorescence assays revealed that the antigens were expressed on the cell surface. These antibodies should prove useful as differentiation markers of human endothelial cells and in studies of endothelial cell function. 相似文献
5.
V. P. Chekhonin K. A. Bulanov G. M. Yusubalieva E. A. Tsibulkina O. I. Gurina Yu. S. Skoblov V. I. Shvets Yu. A. Zhirkov V. P. Baklaushev 《Biochemistry (Moscow) Supplemental Series B: Biomedical Chemistry》2008,2(1):82-87
Employment of radiolabeled antibodies in biological studies, allows their specific accumulation in organs and tissues to be accurately detected. However, stability of such radiolabeled antibodies depends on both the method of labeling and particular experimental conditions. Therefore, stability of labeled antibodies should be determined in every particular experiment. In this study we have investigated stability of the 125I-labeled monoclonal antibodies to gliofibrillary acidic protein (GFAP), endothelial antigen AMVB1, and non-specific mouse IgG at the stage of their synthesis and after their intravenous administration to rats with experimental C6 glioma. Stability of labeled antibodies was determined in blood samples and homogenates of organs by the method of their precipitation with trichloroacetic acid. The 125I-radiolabeled antibodies were characterized by high radiochemical putiry, immunochemical activity and stability of the resultant preparations in blood and tissues, and the brain after administration in vivo. Electrophoretic analysis, thin-layer chromatography, and immunohistochemical tests have demonstrated the radiochemical purity, immunochemical competence, and stability of the labeled antibodies in vivo. 相似文献
6.
J. Bujia M. Sittinger H. Sudhoff A. Fisseler-Eckhoff G. R. Burmester E. Kastenbauer C. Hammer 《In vitro cellular & developmental biology. Animal》1996,32(6):366-371
Summary Cell-specific antigens are mainly found in cells or membrane surfaces rather than in the surrounding matrix. However, until
now it was not possible to produce antibodies specific for cellular structures of chondrocytes. In 1989, Lance (Immunol. Lett.
21:63–73; 1989) first established specific monoclonal antibodies for human articular chondrocytes tested only by immunofluorescence.
Studies describing the specificity of these five antibodies (HUMC 1–5) and their relevance for immunohistological analysis
of cartilage tissue were not available until now. Therefore, the aim of the following study was to investigate the distribution
of HUMC 1, 2, 3, 4, and 5 in mesenchymal cellsin vivo andin vitro immunohistochemically. Further investigations concentrate on the localization of chondrocyte specific antigens using immunoelectron
microscopy. Immunohistological studies showed positive immunostainings with all five antibodies in human chondrocytesin vivo andin vitro. A cross-reaction with human fibroblasts and osteoblasts for the antibodies HUMC 2 and HUMC 5 was observed. furthermore,
a parallel loss of immunoreactivity for HUMC 1, HUMC 3, and HUMC 4 was observed in cultured chondrocytes indicating that the
specific antigens vanish during differentiation observedin vitro. Subsequent immunoblot analysis employing collagens as antigens did not show any reactivity. Using immunoelectron microscopy,
gold particle labeling was observed in intracytoplasmatic vesicles of isolated chondrocytes. Our results indicate that HUMC
1, HUMC 3, and HUMC 4 are specific for cartilage cells and might be suitable for immunohistological analysis of different
cartilage tissues and pathologically altered chondrocytes. 相似文献
7.
8.
The terminal alpha anomeric Ga1NAc residue is an essential sugar for the Tn glycotope, human blood group A determinant, and Forssman antigen. In a previous study [King M.J., Parson S.F., Wu A,M., Jones N., Transfusion 31: 142-149, 1991] we defined two monoclonal antibodies (MoAbs, BRIC66 and BRIC111) reacting with human Tn red blood cells. However, more advanced studies of these two MoAbs were hampered by the lack of availability of Gal/GalNAc related glycotopes. In order to use these antibodies as powerful probes to elucidate structural changes during life processes, we have characterized in detail the combining sites of these two MoAbs using enzyme-linked immunosorbent (ELISA) and inhibition assays with an extended glycan/ligand collection. From the results, it has been established that BRIC66 demonstrated multiple specificities and its reactivity towards glycotopes was defined as: Ga1NAc alpha1-->Ser/Thr (Tn) > or = Ga1NAc alpha1-->3(LFuc alpha1-->2)Gal (Ah) > Ga1NAcalpha1-->3Galbeta1-->4Glc (AL) > Ga1NAalpha1-->3Gal (A) GalNAc alpha1-->3GalNAc > Gal or Glc. Another MoAb, BRIC111, mainly bound Tn-glycophorin. The best ligand for this MoAb was Tn-containing glycopeptides (M.W. < 3.0 x 10(3) Da) from asialo ovine salivary mucin (OSM), which was approximately 70 and 58 times more active than Ga1NAc and monomeric Ga1NAc alpha1-->Ser/Thr (Tn), respectively, suggesting that the active glycotopes present in glycophorin for BRIC111 binding also exist in OSM. The N-acetyl group at carbon-2 and configuration at carbon-2 and carbon-4 of the alpha anomeric Ga1NAc are required for the binding of either MoAb. Identification of these binding properties should aid in the selection of these MoAbs and the conditions required for biological studies and clinical applications. 相似文献
9.
In order to assess the value of immunocytochemical staining as a method of discriminating between benign reactive mesothelial cells and malignant epithelial cells in serous effusions, we have studied the reactions of a panel of commercially available antibodies on cells harvested from 83 pleural and peritoneal fluids and compared the results with the clinical and cytological diagnoses. The antibodies used were raised against cytokeratin (PKK1), epithelial membrane antigen (EMA), carcino-embryonic antigen (CEA), pregnancy specific B1-glycoprotein (SP1) and leucocyte common antigen (LCA). Anti-CEA was positive in 16 of 39 effusions (41%) containing carcinoma cells. Pregnancy specific B1-glycoprotein (SP1) was positive in 33% of the same samples. Mesothelial cells did not stain with these antibodies. Thus anti-CEA and SP1 can be used to discriminate between benign mesothelial and malignant epithelial cells in effusions. Anti-PKK1 stained both benign reactive mesothelial cells and malignant epithelial cells and cannot be used to discriminate between these two cell types. Strong positive staining of malignant cells was noted with anti-EMA. However, as occasional weak staining of mesothelial cells was also noted, strong staining with this antibody may be regarded as suspicious but not conclusive of malignancy. 相似文献
10.
Pharmacological blockade of mGlu2/3 metabotropic glutamate receptors reduces cell proliferation in cultured human glioma cells 总被引:1,自引:0,他引:1
D'Onofrio M Arcella A Bruno V Ngomba RT Battaglia G Lombari V Ragona G Calogero A Nicoletti F 《Journal of neurochemistry》2003,84(6):1288-1295
Glial cell proliferation in culture is under the control of metabotropic glutamate (mGlu) receptors. We have examined whether this control extends to human glioma cells. Primary cultures were prepared from surgically removed human glioblastomas. RT-PCR combined with western blot analysis showed that most of the cultures (eight out of 11) expressed group-II mGlu receptors. In two selected cultures (MZC-12 and FCN-9), the mGlu2/3 receptor antagonist, LY341495, slowed cell proliferation when applied to the growth medium from the second day after plating. This effect was reversible because linear cell growth was restored after washing out the drug. LY341495 reduced glioma cell proliferation at concentrations lower than 100 nm, which are considered as selective for mGlu2/3 receptors. In addition, its action was mimicked by the putative mGlu2/3 receptor antagonist (2S)-alpha-ethylglutamate. The anti-proliferative effect of LY341495 was confirmed by measuring [methyl-3H]-thymidine incorporation in cultures arrested in G0 phase of the cell cycle and then stimulated to proliferate by the addition of 10% fetal calf serum or 100 ng/mL of epidermal growth factor (EGF). In cultures treated with EGF, LY341495 was also able to reduce the stimulation of the mitogen-activated protein kinase (MAPK) pathway, as well as the induction of cyclin D1. Both effects, as well as decreased [methyl-3H]-thymidine incorporation, were partially reduced by co-addition of the potent mGlu2/3 receptor agonist, LY379268. We conclude that activation of group-II mGlu receptors supports the growth of human glioma cells in culture and that antagonists of these receptors should be tested for their ability to reduce tumour growth in vivo. 相似文献
11.
Hans K. Haugland Ole-Bjørn Tysnes 《In vitro cellular & developmental biology. Animal》1996,32(3):159-166
Summary Malignant features in three glioma cell lines were studied in four defined media of various complexity. The cell lines D37MG,
D54MG, and GaMG were able to grow in monolayer culture in all media examined, and as multicellular tumor spheroids in the
two most nutrient-rich media. In the defined media, none of the cell lines were able to migrate in a migration assay on poly-D-lysine-coated
plastic surfaces. Flow cytometric analysis of the GaMG cell line demonstrated no medium-dependent selection of subclones of
glioma cells in spheroids cultured for 30 d. Morphological diversity of spheroids varied according to the supplementation
of the media. The capacity of glioma cells to invade cellular rat brain aggregates was intact in the media examined. However,
glioma migration was severely inhibited by the lack of specific serum components. This study demonstrates that glioma growth
and invasion was heterogenously preserved in the defined media used. Depending on the assay to be used in the study of glioma
cell behavior, the degree of medium supplementation has to be considered. 相似文献
12.
With whole U87MG cells used as antigenic stimulant, two clones 1A5G6 and 1D3A3 secreted monoclonal antibodies which gave intense
staining in monolayer cultures of the cells as ascertained by indirect immunofluorescence. Antibodies from clone 1A5G6 stained
both the cytoplasm and the processes, and that from clone 1D3A3 stained only the cytoplasm and not the processes. 1A5G6 elicited
no cross-reactivity towards human fetal and adult brain and lungs, liver, kidney or spleen, mouse neuroblastoma and melanoma,
rat C6 glioma, neuroblastoma X glioma hybrid and normal rat kidney cells. It gave 58–60% cross reactivity with the human neuroblastoma
and T-cell leukemia cells. The antigenic comPonent has been identified to be a membrane protein of molecular weight 25–30
kilodaltons by immunoblotting. Using C6 glioma cells as antigenic stimulant 19 clones which were positive for C6 glioma cells, but negative for rat liver cells as inferred by indirect immunofluorescence were selected. Antibodies secreted
by all these gave positive reaction towards normal rat kidney and fetal rat kidney cells in culture. Distinct identity of
these clones were ascertained by discernible staining patterns in indirect immunofluorescence on C6 glioma cells. 相似文献
13.
14.
A battery of monoclonal antibodies were produced againstWuchereria bancrofti microfilarial excretory-secretory antigens and their specificity was studied using different filarial antigens. Among the
1116 wells plated out, 42 % of the wells developed hybrids and 5 % of the hybrids showed antiWuchereria bancrofti microfilarial excretory-secretory antigens. Specificity studies on the antibodies produced from 63 cloned and expanded hybrids
showed 10 clones which were specifically positive only toWuchereria bancrofti microfilarial excretory-secretory antigens. 相似文献
15.
This study was performed to evaluate the application of different lectins and monoclonal antibodies against ABH antigens to detect and characterize carbohydrate structures in capillaries of skeletal muscle from humans and laboratory animals. Blood group specific lectins (Griffonia simplicifolia, Griffonia simplicifolia isolectin B4,Lotus tetragonlobus, Ulex europaeus, andDolichos biflorus) and monoclonal antibodies reacting with histo-blood group carbohydrate antigens belonging to type 1 (Lea) and type 2 (H, A and Ley) chains were used as histological markers for capillaries in sections from skeletal muscle. The material consisted of 20 human masseter muscle biopsies from individuals with known blood types: (eight blood group O, nine blood group A, two blood group B, and one blood group AB) and masseter muscles specimens from different laboratory animals (mouse, rat, rabbit, cat, dog, pig, cow, and macaca monkey). Unfixed sections and an avidin alkaline phosphatase method were used to visualize the specific reaction.Ulex lectin stained capillaries in all human biopsies either strongly or moderately. Strong muscle capillary reaction was observed in biopsies from O, B and AB individuals while capillaries from A individuals were only moderately stained.Griffonia simplicifolia marked capillaries in A, B, and AB individuals andGriffonia simplicifolia isolectin B4 stained capillaries in muscle biopsies from B and AB donors.Dolichos biflorus was a weak marker of muscle capillaries from A individuals. Only capillaries from O individuals were stained with the antibody against H type 2. Capillary reaction was not observed with the other antibodies used.Girffonia simplicifolia was an excellent marker for capillaries in mouse muscle whileGriffonia simplicifolia isolectin B4 is recommended for rat muscles. Periodic acid treatment and subsequentLotus tetragonolobus staining is suitable to visualize capillaries in mouse, rat and pig muscle. Using a sensitive histochemical technique for staining with lectins and monoclonal antibodies reacting with blood group related antigens the microvascular density in human skeletal muscle may be estimated. Further, the carbohydrate compounds in the muscle capillaries reflect the individual blood type. A selection of lectins is suitable for demonstration of capillaries in animal skeletal muscle. 相似文献
16.
I. I. Moraru Mioara Manciulea Anca Călugăru Gr. Ghyka L. M. Popescu 《Bioscience reports》1987,7(9):731-736
A novel approach was used to assess the role of phosphoinositide hydrolysis in the mitogenic action of phytohemagglutinin (PHA) or concanavalin A (ConA). The treatment of human peripheral blood leukocytes (PBL) with monospecific antibodies against phospholipase C (PLC) produced a dose-dependent inhibition (up to 100%) of PHA (10 g/ml) or ConA (25 g/ml) proliferative effects. Thus, the activation of membrane-bound PLC is asine-qua-non condition for lectin-induced proliferation of T lymphocytes. The key-role of PLC versus protein kinase C (PKC) is stressed by the fact that the inhibition of PKC with Hidaka's compound H-7 (40 M) produced only a partial blockade (about 25%) of lectin mitogenic effect.To whom correspondence should be addressed. 相似文献
17.
18.
Peigang Ji Liang Wang Jinghui Liu Ping Mao Ruichun Li Haitao Jiang Miao Lou Meng Xu Xiao Yu 《Journal of cellular biochemistry》2019,120(3):3259-3267
Ribosomal protein L34 (RPL34), belonging to the L34E family of ribosomal proteins, was reported to be dysregulated in several types of cancers and plays important roles in tumor progression. However, the expression and roles of RPL34 in human glioma remain largely unknown. Thus, the objective of this study was to investigate the expression and role of RPL34 in glioma. We report here that RPL34 is highly expressed in human glioma tissues and cell lines. Knockdown of RPL34 markedly inhibited the proliferation, migration, and invasion, as well as prevented the epithelial-mesenchymal transition phenotype in glioma cells. Further, mechanistic analysis showed that knockdown of RPL34 significantly downregulated the levels of p-JAK and p-STAT3 in glioma cells. Taken together, our findings indicated that knockdown of RPL34 inhibits the proliferation and migration of glioma cells through the inactivation of JAK/STAT3 signaling pathway. Thus, RPL34 may serve as a potential therapeutic target for the treatment of glioma. 相似文献
19.
John D. Minna Frank Cuttitta Steven Rosen Paul A. Bunn Jr. Desmond N. Carney Adi F. Gazdar Stephen Krasnow 《In vitro cellular & developmental biology. Plant》1981,17(12):1058-1070
Summary We have developed a screening strategy and technology to produce monoclonal antibodies with specificity for human lung cancer
cells. Mice and rats were immunized with well-characterized tissue culture lines of human small cell lung cancer (SCLC), mouse
myeloma x spleen hybrids formed by the technique of Kohler and Milstein, and the resulting culture fluids were screened for
antibody binding phenotype using a radioimmunoassay. To facilitate testing large numbers of culture fluids, a 96-well, microtiter
based, resuable, replicating device was designed. Using this, many hybridoma culture fluids were replica plated for antibody
binding tests on a series of human target cell plates. Hybrids producing antibodies that reacted with the immunizing SCLC
line and another independent SCLC line, but not with autologous B-lymphoblastoid cells derived from one of the patients, were
identified, selected, and then repeatedly recloned using the same screening strategy. With this technology, hybridomas representing
less than 0.5% of all hybrids generated could be isolated and stable antibody producing cultures derived. Such antibodies
reacted with a panel of well-characterized SCLC lines and SCLC samples taken directly from patients but not with a variety
of normal tissues. Using these antibodies we can demonstrate: tumor cell contamination of bone marrow specimens, marked heterogeneity
of antigen expression on cells within individual SCLC lines and individual patients, and inhibition of clonal growth of SCLC
lines in soft agarose assays. All of these findings have potential clinical and cell biologic application.
Presented in the symposium on The Biology of Hybridomas at the 32nd Annual Meeting of the Tissue Culture Association, Washington,
D.C., June 7–11, 1981.
This symposium was supported in part by the following organizations: Bethesda Research Laboratories, Cetus Corporation, Hybritech
Incorporated, MAB-Monoclonal Antibodies, Inc., National Capital Area Branch of the Tissue Culture Association, New England
Nuclear Corporation, and Ortho Pharmaceutical Corporation. 相似文献
20.
Annamaria Sandomenico Annalia Focà Luca Sanguigno Andrea Caporale Giuseppina Focà Angelica Pignalosa 《MABS-AUSTIN》2016,8(8):1575-1589
Post-translational modifications (PTMs) strongly influence the structure and function of proteins. Lysine side chain acetylation is one of the most widespread PTMs, and it plays a major role in several physiological and pathological mechanisms. Protein acetylation may be detected by mass spectrometry (MS), but the use of monoclonal antibodies (mAbs) is a useful and cheaper option. Here, we explored the feasibility of generating mAbs against single or multiple acetylations within the context of a specific sequence. As a model, we used the unstructured N-terminal domain of APE1, which is acetylated on Lys27, Lys31, Lys32 and Lys35. As immunogen, we used a peptide mixture containing all combinations of single or multi-acetylated variants encompassing the 24–39 protein region. Targeted screening of the resulting clones yielded mAbs that bind with high affinity to only the acetylated APE1 peptides and the acetylated protein. No binding was seen with the non-acetylated variant or unrelated acetylated peptides and proteins, suggesting a high specificity for the APE1 acetylated molecules. MAbs could not finely discriminate between the differently acetylated variants; however, they specifically bound the acetylated protein in mammalian cell extracts and in intact cells and tissue slices from both breast cancers and from a patient affected by idiopathic dilated cardiomyopathy. The data suggest that our approach is a rapid and cost-effective method to generate mAbs against specific proteins modified by multiple acetylations or other PTMs. 相似文献