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A monoclonal antibody to cellular transglutaminase 总被引:3,自引:0,他引:3
A cellular enzyme-linked immunosorbent assay was developed for estimating cellular transglutaminase in situ using a monoclonal antibody produced to tissue transglutaminase. The minimum level of detection of TGase was 2-5 ng. The enzyme was present in greater amounts in WI-38 and IMR90 cells than in their simian virus-transformed counterparts. The levels of TGase in the virus-transformed cells increased significantly when the cells were grown in the presence of sodium butyrate to induce enzyme activity. Staining of confluent WI-38 cells by indirect immunofluorescence using the monoclonal antibody showed microscopic fibers suggesting that the enzyme may be associated with detergent-insoluble components. 相似文献
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Stankowiak-Kulpa H Sanders J Depraetere H Jeffreys J Evans M Richards T Furmaniak J Smith BR 《Archivum immunologiae et therapiae experimentalis》2005,53(4):345-351
INTRODUCTION: Mouse monoclonal antibodies (mAbs) with the ability to inhibit thyrotropin (TSH) binding to the TSH receptor (TSHR) are useful tools to study TSH-TSHR interaction. The 3C3 mAb we produced was found to inhibit binding of TSH to human (h)TSHR but not to porcine (p)TSHR. MATERIAL/METHODS: Purified 3C3 immunoglobulin G (IgG) and its antibody-binding fragment were prepared using standard methods and their ability to inhibit TSH binding to hTSHR or pTSHR was analyzed using a coated tube assay. The TSHR epitope reactive with 3C3 IgG was determined using Western blotting, ELISA based on peptides corresponding to the TSHR sequence, and the SPOT synthesis technique. RNA was isolated from 3C3 hybridoma cells and the mAb variable (V) region genes were sequenced and analyzed. RESULTS: 3C3 mAb had a 1 x 108 l/mol binding affinity to the hTSHR as assessed by Scatchard analysis. 3C3 reacted with the hTSHR region between amino acids (aa) 212-230, and two aa differences were found between the corresponding regions in the hTSHR and pTSHR. The light chain (LC) genes of 3C3 were derived from the Vk21 germ-line (97.6% homology) and Jk2 genes. The heavy chain (HC) genes were from the V130 germ-line (94.6% homology) combined with a D gene (not identified) and JH3 gene. The replacement/ silent mutation ratios of 6.0 and 6.5 for the LC and the HC V regions, respectively, indicated that 3C3 underwent antigen-driven maturation. CONCLUSIONS: Mouse mAbs of this type should be useful in studying the interactions between the TSHR, TSH, and mAbs in more detail. 相似文献
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Dominique Bourel Renée Fauchet Bernard Genetet Genevièvre Merdrignac Daniel Hurez 《Immunology letters》1982,5(5):233-237
Monoclonal antibody production recognizing the HLA-A3 antigen is described. The XI-23 antibody reacted with all of the 89 cell suspensions carrying the HLA-A3 antigen (100% cytotoxicity) among a total of 191 suspensions tested. No extra-reactivity or cross-reactivity was observed, particularly with that of HLA-A11. This antibody can thus be considered as a good HLA-typing reagent. 相似文献
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H R Roberts 《The New England journal of medicine》1970,283(10):543-544
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M Pascual E Catana F Spertini K Macon J E Volanakis J A Schifferli 《Journal of immunological methods》1990,127(2):263-269
Factor D is an essential enzyme for activation of complement by the alternative pathway (AP). It has been difficult to obtain mouse monoclonal antibodies (Mabs) which block the function of factor D. We have developed a strategy to obtain such Mabs using a double screening procedure of the initial clones. We selected the clone whose supernatant had the lowest level of anti-factor D Ab by ELISA and abolished factor D haemolytic activity. Addition of this Mab to human serum was shown to abolish conversion of C3 by cobra venom factor, haemolysis of rabbit erythrocytes, and activation of C3 and C5 by cuprophane dialysis membranes. 相似文献
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A K Schr?der A E Gharavi M Steinitz P J Johansson P Christensen 《International archives of allergy and applied immunology》1987,83(1):88-91
Radiolabelled monoclonal IgM rheumatoid factors, four from patients with type II essential cryoglobulinaemia and one originating from a patient with rheumatoid arthritis, were tested for binding to group A, B, C and G streptococci and Escherichia coli. Two of the preparations exhibited different binding patterns for the streptococci, whereas the remaining three were not reactive. The binding of one of the factors to group A streptococci type 15 was inhibited by F(ab')2-fragments of anti-idiotypic antibodies but not anti-IgM Fc or F(ab')2 of normal rabbit IgG, indicating that the reaction was mediated via the antibody combining sites. 相似文献
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A monoclonal antibody related to the human blood group Gerbich 总被引:1,自引:0,他引:1
The monoclonal antibody, GERO, agglutinated all samples of red cells with the exception of Gerbich-negative cells of both Ge and Yus types. Using the antiglobulin test, Gerbich-negative cells reacted as strongly with GERO as did Gerbich-positive cells. 相似文献
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Characterization of a protective monoclonal antibody recognizing Staphylococcus aureus MSCRAMM protein clumping factor A 总被引:1,自引:0,他引:1
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Hall AE Domanski PJ Patel PR Vernachio JH Syribeys PJ Gorovits EL Johnson MA Ross JM Hutchins JT Patti JM 《Infection and immunity》2003,71(12):6864-6870
The Staphylococcus aureus MSCRAMM (microbial surface components recognizing adhesive matrix molecules) protein clumping factor A (ClfA) has been shown to be a critical virulence factor in several experimental models of infection. This report describes the generation, characterization, and in vivo evaluation of a murine monoclonal antibody (MAb) against ClfA. Flow cytometric analysis revealed that MAb 12-9 recognized ClfA protein expressed by all of the clinical S. aureus strains obtained from a variety of sources. In assays measuring whole-cell S. aureus binding to human fibrinogen, MAb 12-9 inhibited S. aureus binding by over 90% and displaced up to 35% of the previously adherent S. aureus bacteria. Furthermore, a single infusion of MAb 12-9 was protective against an intravenous challenge with a methicillin-resistant strain of S. aureus in a murine sepsis model (P < 0.0001). These data suggest that anti-ClfA MAb 12-9 should be further investigated as a novel immunotherapy for the treatment and prevention of life-threatening S. aureus infections. 相似文献
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Tamotsu Takeuchi Ichiro Kubonishi Yuji Ohtsuki Isao Miyoshi 《Virchows Archiv : an international journal of pathology》1991,419(2):147-151
Summary A monoclonal antibody, termed K-20, was generated against an anaplastic thymic carcinoma cell line, Ty-82. Subcapsular thymic epithelial cells of the thymus and blood vessels in various organs were shown to react with the K-20 monoclonal antibody by immunohistochemical staining. Immunofluorescent study revealed that various haematopoietic fresh cells and cell lines did not show any significant reactivity with K-20, except for one Epstein-Barr-virus-carrying lymphoma cell line (SP-50B). Western immunoblotting and affinity purification procedure revealed that K-20 was directed to a protein with a molecular weight of 28 kDa. K-20 is unique in its restrictive reactivity with human subcapsular thymic epithelial cells. 相似文献
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A Borrelia-specific monoclonal antibody binds to a flagellar epitope. 总被引:62,自引:24,他引:62
A G Barbour S F Hayes R A Heiland M E Schrumpf S L Tessier 《Infection and immunity》1986,52(2):549-554
To determine whether expression of type 1 pili varies during the course of Escherichia coli infection in vivo, mice were injected intraperitoneally with 5 X 10(7) CFU of piliated or nonpiliated phase variants per ml, and the degree of piliation was measured in peritoneal exudate by an enzyme-linked immunosorbent assay inhibition method. In the animals challenged with the piliated bacteria, the numbers of organisms increased a log over 9 h and the amount of pilus antigen decreased from 3 to 0.075 micrograms/10 bacteria. After a 4-h delay, nonpiliated bacteria also increased by one log over 9 h; however, the amount of piliation remained virtually undetectable. Piliated E. coli were more virulent than nonpiliated variants in this model (50% lethal dose of 7.5 X 10(6) versus 3 X 10(7), respectively). The difference was significantly reduced by prior passive immunization with rabbit serum containing high titers of antipili antibody. Piliated bacteria adhered in significantly greater numbers to isolated mouse peritoneal membranes than did nonpiliated variants (15,400 +/- 2,700 versus 1,300 +/- 700 bacteria/mm2, respectively; P = 0.05). Adherence was inhibited by the presence of 0.1 M alpha methyl mannose (1,500 +/- 1,800 bacteria/mm2, P = 0.01). These results confirm the results of previous qualitative studies showing that phase variation of type 1 pili occurs in vivo and suggest that these pili may confer an initial advantage for growth of E. coli in the peritoneal cavity, presumably by fostering colonization of the peritoneal serosal surface. 相似文献
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A rat anti-murine granulocyte-macrophage colony-stimulating factor (mGM-CSF) monoclonal antibody, A2, that neutralizes bioactivity in vitro was isolated. The binding epitope recognized by this antibody was identified using human-murine hybrid GM-CSF proteins. A2 was unable to immunoprecipitate a hybrid (hm7) protein containing the human GM-CSF sequence for the first 11 amino terminal amino acids, and the mGM-CSF sequence for amino acids 12-124. In contrast, A2 did recognize a hybrid which substitutes human GM-CSF amino acids 23-36 in the murine sequence. These data suggest that this neutralizing antibody recognizes an epitope at the amino terminus of mGM-CSF. Because hm7 did maintain in vitro bioactivity, it is probable that the epitope recognized by the neutralizing antibody is not itself part of the receptor-binding domain of mGM-CSF; rather, it is likely that neutralization occurs as a result of antibody binding near the receptor-binding site, with steric inhibition of mGM-CSF binding to its receptor. Interestingly, monoclonal antibody A2 does not recognize mGM-CSF glycosylation species corresponding to predicted maximal O-glycosylation variants. The presence of O-glycosylation sites within the antibody-binding epitope was confirmed using site-directed mutagenesis. Potential O-glycosylation sites in native mGM-CSF were removed by introducing conservative amino acid substitutions, and expected molecular weight reductions were obtained. These findings are consistent with previous reports that suggest the importance of the integrity of residues near the amino terminus to GM-CSF bioactivity. 相似文献
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A monoclonal antibody to brush border and passive Heymann nephritis 总被引:19,自引:10,他引:9
P Ronco L Allegri C Melcion E Pirotsky M D Appay J Bariety F Pontillon P Verroust 《Clinical and experimental immunology》1984,55(2):319-332
An IgG2a monoclonal antibody (MoAb) reacting with the brush border of the renal proximal tubule and glomerular capillary wall was produced by fusion of NS1 myeloma cells with spleen cells from BALB/c mice immunized with renal brush border preparations from rat kidney cortex. This antibody reacts with a 90,000 mol. wt protein which can be isolated by immunoprecipitation of radiolabelled brush border or glomerular preparations and localized on these structures by immunoperoxidase electron microscopy, thus demonstrating the presence of common antigenic determinants. Survey of various organs showed that the MoAb reacted with the brush border of the gut, but also with antigens associated with the distal vascular system. In the liver antigenic determinants were located along the sinusoid walls but mainly on bile canaliculi. Specific glomerular binding could be demonstrated in vivo by immunofluorescence after an intravenous injection of 2 mg of antibody or by paired label methodology using tracer amounts. Kinetics however were dramatically different from those observed in classical passive Heymann nephritis since glomerular binding was transient during the first hours after injection. Binding was also found in tubular structures, as well as in lung, liver, spleen and heart. These results identify a well defined antigen-antibody system responsible for the formation of transient extramembranous glomerular deposits and may be relevant to some human cases of glomerulonephritis. They may also provide new models to study glomerular and tubular transfer of membrane bound antibodies. 相似文献
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A monoclonal antibody against a membrane glycoprotein of rat hepatocytes has been produced. The nature of this antibody designated as HAM.4 was analysed by cellular radioimmunoassay, flow cytofluorography and indirect immunoperoxidase procedures. The following characteristics of HAM.4 were elucidated. First, an immunohistochemical study revealed that this antibody stained preferentially the bile canalicular face of hepatocyte membrane. Secondly, HAM.4 cross-reacted with kidney, spleen and thymus as well as liver. The kidney expressed much more the antigen molecules detected by this antibody than the liver did. The antigen was located predominantly on the brush border of proximal tubules in kidney. Thus, HAM.4 would be useful for analysing one of the brush border antigens of renal tubules which has been thought to be a pathogenic antigen for inducing experimental membranous glomerulonephritis. Finally, HAM.4 failed to label the cell membrane of rat hepatoma cell lines examined, indicating that the antigen detected by HAM.4 may disappear from cell surface during the course of hepatocarcinogenesis. 相似文献
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Characterization of a humanized monoclonal antibody recognizing clumping factor A expressed by Staphylococcus aureus 总被引:1,自引:0,他引:1
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Domanski PJ Patel PR Bayer AS Zhang L Hall AE Syribeys PJ Gorovits EL Bryant D Vernachio JH Hutchins JT Patti JM 《Infection and immunity》2005,73(8):5229-5232
We report the humanization and characterization of monoclonal antibody (MAb) T1-2 or tefibazumab, a monoclonal antibody that recognizes clumping factor A expressed on the surface of Staphylococcus aureus. We demonstrate that the binding kinetics of MAb T1-2 is indistinguishable compared to that of its murine parent. Furthermore, MAb T1-2 is shown to enhance the opsonophagocytic uptake of ClfA-coated latex beads, protect against an intravenous challenge in a prophylactic model of rabbit infective endocarditis, and enhance the efficacy of vancomycin therapy in a therapeutic model of established infective endocarditis. 相似文献
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Sprague-Dawley rats were immunized by injection of antigen B-specific T helper factor THF) eluted from Sepharose-antigen D adsorbents. Rat spleen cells from animals immunized with THF were fused with a BALB/c tumour cell (P3x63-Ag8.653) to prepare monoclonal anti-THF antibodies. The hybrids produced were screened for anti-THF antibodies by an enzyme-linked immunoassay (ELISA), and we shall describe the characteristics of one of the hybrid (hybridoma 6-2.2 anti-THF) antibodies produced. (i) The monoclonal hybrid 6-2.2 anti-THF antibody blocks water-soluble timothy extract-induced proliferation of timothy-specific T helper cells when these cells were preincubated with an excess of the anti-THF hybrid 6-2.2 antibody; (ii) incubation of timothy-specific T helper or T suppressor cells with an optimal dose of anti-THF 6-2.2 antibody induces significant levels of [3H]-thymidine incorporation in the absence of antigen; (iii) it binds specifically to the idiotypic determinant expressed on THFk, THFd, TSFk, and antigen B-specific IgE in an ELISA; and (iv) it has no effect upon spleen cells from mice primed with ovalbumin or Ascaris suum antigens. In addition, the monoclonal anti-THF 6-2.2 antibody cultured with normal spleen cells in mini-Marbrook chambers induced significant levels of antigen B-specific T suppressor cells. These studies indicate that the monoclonal anti-THF 6-2.2 antibody has anti-idiotypic antibody properties. 相似文献
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A monoclonal antibody detecting an HLA-DQwl-related determinant 总被引:2,自引:0,他引:2
Joan Raghoebar Geziena M. T. Schreuder Ruud Schuurman Hans Bruning 《Tissue antigens》1985,26(2):100-109
A complement fixing monoclonal antibody (moab) was prepared which reacts with a polymorphic determinant on HLA class II molecules. The moab IIB3 recognises all DQwl (DC1, MB1, LB-E12) positive cells as well as some DR4, DR7, DRw8 and DRw9 positive cells. The moab reacts mainly with B-cells and not or with only a minority of the monocytes. Segregation of the determinant with HLA-DR could be shown. The determinant is strongly expressed on DR2, DR4 and DRw6 positive cell lines but only weakly on DR1 lines. In contrast to a monoclonal antibody against a monomorphic determinant on class II molecules IIB3 did not give a distinct inhibition of the MLC nor did it inhibit the generation of CTLs in MLC as has been described for the DQwl like moab BT 3/4 (Corte et al. 1982). Immunoprecipitation indicates that IIB3 reacts with DQ-like molecules. 相似文献