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21.
Recombinant antibody cloning and phage display technologies were used to produce single-chain antibodies (scFv) against Clostridium difficile toxin B. The starting material was the mouse B cell hybridoma line 5A8, which generates a monoclonal antibody against the toxin. The integrated cloning, screening, and phage display system of Krebber et al. (J. Immunol. Methods 201:35-55, 1997) allowed us to rapidly obtain toxin B-binding scFv sequences derived from the hybridoma cell line. The best candidate scFv sequences, based on preliminary enzyme-linked immunosorbent assay (ELISA) screening data were then subcloned into the compatible expression vector. Recombinant single-chain antibodies were expressed in Escherichia coli. A 29-kDa band was observed on polyacrylamide gel electrophoresis as predicted. The expressed product was characterized by immunoblotting and detection with an anti-FLAG antibody. The toxin B-binding function of the single-chain antibody was shown by a sandwich ELISA. The antibody was highly specific for toxin B and did not cross-react with material isolated from a toxin B-negative C. difficile strain. The sensitivity of the soluble single-chain antibody is significantly higher than the original monoclonal antibody based on ELISA data and could detect a minimum of 10 ng of toxin B/well. Competitive ELISAs established that the affinity of the 5A8 parent antibody and the best representative (clone 10) of the single-chain antibodies were similar and in the range of 10(-8) M. We propose that recombinant antibody technology is a rapid and effective approach to the development of the next generation of immunodiagnostic reagents.  相似文献   
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Age-dependent DNA methylation changes in the ITGAL (CD11a) promoter   总被引:3,自引:0,他引:3  
DNA methylation patterns change with age in a complex fashion, typically with an overall decrease in genomic deoxymethylcytosine (d(m)C) content, but with local increases in some promoters that contain GC-rich sequences known as CpG islands. While the consequences of age-dependent CpG island methylation have recently been studied in organs such as the colon, less is known about the functional significance of the progressive hypomethylation of promoters lacking CpG islands, and the significance of age-dependent changes in T cell DNA methylation is completely unexplored. We asked if age-dependent DNA hypomethylation might contribute to overexpression of the T cell ITGAL gene, which encodes CD11a, a subunit of LFA-1. CD11a mRNA increased with age as well as with experimentally induced DNA hypomethylation. This increase correlated with hypomethylation of sequences flanking the ITGAL promoter in vitro and in aging. 'Patch' methylation of the region suppressed promoter function. DNA methyltransferases 1 and 3a also decreased with aging. These results indicate that hypomethylation of regions flanking the ITGAL promoter may increase CD11a expression, and suggest that age-dependent hypomethylation of promoters lacking CpG islands, perhaps due to decreased DNA methyltransferase expression, may be one mechanism contributing to increased T cell gene expression with aging.  相似文献   
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Characterization of damage modes in dental ceramic bilayer structures   总被引:8,自引:0,他引:8  
Results of contact tests using spherical indenters on flat ceramic coating layers bonded to compliant substrates are reported for selected dental ceramics. Critical loads to produce various damage modes, cone cracking, and quasiplasticity at the top surfaces and radial cracking at the lower (inner) surfaces are measured as a function of ceramic-layer thickness. It is proposed that these damage modes, especially radial cracking, are directly relevant to the failure of all-ceramic dental crowns. The critical load data are analyzed with the use of explicit fracture-mechanics relations, expressible in terms of routinely measurable material parameters (elastic modulus, strength, toughness, hardness) and essential geometrical variables (layer thickness, contact radius). The utility of such analyses in the design of ceramic/substrate bilayer systems for optimal resistance to lifetime-threatening damage is discussed.  相似文献   
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We have examined the mutational basis of adenine phosphoribosyltransferase (APRT, EC 2.4.2.7) deficiency (MIM 102600) in a patient of Polish origin who has been passing 2,8-dihydroxyadenine (DHA) stones since birth, but has considerable residual enzyme activity in lymphocyte extracts. The five exons and flanking regions of APRT were amplified by PCR and then sequenced. A single T insertion was identified at the intron 4 splice donor site (TGgtaa to TGgttaa:IVS4+2insT) in one allele from the proband, his mother, and brother. A G-to-T transversion in exon 5 (GTC-to-TTC:c.448G>T, V150F) was identified in the other allele, and this mutation was also present in one allele from the father and the paternal grandmother. Tru91 and AvaII digestions of PCR products spanning exons 4 and 5, respectively, confirmed the mutations. The mother was heterozygous for an intragenic TaqI site, but all other family members were homozygous for the presence of this site. IVS4+2insT, located on the allele containing the TaqI site, has been identified previously in several families from Europe, suggesting a founder effect, but the substitution in exon 5 is a novel mutation. IVS4+2insT is known to result in complete loss of enzyme activity, and our results suggest that V150F produces an enzyme that is nonfunctional in vivo but has considerable residual activity in vitro.  相似文献   
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Two different hapten designs have been suggested for production of generic antibodies. The first approach was based on the immunogen prepared by conjugating simazine mimic to carrier proteins through the Cl position of the s-triazine herbicide. For sulfonylurea herbicides a single ring hapten strategy was designed in order to produce antibodies with dominant selectivity towards arylsulfonyl or triazine moieties of metsulfuron-methyl. The best monoclonal antibody raised against simazine-derived hapten mimic (clone B10/B8/D2) exhibited in direct ELISA a broad pattern for a group of methylthio and methoxy s-triazines. Cross-reactivities based on simazine ( = 100%) were 417, 125, 25, 50, 28, 42 and 11% for simetryn, ametryn, prometryn, terbutryn, aziprotryn, atraton and atrazine, respectively. The superiour generic pattern was found for monoclonal antibody raised against s-triazine moiety of metsulfuron-methyl herbicide (clone 2C8/C8). This antibody showed in indirect ELISA the cross-reactivity values 100, 142, 95, 60, 40, 167, 83 and 21% for metsulfuronmethyl, cinosulfuron, triasulfuron, primisulfuron-methyl, thifensulfuron-methyl, chlorsulfuron and prosulfuron and tribenuron-methyl, respectively. The assays using both monoclonal and polyclonal antibodies operated within the ppt-ppb calibration ranges.  相似文献   
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Graded porous titanium coatings have been deposited on titanium substrates for dental implants by plasma spraying in an argon atmosphere. X-ray diffraction (XRD), scanning electron microscopy (SEM), surface roughness measurement, and tensile strength tests were performed on graded porous coatings. The results showed that Ti(3)O(5) was formed in the outermost surface of the porous coatings due to oxidation. The graded porous coatings consisted of three layers. The outer layer was full of macropores with a surface roughness of approximately 100 microm. The diameter of many macropores reached and even surpassed 150 microm, which could be beneficial for tissue to grow into the coating. The middle layer consisted of a mixture of micropores and macropores. The inner layer was a very dense and tight interface layer that included mechanical, physical, and metallurgical bonding. In tensile strength tests, testing bars peeled off the coatings, because the adhesive agent fractured, but the coatings remained intact.  相似文献   
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