The Plasmid Mobilome of the Model Plant-Symbiont Sinorhizobium meliloti: Coming up with New Questions and Answers.

Science.gov (United States)

Lagares, Antonio; Sanjuán, Juan; Pistorio, Mariano

2014-10-01

Rhizobia are Gram-negative Alpha- and Betaproteobacteria living in the underground which have the ability to associate with legumes for the establishment of nitrogen-fixing symbioses. Sinorhizobium meliloti in particular-the symbiont of Medicago, Melilotus, and Trigonella spp.-has for the past decades served as a model organism for investigating, at the molecular level, the biology, biochemistry, and genetics of a free-living and symbiotic soil bacterium of agricultural relevance. To date, the genomes of seven different S. meliloti strains have been fully sequenced and annotated, and several other draft genomic sequences are also available. The vast amount of plasmid DNA that S. meliloti frequently bears (up to 45% of its total genome), the conjugative ability of some of those plasmids, and the extent of the plasmid diversity has provided researchers with an extraordinary system to investigate functional and structural plasmid molecular biology within the evolutionary context surrounding a plant-associated model bacterium. Current evidence indicates that the plasmid mobilome in S. meliloti is composed of replicons varying greatly in size and having diverse conjugative systems and properties along with different evolutionary stabilities and biological roles. While plasmids carrying symbiotic functions (pSyms) are known to have high structural stability (approaching that of chromosomes), the remaining plasmid mobilome (referred to as the non-pSym, functionally cryptic, or accessory compartment) has been shown to possess remarkable diversity and to be highly active in conjugation. In light of the modern genomic and current biochemical data on the plasmids of S. meliloti, the current article revises their main structural components, their transfer and regulatory mechanisms, and their potential as vehicles in shaping the evolution of the rhizobial genome.

Crystallization and preliminary crystallographic studies of the recombinant dihydropyrimidinase from Sinorhizobium meliloti CECT4114

International Nuclear Information System (INIS)

Martínez-Rodríguez, Sergio; González-Ramírez, Luis Antonio; Clemente-Jiménez, Josefa María; Rodríguez-Vico, Felipe; Las Heras-Vázquez, Francisco Javier; Gavira, Jose A.; García-Ruíz, Juan Manuel

2006-01-01

The dihydropyrimidinase from S. meliloti CECT4114, with activity towards both hydantoin and dihydrouracil substrates, was crystallized, and diffraction data were collected to 1.85 Å resolution. Dihydropyrimidinases are involved in the reductive pathway of pyrimidine degradation, catalysing the hydrolysis of 5,6-dihydrouracil and 5,6-dihydrothymine to the corresponding N-carbamoyl β-amino acids. This enzyme has often been referred to as hydantoinase owing to its industrial application in the production of optically pure amino acids starting from racemic mixtures of 5-monosubstituted hydantoins. Recombinant dihydropyrimidinase from Sinorhizobium meliloti CECT4114 (SmelDhp) has been expressed, purified and crystallized. Crystallization was performed using the counter-diffusion method with capillaries of 0.3 mm inner diameter. Crystals of SmelDhp suitable for data collection and structure determination were grown in the presence of agarose at 0.1%(w/v) in order to ensure mass transport controlled by diffusion. X-ray data were collected to a resolution of 1.85 Å. The crystal belongs to the orthorhombic space group C222 1 , with unit-cell parameters a = 124.89, b = 126.28, c = 196.10 Å and two molecules in the asymmetric unit. A molecular-replacement solution has been determined and refinement is in progress

Comparative toxicity assessment of CeO{sub 2} and ZnO nanoparticles towards Sinorhizobium meliloti, a symbiotic alfalfa associated bacterium: Use of advanced microscopic and spectroscopic techniques

Energy Technology Data Exchange (ETDEWEB)

Bandyopadhyay, Susmita [Environmental Science and Engineering PhD Program, The University of Texas at El Paso, 500 West University Avenue, El Paso, TX 79968 (United States); University of California Center for Environmental Implications of Nanotechnology (UC CEIN), The University of Texas at El Paso (United States); Peralta-Videa, Jose R. [Department of Chemistry, The University of Texas at El Paso, 500 West University Avenue, El Paso, TX 79968 (United States); Plascencia-Villa, German; Jose-Yacaman, Miguel [Department of Physics and Astronomy, The University of Texas at San Antonio, One UTSA Circle, San Antonio, TX 78249 (United States); Gardea-Torresdey, Jorge L., E-mail: jgardea@utep.edu [Environmental Science and Engineering PhD Program, The University of Texas at El Paso, 500 West University Avenue, El Paso, TX 79968 (United States); Department of Chemistry, The University of Texas at El Paso, 500 West University Avenue, El Paso, TX 79968 (United States); University of California Center for Environmental Implications of Nanotechnology (UC CEIN), The University of Texas at El Paso (United States)

2012-11-30

Highlights: Black-Right-Pointing-Pointer First cytotoxicity study of CeO{sub 2} and ZnO nanoparticles to Sinorhizobium meliloti. Black-Right-Pointing-Pointer First report upon the mechanisms of CeO{sub 2} and ZnO NPs toxicity to S. meliloti. Black-Right-Pointing-Pointer ZnO NPs were found to be bactericidal in lower concentration. Black-Right-Pointing-Pointer CeO{sub 2} NPs had bacteriostatic effect on S. meliloti. - Abstract: Cerium oxide (CeO{sub 2}) and zinc oxide (ZnO) nanoparticles (NPs) are extensively used in a variety of instruments and consumer goods. These NPs are of great concern because of potential toxicity towards human health and the environment. The present work aimed to assess the toxic effects of 10 nm CeO{sub 2} and ZnO NPs towards the nitrogen fixing bacterium Sinorhizobium meliloti. Toxicological parameters evaluated included UV/Vis measurement of minimum inhibitory concentration, disk diffusion tests, and dynamic growth. Ultra high-resolution scanning transmission electron microscopy (STEM) and infrared spectroscopy (FTIR) were utilized to determine the spatial distribution of NPs and macromolecule changes in bacterial cells, respectively. Results indicate that ZnO NPs were more toxic than CeO{sub 2} NPs in terms of inhibition of dynamic growth and viable cells counts. STEM images revealed that CeO{sub 2} and ZnO NPs were found on bacterial cell surfaces and ZnO NPs were internalized into the periplasmic space of the cells. FTIR spectra showed changes in protein and polysaccharide structures of extra cellular polymeric substances present in bacterial cell walls treated with both NPs. The growth data showed that CeO{sub 2} NPs have a bacteriostatic effect, whereas ZnO NPs is bactericidal to S. meliloti. Overall, ZnO NPs were found to be more toxic than CeO{sub 2} NPs.

Symbiotic Nitrogen Fixation in Alfalfa (Medicago Sativa L.) by Sinorhizobium Meliloti at Al-Qassim Regions, Saudi Arabia

International Nuclear Information System (INIS)

Al-Barakah, F. N.; Mridha, M. A. U.

2016-01-01

The nodulation status in alfalfa (Medicago sativa L.) plants by Sinorhizobium meliloti under Saudi field condition was assessed in some selected farms in four seasons for two years. In the present study, we also monitored the introduced S. meliloti strains activity under Saudi soil conditions. The samples were collected at regular seasonal intervals from the selected farms. The total number of nodules, morphology of the nodules and the effectiveness of N/sub 2/-fixation was assessed. In general, it was revealed that soils in the selected areas in Saudi Arabia have sufficient bacteria of the proper types to nodulate the alfalfa plants. These nodules are high in number, small in size and white in color. The nodules obtained from most of the selected farms are ineffective for nitrogen fixation. Inoculation of alfalfa seeds with imported S. meliloti strains failed to fix the atmospheric nitrogen sufficiently and also the growth improvement of alfalfa plants. There was a wide variation in the occurrence of number of nodules among the four seasons in two years. It was also observed that summer season severely affected the nodulation making it nearly zero. This low number of nodules exerts a very slow recovery of nodule formation in the next year. The introduced strains were always over competing with the native strains but they did not survive because of hot and dry summer. Nitrogenase activity of the nodules collected from both the inoculated and non-inoculated farms were always very low in all the collected samples, which indicates that the ability of fixing nitrogen by S. meliloti strains in alfalfa under Saudi soils conditions is very low. (author)

Genome-engineered Sinorhizobium meliloti for the production of poly(lactic-co-3-hydroxybutyric) acid copolymer.

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Tran, Tam T; Charles, Trevor C

2016-02-01

Economically competitive commercial production of biodegradable bioplastics with desirable properties is an important goal. In this study, we demonstrate the use of chromosome engineering of an alternative bacterial host, Sinorhizobium meliloti, for production of the copolymer, poly(lactate-co-3-hydroxybutyrate). Codon-optimized genes for 2 previously engineered enzymes, Clostridium propionicum propionate CoA transferase (Pct532Cp) and Pseudomonas sp. strain MBEL 6-19 polyhydroxyalkanoate (PHA) synthase 1 (PhaC1400Ps6-19), were introduced into S. meliloti Rm1021 by chromosome integration, replacing the native phbC gene. On the basis of phenotypic analysis and detection of polymer product by gas chromatography analysis, synthesis and accumulation of the copolymer was confirmed. The chromosome integrant strain, with the introduced genes under the control of the native phbC promoter, is able to produce over 15% cell dry mass of poly(lactate-co-3-hydroxybutyrate), containing 30 mol% lactate, from growth on mannitol. We were also able to purify the polymer from the culture and confirm the structure by NMR and GC-MS. To our knowledge, this is the first demonstration of production of this copolymer in the Alphaproteobacteria. Further optimization of this system may eventually yield strains that are able to produce economically viable commercial product.

Crystallization and preliminary crystallographic studies of an active-site mutant hydantoin racemase from Sinorhizobium meliloti CECT4114

International Nuclear Information System (INIS)

Martínez-Rodríguez, Sergio; González-Ramírez, Luis Antonio; Clemente-Jiménez, Josefa María; Rodríguez-Vico, Felipe; Las Heras-Vázquez, Francisco Javier; Gavira, Jose Antonio; García-Ruiz, Juan Ma.

2007-01-01

Crystals of an active-site mutated hydantoin racemase from S. meliloti have been obtained in the presence and absence of d,l-5-isopropyl-hydantoin and characterized by X-ray diffraction. A recombinant active-site mutant of hydantoin racemase (C76A) from Sinorhizobium meliloti CECT 4114 (SmeHyuA) has been crystallized in the presence and absence of the substrate d,l-5-isopropyl hydantoin. Crystals of the SmeHyuA mutant suitable for data collection and structure determination were grown using the counter-diffusion method. X-ray data were collected to resolutions of 2.17 and 1.85 Å for the free and bound enzymes, respectively. Both crystals belong to space group R3 and contain two molecules of SmeHyuA per asymmetric unit. The crystals of the free and complexed SmeHyuA have unit-cell parameters a = b = 85.43, c = 152.37 Å and a = b = 85.69, c = 154.38 Å, crystal volumes per protein weight (V M ) of 1.94 and 1.98 Å 3 Da −1 and solvent contents of 36.7 and 37.9%, respectively

Robustness encoded across essential and accessory replicons of the ecologically versatile bacterium Sinorhizobium meliloti

Science.gov (United States)

Walker, Graham C.; Finan, Turlough M.; Mengoni, Alessio; Griffitts, Joel S.

2018-01-01

Bacterial genome evolution is characterized by gains, losses, and rearrangements of functional genetic segments. The extent to which large-scale genomic alterations influence genotype-phenotype relationships has not been investigated in a high-throughput manner. In the symbiotic soil bacterium Sinorhizobium meliloti, the genome is composed of a chromosome and two large extrachromosomal replicons (pSymA and pSymB, which together constitute 45% of the genome). Massively parallel transposon insertion sequencing (Tn-seq) was employed to evaluate the contributions of chromosomal genes to growth fitness in both the presence and absence of these extrachromosomal replicons. Ten percent of chromosomal genes from diverse functional categories are shown to genetically interact with pSymA and pSymB. These results demonstrate the pervasive robustness provided by the extrachromosomal replicons, which is further supported by constraint-based metabolic modeling. A comprehensive picture of core S. meliloti metabolism was generated through a Tn-seq-guided in silico metabolic network reconstruction, producing a core network encompassing 726 genes. This integrated approach facilitated functional assignments for previously uncharacterized genes, while also revealing that Tn-seq alone missed over a quarter of wild-type metabolism. This work highlights the many functional dependencies and epistatic relationships that may arise between bacterial replicons and across a genome, while also demonstrating how Tn-seq and metabolic modeling can be used together to yield insights not obtainable by either method alone. PMID:29672509

Succinoglycan Production Contributes to Acidic pH Tolerance in Sinorhizobium meliloti Rm1021.

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Hawkins, Justin P; Geddes, Barney A; Oresnik, Ivan J

2017-12-01

In this work, the hypothesis that exopolysaccharide plays a role in the survival of Sinorhizobium meliloti at low pH levels is addressed. When S. meliloti was grown at pH 5.75, synthesis of succinoglycan increased, whereas synthesis of galactoglucan decreased. Succinoglycan that was isolated from cultures grown at low pH had a lower degree of polymerization relative to that which was isolated from cultures grown at neutral pH, suggesting that low-molecular weight (LMW) succinoglycan might play a role in adaptation to low pH. Mutants unable to produce succinoglycan or only able to produce high-molecular weight polysaccharide were found to be sensitive to low pH. However, strains unable to produce LMW polysaccharide were 10-fold more sensitive. In response to low pH, transcription of genes encoding proteins for succinoglycan, glycogen, and cyclic β(1-2) glucans biosynthesis increased, while those encoding proteins necessary for the biosynthesis of galactoglucan decreased. While changes in pH did not affect the production of glycogen or cyclic β(1-2) glucan, it was found that the inability to produce cyclic β(1-2) glucan did contribute to pH tolerance in the absence of succinoglycan. Finally, in addition to being sensitive to low pH, a strain carrying mutations in exoK and exsH, which encode the glycanases responsible for the cleavage of succinoglycan to LMW succinoglycan, exhibited a delay in nodulation and was uncompetitive for nodule occupancy. Taken together, the data suggest that the role for LMW succinoglycan in nodule development may be to enhance survival in the colonized curled root hair.

Nodulation by Sinorhizobium meliloti originated from a mining soil alleviates Cd toxicity and increases Cd-phytoextraction in Medicago sativa L.

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Ghnaya, Tahar; Mnassri, Majda; Ghabriche, Rim; Wali, Mariem; Poschenrieder, Charlotte; Lutts, Stanley; Abdelly, Chedly

2015-01-01

Besides their role in nitrogen supply to the host plants as a result of symbiotic N fixation, the association between legumes and Rhizobium could be useful for the rehabilitation of metal-contaminated soils by phytoextraction. A major limitation presents the metal-sensitivity of the bacterial strains. The aim of this work was to explore the usefulness of Sinorhizobium meliloti originated from a mining site for Cd phytoextraction by Medicago sativa. Inoculated and non-inoculated plants were cultivated for 60 d on soils containing 50 and/or 100 mg Cd kg(-1) soil. The inoculation hindered the occurrence of Cd- induced toxicity symptoms that appeared in the shoots of non-inoculated plants. This positive effect of S. meliloti colonization was accompanied by an increase in biomass production and improved nutrient acquisition comparatively to non-inoculated plants. Nodulation enhanced Cd absorption by the roots and Cd translocation to the shoots. The increase of plant biomass concomitantly with the increase of Cd shoot concentration in inoculated plants led to higher potential of Cd-phytoextraction in these plants. In the presence of 50 mg Cd kg(-1) in the soil, the amounts of Cd extracted in the shoots were 58 and 178 μg plant(-1) in non-inoculated and inoculated plants, respectively. This study demonstrates that this association M. sativa-S. meliloti may be an efficient biological system to extract Cd from contaminated soils.

Brucella melitensis MucR, an orthologue of Sinorhizobium meliloti MucR, is involved in resistance to oxidative, detergent, and saline stresses and cell envelope modifications.

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Mirabella, A; Terwagne, M; Zygmunt, M S; Cloeckaert, A; De Bolle, X; Letesson, J J

2013-02-01

Brucella spp. and Sinorhizobium meliloti are alphaproteobacteria that share not only an intracellular lifestyle in their respective hosts, but also a crucial requirement for cell envelope components and their timely regulation for a successful infectious cycle. Here, we report the characterization of Brucella melitensis mucR, which encodes a zinc finger transcriptional regulator that has previously been shown to be involved in cellular and mouse infections at early time points. MucR modulates the surface properties of the bacteria and their resistance to environmental stresses (i.e., oxidative stress, cationic peptide, and detergents). We show that B. melitensis mucR is a functional orthologue of S. meliloti mucR, because it was able to restore the production of succinoglycan in an S. meliloti mucR mutant, as detected by calcofluor staining. Similar to S. meliloti MucR, B. melitensis MucR also represses its own transcription and flagellar gene expression via the flagellar master regulator ftcR. More surprisingly, we demonstrate that MucR regulates a lipid A core modification in B. melitensis. These changes could account for the attenuated virulence of a mucR mutant. These data reinforce the idea that there is a common conserved circuitry between plant symbionts and animal pathogens that regulates the relationship they have with their hosts.

Hydrogen peroxide-regulated genes in the Medicago truncatula-Sinorhizobium meliloti symbiosis.

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Andrio, Emilie; Marino, Daniel; Marmeys, Anthony; de Segonzac, Marion Dunoyer; Damiani, Isabelle; Genre, Andrea; Huguet, Stéphanie; Frendo, Pierre; Puppo, Alain; Pauly, Nicolas

2013-04-01

Reactive oxygen species (ROS), particularly hydrogen peroxide (H(2)O(2)), play an important role in signalling in various cellular processes. The involvement of H(2)O(2) in the Medicago truncatula-Sinorhizobium meliloti symbiotic interaction raises questions about its effect on gene expression. A transcriptome analysis was performed on inoculated roots of M. truncatula in which ROS production was inhibited with diphenylene iodonium (DPI). In total, 301 genes potentially regulated by ROS content were identified 2 d after inoculation. These genes included MtSpk1, which encodes a putative protein kinase and is induced by exogenous H(2)O(2) treatment. MtSpk1 gene expression was also induced by nodulation factor treatment. MtSpk1 transcription was observed in infected root hair cells, nodule primordia and the infection zone of mature nodules. Analysis with a fluorescent protein probe specific for H(2)O(2) showed that MtSpk1 expression and H(2)O(2) were similarly distributed in the nodule infection zone. Finally, the establishment of symbiosis was impaired by MtSpk1 downregulation with an artificial micro-RNA. Several genes regulated by H(2)O(2) during the establishment of rhizobial symbiosis were identified. The involvement of MtSpk1 in the establishment of the symbiosis is proposed. © 2013 The Authors. New Phytologist © 2013 New Phytologist Trust.

The DivJ, CbrA and PleC system controls DivK phosphorylation and symbiosis in Sinorhizobium meliloti

Science.gov (United States)

Pini, Francesco; Frage, Benjamin; Ferri, Lorenzo; De Nisco, Nicole J.; Mohapatra, Saswat S.; Taddei, Lucilla; Fioravanti, Antonella; Dewitte, Frederique; Galardini, Marco; Brilli, Matteo; Villeret, Vincent; Bazzicalupo, Marco; Mengoni, Alessio; Walker, Graham C.; Becker, Anke; Biondi, Emanuele G.

2013-01-01

SUMMARY Sinorhizobium meliloti is a soil bacterium that invades the root nodules it induces on Medicago sativa, whereupon it undergoes an alteration of its cell cycle and differentiates into nitrogen-fixing, elongated and polyploid bacteroid with higher membrane permeability. In Caulobacter crescentus, a related alphaproteobacterium, the principal cell cycle regulator, CtrA, is inhibited by the phosphorylated response regulator DivK. The phosphorylation of DivK depends on the histidine kinase DivJ, while PleC is the principal phosphatase for DivK. Despite the importance of the DivJ in C. crescentus, the mechanistic role of this kinase has never been elucidated in other Alphaproteobacteria. We show here that the histidine kinases DivJ together with CbrA and PleC participate in a complex phosphorylation system of the essential response regulator DivK in S. meliloti. In particular, DivJ and CbrA are involved in DivK phosphorylation and in turn CtrA inactivation, thereby controlling correct cell cycle progression and the integrity of the cell envelope. In contrast, the essential PleC presumably acts as a phosphatase of DivK. Interestingly, we found that a DivJ mutant is able to elicit nodules and enter plant cells, but fails to establish an effective symbiosis suggesting that proper envelope and/or low CtrA levels are required for symbiosis. PMID:23909720

A global analysis of protein expression profiles in Sinorhizobium meliloti: discovery of new genes for nodule occupancy and stress adaptation.

Science.gov (United States)

Djordjevic, Michael A; Chen, Han Cai; Natera, Siria; Van Noorden, Giel; Menzel, Christian; Taylor, Scott; Renard, Clotilde; Geiger, Otto; Weiller, Georg F

2003-06-01

A proteomic examination of Sinorhizobium meliloti strain 1021 was undertaken using a combination of 2-D gel electrophoresis, peptide mass fingerprinting, and bioinformatics. Our goal was to identify (i) putative symbiosis- or nutrient-stress-specific proteins, (ii) the biochemical pathways active under different conditions, (iii) potential new genes, and (iv) the extent of posttranslational modifications of S. meliloti proteins. In total, we identified the protein products of 810 genes (13.1% of the genome's coding capacity). The 810 genes generated 1,180 gene products, with chromosomal genes accounting for 78% of the gene products identified (18.8% of the chromosome's coding capacity). The activity of 53 metabolic pathways was inferred from bioinformatic analysis of proteins with assigned Enzyme Commission numbers. Of the remaining proteins that did not encode enzymes, ABC-type transporters composed 12.7% and regulatory proteins 3.4% of the total. Proteins with up to seven transmembrane domains were identified in membrane preparations. A total of 27 putative nodule-specific proteins and 35 nutrient-stress-specific proteins were identified and used as a basis to define genes and describe processes occurring in S. meliloti cells in nodules and under stress. Several nodule proteins from the plant host were present in the nodule bacteria preparations. We also identified seven potentially novel proteins not predicted from the DNA sequence. Post-translational modifications such as N-terminal processing could be inferred from the data. The posttranslational addition of UMP to the key regulator of nitrogen metabolism, PII, was demonstrated. This work demonstrates the utility of combining mass spectrometry with protein arraying or separation techniques to identify candidate genes involved in important biological processes and niche occupations that may be intransigent to other methods of gene expression profiling.

Evaluation of SinoRhizobium meliloti Efficiency and Qualitative Traits of Alfalfa under Application of Molybdenum

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F Ahmadi Dana

2017-12-01

Full Text Available Introduction Agriculture depends heavily on nitrogen which is biologically fixed through the symbiotic association between rhizobia and legume plants in nodules located on plant roots. Alfalfa is a legume that should fix most of its own N requirement if it is sufficiently nodulated by viable Rhizobium meliloti inoculums. The process of nitrogen fixation is done by the help of an enzyme called nitrogenase and molybdenum which is an important element in the formation of this compound. Molybdenum is required by plants for protein synthesis and is especially important for legumes as it is needed for nitrogen fixation by rhizobia. Therefore the following research was done aimed on studying the effect of different amount of molybdenum and S. rhizobium bacteria on alfalfa’s yield. Material and Methods Alfalfa (Medicago sativa were grown in a field. The experiment was conducted at Karaj in 2013 in split plot arrangement based on completely randomized block design (RCBD, including 2 caring S. rhizobium inoculated seed and non-inoculated as the main plot factorand 3 levels of Molybdenum (0,5,10 kg ha-1 from ammonium molybdate (as the sub plot factor in three replications. Sinorhizobium meliloti bacteria were cultured on plates. Then half of the seeds were inoculated by Sinorhizobium meliloti. Nitrogen fertilizer was added only in one stage before planting up to 50 kg per hectare. Plants were grown until flowering. The data were analyzed by the SAS (9.1 software and mean comparisons were done by Duncan's MRT at the 1% and 5% probability level. Results and Discussion The results showed the effect of different levels of molybdenum and S. Rhizobium bacteria on dry matter yield, molybdenum concentrations in shoots and roots and the number of root nodules was significant. This treatment was significant in comparison to the control treatment with the14.27 ton per hectare. Increasing of molybdenum application, led to increasing of root nodules and showed a

Sinorhizobium meliloti sigma factors RpoE1 and RpoE4 are activated in stationary phase in response to sulfite.

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Bénédicte Bastiat

Full Text Available Rhizobia are soil bacteria able to establish a nitrogen-fixing symbiosis with legume plants. Both in soil and in planta, rhizobia spend non-growing periods resembling the stationary phase of in vitro-cultured bacteria. The primary objective of this work was to better characterize gene regulation in this biologically relevant growth stage in Sinorhizobium meliloti. By a tap-tag/mass spectrometry approach, we identified five sigma factors co-purifying with the RNA polymerase in stationary phase: the general stress response regulator RpoE2, the heat shock sigma factor RpoH2, and three extra-cytoplasmic function sigma factors (RpoE1, RpoE3 and RpoE4 belonging to the poorly characterized ECF26 subgroup. We then showed that RpoE1 and RpoE4 i are activated upon metabolism of sulfite-generating compounds (thiosulfate and taurine, ii display overlapping regulatory activities, iii govern a dedicated sulfite response by controlling expression of the sulfite dehydrogenase SorT, iv are activated in stationary phase, likely as a result of endogenous sulfite generation during bacterial growth. We showed that SorT is required for optimal growth of S. meliloti in the presence of sulfite, suggesting that the response governed by RpoE1 and RpoE4 may be advantageous for bacteria in stationary phase either by providing a sulfite detoxification function or by contributing to energy production through sulfite respiration. This paper therefore reports the first characterization of ECF26 sigma factors, the first description of sigma factors involved in control of sulphur metabolism, and the first indication that endogenous sulfite may act as a signal for regulation of gene expression upon entry of bacteria in stationary phase.

Inter-replicon Gene Flow Contributes to Transcriptional Integration in the Sinorhizobium meliloti Multipartite Genome.

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diCenzo, George C; Wellappili, Deelaka; Golding, G Brian; Finan, Turlough M

2018-05-04

Integration of newly acquired genes into existing regulatory networks is necessary for successful horizontal gene transfer (HGT). Ten percent of bacterial species contain at least two DNA replicons over 300 kilobases in size, with the secondary replicons derived predominately through HGT. The Sinorhizobium meliloti genome is split between a 3.7 Mb chromosome, a 1.7 Mb chromid consisting largely of genes acquired through ancient HGT, and a 1.4 Mb megaplasmid consisting primarily of recently acquired genes. Here, RNA-sequencing is used to examine the transcriptional consequences of massive, synthetic genome reduction produced through the removal of the megaplasmid and/or the chromid. Removal of the pSymA megaplasmid influenced the transcription of only six genes. In contrast, removal of the chromid influenced expression of ∼8% of chromosomal genes and ∼4% of megaplasmid genes. This was mediated in part by the loss of the ETR DNA region whose presence on pSymB is due to a translocation from the chromosome. No obvious functional bias among the up-regulated genes was detected, although genes with putative homologs on the chromid were enriched. Down-regulated genes were enriched in motility and sensory transduction pathways. Four transcripts were examined further, and in each case the transcriptional change could be traced to loss of specific pSymB regions. In particularly, a chromosomal transporter was induced due to deletion of bdhA likely mediated through 3-hydroxybutyrate accumulation. These data provide new insights into the evolution of the multipartite bacterial genome, and more generally into the integration of horizontally acquired genes into the transcriptome. Copyright © 2018 diCenzo, et al.

Inter-replicon Gene Flow Contributes to Transcriptional Integration in the Sinorhizobium meliloti Multipartite Genome

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George C. diCenzo

2018-05-01

Full Text Available Integration of newly acquired genes into existing regulatory networks is necessary for successful horizontal gene transfer (HGT. Ten percent of bacterial species contain at least two DNA replicons over 300 kilobases in size, with the secondary replicons derived predominately through HGT. The Sinorhizobium meliloti genome is split between a 3.7 Mb chromosome, a 1.7 Mb chromid consisting largely of genes acquired through ancient HGT, and a 1.4 Mb megaplasmid consisting primarily of recently acquired genes. Here, RNA-sequencing is used to examine the transcriptional consequences of massive, synthetic genome reduction produced through the removal of the megaplasmid and/or the chromid. Removal of the pSymA megaplasmid influenced the transcription of only six genes. In contrast, removal of the chromid influenced expression of ∼8% of chromosomal genes and ∼4% of megaplasmid genes. This was mediated in part by the loss of the ETR DNA region whose presence on pSymB is due to a translocation from the chromosome. No obvious functional bias among the up-regulated genes was detected, although genes with putative homologs on the chromid were enriched. Down-regulated genes were enriched in motility and sensory transduction pathways. Four transcripts were examined further, and in each case the transcriptional change could be traced to loss of specific pSymB regions. In particularly, a chromosomal transporter was induced due to deletion of bdhA likely mediated through 3-hydroxybutyrate accumulation. These data provide new insights into the evolution of the multipartite bacterial genome, and more generally into the integration of horizontally acquired genes into the transcriptome.

Mining the Sinorhizobium meliloti transportome to develop FRET biosensors for sugars, dicarboxylates and cyclic polyols.

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Alexandre Bourdès

Full Text Available Förster resonance energy transfer (FRET biosensors are powerful tools to detect biologically important ligands in real time. Currently FRET bisosensors are available for twenty-two compounds distributed in eight classes of chemicals (two pentoses, two hexoses, two disaccharides, four amino acids, one nucleobase, two nucleotides, six ions and three phytoestrogens. To expand the number of available FRET biosensors we used the induction profile of the Sinorhizobium meliloti transportome to systematically screen for new FRET biosensors.Two new vectors were developed for cloning genes for solute-binding proteins (SBPs between those encoding FRET partner fluorescent proteins. In addition to a vector with the widely used cyan and yellow fluorescent protein FRET partners, we developed a vector using orange (mOrange2 and red fluorescent protein (mKate2 FRET partners. From the sixty-nine SBPs tested, seven gave a detectable FRET signal change on binding substrate, resulting in biosensors for D-quinic acid, myo-inositol, L-rhamnose, L-fucose, β-diglucosides (cellobiose and gentiobiose, D-galactose and C4-dicarboxylates (malate, succinate, oxaloacetate and fumarate. To our knowledge, we describe the first two FRET biosensor constructs based on SBPs from Tripartite ATP-independent periplasmic (TRAP transport systems.FRET based on orange (mOrange2 and red fluorescent protein (mKate2 partners allows the use of longer wavelength light, enabling deeper penetration of samples at lower energy and increased resolution with reduced back-ground auto-fluorescence. The FRET biosensors described in this paper for four new classes of compounds; (i cyclic polyols, (ii L-deoxy sugars, (iii β-linked disaccharides and (iv C4-dicarboxylates could be developed to study metabolism in vivo.

Influence of different Sinorhizobium meliloti inocula on abundance of genes involved in nitrogen transformations in the rhizosphere of alfalfa (Medicago sativa L.).

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Babić, Katarina Huić; Schauss, Kristina; Hai, Brigitte; Sikora, Sanja; Redzepović, Sulejman; Radl, Viviane; Schloter, Michael

2008-11-01

Inoculation of leguminous seeds with selected rhizobial strains is practised in agriculture to ameliorate the plant yield by enhanced root nodulation and nitrogen uptake of the plant. However, effective symbiosis between legumes and rhizobia does not only depend on the capacity of nitrogen fixation but also on the entire nitrogen turnover in the rhizosphere. We investigated the influence of seed inoculation with two indigenous Sinorhizobium meliloti strains exhibiting different efficiency concerning plant growth promotion on nitrogen turnover processes in the rhizosphere during the growth of alfalfa. Quantification of six target genes (bacterial amoA, nirK, nirS, nosZ, nifH and archaeal amoA) within the nitrogen cycle was performed in rhizosphere samples before nodule formation, at bud development and at the late flowering stage. The results clearly demonstrated that effectiveness of rhizobial inocula is related to abundance of nifH genes in the late flowering phase of alfalfa. Moreover, other genes involved in nitrogen turnover had been affected by the inocula, e.g. higher numbers of amoA copies were observed during flowering when the more effective strain had been inoculated. However, the respective gene abundances differed overall to a greater extent between the three plant development stages than between the inoculation variants.

NCBI nr-aa BLAST: CBRC-DRER-26-0474 [SEVENS

Lifescience Database Archive (English)

Full Text Available CBRC-DRER-26-0474 ref|NP_384172.1| SENSOR HISTIDINE KINASE TRANSMEMBRANE PROTEIN [S...inorhizobium meliloti 1021] emb|CAC41453.1| SENSOR HISTIDINE KINASE TRANSMEMBRANE PROTEIN [Sinorhizobium meliloti] NP_384172.1 1e-159 68% ...

ORF Alignment: NC_003047 [GENIUS II[Archive

Lifescience Database Archive (English)

Full Text Available NC_003047 gi|15964777 >1v4aA 24 435 49 467 e-101 ... emb|CAC45596.1| PUTATIVE GLUTAMATE-AMMONIA...-LIGASE ADENYLYLTRANSFERASE PROTEIN ... [Sinorhizobium meliloti] ref|NP_385130.1| PUTATIVE ... GLUTAMATE-AMMO...NIA-LIGASE ADENYLYLTRANSFERASE PROTEIN ... [Sinorhizobium meliloti 1021] sp|

Transcriptome Response to Heavy Metals in Sinorhizobium meliloti CCNWSX0020 Reveals New Metal Resistance Determinants That Also Promote Bioremediation by Medicago lupulina in Metal-Contaminated Soil.

Science.gov (United States)

Lu, Mingmei; Jiao, Shuo; Gao, Enting; Song, Xiuyong; Li, Zhefei; Hao, Xiuli; Rensing, Christopher; Wei, Gehong

2017-10-15

The symbiosis of the highly metal-resistant Sinorhizobium meliloti CCNWSX0020 and Medicago lupulina has been considered an efficient tool for bioremediation of heavy metal-polluted soils. However, the metal resistance mechanisms of S. meliloti CCNWSX00200 have not been elucidated in detail. Here we employed a comparative transcriptome approach to analyze the defense mechanisms of S. meliloti CCNWSX00200 against Cu or Zn exposure. Six highly upregulated transcripts involved in Cu and Zn resistance were identified through deletion mutagenesis, including genes encoding a multicopper oxidase (CueO), an outer membrane protein (Omp), sulfite oxidoreductases (YedYZ), and three hypothetical proteins (a CusA-like protein, a FixH-like protein, and an unknown protein), and the corresponding mutant strains showed various degrees of sensitivity to multiple metals. The Cu-sensitive mutant (Δ cueO ) and three mutants that were both Cu and Zn sensitive (Δ yedYZ , Δ cusA -like, and Δ fixH -like) were selected for further study of the effects of these metal resistance determinants on bioremediation. The results showed that inoculation with the Δ cueO mutant severely inhibited infection establishment and nodulation of M. lupulina under Cu stress, while inoculation with the Δ yedYZ and Δ fixH -like mutants decreased just the early infection frequency and nodulation under Cu and Zn stresses. In contrast, inoculation with the Δ cusA -like mutant almost led to loss of the symbiotic capacity of M. lupulina to even grow in uncontaminated soil. Moreover, the antioxidant enzyme activity and metal accumulation in roots of M. lupulina inoculated with all mutants were lower than those with the wild-type strain. These results suggest that heavy metal resistance determinants may promote bioremediation by directly or indirectly influencing formation of the rhizobium-legume symbiosis. IMPORTANCE Rhizobium-legume symbiosis has been promoted as an appropriate tool for bioremediation of heavy

Three way interactions between Thymus vulgaris, Medicago truncatula and Sinorhizobium meliloti

DEFF Research Database (Denmark)

Grøndahl, Eva; Ehlers, Bodil Kirstine

2012-01-01

Thymus vulgaris is a dominating component of the Mediterranean garrigue vegetation. It produces aromatic oil, containing monoterpenes, which affects the performance (growth, survival) of other plants, and microorganisms. Annual plant species of the genus Medicago are commonly found in Mediterranean...... shows patterns of adaptation to its thyme neighbor, and 2) if any adaptive response was dependent on the rhizobium, and whether the rhizobium was either "experienced" or "naive" with respect to thyme monoterpenes. Using a G*G*E design, the fitness of 13 genotypes of Medicago truncatula was tested....... Of these genotypes, 7 were ”experienced”, and 6 were ”naive” to thyme. All genotypes were grown on soil either amended with thyme monoterpene or not. In addition, each plant received a rhizobium treatment, which was either: no rhizobium, a mix of thyme experienced Sinorhizobium genotypes, or a mix of thyme naive...

Evaluation of the Effects of Biological and Chemical Fertilizers on Some Physiological and Growth Characteristics of Fenugreek (Trigonella foenum-graecum

Directory of Open Access Journals (Sweden)

Samira Menbari

2017-12-01

Full Text Available Nowadays, the use of soil-born microorganisms as biological fertilizers is considered to be a natural and most desirable solution to maintain sustainability of agricultural soil system. Potassium releasing bacteria, nitrogen fixing and phosphorus dissolving bacteria make mentioned elements available to plants. In order to evaluate the effects of bio-fertilizers Potabarvar 2, Sinorhizobium meliloti, as well as urea fertilizer on physiological properties and yield of Fenugreek, an experiment as complete randomized block design was conducted with five treatments and three replications. Treatments included biofertilizer Potabarvar 2, S. meliloti, inoculation with a mixture of Sinorhizobium+Potabarvar 2, positive control (based on soil analysis and negative control (no fertilization and inoculation.The results showed that all morphological traits were significant at 1%. Most physiological traits except for carotenoid were significantly affected by S. meliloti, and a mixture of Sinorhizobium+Potabarvar 2. Seed inoculation with biofertilizer Sinorhizobium meliloti and Potabarvar 2 lead to increase in growth and eventually shoot yield. Separate application of these biofertilizers led to better results than the integrated application. Symbiotic relationship of Sinorhizobium with Fenugreek increased physiological indices data, especially the absorption of nitrogen and phosphorus, as well as the amount of phenolic antioxidant have been significantly affected. In general, application of S. meliloti resulted in better and more effective increase in yield, quality and plant growth than fertilizer Potabarvar 2 and a mixture of Sinorhizobium+Potabarvar 2.

ORF Alignment: NC_003037 [GENIUS II[Archive

Lifescience Database Archive (English)

Full Text Available NC_003037 gi|16263035 >1nyoA 11 162 8 158 4e-30 ... ref|NP_435828.1| Nex18 Symbiotica...lly induced conserved protein [Sinorhizobium ... meliloti 1021] gb|AAK65240.1| Nex18 Symbiotically ... ... ... induced conserved protein [Sinorhizobium meliloti 1021] ... pir||F95334 Nex18 Symbiotically

Transcriptome Analysis of Polyhydroxybutyrate Cycle Mutants Reveals Discrete Loci Connecting Nitrogen Utilization and Carbon Storage in Sinorhizobium meliloti.

Science.gov (United States)

D'Alessio, Maya; Nordeste, Ricardo; Doxey, Andrew C; Charles, Trevor C

2017-01-01

Polyhydroxybutyrate (PHB) and glycogen polymers are produced by bacteria as carbon storage compounds under unbalanced growth conditions. To gain insights into the transcriptional mechanisms controlling carbon storage in Sinorhizobium meliloti , we investigated the global transcriptomic response to the genetic disruption of key genes in PHB synthesis and degradation and in glycogen synthesis. Under both nitrogen-limited and balanced growth conditions, transcriptomic analysis was performed with genetic mutants deficient in PHB synthesis ( phbA , phbB , phbAB , and phbC ), PHB degradation ( bdhA , phaZ , and acsA2 ), and glycogen synthesis ( glgA1 ). Three distinct genomic regions of the pSymA megaplasmid exhibited altered expression in the wild type and the PHB cycle mutants that was not seen in the glycogen synthesis mutant. An Fnr family transcriptional motif was identified in the upstream regions of a cluster of genes showing similar transcriptional patterns across the mutants. This motif was found at the highest density in the genomic regions with the strongest transcriptional effect, and the presence of this motif upstream of genes in these regions was significantly correlated with decreased transcript abundance. Analysis of the genes in the pSymA regions revealed that they contain a genomic overrepresentation of Fnr family transcription factor-encoding genes. We hypothesize that these loci, containing mostly nitrogen utilization, denitrification, and nitrogen fixation genes, are regulated in response to the intracellular carbon/nitrogen balance. These results indicate a transcriptional regulatory association between intracellular carbon levels (mediated through the functionality of the PHB cycle) and the expression of nitrogen metabolism genes. IMPORTANCE The ability of bacteria to store carbon and energy as intracellular polymers uncouples cell growth and replication from nutrient uptake and provides flexibility in the use of resources as they are available to

Genome-Wide Transcriptional Changes and Lipid Profile Modifications Induced by Medicago truncatula N5 Overexpression at an Early Stage of the Symbiotic Interaction with Sinorhizobium meliloti

Directory of Open Access Journals (Sweden)

Chiara Santi

2017-12-01

Full Text Available Plant lipid-transfer proteins (LTPs are small basic secreted proteins, which are characterized by lipid-binding capacity and are putatively involved in lipid trafficking. LTPs play a role in several biological processes, including the root nodule symbiosis. In this regard, the Medicago truncatula nodulin 5 (MtN5 LTP has been proved to positively regulate the nodulation capacity, controlling rhizobial infection and nodule primordia invasion. To better define the lipid transfer protein MtN5 function during the symbiosis, we produced MtN5-downregulated and -overexpressing plants, and we analysed the transcriptomic changes occurring in the roots at an early stage of Sinorhizobium meliloti infection. We also carried out the lipid profile analysis of wild type (WT and MtN5-overexpressing roots after rhizobia infection. The downregulation of MtN5 increased the root hair curling, an early event of rhizobia infection, and concomitantly induced changes in the expression of defence-related genes. On the other hand, MtN5 overexpression favoured the invasion of the nodules by rhizobia and determined in the roots the modulation of genes that are involved in lipid transport and metabolism as well as an increased content of lipids, especially galactolipids that characterize the symbiosome membranes. Our findings suggest the potential participation of LTPs in the synthesis and rearrangement of membranes occurring during the formation of the infection threads and the symbiosome membrane.

Induction and characterization of monoclonal anti-idiotypic antibodies reactive with idiotopes of canine parvovirus neutralizing monoclonal antibodies.

NARCIS (Netherlands)

G.F. Rimmelzwaan (Guus); J. van Es (Johan); G.A. Drost; F.G.C.M. Uytdehaag (Fons); A.D.M.E. Osterhaus (Albert)

1991-01-01

textabstractMonoclonal anti-idiotypic (anti-Id) antibodies (Ab2) were generated against idiotypes (Id) of canine parvovirus (CPV) specific monoclonal antibodies (MoAbs). The binding of most of these anti-Id antibodies to their corresponding Id could be inhibited by antigen, thus classifying these

A sensitive radioimmunoassay for the detection of monoclonal anti-idiotype antibodies

International Nuclear Information System (INIS)

Morahan, G.

1983-01-01

A radioimmunoassay was developed in order to detect anti-idiotypic antibodies in the supernatants of hybrid cells. This assay is both sensitive and specific for anti-idiotypic (but not anti-allotypic) antibodies. Monoclonal antibodies present in test supernatants are bound by an anti-immunoglobulin coated solid phase. Subsequent incubation with a source of mouse immunoglobulin 'blocks' unreacted anti-immunoglobulin antibodies on the solid phase. Anti-idiotypic antibodies are then detected by their ability to bind 125 I-labelled idiotype-bearing antibody. This paper describes the use of this assay to detect monoclonal anti-idiotypic antibodies in 2 systems; the cross-reactive idiotype of A/J anti-ABA antibodies, and the idiotype expressed by the myeloma protein HOPC 8. Similarly, 125 I-labelled anti-idiotype antibodies may be used in this assay to detect monoclonal idiotype-bearing antibodies. Further modifications are described which would allow the detection of monoclonal anti-allotype antibodies. (Auth.)

Denitrification by Rhizobium meliloti

Energy Technology Data Exchange (ETDEWEB)

Rosen, A

1996-10-01

Rhizobium meliloti strains were investigated for their denitrification activity as free-living cells and in nodules on lucerne (Medicago sativa) roots. They were also investigated for presence of nitrous oxide reductase (nos) activity and for genes using a nosZ probe derived from the Pseudomonas stutzeri. To decide whether R. meliloti strains used as inoculants contribute to the total denitrification activity in a lucerne ley, strains with different denitrifying capacities were used in field and laboratory experiments. The nitrate reduction activity of R. meliloti during anaerobic respiration was compared with that of a strain of Pseudomonas aeruginosa. A great diversity in the denitrification activity was found within strains of R. meliloti, and four of thirteen investigated strains showed an obvious denitrification activity. Two denitrifying bacteria were used as references, one strain each of Bradyrhizobium japonicum and P. aeruginosa. All but one of the R. meliloti strains hybridized to the PstI-fragment of the nosZ-gene from P. stutzeri. Two sizes of the hybridizing fragment, 5 and 7 kb, were noticed. Nos activity was only shown in three R. meliloti strains, and these were all characterized by a high denitrification activity. The potential denitrification activity was about 20, 40, and 80 times higher than the actual denitrification activity for lucerne, fallow, and grass, respectively. The potential denitrification activity was almost the same in lucerne and grass planted soils. Compared with the unplanted soil, the presence of lucerne roots in the soil increased the actual denitrification activity, while roots of both plant species, grass and lucerne, increased the potential denitrification activity in the soil. 32 refs, 7 figs, 1 tab

Evaluation of monoclonal anti-A and anti-B and affinity-purified Ulex europaeus lectin I for forensic blood grouping.

Science.gov (United States)

Gaensslen, R E; Lee, H C; Carroll, J E

1984-01-01

Two different monoclonal anti-A and anti-B and several different affinity purified Ulex europaeus lectin I reagents were evaluated and compared with conventional anti-A and anti-B sera and Ulex anti-H for serologic properties, in inhibition tests with secretor salivas, and in elution tests with bloodstains. The monoclonal and purified reagents were found to be comparable to conventional ones, and accordingly suitable for forensic inhibition and elution procedures.

Anti-idiotypes against a monoclonal anti-haloperidol antibody bind to dopamine receptor

International Nuclear Information System (INIS)

Elazar, Z.; Kanety, H.; Schreiber, M.; Fuchs, S.

1988-01-01

Anti-idiotypic antibodies were raised in rabbits by immunization with a monoclonal anti-haloperidol antibody. Some of these anti-idiotypic antibodies bind in a concentration dependent manner to bovine striatal membranes. Following affinity purification, these antibodies inhibit haloperidol binding to striatal membranes and deplete [ 3 H]-spiperone binding sites from a solubilized preparation of striatal membranes. It is thus concluded that these anti-idiotypic antibodies are an internal image of haloperidol and as such can interact with D 2 -dopamine receptors

Sinorhizobium arboris sp. nov. and Sinorhizobium kostiense sp. nov., isolated from leguminous trees in Sudan and Kenya.

Science.gov (United States)

Nick, G; de Lajudie, P; Eardly, B D; Suomalainen, S; Paulin, L; Zhang, X; Gillis, M; Lindström, K

1999-10-01

SDS-PAGE of total bacterial proteins was applied to the classification of 25 Sudanese and five Kenyan strains isolated from the root nodules of Acacia senegal and Prosopis chilensis. Twenty strains were also studied by multilocus enzyme electrophoresis (MLEE) and the whole 16S rRNA gene was sequenced from two strains representing the two major clusters. These results, together with the previously reported numerical taxonomy analysis, pulsed-field gel electrophoresis studies, DNA-DNA dot-blot hybridization, genomic fingerprinting using repetitive sequence-based PCR, DNA base composition analysis, DNA-DNA reassociation analysis, partial sequencing of the 16S rRNA gene and RFLP analysis of the amplified 16S rRNA gene, showed that all 30 strains belong to the genus Sinorhizobium. Two of the strains grouped with Sinorhizobium saheli and seven with Sinorhizobium terangae, while the rest did not cluster with any of the established species. The majority of the strains formed two phenotypically and genotypically distinct groups and we therefore propose that these strains should be classified as two new species, Sinorhizobium arboris sp. nov. and Sinorhizobium kostiense sp. nov.

Medicago sativa--Sinorhizobium meliloti Symbiosis Promotes the Bioaccumulation of Zinc in Nodulated Roots.

Science.gov (United States)

Zribi, Kais; Nouairi, Issam; Slama, Ines; Talbi-Zribi, Ons; Mhadhbi, Haythem

2015-01-01

In this study we investigated effects of Zn supply on germination, growth, inorganic solutes (Zn, Ca, Fe, and Mg) partitioning and nodulation of Medicago sativa This plant was cultivated with and without Zn (2 mM). Treatments were plants without (control) and with Zn tolerant strain (S532), Zn intolerant strain (S112) and 2 mM urea nitrogen fertilisation. Results showed that M. sativa germinates at rates of 50% at 2 mM Zn. For plants given nitrogen fertilisation, Zn increased plant biomass production. When grown with symbionts, Zn supply had no effect on nodulation. Moreover, plants with S112 showed a decrease of shoot and roots biomasses. However, in symbiosis with S532, an increase of roots biomass was observed. Plants in symbiosis with S. meliloti accumulated more Zn in their roots than nitrogen fertilised plants. Zn supply results in an increase of Ca concentration in roots of fertilised nitrogen plants. However, under Zn supply, Fe concentration decreased in roots and increased in nodules of plants with S112. Zn supply showed contrasting effects on Mg concentrations for plants with nitrogen fertilisation (increase) and plants with S112 (decrease). The capacity of M. sativa to accumulate Zn in their nodulated roots encouraged its use in phytostabilisation processes.

ORF Alignment: NC_003047 [GENIUS II[Archive

Lifescience Database Archive (English)

Full Text Available ... [Sinorhizobium meliloti 1021] ... Length = 101 ... Query: 1 ... MFAVIKTGGKQYRVAANDVITIEKLEGVAGDKIEF...TEILMVGVGADATIGAPFVEGAVVS 60 ... MFAVIKTGGKQYRVAANDVITIEKLEGVAGDKIEFTEILMV...GVGADATIGAPFVEGAVVS Sbjct: 1 ... MFAVIKTGGKQYRVAANDVITIEKLEGVAGDKIEFTEILMVGVGADATIGAPFVEGAVVS 60 ...

ORF Alignment: NC_003047 [GENIUS II[Archive

Lifescience Database Archive (English)

Full Text Available ... [Sinorhizobium meliloti 1021] ... Length = 118 ... Query: 249 ELTCADIMSRDVVTVPGDTTPDHARYLLLKHDIRT...LPVLDENEKLQGTVGLRELAGKEPG 308 ... ELTCADIMSRDVVTVPGDTTPDHARYLLLKHDIRTLP...VLDENEKLQGTVGLRELAGKEPG Sbjct: 1 ... ELTCADIMSRDVVTVPGDTTPDHARYLLLKHDIRTLPVLDENEKLQGTVGLRELAGKEPG 60 ...

ORF Alignment: NC_003047 [GENIUS II[Archive

Lifescience Database Archive (English)

Full Text Available SE ... PROTEIN [Sinorhizobium meliloti 1021] ... Length = 130 ... Query: 751 AREDEGAAEASRLRMEIDELRSILETATDGVVVLGRDGDIRT...MNRSASALFDYDEADMRG 810 ... AREDEGAAEASRLRMEIDELRSILETATDGVVVLGRDGDIRT...MNRSASALFDYDEADMRG Sbjct: 1 ... AREDEGAAEASRLRMEIDELRSILETATDGVVVLGRDGDIRTMNRSASALFDYDEADMRG

ORF Alignment: NC_003047 [GENIUS II[Archive

Lifescience Database Archive (English)

Full Text Available ... [Sinorhizobium meliloti 1021] ... Length = 138 ... Query: 9 ... SKAPRSVLFMCGMNAIRSPMAEALARVALPKGTYVASAGVRQGERD...PFVDVVLEEVGLTI 68 ... SKAPRSVLFMCGMNAIRSPMAEALARVALPKGTYVASAGVRQGERD...PFVDVVLEEVGLTI Sbjct: 1 ... SKAPRSVLFMCGMNAIRSPMAEALARVALPKGTYVASAGVRQGERDPFVDVVLEEVGLTI 60 ... Query: 12

ORF Alignment: NC_003047 [GENIUS II[Archive

Lifescience Database Archive (English)

Full Text Available R ... PROTEIN [Sinorhizobium meliloti 1021] ... Length = 106 ... Query: 12 ... ELTVGEVAERSGLAVSTLHFYEAKGLIRSN...RSRGNQRRYPRSVLRRVAVIKVAQRTGIPL 71 ... ELTVGEVAERSGLAVSTLHFYEAKGLIRSN...RSRGNQRRYPRSVLRRVAVIKVAQRTGIPL Sbjct: 1 ... ELTVGEVAERSGLAVSTLHFYEAKGLIRSNRSRGNQRRYPRSVLRRVAVIKVAQRTGIPL 60 ...

Clearance of 131I-labeled murine monoclonal antibody from patients' blood by intravenous human anti-murine immunoglobulin antibody

International Nuclear Information System (INIS)

Stewart, J.S.; Sivolapenko, G.B.; Hird, V.; Davies, K.A.; Walport, M.; Ritter, M.A.; Epenetos, A.A.

1990-01-01

Five patients treated with intraperitoneal 131I-labeled mouse monoclonal antibody for ovarian cancer also received i.v. exogenous polyclonal human anti-murine immunoglobulin antibody. The pharmacokinetics of 131I-labeled monoclonal antibody in these patients were compared with those of 28 other patients receiving i.p.-radiolabeled monoclonal antibody for the first time without exogenous human anti-murine immunoglobulin, and who had no preexisting endogenous human anti-murine immunoglobulin antibody. Patients receiving i.v. human anti-murine immunoglobulin antibody demonstrated a rapid clearance of 131I-labeled monoclonal antibody from their circulation. The (mean) maximum 131I blood content was 11.4% of the injected activity in patients receiving human anti-murine immunoglobulin antibody compared to 23.3% in patients not given human anti-murine immunoglobulin antibody. Intravenous human anti-murine immunoglobulin antibody decreased the radiation dose to bone marrow (from 131I-labeled monoclonal antibody in the vascular compartment) 4-fold. Following the injection of human anti-murine immunoglobulin antibody, 131I-monoclonal/human anti-murine immunoglobulin antibody immune complexes were rapidly transported to the liver. Antibody dehalogenation in the liver was rapid, with 87% of the injected 131I excreted in 5 days. Despite the efficient hepatic uptake of immune complexes, dehalogenation of monoclonal antibody was so rapid that the radiation dose to liver parenchyma from circulating 131I was decreased 4-fold rather than increased. All patients developed endogenous human anti-murine immunoglobulin antibody 2 to 3 weeks after treatment

Use of radiolabeled monoclonal anti-B1 antibody for B lymphocyte imaging in Rhesus monkeys

International Nuclear Information System (INIS)

Letvin, N.L.; Zalutsky, M.R.; Chalifoux, L.V.; Atkins, H.L.

1987-01-01

Imaging tissues rich in B lymphocytes in man using a radiolabeled monoclonal anti-B cell antibody would be extremely useful in the clinical staging of non-Hodgkins lymphomas. Studies were done in rhesus monkeys using radiolabeled monoclonal anti-B1 antibody to determine the feasibility of such an approach. Immunohistologic studies demonstrated that infused monoclonal anti-B1 binds in vivo with specificity to B cells in lymph nodes and spleen. The kinetics of clearance of 131 I-labeled anti-B1 were determined. The B lymphocyte-rich spleen could be readily visualized by gamma camera scanning without significant background and without the need for image intensification or blood background subtraction techniques. These data support the feasibility of using anti-B1 for staging B cell lymphomas in man. (author)

ORF Alignment: NC_003078 [GENIUS II[Archive

Lifescience Database Archive (English)

Full Text Available lator, ArsR family protein [Sinorhizobium meliloti ... 1021] ... Length = 83 ... Query: 8 ... LSALADPTRRAIVARLAAGEATVNELAAPFEM...SLPAVSKHLKVLERAGLISRGRNAQWRP 67 ... LSALADPTRRAIVARLAAGEATVNELAAPFEMSLPAVSKHL...KVLERAGLISRGRNAQWRP Sbjct: 1 ... LSALADPTRRAIVARLAAGEATVNELAAPFEMSLPAVSKHLKVLERAGLISRGRNAQWRP 60 ...

ORF Sequence: NC_003078 [GENIUS II[Archive

Lifescience Database Archive (English)

Full Text Available etitiveness [Sinorhizobium meliloti 1021] MQLSACARRREAVRYRRRMARILILLFSLLSAFAFPVTPVP... NC_003078 gi|16264863 >gi|16264863|ref|NP_437655.1| probable membrane protein necessary for nodulation comp

ORF Sequence: NC_003047 [GENIUS II[Archive

Lifescience Database Archive (English)

Full Text Available NC_003047 gi|15964332 >gi|15964332|ref|NP_384685.1| PROBABLE PYRAZINAMIDASE/NICOTINAMIDAS...E (INCLUDES: PYRAZINAMIDASE, NICOTINAMIDASE) PROTEIN [Sinorhizobium meliloti 1021] MADAARPDLREAMADEAL

ORF Alignment: NC_003047 [GENIUS II[Archive

Lifescience Database Archive (English)

Full Text Available MOYL-PHOSPHATE SYNTHASE LARGE CHAIN (AMMONIA CHAIN ... ARGININE BIOSYNTHESIS) PROTEIN [Sinorhizobium... meliloti] ... ref|NP_385682.1| PROBABLE CARBAMOYL-PHOSPHATE SYNTHASE ... LARGE CHAIN (AMMONIA

Roles of Extracellular Polysaccharides and Biofilm Formation in Heavy Metal Resistance of Rhizobia

OpenAIRE

Natalia Nocelli; Pablo C. Bogino; Erika Banchio; Walter Giordano

2016-01-01

Bacterial surface components and extracellular compounds, particularly flagella, lipopolysaccharides (LPSs), and exopolysaccharides (EPSs), in combination with environmental signals and quorum-sensing signals, play crucial roles in bacterial autoaggregation, biofilm development, survival, and host colonization. The nitrogen-fixing species Sinorhizobium meliloti (S. meliloti) produces two symbiosis-promoting EPSs: succinoglycan (or EPS I) and galactoglucan (or EPS II). Studies of the S. melilo...

Monoclonal anti-melanoma antibodies and their possible clinical use

International Nuclear Information System (INIS)

Hellstroem, K.E.; Hellstroem, Ingegerd; Washington Univ., Seattle; Washington Univ., Seattle

1985-01-01

Cell surface antigens of human melanoma, as defined by monoclonal antibodies, are discussed and in particular the three antigens p97, a GD3 ganglioside and a proteoglycan. The potential diagnostic uses of antibodies to melanoma antigens are reviewed including in vitro diagnosis by immuno-histology, in vitro diagnosis by serum assays and in vivo diagnosis by tumour imaging using radioactively labelled antibodies. The potential therapeutic uses of monoclonal antibodies to melanoma antigens are also reviewed including targets for antibody therapy, the use of antibodies alone, radiolabelled antibodies, antibody-toxin conjugates, antibody-drug conjugates, anti-idiotypic antibodies and vaccines. (UK)

Monoclonal anti-elastin antibody labelled with technetium-99m

International Nuclear Information System (INIS)

Oliveira, Marcia B.N. de; Silva, Claudia R. da; Araujo, Adriano C. de; Bernardo Filho, Mario; Porto, Luis Cristovao M.S.; Gutfilen, Bianca; Souza, J.E.Q.; Frier, Malcolm

1999-01-01

Technetium-99m ( 99m Tc) is widely employed in nuclear medicine due to its desirable physical, chemical and biological properties. Moreover, it is easily available and normally is inexpensive. A reducing agent is necessary to label cells and molecules with 99m Tc and stannous chloride (Sn C L 2 ) is usually employed. Elastin is the functional protein component of the elastic fiber and it is related with some diseases such as arteriosclerosis, pulmonary emphysema and others. The present study refers to the preparation of the 99m Tc labeled monoclonal anti-elastin antibody. The monoclonal antibody was incubated with an excess of 2-iminothiolane. The free thiol groups created, were capable of binding with the reduced technetium. Labeling was an exchange reaction with 99m Tc-glucoheptonate. The labeled preparation was left at 4 deg C for one hour. Then, it was passed through a Sephadex G50 column. Various fractions were collected and counted. A peak corresponding to the radiolabeled antibody was obtained. Stability studies of the labelled anti-elastin were performed at 0,3 6, 24 hours, at both 4 deg C or room temperature. The biodistribution pattern of the 99m Tc-anti-elastin was studied in healthy male Swiss mice. The immunoreactivity was also determined. An useful labeled-anti-elastin was obtained to future immunoscintigraphic investigations. (author)

ORF Sequence: NC_003047 [GENIUS II[Archive

Lifescience Database Archive (English)

Full Text Available NC_003047 gi|15965329 >gi|15965329|ref|NP_385682.1| PROBABLE CARBAMOYL-PHOSPHATE SYNTHASE LARGE CHAIN (AMMO...NIA CHAIN ARGININE BIOSYNTHESIS) PROTEIN [Sinorhizobium meliloti 1021] MPKRQDIKSILI

Labeling an anti-CD20 monoclonal antibody with 90Y

International Nuclear Information System (INIS)

Perera Pintado, Alejandro; Leyva Montaña, René; Prats Capote, Anaís; Góngora Bravo, Magdiel; Alberti Ramírez, Alejandro; León, Mariela; Hernández González, Ignacio; Dorvignit, Denise

2016-01-01

Lymphomas are among the 10 leading causes of death, both in Cuba and in the world, with an increasing incidence in recent years. Follicular lymphoma low-grade (indolent) is one of the most common in the Western world, representing 1/3 of all non-Hodgkin lymphomas (NHL). More than 90% of patients present with disseminated disease at diagnosis and generally have a slow evolution and good response to conventional treatment; but radically changed its forecast to relapse, resistance to therapeutic and histologic transformation can occur. The monoclonal antibody therapy has been a promising therapeutic. In this respect CD20 antigen it has been considered one of the most attractive targets in the therapy of follicular B cell lymphoma This is expressed in more than 90% of cases, while not present in stem cells and lines progenitors. Despite the success of immunotherapy, the relapse rate is still considerable. In order to increase the cytotoxic potential of immunotherapy, marked with beta emitting radionuclides alpha particles or monoclonal antibodies are used today. Despite encouraging results in patients with non-Hodgkin lymphomas refractory to other treatments, the extremely high costs of these commercial radiopharmaceuticals have greatly limited its application, even in the first world. A sustainable alternative is the marking of other anti-CD20 monoclonal antibodies, so researchers from several countries have concentrated their efforts on rituximaby other similar antibodies labeled with therapeutic radionuclides, as a possible cost-effectively to more problem. Today in Cuba it has an electrolytic generator 90 Sr- 90 Y Isotope Center, which ensures the availability of the radionuclide. In addition, the chimeric MAb rituximab is applied as part of the therapy of NHL in its health system and, recently, the Center for Molecular Immunology has obtained a chimeric monoclonal anti-CD20 antibody biosimilar rituximab, which is in phase clinical trial; which opens prospects for

[Study of anti-idiotype antibodies to human monoclonal antibody].

Science.gov (United States)

Harada, R; Takahashi, N; Owaki, I; Kannagi, R; Endo, N; Morita, N; Inoue, M

1992-02-01

A human monoclonal antibody, ll-50 (IgM, lambda), was generated, which reacted specifically with a major of glycolipid present in LS174T colon cancer cells. The glycolipid antigen which reacted with the ll-50 antibody was expected to four sugar residues from its TLC mobility, and it was ascertained that the glycolipid antigen which reacted with ll-50 antibody might be Lc4 antigen [Gal beta 1----3 GLcNAc beta 1----3 Gal beta 1----4 Glc beta 1----1 Cer] judging from TLC immunostaining and ELISA when the reactivity of ll-50 antibody was tested using various pure glycolipids in 3-5 sugar residues as an antigen. Sera in patients with malignant disorders and healthy individuals were analyzed by Sandwich assay of immobilized and biotinylated ll-50 antibody. The serum of the Lc4 antigen recognized by ll-50 antibody was significantly higher in patients with malignant disorders than that in healthy individuals (p less than 0.05). Three mouse monoclonal anti-idiotype antibodies, G3, B3 and C5 (all IgG1), were generated by the immunization of BALB/c mice with ll-50 antibody. These anti-idiotype antibodies specifically bound to to human monoclonal antibody, ll-50 and had a significant inhibitory activity towards the binding of ll-50 antibody to the Lc4 antigen. This indicated that these anti-idiotype antibodies, G3, B3, and C5, were paratope-related anti-idiotype antibodies. G3, B3, and C5 were expected to define the nearest idiotope because they could mutually inhibit ll-50 antibody. Sera in patients with malignant disorders and healthy individuals were analyzed by Sandwich assay of immobilized and biotinylated anti-idiotype antibodies, G3, B3, and C5. As to the ll-50 like antibodies defined by C5 (Id-C5+), the mean serum level in patients with malignant disorders was significantly higher than that in healthy individuals (p less than 0.05). As to the ll-50 like antibodies defined by B3 (Id-B3+), the mean serum level in patients with malignant disorders was significantly higher

Anti-interleukin-17 monoclonal antibody ixekizumab in chronic plaque psoriasis

DEFF Research Database (Denmark)

Leonardi, Craig; Matheson, Robert; Zachariae, Claus

2012-01-01

Type 17 helper T cells have been suggested to play a pathological role in psoriasis. They secrete several proinflammatory cytokines, including interleukin-17A (also known as interleukin-17). We evaluated the safety and efficacy of ixekizumab (LY2439821), a humanized anti-interleukin-17 monoclonal...... antibody, for psoriasis treatment....

ORF Alignment: NC_003047 [GENIUS II[Archive

Lifescience Database Archive (English)

Full Text Available NC_003047 gi|15966456 >1gkmA 2 501 3 499 e-107 ... emb|CAC47282.1| PUTATIVE HISTIDINE AMMONIA...-LYASE PROTEIN [Sinorhizobium meliloti] ... ref|NP_386809.1| PUTATIVE HISTIDINE AMMONIA-LYASE

Enzyme-linked immunosorbent assay for total sennosides using anti-sennside A and anti-sennoside B monoclonal antibodies.

Science.gov (United States)

Morinaga, Osamu; Uto, Takuhiro; Sakamoto, Seiichi; Tanaka, Hiroyuki; Shoyama, Yukihiro

2009-01-01

Total sennosides concentration is a very important factor when rhubarb and senna will be used as crude drugs. However, one-step analytical technique for total sennosides has not been reported except HPLC. An enzyme-linked immunosorbent assay (ELISA) for total sennosides concentration by using the combination of anti-sennoside A (SA) and anti-sennoside B (SB) monoclonal antibodies (MAbs) in a single assay has been investigated. Total sennosides concentration in rhubarb and senna samples determined by newly developed assay system showed good agreement with those analyzed by ELISA using anti-SA MAb and anti-SB MAb, respectively.

Production of human anti-HLA monoclonal antibodies

Energy Technology Data Exchange (ETDEWEB)

Walker, M.C.; Mercier, F.; Roger, J.; Varin, M.

1986-03-01

Only 40% of the several hundred anti-HLA murine monoclonal antibodies (MAbs) that have been made detect HLA-A,B,C or DR specificities previously defined by human alloantisera, the range of recognized specificities is very narrow, and few of the MAbs have proven useful as tissue typing reagents. In hopes of obtaining HLA typing reagents, the authors are developing a protocol for the production of human anti-HLA MAbs from HLA-antigen (Ag) immunized peripheral blood B cells of volunteering renal patients, immunized to one or more HLA Ags through therapeutic blood transfusions. A simple enrichment of the donor B cells has not been sufficient for anti-HLA MAb production, the authors are currently delineating the conditions necessary for increasing the number of HLA-specific donor B cells by in vitro stimulation with cells expressing the HLA Ag to which the B cell donor is immunized. For the production of MAbs, the stimulated B cells are transformed with Epstein-Barr virus and subsequently fused with KR-4 lymphoblastoid cells. Hybridomas are selected by HAT and Ouabain. Supernatants are screened for anti-HLA activity against lymphocyte targets expressing the original immunizing HLA Ag by complement mediated /sup 51/Cr release assay. Antibody specificity is determined by the complement-dependent microcytotoxicity test used for HLA typing.

A murine monoclonal anti-idiotypic antibody detects a common idiotope on human, mouse and rabbit antibodies to allergen Lol p IV.

Science.gov (United States)

Zhou, E M; Dzuba-Fischer, J M; Rector, E S; Sehon, A H; Kisil, F T

1991-09-01

A syngeneic mouse monoclonal anti-idiotypic antibody (anti-Id), designated as B1/1, was generated against a monoclonal antibody (MoAb 91) specific for Ryegrass pollen allergen Lol p IV. This anti-Id recognized an idiotope (Id) that was also present on other monoclonal antibodies with the same specificity as MoAb 91. Observations that (i) the anti-Id inhibited the binding of MoAb 91 to Lol p IV and (ii) the Id-anti-Id interaction could be inhibited by Lol p IV indicated that the Id was located within or near the antigen combining site. These properties served to characterize B1/1 as an internal image anti-Id. Evidence that an immune response in different species to Lol p IV elicits the formation of antibodies which express a common Id was provided by the observations that (i) the Id-anti-Id interactions could be inhibited by mouse, human and rabbit antisera to Lol p IV and (ii) the binding of these antisera to Lol p IV could be inhibited by the anti-Id. Interestingly, the internal image anti-Id B1/1 also recognized an Id on a monoclonal antibody which was directed to an epitope of Lol p IV, different from that recognized by MoAb 91.

Radioiodination of monoclonal antibody intact anti-CEA

International Nuclear Information System (INIS)

Okada, H.; Souza, I.T.T.; Silva, C.P.G.

1990-11-01

The purpose of this study is to examine a convenient system that can be used to iodinate monoclonal antibodies which is rapid, simple, efficient and reproducible, and which can be accomplished in radiopharmaceutical laboratories. It is important to remember that antibodies are sensitive biochemicals, subject to losses of the activity that is essential to their mode of action, namely the ability to bind specific antigen. The advent of solid phase iodination agents has greatly expanded the range of gentle iodination techniques available for iodinating sensitive biological materials. The agent most widely used is the Iodogen (1,3,4,6 tetrachloro-3a-6a diphenylglycoluril) method. Anti-CEA 4C sub(11) IgG sub(2a,k) (prepared in the Ludwig Institute-Sao Paulo-Brazil ) is used as model to evaluate the Iodogen methodology. The miniature chromatographic system, also rapid, accurate, simple, efficient was elaborated to determine the labelling efficiency incorporation of iodine into immunoglobulin, and the radiochemical purity of sup(131)I-anti-CEA. (author)

Final report for DOE grant FG02-06ER15805

Energy Technology Data Exchange (ETDEWEB)

Gage, Daniel

2012-05-31

DOE funding was used to investigate the role of the phosphotransferase system (PTS) in the symbiotic, nodulating bacterium Sinorhizobium meliloti. This system is well studied in several bacterial species. However, it's organization and function in S. meliloti is substantially different than in the those other, well-studied bacteria. The S. meliloti PTS, through our DOE-funded work, has become a model for how this important signal transduction system works in the a-proteobacteria. We have found that the PTS is relatively simple, used for only signal transduction and not transport, and is involved in regulation of carbon metabolism in response to carbon availability and nitrogen availability.

Evaluación de los reactivos hemoclasificadores monoclonales cubanos HEMO CIM ANTI-A y HEMO CIM ANTI-B en pacientes VIH/SIDA Evaluation of the Cuban anti-A Hemo CIM and anti-B Hemo CIM monoclonal hemoclassifiers in HIV/AIDS patients

Directory of Open Access Journals (Sweden)

Ananidia Rivero

2000-12-01

Full Text Available Se describe un estudio realizado con los hemoclasificadores monoclonales cubanos Hemo CIM anti-A y anti-B producidos por el Centro de Inmunología Molecular (CIM y comercializados por CIMAB S.A, para ser evaluados en pacientes VIH/SIDA. Se analizaron un total de 130 pacientes en el Servicio de Transfusiones del Instituto "Pedro Kourí". Se utilizaron en paralelo los antisueros policlonales de producción nacional (Laboratorios Betera y los reactivos monoclonales comerciales producidos y donados por International Blood Group Reference Laboratory; Bristol, UK. Se demostró que los reactivos Hemo CIM anti-A y Hemo CIM anti-B se comportaron de forma similar a sus homólogos comerciales y policlonales. Al comparar la efectividad de la aglutinación para los 3 reactivos pudimos comprobar que con el reactivo monoclonal, Hemo CIM anti-A y anti-B se obtuvieron los mejores resultados expresados en crucesA study conducted with the Cuban anti-A and anti-B Hemo CIM monoclonal hemoclassifiers produced by the Center of Molecular Immunology (CMI and commercialized by CIMAB S.A. to be evaluated in HIV/AIDS patients is described. 130 patients were analyzed at the Service of Transfusions of "Pedro Kouri" Institute. The polyclonal antisera of national production (Betera Laboratories and the commercial monoclonal reagents produced and donated by the International Blood Group Reference Laboratory; Bristol, UK, were used at the same time. It was proved that the anti-A Hemo CIM and anti-B Hemo CIM reagents behaved similarly to their commercial and polyclonal homologues. On comparing the effectiveness of the agglutination for the 3 reagents, we were able to verify that the best results expressed in crosses were obtained with the anti-A and anti-B Hemo CIM monoclonal reagents

Lymphocyte antibody-dependent cytotoxicity test for evaluation of clinical role of monoclonal anti-D-antibodies for prevention of rhesus sensitization.

Science.gov (United States)

Olovnikova, N I; Belkina, E V; Nikolaeva, T L; Miterev, G Yu; Chertkov, I L

2006-01-01

Monoclonal antibodies to D antigen were studied in the reaction of antibody-dependent cytotoxicity for evaluation of the possibility of using these antibodies for preventing rhesus sensitization. High hemolytic activity of four anti-D-monoclonal antibodies in the antibody-dependent cytotoxicity test, mediated by their interaction with FcgammaRI, and the capacity to accelerate elimination of D+ erythrocytes from circulation did not provide the immunosuppressive effect. It was hypothesized that monoclonal antibodies for prevention of rhesus sensitization should interact with FcgammaRIII on lymphocytes. These monoclonal antibodies are extremely rare: only 4 of 125 studied antibodies mediated hemolysis in the antibody-dependent cytotoxicity test with lymphocytes, while all polyclonal anti-D-preparations exhibited this activity.

The importance of tumor marker titers for the indication of immunoscintigraphy with monoclonal antibodies anti-CEA and anti-CA 19.9

International Nuclear Information System (INIS)

Bouvier, J.F.; Charrie, A.; Fleury-Goyon, M.C.; Chauvot, P. et; Lahneche, B.E.

1986-01-01

In 18 patients operated for malignant tumors 20 immunoscintigraphies were done with a monoclonal antibody cocktail (anti-CEA F(ab') 2 and anti-CA 19.9 F(ab') 2 ). Immediately before scintigraphy tumor marker titers in plasma were determined in all cases. Tumor marker levels corresponding to positive or doubtful scintigraphies are analysed. (Author)

Anti-leukemic activity and tolerability of anti-human CD47 monoclonal antibodies

Science.gov (United States)

Pietsch, E C; Dong, J; Cardoso, R; Zhang, X; Chin, D; Hawkins, R; Dinh, T; Zhou, M; Strake, B; Feng, P-H; Rocca, M; Santos, C Dos; Shan, X; Danet-Desnoyers, G; Shi, F; Kaiser, E; Millar, H J; Fenton, S; Swanson, R; Nemeth, J A; Attar, R M

2017-01-01

CD47, a broadly expressed cell surface protein, inhibits cell phagocytosis via interaction with phagocyte-expressed SIRPα. A variety of hematological malignancies demonstrate elevated CD47 expression, suggesting that CD47 may mediate immune escape. We discovered three unique CD47-SIRPα blocking anti-CD47 monoclonal antibodies (mAbs) with low nano-molar affinity to human and cynomolgus monkey CD47, and no hemagglutination and platelet aggregation activity. To characterize the anti-cancer activity elicited by blocking CD47, the mAbs were cloned into effector function silent and competent Fc backbones. Effector function competent mAbs demonstrated potent activity in vitro and in vivo, while effector function silent mAbs demonstrated minimal activity, indicating that blocking CD47 only leads to a therapeutic effect in the presence of Fc effector function. A non-human primate study revealed that the effector function competent mAb IgG1 C47B222-(CHO) decreased red blood cells (RBC), hematocrit and hemoglobin by >40% at 1 mg/kg, whereas the effector function silent mAb IgG2σ C47B222-(CHO) had minimal impact on RBC indices at 1 and 10 mg/kg. Taken together, our findings suggest that targeting CD47 is an attractive therapeutic anti-cancer approach. However, the anti-cancer activity observed with anti-CD47 mAbs is Fc effector dependent as are the side effects observed on RBC indices. PMID:28234345

Rhizobia from Lanzarote, the Canary Islands, that nodulate Phaseolus vulgaris have characteristics in common with LMW RNA group II Sinorhizobium meliloti of Medicago, Melilotus and Trigonella from soils of mainland Spain

Science.gov (United States)

Several isolates from nodules of Phaseolus vulgaris grown in soil of Lanzarote, an island of the Canaries, had electrophoretic LMW RNA patterns identical with a less common pattern within S. meliloti (assigned as group II) obtained from nodules of alfalfa and alfalfa-related legumes grown in northe...

"Ménage à trois"

DEFF Research Database (Denmark)

Ehlers, Bodil K; Grøndahl, Eva; Ronfort, Joëlle

2012-01-01

of both partners. So far these studies have been typically carried out in a single environment. Here, we ask if the genetic correlation between fitness of the host plant Medicago truncatula (Fabaceae) and its bacterial symbiont Sinorhizobium meliloti is affected by the presence/absence of a monoterpene...

Anti-CEA monoclonal antibody in the diagnosis of colorectal, lung and ovarian carcinoma

International Nuclear Information System (INIS)

Jiang, N.; Lu, B.; Lu, X.; Sha, X.; Yue, D.

2000-01-01

This study evaluated the diagnostic value of radioimmnoimaging (RII) with 99 Tc labeled monoclonal antibody C50, raised originally against carcinoembryonic antigen (anti-CEA) in various tumors. 152 pathologically confirmed patients with a tumor were imaged prior to surgery with an anti-CEA monoclonal antibody labeled with 99 Tc. There were 115 patients with ovarian carcinoma, 26 patients with colorectal carcinoma and 11 patients with lung carcinoma. Images were acquired at 3-6 h post injection and were analyzed by the double blind method. Images of patients with ovarian cancer were compared with B-ultrasound images. Immunohistochemical staining was performed on all cases of colorectal cancer. All RII images demonstrated excellent contrast, clear lesions, and no serious toxic or other side reactions occurred. Transient chills and fever were observed in 3 cases. This study showed a sensitivity=88.2%, specificity=83.2%, and an accuracy=4.0%. The smallest lesion size detected was 2 x 2 cm. The total combined lesion detection rate for primary, metastatic, and recurrence lesions was 84.4%. We conclude that 99 Tc labeled anti-CEA MoAb C50 can be used in the diagnosis of colorectal carcinoma, ovarian carcinoma, and lung carcinoma

The combination of anti-KIR monoclonal antibodies with anti-PD-1/PD-L1 monoclonal antibodies could be a critical breakthrough in overcoming tumor immune escape in NSCLC

Directory of Open Access Journals (Sweden)

He YY

2018-04-01

Full Text Available Yayi He,1,2,* Sangtian Liu,1,* Jane Mattei,3 Paul A Bunn Jr,2 Caicun Zhou,1 Daniel Chan2 1Department of Medical Oncology, Shanghai Pulmonary Hospital, Tongji University Medical School Cancer Institute, Tongji University School of Medicine, Shanghai, China; 2Division of Medical Oncology, Department of Medicine, University of Colorado Anschutz Medical Campus, Aurora, CO, USA; 3Oncology Department, Moinhos de Vento Hospital, Porto Alegre, Brazil *These authors contributed equally to this work Background: The anti-programmed death-1 (PD-1/programmed death ligand-1 (PD-L1 monoclonal antibody has a good effect in the treatment of non-small cell lung cancer (NSCLC, but not all PD-1/PD-L1 positive patients can get benefit from it. Compensatory expression of other immune checkpoints may be correlated with the poor efficacy of anti-PD-1/PD-L1 monoclonal antibodies. The inhibitory human leukocyte antigen (HLA/killer cell Ig-like receptor (KIR can effectively block the killing effect of natural killer (NK cells on tumors. Our previous studies have confirmed that high expression of KIR was correlated with poor prognosis of NSCLC. Inhibitory KIR expression was positively correlated with the expression of PD-1. Methods: The expressions of KIR 2D (L1, L3, L4, S4 (BC032422/ADQ31987/NP_002246/NP_036446, Abcam and PD-1 (NAT 105, Cell marque proteins was assessed by immunohis­tochemistry. Results: The expression of inhibitory KIR in tumor cells or tumor infiltrating lymphocytes (TILs is associated with PD-1 expression. Among PD-1 positive patients, 76.3% were KIR 2D (L1, L3, L4, S4 positive on tumor cells, and 74.6% were KIR 2D (L1, L3, L4, S4 positive on TILs. We compared the expression of inhibitory KIR before and after treatment with nivolumab in 11 patients with NSCLC. We found that five (45.5% patients had positive expression of inhibitory KIR in tumor tissue after being treated with anti-PD-1 monoclonal antibodies, two of whom exhibited a significant

Replicon-dependent differentiation of symbiosis-related genes in Sinorhizobium strains nodulating Glycine max.

Science.gov (United States)

Guo, Hui Juan; Wang, En Tao; Zhang, Xing Xing; Li, Qin Qin; Zhang, Yan Ming; Tian, Chang Fu; Chen, Wen Xin

2014-02-01

In order to investigate the genetic differentiation of Sinorhizobium strains nodulating Glycine max and related microevolutionary mechanisms, three housekeeping genes (SMc00019, truA, and thrA) and 16 symbiosis-related genes on the chromosome (7 genes), pSymA (6 genes), and pSymB (3 genes) were analyzed. Five distinct species were identified among the test strains by calculating the average nucleotide identity (ANI) of SMc00019-truA-thrA: Sinorhizobium fredii, Sinorhizobium sojae, Sinorhizobium sp. I, Sinorhizobium sp. II, and Sinorhizobium sp. III. These species assignments were also supported by population genetics and phylogenetic analyses of housekeeping genes and symbiosis-related genes on the chromosome and pSymB. Different levels of genetic differentiation were observed among these species or different replicons. S. sojae was the most divergent from the other test species and was characterized by its low intraspecies diversity and limited geographic distribution. Intergenic recombination dominated the evolution of 19 genes from different replicons. Intraspecies recombination happened frequently in housekeeping genes and symbiosis-related genes on the chromosome and pSymB, whereas pSymA genes showed a clear pattern of lateral-transfer events between different species. Moreover, pSymA genes were characterized by a lower level of polymorphism and recombination than those on the chromosome and pSymB. Taken together, genes from different replicons of rhizobia might be involved in the establishment of symbiosis with legumes, but these symbiosis-related genes might have evolved differently according to their corresponding replicons.

Reactivity of eleven anti-human leucocyte monoclonal antibodies with lymphocytes from several domestic animals

DEFF Research Database (Denmark)

Aasted, Bent; Blixenkrone-Møller, Merete; Larsen, Else Bang

1988-01-01

Nine commercially available monoclonal antibodies and two monoclonal antibodies from The American Type Culture Collection, raised against various human leucocyte surface antigens, were tested on lymphocytes from cow, sheep, goat, swine, horse, cat, dog, mink, and rabbit as well as man. Four...... antibodies bound to lymphocytes from some of the animals. These were the antibodies against CD8 and CD4 antigen, the antibody to C3b-receptor, and the antibody to the HLA-DR antigen. The CD8 antigen-reactive antibody reacted with lymphocytes from mink, cat, dog, and sheep, while the CD4 antigen......-reactive antibody reacted with lymphocytes from mink. The anti-C3b-R antibody reacted with lymphocytes from horse, swine, dog, and cat, and the anti-HLA-DR reacted with lymphocytes from cow, goat, sheep, horse, dog, cat, and mink....

Colorimetric Detection of Some Highly Hydrophobic Flavonoids Using Polydiacetylene Liposomes Containing Pentacosa-10,12-diynoyl Succinoglycan Monomers

Science.gov (United States)

Yun, Deokgyu; Jeong, Daham; Cho, Eunae; Jung, Seunho

2015-01-01

Flavonoids are a group of plant secondary metabolites including polyphenolic molecules, and they are well known for antioxidant, anti-allergic, anti-inflammatory and anti-viral propertied. In general, flavonoids are detected with various non-colorimetric detection methods such as column liquid chromatography, thin-layer chromatography, and electrochemical analysis. For the first time, we developed a straightforward colorimetric detection system allowing recognition of some highly hydrophobic flavonoids such as alpha-naphthoflavone and beta-naphthoflavone, visually using 10,12-pentacosadiynoic acid (PCDA) derivatized with succinoglycan monomers isolated from Sinorhizobium meliloti. Besides changes in visible spectrum, we also demonstrate fluorescence changes using our detection system in the presence of those flavonoids. The succinoglycan monomers attached to PCDA molecules may function as an unstructured molecular capturer for some highly hydrophobic flavonoids by hydrophobic interactions, and transmit their molecular interactions as a color change throughout the PCDA liposome. PMID:26600071

Effect of exogenous application of rhizopine on lucerne root nodulation

African Journals Online (AJOL)

Rhizopine, 3-0 -methyl scyllo-inosamine was applied to the roots of luceme seedling inoculated with either rhizopine synthesizing Sinorhizobium meliloti strain L530 or the non-rhizopine synthesizing strain Rm 1021 . There was an initial delay in nodule formation. A significant increase in the number of nodules formed in ...

Rituximab chimeric anti-CD20 monoclonal antibody treatment for adult refractory idiopathic thrombocytopenic purpura

DEFF Research Database (Denmark)

Braendstrup, Peter; Bjerrum, Ole W; Nielsen, Ove J

2005-01-01

. Recent studies have shown that rituximab, a chimeric anti-CD20 monoclonal antibody, is useful in the treatment of these patients, with overall response rates of about 50%. Most published reports have included a small number patients including case reports. The present study reports the results...

Ensifer meliloti overexpressing Escherichia coli phytase gene ( appA) improves phosphorus (P) acquisition in maize plants

Science.gov (United States)

Sharma, Vikas; Kumar, Ajit; Archana, G.; Kumar, G. Naresh

2016-10-01

The Escherichia coli phytase gene appA encoding enzyme AppA was cloned in a broad host range plasmid pBBR1MCS2 ( lac promoter), termed pVA1, and transformed into the Ensifer meliloti 1020. Transformation of pVA1 in Ensifer meliloti { E. m (pVA1)} increased its phosphatase and phytase activity by ˜9- and ˜50-fold, respectively, compared to the transformants containing empty plasmid as control { E. m (pBBR1MCS2)}. The western blot experiments using rabbit anti-AppA antibody showed that AppA is translocated into the periplasm of the host after its expression. Ensifer meliloti harboring AppA protein { E. m (pVA1)} and { E. m (pBBR1MCS2)} could acidify the unbuffered phytate minimal media (pH 8.0) containing Ca-phytate or Na-phytate as sole organic P (Po) source to below pH 5.0 and released P. However, both { E. m (pVA1)} and { E. m (pBBR1MCS2)} neither dropped pH of the medium nor released P when the medium was buffered at pH 8.0 using Tris-Cl, indicating that acidification of medium was important for the enzymatic hydrolysis of phytate. Further experiments proved that maize plants inoculated with { E. m. (pVA1)} showed increase in growth under sterile semi solid agar (SSA) medium containing Na-phytate as sole P source. The present study could be helpful in generating better transgenic bioinoculants harboring phosphate mineralization properties that ultimately promote plant growth.

Analysis of nodule meristem persistence and ENOD40 functioning in Medicago truncatula nodule formation

NARCIS (Netherlands)

Wan Xi,

2007-01-01

Medicago root nodules are formed as a result of the interaction of the plant with the soil-borne bacterium Sinorhizobium meliloti. Several plant genes are induced during nodule formation and MtENOD40 is one of the earliest genes activated. The precise function as well as the molecule

Immunotherapy of Alzheimer's disease (AD): from murine models to anti-amyloid beta (Abeta) human monoclonal antibodies.

Science.gov (United States)

Geylis, Valeria; Steinitz, Michael

2006-01-01

The deposition of amyloid beta (Abeta) protein is a key pathological feature in Alzheimer's disease (AD). In murine models of AD, both active and passive immunization against Abeta induce a marked reduction in amyloid brain burden and an improvement in cognitive functions. Preliminary results of a prematurely terminated clinical trial where AD patients were actively vaccinated with aggregated Abeta bear resemblance to those documented in murine models. Passive immunization of AD patients with anti-Abeta antibodies, in particular human antibodies, is a strategy that provides a more cautious management and control of any undesired side effects. Sera of all healthy adults contain anti-Abeta IgG autoimmune antibodies. Hence antigen-committed human B-cells are easily immortalized by Epstein-Barr virus (EBV) into anti-Abeta secreting cell lines. Two anti-Abeta human monoclonal antibodies which we recently prepared bind to the N-terminus of Abeta peptide and were shown to stain amyloid plaques in non-fixed brain sections from an AD patient. It is anticipated that specifically selected anti-Abeta human monoclonal antibodies could reduce and inhibit deposits of amyloid in brain while avoiding the cognitive decline that characterizes AD. In the future, this type of antibody may prove to be a promising immune therapy for the disease.

The symbiotic biofilm of Sinorhizobium fredii SMH12, necessary for successful colonization and symbiosis of Glycine max cv Osumi, is regulated by Quorum Sensing systems and inducing flavonoids via NodD1.

Directory of Open Access Journals (Sweden)

Francisco Pérez-Montaño

Full Text Available Bacterial surface components, especially exopolysaccharides, in combination with bacterial Quorum Sensing signals are crucial for the formation of biofilms in most species studied so far. Biofilm formation allows soil bacteria to colonize their surrounding habitat and survive common environmental stresses such as desiccation and nutrient limitation. This mode of life is often essential for survival in bacteria of the genera Mesorhizobium, Sinorhizobium, Bradyrhizobium, and Rhizobium. The role of biofilm formation in symbiosis has been investigated in detail for Sinorhizobium meliloti and Bradyrhizobium japonicum. However, for S. fredii this process has not been studied. In this work we have demonstrated that biofilm formation is crucial for an optimal root colonization and symbiosis between S. fredii SMH12 and Glycine max cv Osumi. In this bacterium, nod-gene inducing flavonoids and the NodD1 protein are required for the transition of the biofilm structure from monolayer to microcolony. Quorum Sensing systems are also required for the full development of both types of biofilms. In fact, both the nodD1 mutant and the lactonase strain (the lactonase enzyme prevents AHL accumulation are defective in soybean root colonization. The impairment of the lactonase strain in its colonization ability leads to a decrease in the symbiotic parameters. Interestingly, NodD1 together with flavonoids activates certain quorum sensing systems implicit in the development of the symbiotic biofilm. Thus, S. fredii SMH12 by means of a unique key molecule, the flavonoid, efficiently forms biofilm, colonizes the legume roots and activates the synthesis of Nod factors, required for successfully symbiosis.

Nebulized Anti-IL-13 Monoclonal Antibody Fab' Fragment Reduces Allergen-Induced Asthma

OpenAIRE

Hacha, Jonathan; Tomlinson, K; Maertens, Ludovic; Paulissen, Geneviève; Rocks, Natacha; Foidart, Jean-Michel; Noël, Agnès; Palframan, R; Guéders, Maud; Cataldo, Didier

2012-01-01

Rationale: Interleukin-13 (IL-13) is a prototypic Th2 cytokine and a central mediator of the complex cascade of events leading to asthmatic phenotype. Indeed, IL-13 plays key roles in IgE synthesis, bronchial hyperresponsiveness, mucus hypersecretion, subepithelial fibrosis and eosinophil infiltration. Objectives: We assessed the potential efficacy of inhaled anti-IL-13 monoclonal antibody Fab' fragment on allergen-induced airway inflammation, hyperresponsiveness and remodeling in an experime...

Biochemical and Molecular Phylogenetic Study of Agriculturally Useful Association of a Nitrogen-Fixing Cyanobacterium and Nodule Sinorhizobium with Medicago sativa L.

Directory of Open Access Journals (Sweden)

E. V. Karaushu

2015-01-01

Full Text Available Seed inoculation with bacterial consortium was found to increase legume yield, providing a higher growth than the standard nitrogen treatment methods. Alfalfa plants were inoculated by mono- and binary compositions of nitrogen-fixing microorganisms. Their physiological and biochemical properties were estimated. Inoculation by microbial consortium of Sinorhizobium meliloti T17 together with a new cyanobacterial isolate Nostoc PTV was more efficient than the single-rhizobium strain inoculation. This treatment provides an intensification of the processes of biological nitrogen fixation by rhizobia bacteria in the root nodules and an intensification of plant photosynthesis. Inoculation by bacterial consortium stimulates growth of plant mass and rhizogenesis and leads to increased productivity of alfalfa and to improving the amino acid composition of plant leaves. The full nucleotide sequence of the rRNA gene cluster and partial sequence of the dinitrogenase reductase (nifH gene of Nostoc PTV were deposited to GenBank (JQ259185.1, JQ259186.1. Comparison of these gene sequences of Nostoc PTV with all sequences present at the GenBank shows that this cyanobacterial strain does not have 100% identity with any organisms investigated previously. Phylogenetic analysis showed that this cyanobacterium clustered with high credibility values with Nostoc muscorum.

Detection and isolation of novel rhizopine-catabolizing bacteria from the environment

Science.gov (United States)

Gardener; de Bruijn FJ

1998-12-01

Microbial rhizopine-catabolizing (Moc) activity was detected in serial dilutions of soil and rhizosphere washes. The activity observed generally ranged between 10(6) and 10(7) catabolic units per g, and the numbers of nonspecific culture-forming units were found to be approximately 10 times higher. A diverse set of 37 isolates was obtained by enrichment on scyllo-inosamine-containing media. However, none of the bacteria that were isolated were found to contain DNA sequences homologous to the known mocA, mocB, and mocC genes of Sinorhizobium meliloti L5-30. Twenty-one of the isolates could utilize an SI preparation as the sole carbon and nitrogen source for growth. Partial sequencing of 16S ribosomal DNAs (rDNAs) amplified from these strains indicated that five distinct bacterial genera (Arthrobacter, Sinorhizobium, Pseudomonas, Aeromonas, and Alcaligenes) were represented in this set. Only 6 of these 21 isolates could catabolize 3-O-methyl-scyllo-inosamine under standard assay conditions. Two of these, strains D1 and R3, were found to have 16S rDNA sequences very similar to those of Sinorhizobium meliloti. However, these strains are not symbiotically effective on Medicago sativa, and DNA sequences homologous to the nodB and nodC genes were not detected in strains D1 and R3 by Southern hybridization analysis.

A novel anti-GPC3 monoclonal antibody (YP7) | Center for Cancer Research

Science.gov (United States)

Glypican-3 (GPC3) is an emerging therapeutic target in hepatoma. A novel anti-GPC3 monoclonal antibody (YP7) has been generated through a combination of peptide immunization and high-throughput flow cytometry screening. YP7 binds cell-surface-associated GPC3 with high affinity and exhibits significant hepatoma xenograft growth inhibition in nude mice. The new antibody may have

Detection of Signal Regulatory Protein α in Saimiri sciureus (Squirrel Monkey) by Anti-Human Monoclonal Antibody

Science.gov (United States)

de Souza, Hugo Amorim dos Santos; Costa-Correa, Edmar Henrique; Bianco-Junior, Cesare; Andrade, Márcia Cristina Ribeiro; Lima-Junior, Josué da Costa; Pratt-Riccio, Lilian Rose; Daniel-Ribeiro, Cláudio Tadeu; Totino, Paulo Renato Rivas

2017-01-01

Non-human primates (NHP) are suitable models for studying different aspects of the human system, including pathogenesis and protective immunity to many diseases. However, the lack of specific immunological reagents for neo-tropical monkeys, such as Saimiri sciureus, is still a major factor limiting studies in these models. An alternative strategy to circumvent this obstacle has been the selection of immunological reagents directed to humans, which present cross-reactivity with NHP molecules. In this context and considering the key role of inhibitory immunoreceptors—such as the signal regulatory protein α (SIRPα)—in the regulation of immune responses, in the present study, we attempted to evaluate the ability of anti-human SIRPα monoclonal antibodies to recognize SIRPα in antigen-presenting S. sciureus peripheral blood mononuclear cells (PBMC). As shown by flow cytometry analysis, the profile of anti-SIRPα staining as well as the levels of SIRPα-positive cells in PBMC from S. sciureus were similar to those observed in human PBMC. Furthermore, using anti-SIRPα monoclonal antibody, it was possible to detect a decrease of the SIRPα levels on surface of S. sciureus cells after in vitro stimulation with lipopolysaccharides. Finally, using computed-based analysis, we observed a high degree of conservation of SIRPα across six species of primates and the presence of shared epitopes in the extracellular domain between humans and Saimiri genus that could be targeted by antibodies. In conclusion, we have identified a commercially available anti-human monoclonal antibody that is able to detect SIRPα of S. sciureus monkeys and that, therefore, can facilitate the study of the immunomodulatory role of SIRPα when S. sciureus is used as a model. PMID:29312325

Detection of Signal Regulatory Protein α in Saimiri sciureus (Squirrel Monkey by Anti-Human Monoclonal Antibody

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Hugo Amorim dos Santos de Souza

2017-12-01

Full Text Available Non-human primates (NHP are suitable models for studying different aspects of the human system, including pathogenesis and protective immunity to many diseases. However, the lack of specific immunological reagents for neo-tropical monkeys, such as Saimiri sciureus, is still a major factor limiting studies in these models. An alternative strategy to circumvent this obstacle has been the selection of immunological reagents directed to humans, which present cross-reactivity with NHP molecules. In this context and considering the key role of inhibitory immunoreceptors—such as the signal regulatory protein α (SIRPα—in the regulation of immune responses, in the present study, we attempted to evaluate the ability of anti-human SIRPα monoclonal antibodies to recognize SIRPα in antigen-presenting S. sciureus peripheral blood mononuclear cells (PBMC. As shown by flow cytometry analysis, the profile of anti-SIRPα staining as well as the levels of SIRPα-positive cells in PBMC from S. sciureus were similar to those observed in human PBMC. Furthermore, using anti-SIRPα monoclonal antibody, it was possible to detect a decrease of the SIRPα levels on surface of S. sciureus cells after in vitro stimulation with lipopolysaccharides. Finally, using computed-based analysis, we observed a high degree of conservation of SIRPα across six species of primates and the presence of shared epitopes in the extracellular domain between humans and Saimiri genus that could be targeted by antibodies. In conclusion, we have identified a commercially available anti-human monoclonal antibody that is able to detect SIRPα of S. sciureus monkeys and that, therefore, can facilitate the study of the immunomodulatory role of SIRPα when S. sciureus is used as a model.

Novel anti-HER2 monoclonal antibodies: synergy and antagonism with tumor necrosis factor-α

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Ceran Ceyhan

2012-10-01

Full Text Available Abstract Background One-third of breast cancers display amplifications of the ERBB2 gene encoding the HER2 kinase receptor. Trastuzumab, a humanized antibody directed against an epitope on subdomain IV of the extracellular domain of HER2 is used for therapy of HER2-overexpressing mammary tumors. However, many tumors are either natively resistant or acquire resistance against Trastuzumab. Antibodies directed to different epitopes on the extracellular domain of HER2 are promising candidates for replacement or combinatorial therapy. For example, Pertuzumab that binds to subdomain II of HER2 extracellular domain and inhibits receptor dimerization is under clinical trial. Alternative antibodies directed to novel HER2 epitopes may serve as additional tools for breast cancer therapy. Our aim was to generate novel anti-HER2 monoclonal antibodies inhibiting the growth of breast cancer cells, either alone or in combination with tumor necrosis factor-α (TNF-α. Methods Mice were immunized against SK-BR-3 cells and recombinant HER2 extracellular domain protein to produce monoclonal antibodies. Anti-HER2 antibodies were characterized with breast cancer cell lines using immunofluorescence, flow cytometry, immunoprecipitation, western blot techniques. Antibody epitopes were localized using plasmids encoding recombinant HER2 protein variants. Antibodies, either alone or in combination with TNF-α, were tested for their effects on breast cancer cell proliferation. Results We produced five new anti-HER2 monoclonal antibodies, all directed against conformational epitope or epitopes restricted to the native form of the extracellular domain. When tested alone, some antibodies inhibited modestly but significantly the growth of SK-BR-3, BT-474 and MDA-MB-361 cells displaying ERBB2 amplification. They had no detectable effect on MCF-7 and T47D cells lacking ERBB2 amplification. When tested in combination with TNF-α, antibodies acted synergistically on SK-BR-3 cells

High affinity anti-TIM-3 and anti-KIR monoclonal antibodies cloned from healthy human individuals.

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Stefan Ryser

Full Text Available We report here the cloning of native high affinity anti-TIM-3 and anti-KIR IgG monoclonal antibodies (mAbs from peripheral blood mononuclear cells (PBMC of healthy human donors. The cells that express these mAbs are rare, present at a frequency of less than one per 105 memory B-cells. Using our proprietary multiplexed screening and cloning technology CellSpot™ we assessed the presence of memory B-cells reactive to foreign and endogenous disease-associated antigens within the same individual. When comparing the frequencies of antigen-specific memory B-cells analyzed in over 20 screening campaigns, we found a strong correlation of the presence of anti-TIM-3 memory B-cells with memory B-cells expressing mAbs against three disease-associated antigens: (i bacterial DNABII proteins that are a marker for Gram negative and Gram positive bacterial infections, (ii hemagglutinin (HA of influenza virus and (iii the extracellular domain of anaplastic lymphoma kinase (ALK. One of the native anti-KIR mAbs has similar characteristics as lirilumab, an anti-KIR mAb derived from immunization of humanized transgenic mice that is in ongoing clinical trials. It is interesting to speculate that these native anti-TIM-3 and anti-KIR antibodies may function as natural regulatory antibodies, analogous to the pharmacological use in cancer treatment of engineered antibodies against the same targets. Further characterization studies are needed to define the mechanisms through which these native antibodies may function in healthy and disease conditions.

Characterization of the Anti-Bovine Podoplanin Monoclonal Antibody PMab-44.

Science.gov (United States)

Yamada, Shinji; Honma, Ryusuke; Kaneko, Mika K; Nakamura, Takuro; Yanaka, Miyuki; Saidoh, Noriko; Takagi, Michiaki; Konnai, Satoru; Kato, Yukinari

2017-06-01

A type I transmembrane sialoglycoprotein podoplanin (PDPN) is expressed in several normal cells, including podocytes of the kidney, type I alveolar cells of the lung, and lymphatic endothelial cells. We recently produced an anti-bovine PDPN (bovPDPN) monoclonal antibody (mAb), PMab-44, by immunizing mice with recombinant proteins of bovPDPN. In this study, we determined the critical epitope of PMab-44 for the recognition of bovPDPN using many deletion mutants and point mutants of bovPDPN. Flow cytometric analyses revealed that the epitope of PMab-44 was Glu46-Thr50, which corresponds to platelet aggregation-stimulating (PLAG) domain-3. The important amino acids in the PMab-44 epitope were determined to be Glu46, Tyr48, and Thr50. Western blot analysis also confirmed these results, indicating that the PLAG domain of bovPDPN is also important in immunogenicity for producing useful anti-PDPN mAbs.

Durvalumab: an investigational anti-PD-L1 monoclonal antibody for the treatment of urothelial carcinoma

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Faiena I

2018-01-01

Full Text Available Izak Faiena,1,2 Amy L Cummings,3 Anna M Crosetti,3 Allan J Pantuck,1,2 Karim Chamie,1,2 Alexandra Drakaki1–3 1Department of Urology, 2Institute of Urologic Oncology, 3Department of Medicine, Division of Hematology and Oncology, David Geffen School of Medicine at University of California, Los Angeles, CA, USA Abstract: Our expanding knowledge of immunotherapy for solid tumors has led to an explosion of clinical trials aimed at urothelial carcinoma. The primary strategy is centered on unleashing the immune system by releasing the inhibitory signals propagated by programmed cell death-1 (PD-1 and its ligand programmed cell death ligand-1 (PD-L1. Many antibody constructs have been developed to block these interactions and are used in clinical trials. The Food and Drug Administration has already approved a number of checkpoint inhibitors such as anti-cytotoxic T-lymphocyte-associated protein 4 (CTLA4 monoclonal antibodies including ipilimumab; anti-PD-1 monoclonal antibodies including nivolumab and pembrolizumab; anti-PD-L1 antibodies including atezolizumab, avelumab, and durvalumab. One of the latest inhibitors is durvalumab, which is a high-affinity human immunoglobulin G1 kappa monoclonal antibody and blocks the interaction of PD-L1 with PD-1 and CD80. Currently, there are a number of ongoing trials in advanced urothelial carcinoma both using durvalumab monotherapy and in combination with other targeted therapies. In addition, durvalumab is being investigated in the non-muscle-invasive urothelial carcinoma, which is centered around intravenous formulations. These exciting developments have added a significant number of therapies in a previously limited treatment landscape. Keywords: durvalumab, checkpoint inhibitors, metastatic urothelial carcinoma

Prolonged skin graft survival by administration of anti-CD80 monoclonal antibody with cyclosporin A

NARCIS (Netherlands)

Ossevoort, MA; Lorre, K; Boon, L; van den Hout, Y; de Boer, M; De Waele, P; Jonker, M; VandeVoorde, A

Costimulation via the B7/CD28 pathway is an important signal for the activation of T cells. Maximal inhibition of T-cell activation and the induction of alloantigen-specific nonresponsiveness in vitro was achieved using anti-CD80 monoclonal antibody (mAb) in combination with cyclosporin A (CsA).

Immunoscintigraphy of human tumors transplanted in nude mice with radiolabeled anti-ras p21 monoclonal antibodies

International Nuclear Information System (INIS)

Katoh, Y.; Nakata, K.; Kohno, K.; Shima, M.; Satoh, A.; Kusumoto, Y.; Ishii, N.; Kohji, T.; Shiku, H.; Nagataki, S.

1990-01-01

Anti-ras p21 monoclonal antibody (RASK-3) was used for immunoscintigraphy of human cancer cell lines in nude mice. Iodine-125-labeled RASK-3 was injected into nude mice with either human colon cancers (FCC-1 or BM-314) or lung cancer (KNS-62). Clear images were obtained in all three cancers 7 days after the injection of antibody. No localization of 125 I-labeled control monoclonal antibody was observed. The ratio of tissue/blood radioactivity and % ID/g in the tumor were significantly higher than other organs by Day 8. The specific localization index examined by 131 I-RASK-3 and 125 I-control monoclonal antibody was also higher in the tumor than in other tissues. In the in vitro study, binding of RASK-3 to tumor cells increased significantly by treatment of cells with either lysolecithin or periodate-lysine-paraformaldehyde, which confirmed the intracellular localization of ras p21. The mechanism by which anti-ras p21 antibodies accumulate in tumor sites could be the necrotic changes in tumor cells or changes in membrane permeability of non-necrotic cells. These results provide a strong rationale for the utilization of ras p21 as a target antigen in the imaging of a variety of human cancers

Generation, characterization and in vivo biological activity of two distinct monoclonal anti-PEG IgMs

International Nuclear Information System (INIS)

Hashimoto, Yosuke; Shimizu, Taro; Mima, Yu; Abu Lila, Amr S.; Ishida, Tatsuhiro; Kiwada, Hiroshi

2014-01-01

PEGylation, the attachment of polyethylene glycol (PEG) to nanocarriers and proteins, is a widely accepted approach to improving the in vivo efficacy of the non-PEGylated products. However, both PEGylated liposomes and PEGylated proteins reportedly trigger the production of specific antibodies, mainly IgM, against the PEG moiety, which possibly leads to a reduction in safety and therapeutic efficacy of the PEGylated products. In the present study, two monoclonal anti-PEG IgMs — HIK-M09 via immunization with an intravenous injection of PEGylated liposomes (SLs) and HIK-M11 via immunization with a subcutaneous administration of PEGylated ovalbumin (PEG-OVA) were successfully generated. The generated IgMs showed efficient reactivity to mPEG 2000 conjugated to 1,2-distearoyl-sn-glycero-3-phospho-ethanolamine (DSPE), PEGylated liposome (SL) and PEG-OVA. It appears that HIK-M09 recognizes ethoxy (OCH 2 CH 2 ) repeat units along with a terminal motif of PEG, while HIK-M11 recognizes only ethoxy repeat units of PEG. Such unique properties allow HIK-M09 to bind with dense PEG. In addition, their impact on the in vivo clearance of the PEGylated products was investigated. It was found that the generated ant-PEG IgMs induced a clearance of SL as they were intravenously administered with SL. Interestingly, the HIK-M11, generated by PEG-OVA, induced the clearance of both SL and PEG-OVA, while the HIK-M09, generated by SL, induced the clearance of SL only. We here revealed that the presence of serum anti-PEG IgM and the subsequent binding of anti-PEG IgM to the PEGylated products are not necessarily related to the enhanced clearance of the products. It appears that subsequent complement activation following anti-PEG IgM binding is the most important step in dictating the in vivo fate of PEGylated products. This study may have implications for the design, development and clinical application of PEGylated products and therapeutics. - Highlights: • Two monoclonal anti-PEG Ig

Generation, characterization and in vivo biological activity of two distinct monoclonal anti-PEG IgMs

Energy Technology Data Exchange (ETDEWEB)

Hashimoto, Yosuke; Shimizu, Taro; Mima, Yu [Department of Pharmacokinetics and Biopharmaceutics, Subdivision of Biopharmaceutical Sciences, Institute of Health Biosciences, The University of Tokushima, 1-78-1, Sho-machi, Tokushima 770-8505 (Japan); Abu Lila, Amr S. [Department of Pharmacokinetics and Biopharmaceutics, Subdivision of Biopharmaceutical Sciences, Institute of Health Biosciences, The University of Tokushima, 1-78-1, Sho-machi, Tokushima 770-8505 (Japan); Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, Zagazig University, Sharqia Governorate, Zagazig 44519 (Egypt); Ishida, Tatsuhiro, E-mail: ishida@tokushima-u.ac.jp [Department of Pharmacokinetics and Biopharmaceutics, Subdivision of Biopharmaceutical Sciences, Institute of Health Biosciences, The University of Tokushima, 1-78-1, Sho-machi, Tokushima 770-8505 (Japan); Kiwada, Hiroshi [Department of Pharmacokinetics and Biopharmaceutics, Subdivision of Biopharmaceutical Sciences, Institute of Health Biosciences, The University of Tokushima, 1-78-1, Sho-machi, Tokushima 770-8505 (Japan)

2014-05-15

PEGylation, the attachment of polyethylene glycol (PEG) to nanocarriers and proteins, is a widely accepted approach to improving the in vivo efficacy of the non-PEGylated products. However, both PEGylated liposomes and PEGylated proteins reportedly trigger the production of specific antibodies, mainly IgM, against the PEG moiety, which possibly leads to a reduction in safety and therapeutic efficacy of the PEGylated products. In the present study, two monoclonal anti-PEG IgMs — HIK-M09 via immunization with an intravenous injection of PEGylated liposomes (SLs) and HIK-M11 via immunization with a subcutaneous administration of PEGylated ovalbumin (PEG-OVA) were successfully generated. The generated IgMs showed efficient reactivity to mPEG{sub 2000} conjugated to 1,2-distearoyl-sn-glycero-3-phospho-ethanolamine (DSPE), PEGylated liposome (SL) and PEG-OVA. It appears that HIK-M09 recognizes ethoxy (OCH{sub 2}CH{sub 2}) repeat units along with a terminal motif of PEG, while HIK-M11 recognizes only ethoxy repeat units of PEG. Such unique properties allow HIK-M09 to bind with dense PEG. In addition, their impact on the in vivo clearance of the PEGylated products was investigated. It was found that the generated ant-PEG IgMs induced a clearance of SL as they were intravenously administered with SL. Interestingly, the HIK-M11, generated by PEG-OVA, induced the clearance of both SL and PEG-OVA, while the HIK-M09, generated by SL, induced the clearance of SL only. We here revealed that the presence of serum anti-PEG IgM and the subsequent binding of anti-PEG IgM to the PEGylated products are not necessarily related to the enhanced clearance of the products. It appears that subsequent complement activation following anti-PEG IgM binding is the most important step in dictating the in vivo fate of PEGylated products. This study may have implications for the design, development and clinical application of PEGylated products and therapeutics. - Highlights: • Two monoclonal

Detecting fish parvalbumin with commercial mouse monoclonal anti-frog parvalbumin IgG.

Science.gov (United States)

Chen, Lingyun; Hefle, Sue L; Taylor, Steve L; Swoboda, Ines; Goodman, Richard E

2006-07-26

Parvalbumin is a calcium-binding muscle protein that is highly conserved across fish species and amphibians. It is the major cross-reactive allergen associated with both fish and frog allergy. We used two-dimensional electrophoretic and immunoblotting techniques to investigate the utility of a commercial monoclonal anti-frog parvalbumin IgG for detecting parvalbumin present in some commonly consumed fish species. The 2D electrophoresis and immunoblots revealed species-specific differences in proteins that appear to represent various numbers of isoforms of parvalbumin in carp (5), catfish (3), cod (1) and tilapia (2). No parvalbumin was detected in yellowfin tuna. Based on minor differences in relative intensities of protein staining and immunodetection, parvalbumin isoforms may have slight differences in the epitope region recognized by the anti-frog parvalbumin antibody. These results suggest that the frog anti-parvalbumin antibody can be used as a valuable tool to detect parvalbumins from the fish tested in this study, except yellowfin tuna.

Genomic resources for identification of the minimal N2 -fixing symbiotic genome.

Science.gov (United States)

diCenzo, George C; Zamani, Maryam; Milunovic, Branislava; Finan, Turlough M

2016-09-01

The lack of an appropriate genomic platform has precluded the use of gain-of-function approaches to study the rhizobium-legume symbiosis, preventing the establishment of the genes necessary and sufficient for symbiotic nitrogen fixation (SNF) and potentially hindering synthetic biology approaches aimed at engineering this process. Here, we describe the development of an appropriate system by reverse engineering Sinorhizobium meliloti. Using a novel in vivo cloning procedure, the engA-tRNA-rmlC (ETR) region, essential for cell viability and symbiosis, was transferred from Sinorhizobium fredii to the ancestral location on the S. meliloti chromosome, rendering the ETR region on pSymB redundant. A derivative of this strain lacking both the large symbiotic replicons (pSymA and pSymB) was constructed. Transfer of pSymA and pSymB back into this strain restored symbiotic capabilities with alfalfa. To delineate the location of the single-copy genes essential for SNF on these replicons, we screened a S. meliloti deletion library, representing > 95% of the 2900 genes of the symbiotic replicons, for their phenotypes with alfalfa. Only four loci, accounting for < 12% of pSymA and pSymB, were essential for SNF. These regions will serve as our preliminary target of the minimal set of horizontally acquired genes necessary and sufficient for SNF. © 2016 Society for Applied Microbiology and John Wiley & Sons Ltd.

Clinical application of antibody monoclonal humanized anti-EGFrnimotuzumab labeled

International Nuclear Information System (INIS)

Perera Pintado, Alejandro; Peña Quián, Yamilé; Batista Cuéllar, Juan F.; Prats Capote, Anaís; Torres Aroche, Leonel A.; Casacó Santana, Caridad; Sánchez Mendosa, Elvia L.; Sánchez González, Yolaine; Romero Collado, Susana; Quesada Pozo, Rodobaldo; Valladares Oviedo, Lourdes; Masquida García, Elsa M.; Leyva Montaña, René; Casacó, Angel; Ramos Suzarte, Mayra; Crombet, Tania

2016-01-01

Most malignant tumors are of epithelial origin. These are characterized by overexpression of the receptor of epidermal growth factor (EGFR), which the neoplastic cells escape the regulatory mechanisms are allowed, so its high concentration of membrane is generally associated with a poor prognosis . By binding an antibody specifically to this receptor, preventing binding of EGF latter and activation mechanism tyrosine kinase inhibiting cell mitosis and apoptosis causing tumor cell. For this reason, the EGFr has been considered as an attractive target for anti-tumor therapy. The humanized monoclonal antibody anti-EGFr nimotuzumab was developed by the Center of Molecular Immunology (Havana, Cuba). Numerous clinical trials have been developed in the Department of Clinical Research Center Isotopes (Cuba), in which it has been applied this antibody, both labeled with 99mTc for immuno gammagraphic detection of tumors, as labeled with 188 Re for radioimmunotherapy of gliomas high degree of malignancy. The aim of this paper is to show the experience of the Department of Clinical Research of CENTIS in various clinical trials with marking for both immuno gammagraphics detection of tumors, such as for radioimmunotherapy nimotuzumab. (author)

77 FR 9678 - Prospective Grant of Exclusive License: The Development of Human Anti-CD22 Monoclonal Antibodies...

Science.gov (United States)

2012-02-17

.../patent applications for the technology family, to Sanomab, Ltd. The patent rights in these inventions... DEPARTMENT OF HEALTH AND HUMAN SERVICES National Institutes of Health Prospective Grant of Exclusive License: The Development of Human Anti-CD22 Monoclonal Antibodies for the Treatment of Human...

77 FR 41792 - Prospective Grant of Co-Exclusive License: The Development of Human Anti-CD22 Monoclonal...

Science.gov (United States)

2012-07-16

... applications for the technology family, to Customized Therapeutics. The patent rights in these inventions have... DEPARTMENT OF HEALTH AND HUMAN SERVICES National Institutes of Health Prospective Grant of Co-Exclusive License: The Development of Human Anti-CD22 Monoclonal Antibodies for the Treatment of Human...

Monoclonal anti-human factor VII antibodies. Detection in plasma of a second protein antigenically and genetically related to factor VII.

OpenAIRE

Broze, G J; Hickman, S; Miletich, J P

1985-01-01

Several murine monoclonal anti-human Factor VII antibodies were produced using hybridoma technology. Two noncompetitive monoclonal antibodies were used to examine by Western blotting the Factor VII cross-reactive material (CRM) in normal human plasma and three commercially available congenitally Factor VII-deficient plasmas, and to construct a facile "sandwich" immunoassay for plasma Factor VII. A second, previously undescribed, form of Factor VII CRM was detected in human plasma, which on We...

Individual and combining effects of anti-RANKL monoclonal antibody and teriparatide in ovariectomized mice

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Naoto Tokuyama

2015-06-01

Full Text Available We examined the individual and combined effects of teriparatide and anti-RANKL (receptor activator of nuclear factor κB ligand monoclonal antibody in ovariectomized mice. Three-month-old female C57BL/6 mice were ovariectomized (OVX or sham operated. Four weeks after OVX, they were assigned to 3 different groups to receive anti-RANKL monoclonal antibody (Ab alone (5 mg/kg single injection at 4 weeks after OVX, Ab group, teriparatide alone (80 μg/kg daily injection for 4 weeks from 4 weeks after OVX, PTH group, or mAb plus teriparatide (Ab + PTH group. Mice were sacrificed 8 weeks after OVX. Bone mineral density (BMD was measured at the femur and lumbar spine. Hind limbs were subjected to histological and histomorphometric analysis. Serum osteocalcin and CTX-I levels were measured to investigate the bone turnover. Compared with Ab group, Ab + PTH group showed a significant increase in BMD at distal femur and femoral shaft. Cortical bone volume was significantly increased in PTH and Ab + PTH groups compared with Ab group. Bone turnover in Ab + PTH group was suppressed to the same degree as in Ab group. The number of TRAP-positive multinucleated cells was markedly reduced in Ab and Ab + PTH groups. These results suggest that combined treatment of teriparatide with anti-RANKL antibody has additive effects on BMD in OVX mice compared with individual treatment.

The Role of Anti-Drug Antibodies in the Pharmacokinetics, Disposition, Target Engagement, and Efficacy of a GITR Agonist Monoclonal Antibody in Mice.

Science.gov (United States)

Brunn, Nicholas D; Mauze, Smita; Gu, Danling; Wiswell, Derek; Ueda, Roanna; Hodges, Douglas; Beebe, Amy M; Zhang, Shuli; Escandón, Enrique

2016-03-01

Administration of biologics to enhance T-cell function is part of a rapidly growing field of cancer immunotherapy demonstrated by the unprecedented clinical success of several immunoregulatory receptor targeting antibodies. While these biologic agents confer significant anti-tumor activity through targeted immune response modulation, they can also elicit broad immune responses potentially including the production of anti-drug antibodies (ADAs). DTA-1, an agonist monoclonal antibody against GITR, is a highly effective anti-tumor treatment in preclinical models. We demonstrate that repeated dosing with murinized DTA-1 (mDTA-1) generates ADAs with corresponding reductions in drug exposure and engagement of GITR on circulating CD3(+) CD4(+) T cells, due to rapid hepatic drug uptake and catabolism. Mice implanted with tumors after induction of preexisting mDTA-1 ADA show no anti-tumor efficacy when given 3 mg/kg mDTA-1, an efficacious dose in naive mice. Nonetheless, increasing mDTA-1 treatment to 30 mg/kg in ADA-positive mice restores mDTA-1 exposure and GITR engagement on circulating CD3(+) CD4(+) T cells, thereby partially restoring anti-tumor efficacy. Formation of anti-mDTA-1 antibodies and changes in drug exposure and disposition does not occur in GITR(-/-) mice, consistent with a role for GITR agonism in humoral immunity. Finally, the administration of muDX400, a murinized monoclonal antibody against the checkpoint inhibitor PD-1, dosed alone or combined with mDTA-1 did not result in reduced muDX400 exposure, nor did it change the nature of the anti-mDTA-1 response. This indicates that anti-GITR immunogenicity may not necessarily impact the pharmacology of coadministered monoclonal antibodies, supporting combination immunomodulatory strategies. Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.

A novel rabbit anti-hepatocyte growth factor monoclonal neutralizing antibody inhibits tumor growth in prostate cancer cells and mouse xenografts

International Nuclear Information System (INIS)

Yu, Yanlan; Chen, Yicheng; Ding, Guoqing; Wang, Mingchao; Wu, Haiyang; Xu, Liwei; Rui, Xuefang; Zhang, Zhigen

2015-01-01

The hepatocyte growth factor and its receptor c-Met are correlated with castration-resistance in prostate cancer. Although HGF has been considered as an attractive target for therapeutic antibodies, the lack of cross-reactivity of monoclonal antibodies with human/mouse HGFs is a major obstacle in preclinical developments. We generated a panel of anti-HGF RabMAbs either blocking HGF/c-Met interaction or inhibiting c-Met phosphorylation. We selected one RabMAb with mouse cross-reactivity and demonstrated that it blocked HGF-stimulated downstream activation in PC-3 and DU145 cells. Anti-HGF RabMAb inhibited not only the growth of PC-3 cells but also HGF-dependent proliferation in HUVECs. We further demonstrated the efficacy and potency of the anti-HGF RabMAb in tumor xenograft mice models. Through these in vitro and in vivo experiments, we explored a novel therapeutic antibody for advanced prostate cancer. - Highlights: • HGF is an attractive target for castration-refractory prostate cancer. • We generated and characterized a panel of anti-HGF rabbit monoclonal antibodies. • More than half of these anti-HGF RabMAbs was cross-reactive with mouse HGF. • Anti-HGF RabMAb blocks HGF-stimulated phosphorylation and cell growth in vitro. • Anti-HGF RabMAb inhibits tumor growth and angiogenesis in xenograft mice

A novel rabbit anti-hepatocyte growth factor monoclonal neutralizing antibody inhibits tumor growth in prostate cancer cells and mouse xenografts

Energy Technology Data Exchange (ETDEWEB)

Yu, Yanlan; Chen, Yicheng; Ding, Guoqing; Wang, Mingchao; Wu, Haiyang; Xu, Liwei; Rui, Xuefang; Zhang, Zhigen, E-mail: srrshurology@163.com

2015-08-14

The hepatocyte growth factor and its receptor c-Met are correlated with castration-resistance in prostate cancer. Although HGF has been considered as an attractive target for therapeutic antibodies, the lack of cross-reactivity of monoclonal antibodies with human/mouse HGFs is a major obstacle in preclinical developments. We generated a panel of anti-HGF RabMAbs either blocking HGF/c-Met interaction or inhibiting c-Met phosphorylation. We selected one RabMAb with mouse cross-reactivity and demonstrated that it blocked HGF-stimulated downstream activation in PC-3 and DU145 cells. Anti-HGF RabMAb inhibited not only the growth of PC-3 cells but also HGF-dependent proliferation in HUVECs. We further demonstrated the efficacy and potency of the anti-HGF RabMAb in tumor xenograft mice models. Through these in vitro and in vivo experiments, we explored a novel therapeutic antibody for advanced prostate cancer. - Highlights: • HGF is an attractive target for castration-refractory prostate cancer. • We generated and characterized a panel of anti-HGF rabbit monoclonal antibodies. • More than half of these anti-HGF RabMAbs was cross-reactive with mouse HGF. • Anti-HGF RabMAb blocks HGF-stimulated phosphorylation and cell growth in vitro. • Anti-HGF RabMAb inhibits tumor growth and angiogenesis in xenograft mice.

Novel anti-Sialyl-Tn monoclonal antibodies and antibody-drug conjugates demonstrate tumor specificity and anti-tumor activity.

Science.gov (United States)

Prendergast, Jillian M; Galvao da Silva, Ana Paula; Eavarone, David A; Ghaderi, Darius; Zhang, Mai; Brady, Dane; Wicks, Joan; DeSander, Julie; Behrens, Jeff; Rueda, Bo R

Targeted therapeutics that can differentiate between normal and malignant tumor cells represent the ideal standard for the development of a successful anti-cancer strategy. The Sialyl-Thomsen-nouveau antigen (STn or Sialyl-Tn, also known as CD175s) is rarely seen in normal adult tissues, but it is abundantly expressed in many types of human epithelial cancers. We have identified novel antibodies that specifically target with high affinity the STn glycan independent of its carrier protein, affording the potential to recognize a wider array of cancer-specific sialylated proteins. A panel of murine monoclonal anti-STn therapeutic antibodies were generated and their binding specificity and efficacy were characterized in vitro and in in vivo murine cancer models. A subset of these antibodies were conjugated to monomethyl auristatin E (MMAE) to generate antibody-drug conjugates (ADCs). These ADCs demonstrated in vitro efficacy in STn-expressing cell lines and significant tumor growth inhibition in STn-expressing tumor xenograft cancer models with no evidence of overt toxicity.

A comparative genomics screen identifies a Sinorhizobium meliloti 1021 sodM-like gene strongly expressed within host plant nodules

Directory of Open Access Journals (Sweden)

Queiroux Clothilde

2012-05-01

Full Text Available Abstract Background We have used the genomic data in the Integrated Microbial Genomes system of the Department of Energy’s Joint Genome Institute to make predictions about rhizobial open reading frames that play a role in nodulation of host plants. The genomic data was screened by searching for ORFs conserved in α-proteobacterial rhizobia, but not conserved in closely-related non-nitrogen-fixing α-proteobacteria. Results Using this approach, we identified many genes known to be involved in nodulation or nitrogen fixation, as well as several new candidate genes. We knocked out selected new genes and assayed for the presence of nodulation phenotypes and/or nodule-specific expression. One of these genes, SMc00911, is strongly expressed by bacterial cells within host plant nodules, but is expressed minimally by free-living bacterial cells. A strain carrying an insertion mutation in SMc00911 is not defective in the symbiosis with host plants, but in contrast to expectations, this mutant strain is able to out-compete the S. meliloti 1021 wild type strain for nodule occupancy in co-inoculation experiments. The SMc00911 ORF is predicted to encode a “SodM-like” (superoxide dismutase-like protein containing a rhodanese sulfurtransferase domain at the N-terminus and a chromate-resistance superfamily domain at the C-terminus. Several other ORFs (SMb20360, SMc01562, SMc01266, SMc03964, and the SMc01424-22 operon identified in the screen are expressed at a moderate level by bacteria within nodules, but not by free-living bacteria. Conclusions Based on the analysis of ORFs identified in this study, we conclude that this comparative genomics approach can identify rhizobial genes involved in the nitrogen-fixing symbiosis with host plants, although none of the newly identified genes were found to be essential for this process.

{sup 99m}Tc-labeled chimeric anti-NCA 95 antigranulocyte monoclonal antibody for bone marrow imaging

Energy Technology Data Exchange (ETDEWEB)

Sarwar, M.; Higuchi, Tetsuya; Tomiyoshi, Katsumi [Gunma Univ., Maebashi (Japan). School of Medicine] [and others

1998-09-01

Chimeric mouse-human antigranulocyte monoclonal antibody (ch MAb) against non-specific cross-reacting antigen (NCA-95) was labeled with {sup 99m}Tc (using a direct method) and {sup 125}I (using the chloramine T method), and its binding to human granulocytes and LS-180 colorectal carcinoma cells expressing carcinoembryonic antigen on their surfaces, cross-reactive with anti-NCA-95 chimeric monoclonal antibody, increased in proportion to the number of cells added and reached more than 80% and 90%, respectively. In biodistribution studies, {sup 99m}Tc and {sup 125}I-labeled ch anti-NCA-95 MAb revealed high tumor uptake, and the tumor-to-blood ratio was 2.9 after 24 hours. The tumor-to-normal-organ ratio was also more than 3.0 in all organs except for the tumor-to-kidney ratio. Scintigrams of athymic nude mice confirmed the results of biodistribution studies that showed higher radioactivity in tumor and kidney of the mice administered with {sup 99m}Tc-labeled ch MAb. A normal volunteer injected with {sup 99m}Tc-labeled ch anti-NCA-95 antigranulocyte MAb showed clear bone marrow images, and a patient with aplastic anemia revealed irregular uptake in his lumbar spine, suggesting its utility for bone marrow scintigraphy and for the detection of hematological disorders, infections, and bone metastasis. (author)

Effect of producer cell line on functional activity of anti-D monoclonal antibodies destined for prevention of rhesus sensitization.

Science.gov (United States)

Olovnikova, N I; Ershler, M A; Belkina, E V; Nikolaeva, T L; Miterev, G Yu

2009-04-01

The ability of anti-D antibodies to cause antigen-specific immunosuppression depends on their interaction with low-affinity Fcgamma-receptors. Human monoclonal antibodies to D antigen of the rhesus system were investigated by antibody-dependent cytotoxicity assay in order to estimate their ability to induce hemolysis mediated by low-affinity Fcgamma receptors. We demonstrate that affinity of monoclonal antibodies to receptors of this type does not depend on primary structure of Fc-fragment, but depends on the producer cell line which expresses the antibodies. Monoclonal IgG1 antibodies interacting with FcgammaRIIa and FcgammaRIII lost this property, if they were secreted by human-mouse heterohybridoma, but not by human B-cell line. On the opposite, monoclonal antibodies that could not activate low-affinity Fcgamma receptors were highly active after human cells fusion with rat myeloma YB2/0. Hemolytic activity of IgG3 remained unchanged after fusion of human cells with rodent cells.

Characterization of a lipopolysaccharide mutant of Leptospira derived by growth in the presence of an anti-lipopolysaccharide monoclonal antibody

NARCIS (Netherlands)

Zapata, Sonia; Trueba, Gabriel; Bulach, Dieter M.; Boucher, David; Adler, Ben; Hartskeerl, Rudy

2010-01-01

A lipopolysaccharide mutant of Leptospira interrogans (LaiMut) was obtained by growth in the presence of an agglutinating monoclonal antibody (mAb) against lipopolysaccharide. Agglutination reactions with anti-lipopolysaccharide mAbs and polyclonal antibodies showed that LaiMut had lost some

Development of new versions of anti-human CD34 monoclonal antibodies with potentially reduced immunogenicity

International Nuclear Information System (INIS)

Qian Weizhu; Wang Ling; Li Bohua; Wang Hao; Hou Sheng; Hong Xueyu; Zhang Dapeng; Guo Yajun

2008-01-01

Despite the widespread clinical use of CD34 antibodies for the purification of human hematopoietic stem/progenitor cells, all the current anti-human CD34 monoclonal antibodies (mAbs) are murine, which have the potential to elicit human antimouse antibody (HAMA) immune response. In the present study, we developed three new mouse anti-human CD34 mAbs which, respectively, belonged to class I, class II and class III CD34 epitope antibodies. In an attempt to reduce the immunogenicity of these three murine mAbs, their chimeric antibodies, which consisted of mouse antibody variable regions fused genetically to human antibody constant regions, were constructed and characterized. The anti-CD34 chimeric antibodies were shown to possess affinity and specificity similar to that of their respective parental murine antibodies. Due to the potentially better safety profiles, these chimeric antibodies might become alternatives to mouse anti-CD34 antibodies routinely used for clinical application

Generation of chimeric bispecific G250/anti-CD3 monoclonal antibody, a tool to combat renal cell carcinoma

NARCIS (Netherlands)

Luiten, R. M.; Coney, L. R.; Fleuren, G. J.; Warnaar, S. O.; Litvinov, S. V.

1996-01-01

The monoclonal antibody (MAb) G250 binds to a tumour-associated antigen, expressed in renal cell carcinoma (RCC), which has been demonstrated to be a suitable target for antibody-mediated immunotherapy. A bispecific antibody having both G250 and anti-CD3 specificity can cross-link G250

Micrometastatic cancer cells in bone marrow: in vitro detection with anti-cytokeratin and in vivo labeling with anti-17-1A monoclonal antibodies

International Nuclear Information System (INIS)

Schlimok, G.; Funke, I.; Holzmann, B.

1987-01-01

The detection of early micrometastasis or disseminated single tumor cells poses a problem for conventional diagnosis procedures. Using a panel of monoclonal antibodies against cytokeratin and the 17-1A epithelial antigen the authors identified immunocytochemically tumor cells in bone marrow of patients with breast cancer and colorectal cancer at the time of surgery of the primary tumor. Monoclonal antibody CK2, recognizing the human cytokeratin component 18 in simple epithelia, appeared to be the most suitable reagent because of its negative reaction with bone marrow samples of the noncarcinoma patients. Its specificity was further demonstrated in a double-marker staining procedure using an anti-leukocyte common antigen monoclonal antibody (T200) as counterstain. A comparative analysis showed that immunocytology was clearly superior to conventional cytology and histology. In 9.5-20.5% of patients without distant metastasis, tumor cells could be detected in bone marrow. They found a significant correlation between tumor cells in bone marrow and conventional risk factors, such as distant metastasis or lymph node involvement. In a first approach toward immunotherapy they demonstrated in 3 patients that infused monoclonal antibody 17-1A can label single tumor cells in bone marrow in vivo. They then used this single approach to follow up on 7 patients undergoing 17-1A therapy in an adjuvant clinical trial

Therapeutic effect of anti-feline TNF-alpha monoclonal antibody for feline infectious peritonitis.

Science.gov (United States)

Doki, Tomoyoshi; Takano, Tomomi; Kawagoe, Kohei; Kito, Akihiko; Hohdatsu, Tsutomu

2016-02-01

Feline infectious peritonitis virus (FIPV) replication in macrophages/monocytes induced tumor necrosis factor (TNF)-alpha production, and that the TNF-alpha produced was involved in aggravating the pathology of FIP. We previously reported the preparation of a feline TNF-alpha (fTNF-alpha)-neutralizing mouse monoclonal antibody (anti-fTNF-alpha mAb). This anti-fTNF-alpha mAb 2-4 was confirmed to inhibit the following fTNF-alpha-induced conditions in vitro. In the present study, we investigated whether mAb 2-4 improved the FIP symptoms and survival rate of experimentally FIPV-inoculated SPF cats. Progression to FIP was prevented in 2 out of 3 cats treated with mAb 2-4, whereas all 3 cats developed FIP in the placebo control group. Plasma alpha1-glycoprotein and vascular endothelial growth factor levels were improved by the administration of mAb 2-4, and the peripheral lymphocyte count also recovered. These results strongly suggested that the anti-fTNF-alpha antibody is effective for the treatment of FIP. Copyright © 2015 Elsevier Ltd. All rights reserved.

Therapeutic potential of an anti-high mobility group box-1 monoclonal antibody in epilepsy.

Science.gov (United States)

Zhao, Junli; Wang, Yi; Xu, Cenglin; Liu, Keyue; Wang, Ying; Chen, Liying; Wu, Xiaohua; Gao, Feng; Guo, Yi; Zhu, Junming; Wang, Shuang; Nishibori, Masahiro; Chen, Zhong

2017-08-01

Brain inflammation is a major factor in epilepsy, and the high mobility group box-1 (HMGB1) protein is known to contribute significantly to the generation of seizures. Here, we investigated the therapeutic potential of an anti-HMGB1 monoclonal antibody (mAb) in epilepsy. anti-HMGB1 mAb attenuated both acute seizure models (maximal electroshock seizure, pentylenetetrazole-induced and kindling-induced), and chronic epilepsy model (kainic acid-induced) in a dose-dependent manner. Meanwhile, the anti-HMGB1 mAb also attenuated seizure activities of human brain slices obtained from surgical resection from drug-resistant epilepsy patients. The mAb showed an anti-seizure effect with a long-term manner and appeared to be minimal side effects at even very high dose (no disrupted physical EEG rhythm and no impaired basic physical functions, such as body growth rate and thermoregulation). This anti-seizure effect of mAb results from its inhibition of translocated HMGB1 from nuclei following seizures, and the anti-seizure effect was absent in toll-like receptor 4 knockout (TLR4 -/- ) mice. Interestingly, the anti-HMGB1 mAb also showed a disease-modifying anti-epileptogenetic effect on epileptogenesis after status epileptics, which is indicated by reducing seizure frequency and improving the impaired cognitive function. These results indicate that the anti-HMGB1 mAb should be viewed as a very promising approach for the development of novel therapies to treat refractory epilepsy. Copyright © 2017 Elsevier Inc. All rights reserved.

Radioimmunodetection of colorectal cancer, using anti-CEA monoclonal antibodies

International Nuclear Information System (INIS)

Murayama, Hiroki; Watanabe, Tadashi; Tadokoro, Masanori; Takagi, Hiroshi; Sakuma, Sadayuki; Sakamoto, Junichi.

1989-01-01

Aiming at radioimmunodetection of colorectal cancer, anti-CEA monoclonal antibodies (CEA102) were produced by immunization with purified CEA. CEA102 showed high specificity with clorectal cancer by mixed hemadsorption assay and immunoperoxidase technique. The antigen detected by CEA102 was confirmed to be carcinoembryonic antigen (CEA) and its molecular weight was estimated to be ca. 180,000 by biochemical analysis. The in vivo study using nude mice grafted a human colorectal cancer or a human malignant melanoma showed greater accumulation of 125 I-labeled CEA102 in CEA-positive colorectal cancer than in nude mouse tissues and CEA-negative malignant melanoma. Moreover we successfully obtained scans with good localization of the grafted colorectal cancer on FCR (Fuji Computed Radiography). Using 131 I-labeled CEA102 liver metastasis in the patient with colorectal cancer was successfully detected by external scanning with γ-camera. These results suggest that radiolabeled CEA102 is useful for the detection of colorectal cancer. (author)

Anti-respiratory syncytial virus (RSV) G monoclonal antibodies reduce lung inflammation and viral lung titers when delivered therapeutically in a BALB/c mouse model.

Science.gov (United States)

Caidi, Hayat; Miao, Congrong; Thornburg, Natalie J; Tripp, Ralph A; Anderson, Larry J; Haynes, Lia M

2018-06-01

RSV continues to be a high priority for vaccine and antiviral drug development. Unfortunately, no safe and effective RSV vaccine is available and treatment options are limited. Over the past decade, several studies have focused on the role of RSV G protein on viral entry, viral neutralization, and RSV-mediated pathology. Anti-G murine monoclonal antibody (mAb) 131-2G treatment has been previously shown to reduce weight loss, bronchoalveolar lavage (BAL) cell number, airway reactivity, and Th2-type cytokine production in RSV-infected mice more rapidly than a commercial humanized monoclonal antibody (mAb) against RSV F protein (Palivizumab). In this study, we have tested two human anti-RSV G mAbs, 2B11 and 3D3, by both prophylactic and therapeutic treatment for RSV in the BALB/c mouse model. Both anti-G mAbs reduced viral load, leukocyte infiltration and IFN-γ and IL-4 expression in cell-free BAL supernatants emphasizing the potential of anti-G mAbs as anti-inflammatory and antiviral strategies. Published by Elsevier B.V.

Sequences of 12 monoclonal anti-dinitrophenyl spin-label antibodies for NMR studies

International Nuclear Information System (INIS)

Leahy, D.J.; Rule, G.S.; Whittaker, M.M.; McConnell, H.M.

1988-01-01

Eleven monoclonal antibodies specific for a spin-labeled dinitrophenyl hapten (DNP-SL) have been produces for use in NMR studies. They have been named AN01 and ANO3-AN12. The stability constants for the association of these antibodies with DNP-SL and related haptens were measured by fluorescence quenching. cDNA clones coding for the heavy and light chains of each antibody and of an additional anti-DNP-SL monoclonal antibody, ANO2, have been isolated. The nucleic acid sequence of the 5' end of each clone has been determined, and the amino acid sequence of the variable regions of each antibody has been deduced from the cDNA sequence. The sequences are relatively heterogeneous, but both the heavy and the light chains of ANO1 and ANO3 are derived from the same variable-region gene families as those of the ANO2 antibody. ANO7 has a heavy chain that is related to that of ANO2, and ANO9 has a related light chain. ANO5 and ANO6 are unrelated to ANO2 but share virtually identical heavy and light chains. Preliminary NMR difference spectra comparing related antibodies show that sequence-specific assignment of resonances is possible. Such spectra also provide a measure of structural relatedness

Elucidation of the critical epitope of an anti-EGFR monoclonal antibody EMab-134.

Science.gov (United States)

Kaneko, Mika K; Yamada, Shinji; Itai, Shunsuke; Chang, Yao-Wen; Nakamura, Takuro; Yanaka, Miyuki; Kato, Yukinari

2018-07-01

The epidermal growth factor receptor (EGFR) is a type-1 transmembrane receptor tyrosine kinase, which activates the downstream signaling cascades in many tumors, such as oral and lung cancers. We previously developed EMab-134, a novel anti-EGFR monoclonal antibody (mAb), which reacts with endogenous EGFR-expressing cancer cell lines and normal cells independent of glycosylation in Western blotting, flow cytometry, and immunohistochemical analysis. EMab-134 showed very high sensitivity (94.7%) to oral squamous cell carcinomas in immunohistochemical analysis. In this study, we performed enzyme-linked immunosorbent assay (ELISA), flow cytometry, and immunohistochemical analysis to determine the epitope of EMab-134. A blocking peptide (375-394 amino acids of EGFR) neutralized the EMab-134 reaction against oral cancer cells in flow cytometry and immunohistochemistry. The minimum epitope of EMab-134 was found to be the 377- RGDSFTHTPP -386 sequence. Our findings can be applied for the production of more functional anti-EGFR mAbs that in turn can be used for antitumor treatments.

Human anti-plague monoclonal antibodies protect mice from Yersinia pestis in a bubonic plague model.

Directory of Open Access Journals (Sweden)

Xiaodong Xiao

2010-10-01

Full Text Available Yersinia pestis is the etiologic agent of plague that has killed more than 200 million people throughout the recorded history of mankind. Antibiotics may provide little immediate relief to patients who have a high bacteremia or to patients infected with an antibiotic resistant strain of plague. Two virulent factors of Y. pestis are the capsid F1 protein and the low-calcium response (Lcr V-protein or V-antigen that have been proven to be the targets for both active and passive immunization. There are mouse monoclonal antibodies (mAbs against the F1- and V-antigens that can passively protect mice in a murine model of plague; however, there are no anti-Yersinia pestis monoclonal antibodies available for prophylactic or therapeutic treatment in humans. We identified one anti-F1-specific human mAb (m252 and two anti-V-specific human mAb (m253, m254 by panning a naïve phage-displayed Fab library against the F1- and V-antigens. The Fabs were converted to IgG1s and their binding and protective activities were evaluated. M252 bound weakly to peptides located at the F1 N-terminus where a protective mouse anti-F1 mAb also binds. M253 bound strongly to a V-antigen peptide indicating a linear epitope; m254 did not bind to any peptide from a panel of 53 peptides suggesting that its epitope may be conformational. M252 showed better protection than m253 and m254 against a Y, pestis challenge in a plague mouse model. A synergistic effect was observed when the three antibodies were combined. Incomplete to complete protection was achieved when m252 was given at different times post-challenge. These antibodies can be further studied to determine their potential as therapeutics or prophylactics in Y. pestis infection in humans.

Appraisal of radioimmunotherapy with 131I anti-alpha fetoprotein monoclonal antibody in patients with primary liver cancer

International Nuclear Information System (INIS)

Huang Yaozhang

1992-01-01

Mixed anti-alpha-fetoprotein monoclonal antibodies (AFPMcAb) labeled with 131 I were used in the treatment of 23 patients of moderate to advanced primary liver cancer. In 16 cases treated with 24 doses, the survival periods were 18-605 days with a mean of 135 days. Two patients with moderately advanced liver cancer had a mean survival period of 465 days. According to our experience, the larger dose of 131 I and of anti-AFPMcAb, the longer the survival period and the better the therapeutic results were observed. The relationship between the ratio of cancer/liver radioactivity and the survival period remains to be elucidated

Efficacy of Wnt-1 monoclonal antibody in sarcoma cells

International Nuclear Information System (INIS)

Mikami, Iwao; Koizumi, Kiyoshi; Jablons, David M; You, Liang; He, Biao; Xu, Zhidong; Batra, Sonny; Lee, Amie Y; Mazieres, Julien; Reguart, Noemi; Uematsu, Kazutsugu

2005-01-01

Sarcomas are one of the most refractory diseases among malignant tumors. More effective therapies based on an increased understanding of the molecular biology of sarcomas are needed as current forms of therapy remain inadequate. Recently, it has been reported that Wnt-1/β-catenin signaling inhibits apoptosis in several cancers. In this study, we investigated the efficacy of a monoclonal anti-Wnt-1 antibody in sarcoma cells. We treated cell lines A-204, SJSA-1, and fresh primary cultures of lung metastasis of sarcoma with a monoclonal anti-Wnt-1 antibody. Wnt-1 siRNA treatment was carried out in A-204. We assessed cell death using Crystal Violet staining. Apoptosis induction was estimated by flow cytometry analysis (Annexin V and PI staining). Cell signaling changes were determined by western blotting analysis. We detected Wnt-1 expression in all tissue samples and cell lines. Significant apoptosis induction was found in monoclonal anti-Wnt-1 antibody treated cells compared to control monoclonal antibody treated cells (p < 0.02). Similarly, we observed increased apoptosis in Wnt-1 siRNA treated cells. Blockade of Wnt-1 signaling in both experiments was confirmed by analyzing intracellular levels of Dishevelled-3 and of cytosolic β-catenin. Furthermore, the monoclonal anti-Wnt-1 antibody also induced cell death in fresh primary cultures of metastatic sarcoma in which Wnt-1 signaling was active. Our results indicate that Wnt-1 blockade by either monoclonal antibody or siRNA induces cell death in sarcoma cells. These data suggest that Wnt-1 may be a novel therapeutic target for the treatment of a subset of sarcoma cells in which Wnt-1/β-catenin signaling is active

Demonstration of monoclonal anti-carcinoembryonic antigen (CEA) antibody internalization by electron microscopy, western blotting and radioimmunoassay.

Science.gov (United States)

Tsaltas, G; Ford, C H; Gallant, M

1992-01-01

One of the important factors affecting the action of monoclonal antibodies (Mabs) or immunoconjugates on tumour sites depends on whether the Mab is internalized by the cancer cells in question. The underexplored subject of internalization is discussed in this paper, and a number of in vitro techniques for investigating internalization are evaluated, using a model which consists of a well characterized anti-carcinoembryonic antigen (anti-CEA) Mab and a number of CEA expressing human cancer cell lines. Employing two alternative radiolabeling assays, evidence for internalization of the anti-CEA Mab by a CEA-positive colorectal cancer cell line (LS174T) was obtained throughout the time intervals examined (5 min to 150 min). Electronmicroscopy employing horseradish-peroxidase labeled anti-CEA Mab and control antibody permitted direct visualization of anti-CEA Mab-related staining in intracellular compartments of a high CEA-expressor human colorectal cell line (SKCO1). Finally Western blots of samples derived from cytosolic and membrane components of solubilized cells from lung and colonic cancer cell lines provided evidence for internalized anti-CEA Mab throughout seven half hour intervals, starting at 5 minutes. Internalized anti-CEA was detected in all CEA expressing cell lines (LS174T, SKCO1, BENN) but not in the case of a very low CEA expressor line (COLO 320).

First clinical evaluation of radioimmunoimaging using anti-human lung cancer monoclonal antibodies

International Nuclear Information System (INIS)

Zhou Qian

1991-01-01

Anti-human large cell lung cancer monoclonal antibodies (McAb) 2E3 and 6D1 were produced in the laboratory. Immunohistochemical studies and radiobinding assay showed these antibodies possessed high specificity against lung cancer cells. 28 patients with lung masses were investigated with 131 I-labeled McAb 6D1 and/or 2E3 scintigraphy. 19 of them were histologically proven and 13 were diagnosed primary lung carcinoma. Radioimmunoimaging visualized 10/13 of the primary lung cancers with a detection rate of 77%. Only 1 case of the non-cancer patients and a false localization, giving a true negative rate of 83%. Pathologically the squamous cell lung carcinoma had the highest localization and the small cell lung carcinoma next, but the detection rate was 100% for both. The adenocarcinoma of lung was less sensitive to these McAbs, with a detection rate of only 33% (1 of 3 cases). We conclude that radioimmunoimaging with anti-human large cell lung cancer McAbs is more specific and effective in detecting primary lung cancers and differentiating lung masses than with antibodies against other tumor associated antigens

[Comparative Study for Anti-Hepatitis B Surface Antigen Titers Based on Two Measurement Methods: Using Monoclonal Antibodies Isolated from Hepatitis B Vaccinated Recipients].

Science.gov (United States)

Oone, Kumiko; Kani, Satomi; Oohashi, Minoru; Shinkai, Noboru; Inoue, Takako; Wakimoto, Yukio; Tanaka, Yasuhito

2015-08-01

As anti-hepatitis B surface antigen (anti-HBs) titers vary depending on the measurement methods, we compared two different methods to measure anti-HBs titers in sera and HBs monoclonal antibodies. The sera from 182 HB virus-resolved patients who were negative for HBsAg but positive for antiHB core protein (HBc) and/or anti-HBs were obtained. The measurement of anti-HBs was compared using either Lumipulse G1200 or Architect i2000SR. Six different monoclonal antibody (mAbs) clones isolated from healthy individuals inoculated with hepatitis B vaccine Bimmugen (genotype C) were used. A statistically significant correlation in anti-HBs titers was found between the two methods tested (Y = 0.951X + 100.7, R = 0.813, p Lumipulse and 12 (6.6%) were opposite results. Measuring 2 mAbs with HBV neutralizing activity, the titers of the 116 antibody (1.0 μg/mL) were comparable (689.3 mIU/mL by Lumipulse and 440.7 mIU/mL by Architect), whereas those of the 478 antibody (1.0 μg/mL) were much lower by Architect than by Lumipulse (42.6 vs. 818.6 mIU/mL, respectively). Of four other mAbs without HBV neutralizing activity, equal titers were observed for one; two mAbs had less anti-HB titers by Architect; and one was below the cut-off index (Lumipulse, and the potential ability to detect the 478 antibody with neutralizing activity is low, indicating that Architect might underestimate anti-HBs titers. Future studies should standardize the anti-HBs titer measurement system.

Elucidation of the critical epitope of an anti-EGFR monoclonal antibody EMab-134

Directory of Open Access Journals (Sweden)

Mika K. Kaneko

2018-07-01

Full Text Available The epidermal growth factor receptor (EGFR is a type-1 transmembrane receptor tyrosine kinase, which activates the downstream signaling cascades in many tumors, such as oral and lung cancers. We previously developed EMab-134, a novel anti-EGFR monoclonal antibody (mAb, which reacts with endogenous EGFR-expressing cancer cell lines and normal cells independent of glycosylation in Western blotting, flow cytometry, and immunohistochemical analysis. EMab-134 showed very high sensitivity (94.7% to oral squamous cell carcinomas in immunohistochemical analysis. In this study, we performed enzyme-linked immunosorbent assay (ELISA, flow cytometry, and immunohistochemical analysis to determine the epitope of EMab-134. A blocking peptide (375–394 amino acids of EGFR neutralized the EMab-134 reaction against oral cancer cells in flow cytometry and immunohistochemistry. The minimum epitope of EMab-134 was found to be the 377-RGDSFTHTPP−386 sequence. Our findings can be applied for the production of more functional anti-EGFR mAbs that in turn can be used for antitumor treatments.

Immobilization of Murine Anti-BMP-2 Monoclonal Antibody on Various Biomaterials for Bone Tissue Engineering

Directory of Open Access Journals (Sweden)

Sahar Ansari

2014-01-01

Full Text Available Biomaterials are widely used as scaffolds for tissue engineering. We have developed a strategy for bone tissue engineering that entails application of immobilized anti-BMP-2 monoclonal antibodies (mAbs to capture endogenous BMPs in vivo and promote antibody-mediated osseous regeneration (AMOR. The purpose of the current study was to compare the efficacy of immobilization of a specific murine anti-BMP-2 mAb on three different types of biomaterials and to evaluate their suitability as scaffolds for AMOR. Anti-BMP-2 mAb or isotype control mAb was immobilized on titanium (Ti microbeads, alginate hydrogel, and ACS. The treated biomaterials were surgically implanted in rat critical-sized calvarial defects. After 8 weeks, de novo bone formation was assessed using micro-CT and histomorphometric analyses. Results showed de novo bone regeneration with all three scaffolds with immobilized anti-BMP-2 mAb, but not isotype control mAb. Ti microbeads showed the highest volume of bone regeneration, followed by ACS. Alginate showed the lowest volume of bone. Localization of BMP-2, -4, and -7 antigens was detected on all 3 scaffolds with immobilized anti-BMP-2 mAb implanted in calvarial defects. Altogether, these data suggested a potential mechanism for bone regeneration through entrapment of endogenous BMP-2, -4, and -7 proteins leading to bone formation using different types of scaffolds via AMOR.

The trehalose utilization gene thuA ortholog in Mesorhizobium loti does not influence competitiveness for nodulation on Lotus spp.

Science.gov (United States)

Ampomah, Osei Yaw; Jensen, John Beck

2014-03-01

Competitiveness for nodulation is a desirable trait in rhizobia strains used as inoculant. In Sinorhizobium meliloti 1021 mutation in either of the trehalose utilization genes thuA or thuB influences its competitiveness for root colonization and nodule occupancy depending on the interacting host. We have therefore investigated whether mutation in the thuA ortholog in Mesorhizobium loti MAFF303099 also leads to a similar competitive phenotype on its hosts. The results show that M. loti thuA mutant Ml7023 was symbiotically effective and was as competitive as the wild type in colonization and nodule occupancy on Lotus corniculatus and Lotus japonicus. The thuA gene in M. loti was not induced during root colonization or in the infection threads unlike in S. meliloti, despite its induction by trehalose and high osmolarity in in vitro assays.

Transient human anti-mouse antibodies (HAMA) interference in CA 125 measurements during monitoring of ovarian cancer patients treated with murine monoclonal antibody.

NARCIS (Netherlands)

Oei, A.L.M.; Sweep, F.C.; Massuger, L.F.A.G.; Olthaar, A.J.; Thomas, C.M.G.

2008-01-01

OBJECTIVE: To investigate the influence of human anti-mouse antibodies (HAMA) on serial CA 125 measurements in serum of patients with epithelial ovarian cancer following single intraperitoneal (IP) therapy with Yttrium-90-labeled human milk fat globule 1 murine monoclonal antibody ((90)Y-muHMFG1) as

The biodistribution study of 99mTc labelled anti-CEA monoclonal antibody in tumor bearing nude mice

International Nuclear Information System (INIS)

Lou Zongxin

1992-01-01

The author report the optimal condition of 99m Tc labelling with anti-CEA monoclonal antibody using chelating of 99m Tc with dimethylformamide. The labelling rate of this method is 60%-80%, the radiochemical purity of labelling antibody over 90% and maintain its better immuno activity. The biodistribution of the tumor bearing nude mice demonstrates that as compared with the control group, 24 hours after the intraperitoneal injection the injected labelled antibody has its specific concentration in tumor tissue

Enhancement of retroviral infection in vitro by anti-Le(y) IgG: reversal by humanization of monoclonal mouse antibody

DEFF Research Database (Denmark)

Hansen, J E; Sørensen, A M; Arendrup, M

1993-01-01

Monoclonal mouse IgG3 antibody (ABL 364) against the carbohydrate Le(y) antigen enhanced infection in vitro with HTLV-1 and with HIV-1 when propagated in both transformed and normal lymphocytes. Enhancement was independent of complement, occurred with both lymphocytes and monocytes as target cells...... with no indication of any alternative pathway of infection, as evidenced by abrogation of enhancement by anti-CD4 MAb or soluble recombinant CD4, and also the inability of anti-Le(y) MAb to mediate HIV infection of HSB-2 cells in which HTLV-1/HIV pseudovirus infection was enhanced. While F(ab)2 fragments of ABL 364...

Analyses of the peripheral immunome following multiple administrations of avelumab, a human IgG1 anti-PD-L1 monoclonal antibody

OpenAIRE

Donahue, Renee N.; Lepone, Lauren M.; Grenga, Italia; Jochems, Caroline; Fantini, Massimo; Madan, Ravi A.; Heery, Christopher R.; Gulley, James L.; Schlom, Jeffrey

2017-01-01

Background Multiple anti-PD-L1/PD-1 checkpoint monoclonal antibodies (MAb) have shown clear evidence of clinical benefit. All except one have been designed or engineered to omit the possibility to mediate antibody-dependent cell-mediated cytotoxicity (ADCC) as a second potential mode of anti-tumor activity; the reason for this is the concern of lysis of PD-L1 positive immune cells. Avelumab is a fully human IgG1 MAb which has been shown in prior in vitro studies to mediate ADCC versus a range...

Demonstration of two distinct antigenic determinants on hepatitis B e antigen by monoclonal antibodies

International Nuclear Information System (INIS)

Imai, M.; Nomura, M.; Gotanda, T.; Sano, T.; Tachibana, K.; Miyamoto, H.; Takahashi, K.; Toyama, S.; Miyakawa, Y.; Mayumi, M.

1982-01-01

Mice were immunized against hepatitis B e antigen (HBeAg) isolated from sera of asymptomatic carriers of hepatitis B virus. Their spleen cells were fused with mouse myeloma (NS-1) cells, and 5 clones of hybridoma cells secreting antibody against HBeAg (anti-HBe) were isolated. For the production of anti-HBe in large scale, cells were cultivated both in vitro and in the peritoneal cavity of ascitic mice. Although monoclonal antibodies produced by these clones showed a strong reactivity of anti-HBe in hemagglutination tests, individual monoclonal anti-HBe did not reveal any precipitin line in immunodiffusion. When 2 of the 5 monoclonal antibodies were mixed together, however, some combinations showed a precipitin line against HBeAg, whereas others did not. Utilizing solid-phase radioimmunoassay involving a number of combinations of monoclonal antibodies used for solid-phase and radiolabeling, the 5 antibodies were classified into 2 groups. Three of the anti-HBe antibodies were found to be directed to 1 determinant of HBeAg (determinant a); the remaining 2 to the other determinant (determinant b). Determinants a and b were detected on HBeAg in the serum, as well as on the polypeptide of 19,000 daltons (P19) derived from the nucleocapsid of hepatitis B virus. Monoclonal anti-HBe antibodies with different specificities may provide useful tools in delineating the antigenic structure of HBeAg and also in evaluating immune responses of the host directed to its subdeterminants

A monoclonal antibody that specifically recognizes m6A nucleoside

OpenAIRE

Espuny, Ruth; Castro, Ana; Codony, Carles; Eritja Casadellà, Ramón; Bach-Elias, Montse

1998-01-01

A hybridoma against the nucleoside m6A has been obtained from mouse spleen. This hybridoma was named H65 and it secretes monoclonal antibodies anti-m6A. The competition assays showed that the monoclonal antibody was highly specific for m6A nucleoside.

Homology of ab1 and ab3 monoclonal antibodies that neutralize Semliki Forest virus

NARCIS (Netherlands)

Fernandez, IM; Bos, NA; Harmsen, M; Verheul, AFM; Snippe, H; Kraaijeveld, CA

2001-01-01

A noninternal image monoclonal antiidiotypic antibody (ab2 mAb), designated 1,13A321, that had proved its efficacy as vaccine against infection with Semliki Forest virus (SFV) in BALB/c mice, was used as immunogen to generate a panel of SFV-neutralizing monoclonal anti-anti-idiotypic antibodies (ab3

Improved radioimaging and tumor localization with monoclonal F(ab')2

International Nuclear Information System (INIS)

Wahl, R.L.; Parker, C.W.; Philpott, G.W.

1983-01-01

Monoclonal anti-tumor antibodies have great promise for radioimmunodetection and localization of tumors. Fab and F(ab')2 fragments, which lack the Fc fragment of antibody (Ab), are cleared more rapidly from the circulation and may have less nonspecific tissue binding than intact Ab. In radioimaging studies using a murine monoclonal antibody to carcinoembryonic antigen in a human colon carcinoma xenografted into hamsters, F(ab')2 fragments were shown superior to Fab fragments and intact antibody for scintiscanning. In double-label experiments with anti-CEA antibody and control monoclonal IgG, F(ab')2 fragments were found to give better and more rapid specific tumor localization than intact antibody or Fab fragments. F(ab')2 fragments offer significant promise for tumor imaging and possibly therapy

Facilitation of syngeneic stem cell engraftment by anti-class I monoclonal antibody pretreatment of unirradiated recipients

International Nuclear Information System (INIS)

Voralia, M.; Semeluk, A.; Wegmann, T.G.

1987-01-01

We have established a murine model of syngeneic bone marrow transplantation based on the use of monoclonal antibody as the sole conditioning regimen in unirradiated recipients. Administration of a single injection of monoclonal antibody directed against major histocompatibility complex-encoded class I determinants facilitated permanent hemopoietic stem cell engraftment without any apparent side-effects. Whereas untreated hosts exhibited a maximal chimerism of 15% at donor cell doses of up to 12 X 10(7) bone marrow cells, pretreatment by 2 mg of anti-class I antibody one week prior to transplantation of 3 X 10(7) syngeneic bone marrow cells resulted in a mean donor representation of about 80%. The antibody can be given up to four weeks prior to transplantation, and the degree of donor engraftment observed is a function of the dose of antibody administered. The fact that specific antibody enhanced engraftment in two strain combinations indicates that antibody is the active agent in facilitating engraftment and that facilitation is not strain-restricted. Anti-class I antibodies of the IgG2a, but not IgG1, isotype are effective in promoting engraftment. Although the isotype requirement suggests a role for antibody-mediated cytotoxicity in promoting stem cell engraftment, the extensive time-frame of facilitation suggests that other effects of the antibody may also be involved. The model of syngeneic bone marrow transplantation we describe here will be useful in studying the mechanisms regulating stem cell engraftment and may have potential clinical application as an approach to autologous marrow transplantation

Immunotherapy for the treatment of colorectal tumors: focus on approved and in-clinical-trial monoclonal antibodies

Directory of Open Access Journals (Sweden)

Françoso A

2017-01-01

Full Text Available Alex Françoso,1 Patricia Ucelli Simioni1–3 1Department of Biomedical Science, Faculty of Americana, Americana, 2Department of Genetics, Evolution and Bioagents, Institute of Biology, University of Campinas, Campinas, 3Department of Biochemistry and Microbiology, Institute of Biosciences, Universidade Estadual Paulista, Rio Claro, São Paulo, Brazil Abstract: Colorectal cancer is considered a disease of the elderly population. Since the number of geriatric patients continues to rise, monoclonal antibody therapy is the most promising therapy in the recent research. Presently, the monoclonal antibodies most frequently used in the treatment of colorectal tumors are bevacizumab, cetuximab, panitumumab, and ramucirumab. Bevacizumab is a monoclonal antibody that acts on VEGF. Cetuximab and panitumumab act on EGFR. Ramucirumab binds directly to the ligand-binding pocket of VEGFR-2 to block the binding of VEGF-A, VEGF-C, and VEGF-D. These monoclonal antibodies, alone or in association with radiotherapy or chemotherapy, are presenting good results and are increasing patient survival, despite the side effects. Due to the limited number of molecules available, several studies are trying to develop new monoclonal antibodies for the treatment of colorectal tumors. Among those being studied, some recent molecules are in phase I and/or II trials and are yielding advantageous results, such as anti-DR5, anti-Fn14, anti-IGF-1R, anti-EGFR, anti-NRP1, and anti-A33 antibodies. This has been successful in reducing side effects and in treating nonresponsive patients. Keywords: monoclonal antibodies, colorectal tumor, bevacizumab, cetuximab, panitumumab, ramucirumab

Evaluation of dry blood spot technique for quantification of an Anti-CD20 monoclonal antibody drug in human blood samples.

Science.gov (United States)

Lin, Yong-Qing; Zhang, Yilu; Li, Connie; Li, Louis; Zhang, Kelley; Li, Shawn

2012-01-01

To evaluate the dried blood spot (DBS) technique in ELISA quantification of larger biomolecular drugs, an anti-CD20 monoclonal antibody drug was used as an example. A method for the quantification of the anti-CD20 drug in human DBS was developed and validated. The drug standard and quality control samples prepared in fresh human blood were spotted on DBS cards and then extracted. A luminescent ELISA was used for quantification of the drug from DBS samples. The assay range of the anti-CD20 drug standards in DBS was 100-2500ng/mL. The intra-assay precision (%CV) ranged from 0.4% to 10.1%, and the accuracy (%Recovery) ranged from 77.9% to 113.9%. The inter assay precision (%CV) ranged from 5.9% to 17.4%, and the accuracy ranged from 81.5% to 110.5%. The DBS samples diluted 500 and 50-fold yielded recovery of 88.7% and 90.7%, respectively. The preparation of DBS in higher and lower hematocrit (53% and 35%) conditions did not affect the recovery of the drug. Furthermore, the storage stability of the anti-CD20 drug on DBS cards was tested at various conditions. It was found that the anti-CD20 drug was stable for one week in DBS stored at room temperature. However, it was determined that the stability was compro]mised in DBS stored at high humidity, high temperature (55°C), and exposed to direct daylight for a week, as well as for samples stored at room temperature and high humidity conditions for a month. Stability did not change significantly in samples that underwent 3 freeze/thaw cycles. Our results demonstrated a successful use of DBS technique in ELISA quantification of an anti-CD20 monoclonal antibody drug in human blood. The stability data provides information regarding sample storage and shipping for future clinical studies. It is, therefore, concluded that the DBS technique is applicable in the quantification of other large biomolecule drugs or biomarkers. Copyright © 2011 Elsevier Inc. All rights reserved.

The transcriptional activator LdtR from 'Candidatus Liberibacter asiaticus' mediates osmotic stress tolerance.

Directory of Open Access Journals (Sweden)

Fernando A Pagliai

2014-04-01

Full Text Available The causal agent of Huanglongbing disease, 'Candidatus Liberibacter asiaticus', is a non-culturable, gram negative, phloem-limited α-proteobacterium. Current methods to control the spread of this disease are still limited to the removal and destruction of infected trees. In this study, we identified and characterized a regulon from 'Ca. L. asiaticus' involved in cell wall remodeling, that contains a member of the MarR family of transcriptional regulators (ldtR, and a predicted L,D-transpeptidase (ldtP. In Sinorhizobium meliloti, mutation of ldtR resulted in morphological changes (shortened rod-type phenotype and reduced tolerance to osmotic stress. A biochemical approach was taken to identify small molecules that modulate LdtR activity. The LdtR ligands identified by thermal shift assays were validated using DNA binding methods. The biological impact of LdtR inactivation by the small molecules was then examined in Sinorhizobium meliloti and Liberibacter crescens, where a shortened-rod phenotype was induced by growth in presence of the ligands. A new method was also developed to examine the effects of small molecules on the viability of 'Ca. Liberibacter asiaticus', using shoots from HLB-infected orange trees. Decreased expression of ldtRLas and ldtPLas was observed in samples taken from HLB-infected shoots after 6 h of incubation with the LdtR ligands. These results provide strong proof of concept for the use of small molecules that target LdtR, as a potential treatment option for Huanglongbing disease.

Safety and efficacy of ofatumumab, a fully human monoclonal anti-CD20 antibody, in patients with relapsed or refractory B-cell chronic lymphocytic leukemia

DEFF Research Database (Denmark)

Coiffier, Bertrand; Lepretre, Stéphane; Pedersen, Lars Møller

2008-01-01

Safety and efficacy of the fully human anti-CD20 monoclonal antibody, ofatumumab, was analyzed in a multicenter dose-escalating study including 33 patients with relapsed or refractory chronic lymphocytic leukemia. Three cohorts of 3 (A), 3 (B), and 27 (C) patients received 4, once weekly, infusio...

Regulation of Long-Chain N-Acyl-Homoserine Lactones in Agrobacterium vitis

OpenAIRE

Hao, Guixia; Burr, Thomas J.

2006-01-01

Homologs of quorum-sensing luxR and luxI regulatory genes, avsR and avsI, were identified in Agrobacterium vitis strain F2/5. Compared to other LuxI proteins from related species, the deduced AvsI shows the greatest identity to SinI (71%) from Sinorhizobium meliloti Rm1021. AvsR possesses characteristic autoinducer binding and helix-turn-helix DNA binding domains and shares a high level of identity with SinR (38%) from Rm1021. Site-directed mutagenesis of avsR and avsI was performed, and both...

Clearance of a monoclonal anti-DNA antibody following administration of DNA in normal and autoimmune mice

International Nuclear Information System (INIS)

Jones, F.S.; Pisetsky, D.S.; Kurlander, R.J.

1986-01-01

To study the assembly of DNA-anti-DNA complexes in vivo, we have measured the clearance from blood and organ localization of a murine IgG2a monoclonal anti-DNA antibody, called 6/0, following the infusion of DNA intravenously or intraperitoneally. Intraperitoneal DNA caused a profound acceleration of 6/0 anti-DNA clearance that was dose dependent and demonstrable after the infusion of as little as 1.9 microgram per gram of body weight of single-stranded DNA. The antibody was cleared primarily in the liver without increased deposition in the kidney. Intraperitoneal infusions of DNA also accelerated the clearance of 6/0 in autoimmune MRL-lpr/lpr mice. In contrast, intravenous DNA given in comparable doses caused only a slight increase in 6/0 antibody clearance; this accelerated clearance was seen only at low antigen doses and only during the first 10 min following DNA infusion. Using double-radiolabeling techniques, 6/0 and Cl.18, an IgG2ak myeloma protein without anti-DNA activity, were found to disappear from blood at a comparable rate in both B6D2 mice and MRL-lpr/lpr mice. These results suggest that the DNA-anti-DNA immune complexes can form in vivo but that this process is profoundly affected by the manner in which DNA enters the circulation. In addition, the results suggest that DNA-dependent clearance is not a major pathway for anti-DNA metabolism in normal or at least one strain of autoimmune mice

Purification of immunoreactive radiolabeled moniclonal antibodies with anti-iodiotypic moniclonal antibodies

International Nuclear Information System (INIS)

Temponi, M.; Pupa, S.; Ferrone, S.

1990-01-01

A method is described to purify immunoreactive moniclonal antibodies from radiolabeled monoclonal antibody preparations. The method is based on incubation of radiolabeled monoclonal antibodies with insolubilized anti-idiotypic monoclonal antibodies to idiotopes within the antigen-combining site of monoclonal antibodies to be purified an elution of bound monoclonal antibodies with a low pH buffer. The immunoreactive fraction of the purified monoclonal antibodies was at least 82%; the yeald was at least 73%. The purification procedure did not cause any detectable change in the affinity constant of the eluted monoclonal antibodies. The method is simple and rapid; the requirement for anti-idiotypic monoclonal antibodies to idiotopes within the antigen-combining site of the antibodies to be purified is not likely to represent a major limitation in the broad application of the present method, since the hybridoma technology has greatly facilitated the development of anti-idiotypic monoclonal antibodies. (author). 12 refs.; 4 figs.; 1 tab

Secukinumab, a human anti-interleukin-17A monoclonal antibody, in patients with psoriatic arthritis (FUTURE 2): a randomised, double-blind, placebo-controlled, phase 3 trial

NARCIS (Netherlands)

McInnes, Iain B.; Mease, Philip J.; Kirkham, Bruce; Kavanaugh, Arthur; Ritchlin, Christopher T.; Rahman, Proton; van der Heijde, Désirée; Landewé, Robert; Conaghan, Philip G.; Gottlieb, Alice B.; Richards, Hanno; Pricop, Luminita; Ligozio, Gregory; Patekar, Manmath; Mpofu, Shephard; Bird, Paul; Hall, Stephen; Nash, Peter; Zochling, Jane; de Vlam, Kurt; Langenaken, Christine; Geusens, Piet; Beaulieu, Andre; Tremblay, Jean-Luc; McCarthy, Tim; Papp, Kim; Poulin, Yves; Cohen, Martin; Galatikova, Dagmar; Dokoupilova, Eva; Dvorak, Zdenek; Mann, Herman; Sieper, Joachim; Spieler, Wolfgang; Kurthen, Reiner; Braun, Juergen; Wollenhaupt, Juergen; Tony, Hans-Peter; Schuch, Florian; Schulze-Koops, Hendrik; Rech, Juergen; Leszczynski, Piotr; Adamski, Zygmunt; Szepietowski, Jacek; Tlustochowicz, Witold; Kaszuba, Andrzej; Szymanska, Malgorzata; Stanislav, Marina; Nesmeyanova, Olga; Vezikova, Natalia; Ershova, Olga; Izmozherova, Nadezda; Zotkin, Eugeny; Petrova, Marianna; Kastanayan, Alexander; Yakupova, Svetlana; Agafina, Alina; Asavatanabodee, Paijit; Suwannalai, Parawee; Kerrane, Jerome; Tahir, Hasan; McInnes, Iain; Edwards, Christopher; Chinoy, Hector; Marzo-Ortega, Helena; Kaul, Arvind; Sheeran, Thomas; Clunie, Gavin; Schechtman, Joy; Gaylis, Norman; Kaine, Jeffrey; Lawson, Jeffrey; El-Kadi, Hisham; Flint, Kathleen; Kivitz, Alan; Churchhill, Melvin; Sikes, David; Lowenstein, Mitchell; Halpert, Elias; Abdulky, Mary; Palmer, William; Codding, Christine; Legerton, Clarence; Singhal, Atul; Sunkureddi, Prashanth; Gough, William; Forman, Seth; Box, Jane; Khan, Mohamed; Barranco, Elizabeth

2015-01-01

Background Interleukin 17A is a proinflammatory cytokine that is implicated in the pathogenesis of psoriatic arthritis. We assessed the efficacy and safety of subcutaneous secukinumab, a human anti-interleukin-17A monoclonal antibody, in patients with psoriatic arthritis. Methods In this phase 3,

Monoclonal antibody therapy of inflammatory bowel disease

NARCIS (Netherlands)

van Deventer, S. J.; Camoglio, L.

1996-01-01

Several anti-inflammatory drugs have therapeutic efficacy in inflammatory bowel disease, but their targets remain incompletely characterized. The development of monoclonal antibodies that either recognize epitopes on immune-competent cells, or neutralize pro-inflammatory cytokines, has helped to

Human peripheral blood monocytes display surface antigens recognized by monoclonal antinuclear antibodies

International Nuclear Information System (INIS)

Holers, V.M.; Kotzin, B.L.

1985-01-01

The authors used monoclonal anti-nuclear autoantibodies and indirect immunofluorescence to examine normal human peripheral blood mononuclear leukocytes for the presence of cell surface nuclear antigens. Only one monoclonal anti-histone antibody (MH-2) was found to bind to freshly isolated PBL, staining approximately 10% of large cells. However, after cells were placed into culture for 16-24 h, a high percentage (up to 60%) of large-sized cells were recognized by an anti-DNA (BWD-1) and several different antihistone monoclonal antibodies (BWH-1, MH-1, and MH-2). These antibodies recognize separate antigenic determinants on chromatin and histones extracted from chromatin. The histone antigen-positive cells were viable, and the monoclonal antibodies could be shown to be binding to the cell surface and not to the nucleus. Using monoclonal antibodies specific for monocytes and T cells, and complement-mediated cytotoxicity, the cells bearing histone antigens were shown to be primarily monocytes. The appearance of histone and DNA antigen-positive cells was nearly completely inhibited by the addition of low concentrations of cycloheximide at initiation of the cultures. In contrast, little effect on the percentage of positive cells was detected if cells were exposed to high doses of gamma irradiation before culture. These data further support the existence of cell surface nuclear antigens on selected cell subsets, which may provide insight into the immunopathogenesis of systemic lupus erythematosus and related autoimmune diseases

Monoclonal antibodies for radioimmunodetection of tumours and for targeting

International Nuclear Information System (INIS)

Baldwin, R.W.; Embleton, M.J.; Pimm, M.V.

1983-01-01

A monoclonal antibody 791T/36 prepared against human osteogenic sarcoma has been used to detect primary and metastatic colorectal carcinomas by external imaging of patients following injection of 131 I-labelled antibody. In 10 of 11 patients radiolabelled 791T/36 antibody localized in tumours, the tumour/non tumour ratio of radioactivity ranging from 1.5:1 to 8.1. 791T/36 antibody was also evaluated for its potential for targeting anti-tumour agents including cytotoxic drugs (Vindesine) and immunomodulating agents (interferon). Vindesine-791T/36 conjugates were preferentially cytotoxic in vitro for target cells expressing the 791T/36 anti-body defined antigen. Also interferon conjugated to 791T/36 antibody, like free interferon activated peripheral blood natural killer cell activity. These in vitro tests together with related studies on antibody localization in vivo indicate the potential of monoclonal antibody targeting of anti-tumour agents

A fundamental study of immunoscintigraphy with sup 131 I-labeled anti-CA 19-9 and anti-CEA monoclonal antibodies; Imaging of tumor-bearing mice by IMACIS-1 and cell ELISA with human tumor cells

Energy Technology Data Exchange (ETDEWEB)

Nogami, Toshihiko; Miura, Hiroshi; Ohmi, Shoichi; Kazahaya, Yasuhiro [CIS DIAGNOSTIC K.K., Chiba (Japan)

1990-05-01

A study was made on 2 types of {sup 131}I-labeled anti-CA 19-9 and anti-CEA mouse monoclonal antibodies (IMACIS-1) against human cancer related antigen as to their usefulness in radioimmunoimaging. Tumor-bearing nude mice were used for comparison. The transplanted tumors (SW948, COLO 201) were clearly visualized 48-72 hours after administration of IMACIS-1. Tumor/blood ratio 72 hours after administration: 8.69 in COLO 201 and 5.70 in SW948, showing ca. 10-15 times as high as those in PC-3 and HEp-2. IMACIS-1 therefore is considered useful in radioimmunoimaging of cancer. Analysis was made by in vitro cell ELISA. As a result, both of the cells specifically reacted with anti-CA 19-9 but not anti-CEA. (author).

Combination of two anti-CD5 monoclonal antibodies synergistically induces complement-dependent cytotoxicity of chronic lymphocytic leukaemia cells.

Science.gov (United States)

Klitgaard, Josephine L; Koefoed, Klaus; Geisler, Christian; Gadeberg, Ole V; Frank, David A; Petersen, Jørgen; Jurlander, Jesper; Pedersen, Mikkel W

2013-10-01

The treatment of chronic lymphocytic leukaemia (CLL) has been improved by introduction of monoclonal antibodies (mAbs) that exert their effect through secondary effector mechanisms. CLL cells are characterized by expression of CD5 and CD23 along with CD19 and CD20, hence anti-CD5 Abs that engage secondary effector functions represent an attractive opportunity for CLL treatment. Here, a repertoire of mAbs against human CD5 was generated and tested for ability to induce complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC) both as single mAbs and combinations of two mAbs against non-overlapping epitopes on human CD5. The results demonstrated that combinations of two mAbs significantly increased the level of CDC compared to the single mAbs, while no enhancement of ADCC was seen with anti-CD5 mAb combinations. High levels of CDC and ADCC correlated with low levels of Ab-induced CD5 internalization and degradation. Importantly, an anti-CD5 mAb combination enhanced CDC of CLL cells when combined with the anti-CD20 mAbs rituximab and ofatumumab as well as with the anti-CD52 mAb alemtuzumab. These results suggest that an anti-CD5 mAb combination inducing CDC and ADCC may be effective alone, in combination with mAbs against other targets or combined with chemotherapy for CLL and other CD5-expressing haematological or lymphoid malignancies. © 2013 John Wiley & Sons Ltd.

Characterization of a recombinant humanized anti-cocaine monoclonal antibody and its Fab fragment.

Science.gov (United States)

Kirley, Terence L; Norman, Andrew B

2015-01-01

Variations of post-translational modifications are important for stability and in vivo behavior of therapeutic antibodies. A recombinant humanized anti-cocaine monoclonal antibody (h2E2) was characterized for heterogeneity of N-linked glycosylation and disulfide bonds. In addition, charge heterogeneity, which is partially due to the presence or absence of C-terminal lysine on the heavy chains, was examined. For cocaine overdose therapy, Fab fragments may be therapeutic, and thus, a simplified method of generation, purification, and characterization of the Fab fragment generated by Endoproteinase Lys-C digestion was devised. Both the intact h2E2 antibody and purified Fab fragments were analyzed for their affinities for cocaine and 2 of its metabolites, benzoylecgonine and cocaethylene, by fluorescence quenching of intrinsic antibody tyrosine and tryptophan fluorescence resulting from binding of these drugs. Binding constants obtained from fluorescence quenching measurements are in agreement with recently published radioligand and ELISA binding assays. The dissociation constants determined for the h2E2 monoclonal and its Fab fragment are approximately 1, 5, and 20 nM for cocaethylene, cocaine, and benzoylecgonine, respectively. Tryptophan fluorescence quenching (emission at 330 nm) was measured after either excitation of tyrosine and tryptophan (280 nm) or selective excitation of tryptophan alone (295 nm). More accurate binding constants are obtained using tryptophan selective excitation at 295 nm, likely due to interfering absorption of cocaine and metabolites at 280 nm. These quenching results are consistent with multiple tryptophan and tyrosine residues in or near the predicted binding location of cocaine in a previously published 3-D model of this antibody's variable region.

Development of radiolabelling techniques of anti-CEA monoclonal antibody

International Nuclear Information System (INIS)

Castiglia, S.G. de

1998-01-01

The purpose of this work was to label monoclonal and polyclonal antibodies with 99 Tc m such as the ior-CEA-1 antibody and polyclonal IgG using a direct method, to check the radiochemical and biological behavior of labelled products, to prepare it under sterile and apyrogenic conditions as a lyophilized kit and to employ it in clinical trials. In addition, a photoactivation method was used to label polyclonal IgG with 99 Tc m and to compare with the established method using mercaptoethanol (2-ME) as the reducing agent. Finally polyclonal IgG was labelled using an indirect method in which a chelator was covalently attached to the protein and the 99 Tc m added as glucoheptonate complex. The properties of 99 Tc m when labelled with monoclonal and polyclonal antibodies by different methods were assessed by in vitro and in vivo studies

Genome sequence of Ensifer meliloti strain WSM1022; a highly effective microsymbiont of the model legume Medicago truncatula A17.

Science.gov (United States)

Terpolilli, Jason; Hill, Yvette; Tian, Rui; Howieson, John; Bräu, Lambert; Goodwin, Lynne; Han, James; Liolios, Konstantinos; Huntemann, Marcel; Pati, Amrita; Woyke, Tanja; Mavromatis, Konstantinos; Markowitz, Victor; Ivanova, Natalia; Kyrpides, Nikos; Reeve, Wayne

2013-12-20

Ensifer meliloti WSM1022 is an aerobic, motile, Gram-negative, non-spore-forming rod that can exist as a soil saprophyte or as a legume microsymbiont of Medicago. WSM1022 was isolated in 1987 from a nodule recovered from the roots of the annual Medicago orbicularis growing on the Cyclades Island of Naxos in Greece. WSM1022 is highly effective at fixing nitrogen with M. truncatula and other annual species such as M. tornata and M. littoralis and is also highly effective with the perennial M. sativa (alfalfa or lucerne). In common with other characterized E. meliloti strains, WSM1022 will nodulate but fixes poorly with M. polymorpha and M. sphaerocarpos and does not nodulate M. murex. Here we describe the features of E. meliloti WSM1022, together with genome sequence information and its annotation. The 6,649,661 bp high-quality-draft genome is arranged into 121 scaffolds of 125 contigs containing 6,323 protein-coding genes and 75 RNA-only encoding genes, and is one of 100 rhizobial genomes sequenced as part of the DOE Joint Genome Institute 2010 Genomic Encyclopedia for Bacteria and Archaea-Root Nodule Bacteria (GEBA-RNB) project.

Competitive Advantage Provided by Bacterial Motility in the Formation of Nodules by Rhizobium meliloti

Science.gov (United States)

Ames, Peter; Bergman, Kostia

1981-01-01

The effect of motility on the competitive success of Rhizobium meliloti in nodule production was investigated. A motile strain formed more nodules than expected when mixed at various unfavorable ratios with either flagellated or nonflagellated nonmotile derivatives. We conclude that motility confers a selective advantage on rhizobia when competing with nonmotile strains. PMID:7298580

Novel flow cytometric analysis of the progress and route of internalization of a monoclonal anti-carcinoembryonic antigen (CEA) antibody.

Science.gov (United States)

Ford, C H; Tsaltas, G C; Osborne, P A; Addetia, K

1996-03-01

A flow cytometric method of studying the internalization of a monoclonal antibody (Mab) directed against carcinoembryonic antigen (CEA) has been compared with Western blotting, using three human colonic cancer cell lines which express varying amounts of the target antigen. Cell samples incubated for increasing time intervals with fluoresceinated or unlabelled Mab were analyzed using flow cytometry or polyacrylamide gel electrophoresis and Western blotting. SDS/PAGE analysis of cytosolic and membrane components of solubilized cells from the cell lines provided evidence of non-degraded internalized anti-CEA Mab throughout seven half hour intervals, starting at 5 min. Internalized anti-CEA was detected in the case of high CEA expressing cell lines (LS174T, SKCO1). Very similar results were obtained with an anti-fluorescein flow cytometric assay. Given that these two methods consistently provided comparable results, use of flow cytometry for the detection of internalized antibody is suggested as a rapid alternative to most currently used methods for assessing antibody internalization. The question of the endocytic route followed by CEA-anti-CEA complexes was addressed by using hypertonic medium to block clathrin mediated endocytosis.

Pharmacokinetics of 111In-labeled anti-p97 monoclonal antibody in patients with metastatic malignant melanoma

International Nuclear Information System (INIS)

Rosenblum, M.G.; Murray, J.L.; Haynie, T.P.; Glenn, H.J.; Jahns, M.F.; Benjamin, R.S.; Frincke, J.M.; Carlo, D.J.; Hersh, E.M.

1985-01-01

Twenty-eight patients with metastatic malignant melanoma received anti-p97 murine monoclonal antibody (96.5) infused over 2 h at doses between 1 and 20 mg coupled to either 2.5 or 5.0 mCi of 111 In by the bifunctional chelating agent diethyltriaminepentaacetic acid. Clearance of 111 In from plasma closely fit an open, one-compartment mathematical model (r2 greater than 0.90). The overall half-life of 111 In plasma was approximately 31 h and did not appear to be dependent on the total dose of antibody administered. The apparent volume of distribution of the 111 In label approximated the total blood volume (7.8 +/- 0.7 liters) at the 1-mg dose and decreased to 3.0 +/- 0.14 liters at the 20-mg dose, suggesting saturation of antigenic or other extravascular binding sites at higher antibody doses. The clearance of the murine monoclonal antibody itself from plasma was measured by an enzyme-linked immunosorbent assay. The pharmacokinetics for the murine antibody in plasma also fit an open, one-compartment mathematical model. All pharmacokinetic parameters for unlabeled antibody closely paralleled those found for 111 In-labeled antibody pharmacokinetics. This suggests that the 111 In radiolabel remains complexed to the monoclonal antibody after in vivo administration. The cumulative urinary excretion of the 111 In label over 48 h was between 12 and 23% of the total administered dose and is assumed to represent 111 In-labeled chelate complex unattached to antibody. Analysis of the 111 In label in spleen, liver, heart, and kidney showed that the concentration of label in liver tissue was reduced with increasing antibody doses and coincided with changes in the apparent volume of distribution

A Neutralizing Anti-gH/gL Monoclonal Antibody Is Protective in the Guinea Pig Model of Congenital CMV Infection

Science.gov (United States)

Auerbach, Marcy R.; Yan, Donghong; Vij, Rajesh; Hongo, Jo-Anne; Nakamura, Gerald; Vernes, Jean-Michel; Meng, Y. Gloria; Lein, Samantha; Chan, Pamela; Ross, Jed; Carano, Richard; Deng, Rong; Lewin-Koh, Nicholas; Xu, Min; Feierbach, Becket

2014-01-01

Human cytomegalovirus (HCMV) is the most common cause of congenital virus infection. Congenital HCMV infection occurs in 0.2–1% of all births, and causes birth defects and developmental abnormalities, including sensorineural hearing loss and developmental delay. Several key studies have established the guinea pig as a tractable model for the study of congenital HCMV infection and have shown that polyclonal antibodies can be protective [1]–[3]. In this study, we demonstrate that an anti-guinea pig CMV (GPCMV) glycoprotein H/glycoprotein L neutralizing monoclonal antibody protects against fetal infection and loss in the guinea pig. Furthermore, we have delineated the kinetics of GPCMV congenital infection, from maternal infection (salivary glands, seroconversion, placenta) to fetal infection (fetus and amniotic fluid). Our studies support the hypothesis that a neutralizing monoclonal antibody targeting an envelope GPCMV glycoprotein can protect the fetus from infection and may shed light on the therapeutic intervention of HCMV congenital infection in humans. PMID:24722349

Extended region of nodulation genes in Rhizobium meliloti 1021. II. Nucleotide sequence, transcription start sites and protein products

International Nuclear Information System (INIS)

Fisher, R.F.; Swanson, J.A.; Mulligan, J.T.; Long, S.R.

1987-01-01

The authors have established the DNA sequence and analyzed the transcription and translation products of a series of putative nodulation (nod) genes in Rhizobium meliloti strain 1021. Four loci have been designated nodF, nodE, nodG and nodH. The correlation of transposon insertion positions with phenotypes and open reading frames was confirmed by sequencing the insertion junctions of the transposons. The protein products of these nod genes were visualized by in vitro expression of cloned DNA segments in a R. meliloti transcription-translation system. In addition, the sequence for nodG was substantiated by creating translational fusions in all three reading frames at several points in the sequence; the resulting fusions were expressed in vitro in both E. coli and R. meliloti transcription-translation systems. A DNA segment bearing several open reading frames downstream of nodG corresponds to the putative nod gene mutated in strain nod-216. The transcription start sites of nodF and nodH were mapped by primer extension of RNA from cells induced with the plant flavone, luteolin. Initiation of transcription occurs approximately 25 bp downstream from the conserved sequence designated the nod box, suggesting that this conserved sequence acts as an upstream regulator of inducible nod gene expression. Its distance from the transcription start site is more suggestive of an activator binding site rather than an RNA polymerase binding site

Stable symbiotic nitrogen fixation under water-deficit field conditions by a stress-tolerant alfalfa microsymbiont and its complete genome sequence.

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Jozefkowicz, Cintia; Brambilla, Silvina; Frare, Romina; Stritzler, Margarita; Piccinetti, Carlos; Puente, Mariana; Berini, Carolina Andrea; Pérez, Pedro Reyes; Soto, Gabriela; Ayub, Nicolás

2017-12-10

We here characterized the stress-tolerant alfalfa microsymbiont Sinorhizobium meliloti B401. B401-treated plants showed high nitrogen fixation rates under humid and semiarid environments. The production of glycine betaine in isolated bacteroids positively correlated with low precipitation levels, suggesting that this compound acts as a critical osmoprotectant under field conditions. Genome analysis revealed that strain B401 contains alternative pathways for the biosynthesis and uptake of glycine betaine and its precursors. Such genomic information will offer substantial insight into the environmental physiology of this biotechnologically valuable nitrogen-fixing bacterium. Copyright © 2017 Elsevier B.V. All rights reserved.

The catabolism of radioiodinated anti-lung-cancer monoclonal antibodies in tumor-bearing nude mice

International Nuclear Information System (INIS)

Shi Xubao

1991-01-01

Nude mice bearing humor lung cancer xenografts were injected intravenously or intraperitoneally with a mixture of radioiodinated anti-lung-cancer monoclonal antibodies, 2E3 and 6D1. The blood radioactivity versus time curve was fitted to a two-compartment open model with a 3.4 day blood radioactivity clearance half-life and a 636 ml/kg apparent distribution volume. Radioiodinated 2E3 and 6D1 given intraperitoneally were rapidly absorbed, with a 2.08 absorption half-life and 89% bioavailability. The highest radioactivity levels were found in the tumor, blood, liver and spleen 1-3 days after injection; next came the lung, kidney, stomach and intestine. The relative radioactivity increased in the tumor as levels in blood and normal tissues decreased. The in vivo deiodination of radioiodinated 2E3 and 6D1 was about 18.6% and free radioiodine was excreted in the urine

Improved tumor imaging with radiolabeled monoclonal antibodies by plasma clearance with anti-antibody column

International Nuclear Information System (INIS)

Lear, J.L.; Kasliwal, R.; Feyerabend, A.; Bunn, P.; Dienhart, D.G.; Johnson, T.K.; Glenn, S.D.; Maddock, S.W.

1990-01-01

This paper reports on imaging of tumors with use of radiolabeled monoclonal antibodies (MoAs) that often hindered by high levels of background activity. The ability to lower blood pool MoA activity at a selected time after injection offers a potential method to reduce background while preserving tumor uptake. Toward this goal, the authors investigated the process of clearing MoA from patients' plasma with use of an anti-antibody column. One patient with breast cancer and four with lung cancer were given intravenous injection of 5 mCi of indium-111 KC4 (Coulter Immunology) and imaged at 20, 24, 48, and 72 hours with use of a whole-body canner coupled to a computer. Plasma clearance was performed between the 20- and 24-hour images with use of a COBEIA system. Images were inspected visually and analyzed by region-of-interest quantification

Characterization of anti-P monoclonal antibodies directed against the ribosomal protein-RNA complex antigen and produced using Murphy Roths large autoimmune-prone mice.

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Sato, H; Onozuka, M; Hagiya, A; Hoshino, S; Narita, I; Uchiumi, T

2015-02-01

Autoantibodies, including anti-ribosomal P proteins (anti-P), are thought to be produced by an antigen-driven immune response in systemic lupus erythematosus (SLE). To test this hypothesis, we reconstituted the ribosomal antigenic complex in vitro using human P0, phosphorylated P1 and P2 and a 28S rRNA fragment covering the P0 binding site, and immunized Murphy Roths large (MRL)/lrp lupus mice with this complex without any added adjuvant to generate anti-P antibodies. Using hybridoma technology, we subsequently obtained 34 clones, each producing an anti-P monoclonal antibody (mAb) that recognized the conserved C-terminal tail sequence common to all three P proteins. We also obtained two P0-specific monoclonal antibodies, but no antibody specific to P1, P2 or rRNA fragment. Two types of mAbs were found among these anti-P antibodies: one type (e.g. 9D5) reacted more strongly with the phosphorylated P1 and P2 than that with their non-phosphorylated forms, whereas the other type (e.g. 4H11) reacted equally with both phosphorylated and non-phosphorylated forms of P1/P2. Both 9D5 and 4H11 inhibited the ribosome/eukaryotic elongation factor-2 (eEF-2)-coupled guanosine triphosphate (GTP)ase activity. However, preincubation with a synthetic peptide corresponding to the C-terminal sequence common to all three P proteins, but not the peptide that lacked the last three C-terminal amino acids, mostly prevented the mAb-induced inhibition of GTPase activity. Thus, at least two types of anti-P were produced preferentially following the immunization of MRL mice with the reconstituted antigenic complex. Presence of multiple copies of the C-termini, particularly that of the last three C-terminal amino acid residues, in the antigenic complex appears to contribute to the immunogenic stimulus. © 2014 British Society for Immunology.

Characterization of anti-P monoclonal antibodies directed against the ribosomal protein–RNA complex antigen and produced using Murphy Roths large autoimmune-prone mice

Science.gov (United States)

Sato, H; Onozuka, M; Hagiya, A; Hoshino, S; Narita, I; Uchiumi, T

2015-01-01

Autoantibodies, including anti-ribosomal P proteins (anti-P), are thought to be produced by an antigen-driven immune response in systemic lupus erythematosus (SLE). To test this hypothesis, we reconstituted the ribosomal antigenic complex in vitro using human P0, phosphorylated P1 and P2 and a 28S rRNA fragment covering the P0 binding site, and immunized Murphy Roths large (MRL)/lrp lupus mice with this complex without any added adjuvant to generate anti-P antibodies. Using hybridoma technology, we subsequently obtained 34 clones, each producing an anti-P monoclonal antibody (mAb) that recognized the conserved C-terminal tail sequence common to all three P proteins. We also obtained two P0-specific monoclonal antibodies, but no antibody specific to P1, P2 or rRNA fragment. Two types of mAbs were found among these anti-P antibodies: one type (e.g. 9D5) reacted more strongly with the phosphorylated P1 and P2 than that with their non-phosphorylated forms, whereas the other type (e.g. 4H11) reacted equally with both phosphorylated and non-phosphorylated forms of P1/P2. Both 9D5 and 4H11 inhibited the ribosome/eukaryotic elongation factor-2 (eEF-2)-coupled guanosine triphosphate (GTP)ase activity. However, preincubation with a synthetic peptide corresponding to the C-terminal sequence common to all three P proteins, but not the peptide that lacked the last three C-terminal amino acids, mostly prevented the mAb-induced inhibition of GTPase activity. Thus, at least two types of anti-P were produced preferentially following the immunization of MRL mice with the reconstituted antigenic complex. Presence of multiple copies of the C-termini, particularly that of the last three C-terminal amino acid residues, in the antigenic complex appears to contribute to the immunogenic stimulus. PMID:25255895

Bioactivity assays and application of 125I labeled human mouse chimeric anti-CD22 monoclonal antibody SM03

International Nuclear Information System (INIS)

Lu Pingping; Meng Zhiyun; Dou Guifang; Wu Yingliang; Wang Minwei

2008-01-01

To investigate the bioactivity and application of 125 I labeled human mouse chimeric monoclonal SM03, SM03 was labeled with 125 I using Indogen method. The labeled mixture was purified by Sephacryl S-300 HR separation chromospectry. The purity and concentration of separated fractions were determined by HPLC and Protein Assay Kit, respectively. Competitive binding method and ELISA method were used for bioactivity assays. 125 I-SM03 was applied to screen cell lines which express the most abundant CD22 antigen. The purity and recovery of 125 I-SM03 were >99% and >47%, respectively. The bioactivity of 125 I- SM03 and SM03 hasn't significant difference in statistics. Ramos cell line had the strongest special radioactivity when 125 I-SM03 bound with in Raji, Daudi and Ramos cell lines. Indogen method is a good way to label Human mouse chimeric anti-CD22 monoclonal antibody SM03 and the label will not affect the activity of SM03. The 125 I-SM03 not only can be used for detect agent, but also may be put into market for NHL therapy. (authors)

C-kit-targeted imaging of gastrointestinal stromal tumor using radiolabeled anti-c-kit monoclonal antibody in a mouse tumor model

International Nuclear Information System (INIS)

Sogawa, Chizuru; Tsuji, Atsushi B.; Sudo, Hitomi; Sugyo, Aya; Yoshida, Chisato; Odaka, Kenichi; Uehara, Tomoya; Arano, Yasushi; Koizumi, Mitsuru; Saga, Tsuneo

2010-01-01

Introduction: Gastrointestinal stromal tumor (GIST) is the most common mesenchymal tumor arising from the gastrointestinal tract and highly expresses mutated c-kit. We aimed to develop a specific and sensitive method for detecting GISTs using radiolabeled anti-c-kit monoclonal antibody. Methods: A mutated c-kit-expressing cell clone was established by transfecting an expressing vector of mutated c-kit gene into HEK293 human embryonic kidney cells. The tumors were developed by inoculating c-kit-expressing cells into nude mice. 125 I- and 111 In-labeled anti-c-kit antibodies (12A8 and 41A11) were evaluated in vitro by cell binding, competitive inhibition and cellular internalization assays, and in vivo by biodistribution and imaging studies in tumor-bearing mice. Results: Both 125 I- and 111 In-labeled antibodies showed specific binding with c-kit-expressing cells with high affinity (dissociation constants = 2.2-7.1x10 9 M -1 ). Internalization assay showed that 125 I-labeled antibodies were rapidly internalized and dehalogenated, with the release of 125 I from the cells, resulting in reduction of cell-associated radioactivity with time. In contrast, 111 In-labeled antibody was internalized but did not result in the reduced radioactivity associated with tumor cells. Reflecting this phenomenon, the in vivo tumor uptake of 125 I-labeled antibody was low on Day 1, further decreasing with time, while tumor uptake of 111 In-labeled antibody was high on Day 1, further increasing with time. The xenografted tumor was clearly visualized by scintigraphy after injection of 111 In-labeled antibody. Conclusion: The anti-c-kit monoclonal antibody labeled with a metal radionuclide would be promising for c-kit-targeted imaging of GISTs.

ERBB oncogene proteins as targets for monoclonal antibodies.

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Polanovski, O L; Lebedenko, E N; Deyev, S M

2012-03-01

General properties of the family of tyrosine kinase ERBB receptors are considered in connection with their role in the generation of cascades of signal transduction in normal and tumor cells. Causes of acquisition of oncogene features by genes encoding these receptors and their role in tumorigenesis are analyzed. Anti-ERBB monoclonal antibodies approved for therapy are described in detail, and mechanisms of their antitumor activity and development of resistance to them are reviewed. The existing and the most promising strategies for creating and using monoclonal antibodies and their derivatives for therapy of cancer are discussed.

Roles of Extracellular Polysaccharides and Biofilm Formation in Heavy Metal Resistance of Rhizobia

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Natalia Nocelli

2016-05-01

Full Text Available Bacterial surface components and extracellular compounds, particularly flagella, lipopolysaccharides (LPSs, and exopolysaccharides (EPSs, in combination with environmental signals and quorum-sensing signals, play crucial roles in bacterial autoaggregation, biofilm development, survival, and host colonization. The nitrogen-fixing species Sinorhizobium meliloti (S. meliloti produces two symbiosis-promoting EPSs: succinoglycan (or EPS I and galactoglucan (or EPS II. Studies of the S. meliloti/alfalfa symbiosis model system have revealed numerous biological functions of EPSs, including host specificity, participation in early stages of host plant infection, signaling molecule during plant development, and (most importantly protection from environmental stresses. We evaluated functions of EPSs in bacterial resistance to heavy metals and metalloids, which are known to affect various biological processes. Heavy metal resistance, biofilm production, and co-culture were tested in the context of previous studies by our group. A range of mercury (Hg II and arsenic (As III concentrations were applied to S. meliloti wild type strain and to mutant strains defective in EPS I and EPS II. The EPS production mutants were generally most sensitive to the metals. Our findings suggest that EPSs are necessary for the protection of bacteria from either Hg (II or As (III stress. Previous studies have described a pump in S. meliloti that causes efflux of arsenic from cells to surrounding culture medium, thereby protecting them from this type of chemical stress. The presence of heavy metals or metalloids in culture medium had no apparent effect on formation of biofilm, in contrast to previous reports that biofilm formation helps protect various microorganism species from adverse environmental conditions. In co-culture experiments, EPS-producing heavy metal resistant strains exerted a protective effect on AEPS-non-producing, heavy metal-sensitive strains; a phenomenon

Development of an Anti-HER2 Monoclonal Antibody H2Mab-139 Against Colon Cancer.

Science.gov (United States)

Kaneko, Mika K; Yamada, Shinji; Itai, Shunsuke; Kato, Yukinari

2018-02-01

Human epidermal growth factor receptor 2 (HER2) expression has been reported in several cancers, such as breast, gastric, lung, pancreatic, and colorectal cancers. HER2 is overexpressed in those cancers and is associated with poor clinical outcomes. Trastuzumab, a humanized anti-HER2 antibody, provides significant survival benefits for patients with HER2-overexpressing breast cancers and gastric cancers. In this study, we developed a novel anti-HER2 monoclonal antibody (mAb), H 2 Mab-139 (IgG 1 , kappa) and investigated it against colon cancers using flow cytometry, western blot, and immunohistochemical analyses. Flow cytometry analysis revealed that H 2 Mab-139 reacted with colon cancer cell lines, such as Caco-2, HCT-116, HCT-15, HT-29, LS 174T, COLO 201, COLO 205, HCT-8, SW1116, and DLD-1. Although H 2 Mab-139 strongly reacted with LN229/HER2 cells on the western blot, we did not observe a specific signal for HER2 in colon cancer cell lines. Immunohistochemical analyses revealed sensitive and specific reactions of H 2 Mab-139 against colon cancers, indicating that H 2 Mab-139 is useful in detecting HER2 overexpression in colon cancers using flow cytometry and immunohistochemical analyses.

Characterization of monoclonal antibodies against human thyrotropin and use in an immunoradiometric assay and immunohistochemistry

International Nuclear Information System (INIS)

Benkirane, M.; Bon, D.; Bellot, F.; Prince, P.; Delori, P.; Hassoun, J.; Carayon, P.

1987-01-01

Monoclonal antibodies were prepared against human thyrotropin. 13 different antibodies were characterized. Ten antibodies were of the IgG1 subclass. The affinities of the antibodies were in the range 10 9 -10 11 mol -1 .l. Four of them were specific for hTSH and did not react with hLH, hFSH, hCG or αhCG. Four reacted with these hormones and recognized the α subunit of hCG. One cross-reacted only with HFSH. The remaining four antibodies recognized the holo-hTSH only, and thus were designated as anti-conformational determinants. Monoclonal antibodies reacting with different antigenic determinants on the hTSH molecule defined seven clusters. Two of them were used to develop a simplified two-site sandwich radioimmunoassay in which one monoclonal antibody was immobilized on tubes (anti-βTSH) and another (anti-α) labelled with 125 I. This assay was highly specific and demonstrated a sensitivity level of 0.1 μIU/ml. Two monoclonal antibodies were used in immunohistochemistry and their quality and specificity was assessed in the detection of hTSH immunoreactivity in human pituitary biological sections. 20 refs.; 6 figs.; 2 tabs

Two-site sandwich radioimmunoassay of human gamma interferon with monoclonal antibodies

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Tanaka, E; Imai, M; Usuda, S; Tachibana, K; Okamoto, H; Ohike, Y; Nakamura, T; Miyakawa, Y; Mayumi, M [Jichi Medical School, Minamikawachi, Tochigi (Japan)

1985-03-18

Two monoclonal antibodies were raised against human gamma interferon (IFN-..gamma..) derived from E. coli harboring the recombinant cDNA for IFN-..gamma.., and one against a synthetic peptide representing its C-terminus amino acid sequence of 20 residues. The monoclonal antibody against the synthetic peptide reacted either with IFN-..gamma.. or the synthetic peptide. One monoclonal anti-IFN-..gamma.. did not react with the synthetic peptide, while the other showed a weak binding with the peptide. A 2-site '1-step' radioimmunoassay was developed. The assay was rapid with a sensitivity capable of detecting a few ng/ml of IFN-..gamma...

Anti-Neuroblastoma Activity of Gold Nanorods Bound with GD2 Monoclonal Antibody under Near-Infrared Laser Irradiation

International Nuclear Information System (INIS)

Peng, Ching-An; Wang, Chung-Hao

2011-01-01

High-risk neuroblastoma is one of the most common deaths in pediatric oncology. Current treatment of this disease involves a coordinated sequence of chemotherapy, surgery, and radiation. Further advances in therapy will require the targeting of tumor cells in a more selective and efficient way so that survival can be improved without substantially increasing toxicity. To achieve tumor-selective delivery, disialoganglioside (GD2) expressed by almost all neuroblastoma tumors represents a potential molecular target that can be exploited for tumor-selective delivery. In this study, GD2 monoclonal antibody (anti-GD2) was conjugated to gold nanorods (GNRs) which are one of anisotropic nanomaterials that can absorb near-infrared (NIR) laser light and convert it to energy for photothermolysis of tumor cells. Thiolated chitosan, due to its biocompatibility, was used to replace cetyltrimethylammonium bromide (CTAB) originally used in the synthesis of gold nanorods. In order to specifically target GD2 overexpressed on the surface of neuroblastoma stNB-V1 cells, anti-GD2 was conjugated to chitosan modified GNRs (CGNRs). To examine the fate of CGNRs conjugated with anti-GD2 after incubation with neuroblastoma cells, rhadoamine B was labeled on CGNRs functionalized with anti-GD2. Our results illustrated that anti-GD2-conjugated CGNRs were extensively endocytosed by GD2 + stNB-V1 neuroblastoma cells via antibody-mediated endocytosis. In addition, we showed that anti-GD2 bound CGNRs were not internalized by GD2 − SH-SY5Y neuroblastoma cells. After anti-GD2-linked CGNRs were incubated with neuroblatoma cells for six hours, the treated cells were further irradiated with 808 nm NIR laser. Post-NIR laser exposure, when examined by calcein-AM dye, stNB-V1 cells all underwent necrosis, while non-GD2 expressing SH-SY5Y cells all remained viable. Based on the in vitro study, CGNRs bound with anti-GD2 has the potential to be utilized as a therapeutic thermal coupling agent that generates

In ovo injection of anti-chicken CD25 monoclonal antibodies depletes CD4+CD25+ T cells in chickens.

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Shanmugasundaram, Revathi; Selvaraj, Ramesh K

2013-01-01

The CD4(+)CD25(+) cells have T regulatory cell properties in chickens. This study investigated the effect of in ovo injection of anti-chicken CD25 monoclonal antibodies (0.5 mg/egg) on CD4(+)CD25(+) cell depletion and on amounts of interleukin-2 mRNA and interferon-γ mRNA in CD4(+)CD25(-) cells posthatch. Anti-chicken CD25 or PBS (control) was injected into 16-d-old embryos. Chicks hatched from eggs injected with anti-chicken CD25 antibodies had a lower CD4(+)CD25(+) cell percentage in the blood until 25 d posthatch. The anti-chicken CD25 antibody injection nearly depleted CD4(+)CD25(+) cells in the blood until 16 d posthatch. At 30 d posthatch, the CD4(+)CD25(+) cell percentage in the anti-CD25-antibody-injected group was comparable with the percentage in the control group. At 16 d posthatch, the anti-chicken CD25 antibody injection decreased CD4(+)CD25(+) cell percentages in the thymus, spleen, and cecal tonsils. Chickens hatched from anti-CD25-antibody-injected eggs had approximately 25% of CD4(+)CD25(+) cells in the cecal tonsils and thymus compared with those in the cecal tonsils and thymus of the control group. The CD4(+)CD25(-) cells from the spleen and cecal tonsils of chicks hatched from anti-chicken-CD25-injected eggs had higher amounts of interferon-γ and interleukin-2 mRNA than CD4(+)CD25(-) cells from the control group. It could be concluded that injecting anti-chicken CD25 antibodies in ovo at 16 d of incubation nearly depleted the CD4(+)CD25(+) cells until 25 d posthatch.

Three-site sandwich radioimmunoassay with monoclonal antibodies for a sensitive determination of human alpha-fetoprotein

International Nuclear Information System (INIS)

Nomura, M.; Imai, M.; Takahashi, K.; Kumakura, T.; Tachibana, K.; Aoyagi, S.; Usuda, S.; Nakamura, T.; Miyakawa, Y.; Mayumi, M.

1983-01-01

Utilizing monoclonal antibodies against human alpha-fetoprotein, 3 distinct antigenic determinants were identified. These antigenic determinants, provisionally designated a, b and c, were arranged in such a manner that the binding of one determinant with the corresponding antibody did not inhibit, or only barely inhibited the binding of antibodies directed to the other 2 determinants. Monoclonal antibodies with 3 different specificities were, therefore, applied to develop a sandwich-type solid-phase radioimmunoassay of the antigen in which wells were coated with anti-a, and radiolabeled anti-b together with radiolabeled anti-c was employed to detect the bound antigen. The 3-site sandwich radioimmunoassay involving 3 different determinants gave a higher sensitivity than 2-site assays in which only anti-b or anti-c was employed as a radiolabeled reagent, because the radioactivity of the 2 labeled antibodies was added on the antigen bound to immobilized anti-a. (Auth.)

Monoclonal Idiotope Vaccine against Streptococcus pneumoniae Infection

Science.gov (United States)

McNamara, Mary K.; Ward, Ronald E.; Kohler, Heinz

1984-12-01

A monoclonal anti-idiotope antibody coupled to a carrier protein was used to immunize BALB/c mice against a lethal Streptococcus pneumoniae infection. Vaccinated mice developed a high titer of antibody to phosphorylcholine, which is known to protect against infection with Streptococcus pneumoniae. Measurement of the median lethal dose of the bacteria indicated that anti-idiotope immunization significantly increased the resistance of BALB/c mice to the bacterial challenge. Antibody to an idiotope can thus be used as an antigen substitute for the induction of protective immunity.

In-vitro inhibition of IFNγ+ iTreg mediated by monoclonal antibodies against cell surface determinants essential for iTreg function

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Daniel Volker

2012-08-01

Full Text Available Abstract Background IFNγ-producing CD4+CD25+Foxp3+ PBL represent a subtype of iTreg that are associated with good long-term graft outcome in renal transplant recipients and suppress alloresponses in-vitro. To study the mechanism of immunosuppression, we attempted to block cell surface receptors and thereby inhibited the function of this iTreg subset in-vitro using monoclonal antibodies and recombinant proteins. Methods PBL of healthy control individuals were stimulated polyclonally in-vitro in the presence of monoclonal antibodies or recombinant proteins against/of CD178, CD152, CD279, CD28, CD95, and HLA-DR. Induction of IFNγ+ iTreg and proliferation of effector cells was determined using four-color fluorescence flow cytometry. Blockade of iTreg function was analyzed using polyclonally stimulated co-cultures with separated CD4+CD25+CD127-IFNγ+ PBL. Results High monoclonal antibody concentrations inhibited the induction of CD4+CD25+Foxp3+IFNγ+ PBL (anti-CD152, anti-CD279, anti-CD95: p +CD25+CD127-IFNγ+ PBL (anti-CD178, anti-CD152, anti-CD279, anti-CD95: p +CD25+Foxp3+IFNγ+ PBL (rCD152 and rCD95: p +CD25+CD127-IFNγ+ PBL showed lower cell proliferation than co-cultures with CD4+CD25+CD127-IFNγ- PBL (p +CD25+CD127-IFNγ- PBL-containing co-cultures in the presence of monoclonal antibody (anti-CD28, anti-CD152, anti-CD279: p +CD25+CD127-IFNγ+ PBL (with the exception anti-CD28 monoclonal antibody: p +CD25+CD127-IFNγ- PBL but do not efficiently block suppressive iTreg function in co-cultures with CD4+CD25+CD127-IFNγ+ PBL. Conclusions CD178, CD152, CD279, CD28, CD95, and HLA-DR determinants are important for induction and suppressive function of IFNγ+ iTreg.

Itolizumab – a humanized anti-CD6 monoclonal antibody with a better side effects profile for the treatment of psoriasis

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Menon R

2015-04-01

Full Text Available Roshni Menon, Brinda G David Department of Dermatology, Venereology and Leprosy, Sri Venkateshwaraa Medical College Hospital and Research Centre, Ariyur, Pondicherry, India Abstract: Management of psoriasis is a challenge to the treating physician. The chronic inflammatory state of psoriasis with exacerbations and remissions necessitate “on-and-off” treatment schedules. The safety profiles of drugs and tolerability issues for patients are important factors to be considered during treatment. Various biological agents targeting T-cells and the inflammatory cytokines are available for systemic treatment of psoriasis. However, major causes of concern while using these drugs are risk of susceptibility to infection and development of anti-drug antibodies, which will affect the pharmacokinetic properties, efficacy, and safety profile of the drug. Itolizumab, a humanized anti-CD6 monoclonal antibody, is a new molecule that acts by immunomodulating the CD6 molecule. CD6 is a co-stimulatory molecule required for optimal T-cell stimulation by the antigen-presenting cells. This step is crucial in T-cell proliferation to form Th1 and Th17 cells, which play a major role in the pathogenesis of psoriasis. This article deals with the properties of Itolizumab and its role in the treatment of psoriasis. Based on the available published data, Itolizumab seems to have a better adverse effects profile and at the same time comparatively less efficacy when compared to other biological agents available for treating psoriasis. Larger studies with longer duration are required to clearly depict the long-term side effects profile. Keywords: Itolizumab, CD6, psoriasis, monoclonal antibody, biologicals 

Role of immunoscintigraphy using Tc-99 m labelled monoclonal anti-CEA antibodies in the detection of Colorectal-rectal carcinoma

Energy Technology Data Exchange (ETDEWEB)

Ziada, G; Moustafa, H; Elhaddad, Sh; Elasser, M [Center of oncology and nuclear medicine of Kasr El-Eini Hospital, Cairo university, (Egypt)

1995-10-01

Twelve patients with colorectal carcinoma confirmed histopathologically and associated with high serum CEA level and underwent preoperative immunoscintigraphy (planar and SPECT projections) followed by operative resection with able to detect the primary tumor in colorectal region with 100% sensitivity, specificity and accuracy. Immunoscintigraphy showed sensitivity of 85%, specificity of 60% and accuracy of 66.6% in detection of para-aortic lymph nodes involved, as compared to 0%, 25% and 25% in abdominal sonography respectively. Concerning the liver involvement, there was higher sensitivity of 100% and accuracy of 91% of immunoscintigraphy in comparison to 50% and 66.6% abdominal sonography respectively. Imaging procedure at 4 and 24 hors postinjection with planar and SPECT studies were useful for proper localization of involved sites and to differentiate between malignant and benign lesions. Immunoscintigraphy is a safe procedure and the preparation of the kit of monoclonal anti-CEA anti-body is rapid with labelling efficiency more than 95% and with no adverse reaction. 3 figs.,2 tabs.

Role of immunoscintigraphy using Tc-99 m labelled monoclonal anti-CEA antibodies in the detection of Colorectal-rectal carcinoma

International Nuclear Information System (INIS)

Ziada, G.; Moustafa, H.; Elhaddad, Sh.; Elasser, M.

1995-01-01

Twelve patients with colorectal carcinoma confirmed histopathologically and associated with high serum CEA level and underwent preoperative immunoscintigraphy (planar and SPECT projections) followed by operative resection with able to detect the primary tumor in colorectal region with 100% sensitivity, specificity and accuracy. Immunoscintigraphy showed sensitivity of 85%, specificity of 60% and accuracy of 66.6% in detection of para-aortic lymph nodes involved, as compared to 0%, 25% and 25% in abdominal sonography respectively. Concerning the liver involvement, there was higher sensitivity of 100% and accuracy of 91% of immunoscintigraphy in comparison to 50% and 66.6% abdominal sonography respectively. Imaging procedure at 4 and 24 hors postinjection with planar and SPECT studies were useful for proper localization of involved sites and to differentiate between malignant and benign lesions. Immunoscintigraphy is a safe procedure and the preparation of the kit of monoclonal anti-CEA anti-body is rapid with labelling efficiency more than 95% and with no adverse reaction. 3 figs.,2 tabs

A Unique Report: Development of Super Anti-Human IgG Monoclone with Optical Density Over Than 3

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Leili Aghebati Maleki

2013-08-01

Full Text Available Purpose: Monoclonal antibodies and related conjugates are key reagents used in biomedical researches as well as, in treatment, purification and diagnosis of infectious and non- infectious diseases. Methods: Balb/c mice were immunized with purified human IgG. Spleen cells of the most immune mouse were fused with SP2/0 in the presence of Poly Ethylene Glycol (PEG. Supernatant of hybridoma cells was screened for detection of antibody by ELISA. Then, the sample was assessed for cross-reactivity with IgM & IgA by ELISA and confirmed by immunoblotting. The subclasses of the selected mAbs were determined. The best clone was injected intraperitoneally to some pristane-injected mice. Anti-IgG mAb was purified from the animals' ascitic fluid by Ion exchange chromatography and then, mAb was conjugated with HRP. Results: In the present study, over than 50 clones were obtained that 1 clone had optical density over than 3. We named this clone as supermonoclone which was selected for limiting dilution. The result of the immunoblotting, showed sharp band in IgG position and did not show any band in IgM&IgA position. Conclusion: Based on the findings of this study, the conjugated monoclonal antibody could have application in diagnosis of infectious diseases like Toxoplasmosis, Rubella and IgG class of other infectious and non- infectious diseases.

A Unique Report: Development of Super Anti-Human IgG Monoclone with Optical Density Over Than 3

Science.gov (United States)

Aghebati Maleki, Leili; Baradaran, Behzad; Abdolalizadeh, Jalal; Ezzatifar, Fatemeh; Majidi, Jafar

2013-01-01

Purpose: Monoclonal antibodies and related conjugates are key reagents used in biomedical researches as well as, in treatment, purification and diagnosis of infectious and non- infectious diseases. Methods: Balb/c mice were immunized with purified human IgG. Spleen cells of the most immune mouse were fused with SP2/0 in the presence of Poly Ethylene Glycol (PEG). Supernatant of hybridoma cells was screened for detection of antibody by ELISA. Then, the sample was assessed for cross-reactivity with IgM & IgA by ELISA and confirmed by immunoblotting. The subclasses of the selected mAbs were determined. The best clone was injected intraperitoneally to some pristane-injected mice. Anti-IgG mAb was purified from the animals' ascitic fluid by Ion exchange chromatography and then, mAb was conjugated with HRP. Results: In the present study, over than 50 clones were obtained that 1 clone had optical density over than 3. We named this clone as supermonoclone which was selected for limiting dilution. The result of the immunoblotting, showed sharp band in IgG position and did not show any band in IgM&IgA position. Conclusion: Based on the findings of this study, the conjugated monoclonal antibody could have application in diagnosis of infectious diseases like Toxoplasmosis, Rubella and IgG class of other infectious and non- infectious diseases. PMID:24312857

Strigolactones in the Rhizobium-legume symbiosis: Stimulatory effect on bacterial surface motility and down-regulation of their levels in nodulated plants.

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Peláez-Vico, María A; Bernabéu-Roda, Lydia; Kohlen, Wouter; Soto, María J; López-Ráez, Juan A

2016-04-01

Strigolactones (SLs) are multifunctional molecules acting as modulators of plant responses under nutrient deficient conditions. One of the roles of SLs is to promote beneficial association with arbuscular mycorrhizal (AM) fungi belowground under such stress conditions, mainly phosphorus shortage. Recently, a role of SLs in the Rhizobium-legume symbiosis has been also described. While SLs' function in AM symbiosis is well established, their role in the Rhizobium-legume interaction is still emerging. Recently, SLs have been suggested to stimulate surface motility of rhizobia, opening the possibility that they could also act as molecular cues. The possible effect of SLs in the motility in the alfalfa symbiont Sinorhizobium meliloti was investigated, showing that the synthetic SL analogue GR24 stimulates swarming motility in S. meliloti in a dose-dependent manner. On the other hand, it is known that SL production is regulated by nutrient deficient conditions and by AM symbiosis. Using the model alfalfa-S. meliloti, the impact of phosphorus and nitrogen deficiency, as well as of nodulation on SL production was also assessed. The results showed that phosphorus starvation promoted SL biosynthesis, which was abolished by nitrogen deficiency. In addition, a negative effect of nodulation on SL levels was detected, suggesting a conserved mechanism of SL regulation upon symbiosis establishment. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Immunohistochemical Analysis of Inflammatory Rheumatoid Synovial Tissues Using Anti-Human Podoplanin Monoclonal Antibody Panel.

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Suzuki, Tomoto; Takakubo, Yuya; Oki, Hiroharu; Liu, Xing; Honma, Ryusuke; Naganuma, Yasushi; Goodman, Stuart B; Kaneko, Mika K; Kato, Yukinari; Takagi, Michiaki

2018-02-01

Podoplanin (PDPN) is a transmembrane sialoglycoprotein, which is expressed in several normal tissues and malignant tumors. Although PDPN expression in rheumatoid arthritis (RA) has been reported, the role of PDPN in RA and other arthritic conditions has not been fully elucidated. In this study, we examined PDPN expression in inflammatory synovial tissues using an anti-human PDPN (hPDPN) monoclonal antibody (mAb) panel to select the most useful one for evaluation of synovitis. Synovial tissue samples were obtained from 11 RA patients and 9 osteoarthritis (OA) patients undergoing joint surgery. PDPN-positive cells were immunostained by a panel of PDPN mAbs (NZ-1, LpMab-3, LpMab-7, LpMab-10, LpMab-12, LpMab-13, and LpMab-17), followed by cell grading of inflammation and cell counting of PDPN-positivity by a quantitative analyzer. Immunohistochemistry showed that PDPN was markedly expressed in both macrophage-like type A and fibroblast-like type B lining cells of the hyperplastic synovial lining cell layer, and macrophages and fibroblasts in the stroma of RA. Among anti-PDPN mAbs, LpMab-12 showed the highest score. In inflammatory OA synovium, PDPN expression was also detectable. Although LpMab-12 also showed the highest score in OA, the difference was not statistically significant. The inflammatory synovitis score of RA was significantly higher than that of OA. PDPN was expressed in inflammatory lining cells and sublining stroma of RA and OA synovium. In the seven anti-hPDPN antibodies examined, LpMab-12 was the most stainable antibody for PDPN in RA synovitis. Thus, LpMab-12 for PDPN has a possible and promising specific biomarker for evaluating synovitis in RA and inflammatory OA.

At least two Fc Neu5Gc residues of monoclonal antibodies are required for binding to anti-Neu5Gc antibody

OpenAIRE

Yu, Chuanfei; Gao, Kai; Zhu, Lei; Wang, Wenbo; Wang, Lan; Zhang, Feng; Liu, Chunyu; Li, Meng; Wormald, Mark R.; Rudd, Pauline M.; Wang, Junzhi

2016-01-01

Two non-human glycan epitopes, galactose-Į-1,3-galactose (Į-gal) and Neu5Gc-Į-2-6-galactose (Neu5Gc) have been shown to be antigenic when attached to Fab oligosaccharides of monoclonal antibodies (mAbs) , while Į-gal attached to Fc glycans were not. However, the antigenicity of Neu5Gc on the Fc glycans remains unclear in the context that most mAbs carry only Fc glycans. After studying two clinical mAbs carrying significant amounts of Fc Neu5Gc, we show that their binding activity with anti-Ne...

Negative hyper-selection of metastatic colorectal cancer patients for anti-EGFR monoclonal antibodies: the PRESSING case-control study.

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Cremolini, C; Morano, F; Moretto, R; Berenato, R; Tamborini, E; Perrone, F; Rossini, D; Gloghini, A; Busico, A; Zucchelli, G; Baratelli, C; Tamburini, E; Tampellini, M; Sensi, E; Fucà, G; Volpi, C; Milione, M; Di Maio, M; Fontanini, G; De Braud, F; Falcone, A; Pietrantonio, F

2017-12-01

Refining the selection of metastatic colorectal cancer patients candidates for anti-epidermal growth factor receptor (EGFR) monoclonal antibodies beyond RAS and BRAF testing is a challenge of precision oncology. Several uncommon genomic mechanisms of primary resistance, leading to activation of tyrosine kinase receptors other than EGFR or downstream signalling pathways, have been suggested by preclinical and retrospective studies. We conducted this multicentre, prospective, case-control study to demonstrate the negative predictive impact of a panel of rare genomic alterations [PRESSING (PRimary rESiStance IN RAS and BRAF wild-type metastatic colorectal cancer patients treated with anti-eGfr monoclonal antibodies) panel], including HER2/MET amplifications, ALK/ROS1/NTRK1-3/RET fusions and PIK3CA mutations. Hypothesizing a prevalence of candidate alterations of 15% and 0% in resistant and sensitive RAS and BRAF wild-type patients, respectively, with two-sided α and β errors of 0.05 and 0.20, 47 patients per group were needed. Forty-seven patients per group were included. PRESSING panel alterations were significantly more frequent in resistant (24 out of 47, 51.1%) than in sensitive (1 out of 47, 2.1%) patients (P < 0.001) and in right- (12 out of 29, 41.4%) than left-sided (13 out of 65, 20.0%) tumours (P = 0.03). The predictive accuracy of PRESSING panel and sidedness was 75.3% and 70.2%, respectively. Among hyper-selected patients, right-sidedness was still associated with resistance (P = 0.002). The predictive accuracy of the combined evaluation of PRESSING panel and sidedness was 80.4%. As a secondary analysis, 8 (17.0%) resistant and 0 sensitive patients showed microsatellite instability (P < 0.001). The investigated panel of genomic alterations allows refining the selection of RAS and BRAF wild-type metastatic colorectal cancer patients candidates for anti-EGFRs, partially explaining and further corroborating the predictive ability of primary

The study of labeling with Iodine-131 of monoclonal antibody anti-CD20 used for the treatment of non-Hodgkin lymphoma

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Akanji, Akinkunmi Ganiyu

2006-01-01

Lymphomas are malignancies of the lymphatic system, described by Thomas Hodgkin in 1932. Traditionally, lymphomas are classified in two basic groups: Hodgkin disease and non-Hodgkin lymphoma (NHL). Patients with NHL were earlier treated with radiotherapy alone or in combination with immunotherapy using monoclonal antibody anti-CD20 (ex., Rituximab-Mabthera, Roche). However, Radioimmunotherapy is a new modality of treatment for patients with NHL, in which cytotoxic radiation from therapeutic radioisotopes is delivered to tumors through monoclonal antibodies. This study focused on labeling conditions of monoclonal antibody anti-CD20 (Rituximab-Mabthera, Roche) with iodine-131, by direct radioiodination method using Chloramine-T as oxidizing agent. Labeling parameters investigated were: Radiochemical purity (RP), method of purification, incubation time, antibody mass, oxidative agent mass, stability in vitro, stability in vivo, immunoreactivity and biological distribution performed in normal Swiss mouse. Product of high radiochemical purity was obtained with no notable difference between the methods applied. No clear evidence of direct influence of incubation time on radiochemical purity of the labeled antibody was observed. Whereas, a clear evidence of direct influence of activity on radiochemical purity of the labeled antibody was observed when antibody mass was varied. After purification, the labeled product presented radiochemical purity of approximately 100 %. Product of superior radiochemical yield was observed when standard condition of labeling was used. The labeled product presented variation in radiochemical purity using five different stabilizer conditions. The condition in which gentisic acid was combined with freeze appears more suitable and capable of minimizing autoradiolysis of the antibody labeled with high therapeutic activity of iodine-131. The labeled product presented low immunoreactivity when compared to the literature. Biological distribution in

The study of labeling with iodine-131 of monoclonal antibody anti-CD20 used for the treatment of non-Hodgkin lymphoma

International Nuclear Information System (INIS)

Akanji, Akinkunmi Ganiyu

2006-01-01

Lymphomas are malignancies of the lymphatic system, described by Thomas Hodgkin in 1932. Traditionally, lymphomas are classified in two basic groups: Hodgkin disease and non-Hodgkin lymphoma (NHL). Patients with NHL were earlier treated with radiotherapy alone or in combination with immunotherapy using monoclonal antibody anti-CD20 (ex., Rituximab-Mabthera, Roche). However, Radioimmunotherapy is a new modality of treatment for patients with NHL, in which cytotoxic radiation from therapeutic radioisotopes is delivered to tumors through monoclonal antibodies. This study focused on labeling conditions of monoclonal anti-CD20 (ex., Rituximab-Mabthera, Roche) with iodine-131, by direct radioiodination method using Chloramine-T as oxidizing agent. Labeling parameters investigated were: Radiochemical purity (RP), method of purification, incubation time, antibody mass, oxidative agent mass, stability in vitro, immunoreactivity and biological distribution performed in normal Swiss mouse. Product of high radiochemical purity was obtained with no notable difference between the methods applied. No clear evidence of direct influence of incubation time on radiochemical purity of the labeled antibody was observed. Whereas, a clear evidence of direct influence of activity on radiochemical purity of the labeled antibody was varied. After purification the labeled product presented radiochemical purity of approximately 100 %. Product of superior radiochemical yield was observed when standard condition of labeling was used. The labeled product presented variation in radiochemical purity using five different stabilizer conditions. The condition in which gentisic acid combined with freeze appears more suitable and capable of minimizing autoradiolysis of the antibody labeled with freeze appears more suitable and capable of minimizing autoradiolysis of the antibody labeled with high therapeutic activity of iodine-131. The labeled product presented low immunoreactivity when compared to the

The clinical application of radioimmunoimaging with 99mTc labeled anti-CEA monoclonal antibody C50

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Jiang Ningyi; Sha Xiaozhen; Zhang Hua

1996-01-01

To evaluate the clinical value of the radioimmunoimaging (RII) for the diagnosis of tumor with 99m Tc labeled anti-CEA monoclonal antibody C50, C50 was labeled with 99m Tc using modified 2-mercaptoethanol method. 99m Tc-C50 RII was performed in 70 patients with tumor. All were pathologically proved after operation. The sensitivity of 99m Tc-C50 RII for tumor was 82.9%, the specificity was 86.2%, the false negative rate was 17.1%, and the false positive rate was 13.8%. The positive predictive value was 89.5%, the negative predictive value was 78.1%. The coincidence rates was 82.4%, 84.6% and 80.0% for the ovarial intestinal and lung tumor respectively. 99m Tc-C50 RII was useful in clinical diagnosis of tumor

Methods for the Isolation of Genes Encoding Novel PHA Metabolism Enzymes from Complex Microbial Communities.

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Cheng, Jiujun; Nordeste, Ricardo; Trainer, Maria A; Charles, Trevor C

2017-01-01

Development of different PHAs as alternatives to petrochemically derived plastics can be facilitated by mining metagenomic libraries for diverse PHA cycle genes that might be useful for synthesis of bio-plastics. The specific phenotypes associated with mutations of the PHA synthesis pathway genes in Sinorhizobium meliloti and Pseudomonas putida, allows the use of powerful selection and screening tools to identify complementing novel PHA synthesis genes. Identification of novel genes through their function rather than sequence facilitates the functional proteins that may otherwise have been excluded through sequence-only screening methodology. We present here methods that we have developed for the isolation of clones expressing novel PHA metabolism genes from metagenomic libraries.

Methods for the isolation of genes encoding novel PHB cycle enzymes from complex microbial communities.

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Nordeste, Ricardo F; Trainer, Maria A; Charles, Trevor C

2010-01-01

Development of different PHAs as alternatives to petrochemically derived plastics can be facilitated by mining metagenomic libraries for diverse PHA cycle genes that might be useful for synthesis of bioplastics. The specific phenotypes associated with mutations of the PHA synthesis pathway genes in Sinorhizobium meliloti allows for the use of powerful selection and screening tools to identify complementing novel PHA synthesis genes. Identification of novel genes through their function rather than sequence facilitates finding functional proteins that may otherwise have been excluded through sequence-only screening methodology. We present here methods that we have developed for the isolation of clones expressing novel PHA metabolism genes from metagenomic libraries.

Establishment of CMab-43, a Sensitive and Specific Anti-CD133 Monoclonal Antibody, for Immunohistochemistry.

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Itai, Shunsuke; Fujii, Yuki; Nakamura, Takuro; Chang, Yao-Wen; Yanaka, Miyuki; Saidoh, Noriko; Handa, Saori; Suzuki, Hiroyoshi; Harada, Hiroyuki; Yamada, Shinji; Kaneko, Mika K; Kato, Yukinari

2017-10-01

CD133, also known as prominin-1, was first described as a cell surface marker on early progenitor and hematopoietic stem cells. It is a five-domain transmembrane protein composed of an N-terminal extracellular tail, two small cytoplasmic loops, two large extracellular loops containing seven potential glycosylation sites, and a short C-terminal intracellular tail. CD133 has been used as a marker to identify cancer stem cells derived from primary solid tumors and as a prognostic marker of gliomas. Herein, we developed novel anti-CD133 monoclonal antibodies (mAbs) and characterized their efficacy in flow cytometry, Western blot, and immunohistochemical analyses. We expressed the full length of CD133 in LN229 glioblastoma cells, immunized mice with LN229/CD133 cells, and performed the first screening using flow cytometry. After limiting dilution, we established 100 anti-CD133 mAbs, reacting with LN229/CD133 cells but not with LN229 cells. Subsequently, we performed the second and third screening with Western blot and immunohistochemical analyses, respectively. Among 100 mAbs, 11 strongly reacted with CD133 in Western blot analysis. One of 11 clones, CMab-43 (IgG 2a , kappa), showed a sensitive and specific reaction against colon cancer cells, warranting the use of CMab-43 in detecting CD133 in pathological analyses of CD133-expressing cancers.

The feasibility research of galactosyl-anti-mouse CD3 monoclonal antibody being used as carrier of immunotherapy after surgical operation of liver cancer

International Nuclear Information System (INIS)

Li Yunchun; Guan Changtian; Yang Xiaochuan; He Sheng; Jiang Ping; Yuan Lin

2000-01-01

Objective: To probe into the feasibility of galactosyl-anti-mouse CD 3 monoclonal antibody (Gal-Ant-CD 3 McAb) being used as carrier of immunotherapy after surgical operation of liver cancer. Methods: Gal-Ant-CD 3 McAb was prepared by the covalent coupling of anti-mouse CD 3 monoclonal antibody (Ant-CD 3 McAb) with a bifunctional reagent, 2-imino-2-methoxyethyl-1-thio-galactose. After Gal-Ant-CD 3 McAb and Ant-CD 3 McAb were labelled with 131 I or 125 I, the data of biodistribution in mice, and of imaging in rabbit were obtained. After tumour infiltrating lymphocytes (TIL) and Gal-Ant-CD 3 McAb were coupled into Gal-Ant-CD 3 McAb-TIL, its liver taxis and cytotoxic activity against autologous cancer cells were measured in vitro. Results: Gal-Ant-CD 3 McAb had remarkable livertaxis and its uptake in per gram liver was (59.0 +- 2.1)% that was more than two-fold higher than that of Ant-CD 3 McAb. Gal-Ant-CD 3 McAb-TIL had an obvious liver taxis and cytotoxic activity against autologous cancer cells in vitro. Conclusion: Gal-Ant-CD 3 McAb can be used as the carrier of immunotherapy after surgical operation of liver cancer

Antilymphocytic antibodies and marrow transplantation. VIII. Recipient conditioning with Clq-affine monoclonal anti-pan T antibodies prevents GVHD in homozygous fully mismatched mice

International Nuclear Information System (INIS)

Thierfelder, S.; Kummer, U.; Schuh, R.; Mysliwietz, J.

1986-01-01

An approach to suppressing secondary disease with antibodies was studied that differed from conventional antibody treatment of donor marrow in vitro. It consisted of the selection of anti-Thy-1 antibodies with high affinity for Clq, the first subunit of the complement cascade, and a single injection of such antibodies into prospective irradiated marrow recipients. Monoclonal mouse IgM and rat IgG 2c antibodies of high titers in complement-dependent test systems but with low affinity for Clq caused little immunosuppression. Monoclonal rat IgG2b or mouse IgG2a anti-Thy-1 antibodies with high affinity for Clq prevented acute and chronic mortality of graft-v-host disease (GVHD), however, when injected in irradiated CBA or AKR mice prior to C57BL/6 spleen and/or bone marrow cell transfusion. This treatment simultaneously suppressed residual host-v-graft reactivity of the irradiated mice, so that permanent hematopoietic engraftment ensued even at 5 or 6 Gy. Full chimerism and specific tolerance were obtained. Primary immune response to SRBC was clearly depressed in the chimeras; secondary immune response was not. Clearance of T cell antibody activity (greater than 6 days), timing, and dose of injected antibody, as well as other modalities of the conditioning treatment that may have contributed to the remarkable immunosuppression, are discussed

Reactivo hemoclasificador monoclonal cubano hemo-cim anti-a: Estudio de estabilidad Cuban anti-A Hemo-CIM hemoclassifier monoclonal reagent: Stability study

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Lilia Suárez Batista

1999-08-01

Full Text Available Los estudios de estabilidad de los reactivos fabricados a partir de anticuerpos monoclonales (AcM de origen murino, son esenciales para obtener resultados adecuados en su aplicación práctica y constituyen un requisito indispensable en las buenas prácticas de producción, lo que permite establecer adecuadamente la vida de estos productos. Se usaron los métodos de hemaglutinación recomendados para medir la actividad biológica del producto Hemo-CIM anti-A expresada en la potencia, la avidez y la intensidad frente a un panel de eritrocitos del grupo A1 y A2B, que se incluyó por su baja expresión del antígeno A para demostrar más efectivamente cualquier deterioro que sufriera el reactivo. Se estableció que la vida útil del reactivo hemoclasificador producido en el Centro de Inmunología Molecular es de 2 años y se determinó que la temperatura de almacenamiento del producto está entre 2 y 8 °C. Además, los lotes que se colocaron diariamente en la meseta de trabajo durante 5 horas a 21 °C a partir del mes 0 y a los 8, 14 y 22 meses después de su producción, simulando las condiciones a la que los usuarios someten a estos reactivos, mantuvieron las características de calidad que se requieren para su uso, lo que demostró que con este producto se puede trabajar en esas condicionesThe stability studies of the reactives obtained from monoclonal antibodies of murine origin are essential to attain adequate results in their practical application and are also an indispensable requirement for good production practices, which allow to establish the life of these products adequately. The hemagglutination methods were used to measure the biological activity of the anti-A Hemo-CIM product expressed in power, avidity and intesity against a set of erythrocytes of group A1 and A2B that was included due to its low antigen A expression to show more effectively any deterioration suffered by the reagent. It was determined that the useful life of the

Plasma exchanges for severe acute neurological deterioration in patients with IgM anti-myelin-associated glycoprotein (anti-MAG) neuropathy.

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Baron, M; Lozeron, P; Harel, S; Bengoufa, D; Vignon, M; Asli, B; Malphettes, M; Parquet, N; Brignier, A; Fermand, J P; Kubis, N; Arnulf, Bertrand

2017-06-01

Monoclonal IgM anti-myelin-associated glycoprotein (MAG) antibody-related peripheral neuropathy (anti-MAG neuropathy) is predominantly a demyelinating sensory neuropathy with ataxia and distal paresthesia. The clinical course of anti-MAG neuropathy is usually slowly progressive making difficult the identification of clear criteria to start a specific treatment. Although no consensus treatment is yet available, a rituximab-based regimen targeting the B-cell clone producing the monoclonal IgM may be proposed, alone or in combination with alkylating agents or purine analogs. However, in some rare cases, an acute and severe neurological deterioration can occur in few days leading to a rapid loss of autonomy. In these cases, a treatment rapidly removing the monoclonal IgM from the circulation might be useful before initiating a specific therapy. We report successful treatment with plasma exchanges (PE) in four patients presenting with acute neurological deterioration. PE allowed a dramatic and rapid neurological improvement in all patients. PE are safe and may be useful at the initial management of these cases of anti-MAG neuropathy.

Clinical significance of BRAF non-V600E mutations on the therapeutic effects of anti-EGFR monoclonal antibody treatment in patients with pretreated metastatic colorectal cancer: the Biomarker Research for anti-EGFR monoclonal Antibodies by Comprehensive Cancer genomics (BREAC) study.

Science.gov (United States)

Shinozaki, Eiji; Yoshino, Takayuki; Yamazaki, Kentaro; Muro, Kei; Yamaguchi, Kensei; Nishina, Tomohiro; Yuki, Satoshi; Shitara, Kohei; Bando, Hideaki; Mimaki, Sachiyo; Nakai, Chikako; Matsushima, Koutatsu; Suzuki, Yutaka; Akagi, Kiwamu; Yamanaka, Takeharu; Nomura, Shogo; Fujii, Satoshi; Esumi, Hiroyasu; Sugiyama, Masaya; Nishida, Nao; Mizokami, Masashi; Koh, Yasuhiro; Abe, Yukiko; Ohtsu, Atsushi; Tsuchihara, Katsuya

2017-11-07

Patients with BRAF V600E -mutated metastatic colorectal cancer (mCRC) have a poorer prognosis as well as resistance to anti-EGFR antibodies. However, it is unclear whether BRAF mutations other than BRAF V600E (BRAF non-V600E mutations) contribute to anti-EGFR antibody resistance. This study was composed of exploratory and inference cohorts. Candidate biomarkers identified by whole exome sequencing from super-responders and nonresponders in the exploratory cohort were validated by targeted resequencing for patients who received anti-EGFR antibody in the inference cohort. In the exploratory cohort, 31 candidate biomarkers, including KRAS/NRAS/BRAF mutations, were identified. Targeted resequencing of 150 patients in the inference cohort revealed 40 patients with RAS (26.7%), 9 patients with BRAF V600E (6.0%), and 7 patients with BRAF non-V600E mutations (4.7%), respectively. The response rates in RAS, BRAF V600E , and BRAF non-V600E were lower than those in RAS/BRAF wild-type (2.5%, 0%, and 0% vs 31.9%). The median PFS in BRAF non-V600E mutations was 2.4 months, similar to that in RAS or BRAF V600E mutations (2.1 and 1.6 months) but significantly worse than that in wild-type RAS/BRAF (5.9 months). Although BRAF non-V600E mutations identified were a rare and unestablished molecular subtype, certain BRAF non-V600E mutations might contribute to a lesser benefit of anti-EGFR monoclonal antibody treatment.

Anti-Podocalyxin Monoclonal Antibody 47-mG2a Detects Lung Cancers by Immunohistochemistry.

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Yamada, Shinji; Itai, Shunsuke; Kaneko, Mika K; Kato, Yukinari

2018-04-01

Lung cancer is one of the leading causes of cancer-related deaths in the world. Regardless of the advances in lung cancer treatments, the prognosis is still poor. Podocalyxin (PODXL) is a highly glycosylated type I transmembrane protein that is expressed in normal tissues, including the heart, pancreas, and breast. It is also found and used as a diagnostic marker in many cancers, such as renal, brain, breast, oral, and lung cancers. We previously developed specific and sensitive anti-PODXL monoclonal antibodies, PcMab-47 (mouse IgG 1 , kappa) and its mouse IgG 2a -type (47-mG 2a ), both of which were suitable for immunohistochemical analyses of oral cancers. In this study, we investigated the utility of PcMab-47 and 47-mG 2a for the immunohistochemical analyses of lung cancers. PcMab-47 stained 51/70 (72.9%) cases of lung cancer, whereas 47-mG 2a stained 59/70 (84.3%) cases, indicating that the latter antibody is more sensitive and is useful for detecting PODXL in lung cancers.

B cell-targeted therapy with anti-CD20 monoclonal antibody in a mouse model of Graves' hyperthyroidism.

Science.gov (United States)

Ueki, I; Abiru, N; Kobayashi, M; Nakahara, M; Ichikawa, T; Eguchi, K; Nagayama, Y

2011-03-01

Graves' disease is a B cell-mediated and T cell-dependent autoimmune disease of the thyroid which is characterized by overproduction of thyroid hormones and thyroid enlargement by agonistic anti-thyrotrophin receptor (TSHR) autoantibody. In addition to antibody secretion, B cells have recently been recognized to function as antigen-presenting/immune-modulatory cells. The present study was designed to evaluate the efficacy of B cell depletion by anti-mouse (m) CD20 monoclonal antibody (mAb) on Graves' hyperthyroidism in a mouse model involving repeated injection of adenovirus expressing TSHR A-subunit (Ad-TSHR289). We observe that a single injection of 250 µg/mouse anti-mCD20 mAb eliminated B cells efficiently from the periphery and spleen and to a lesser extent from the peritoneum for more than 3 weeks. B cell depletion before immunization suppressed an increase in serum immunoglobulin (Ig)G levels, TSHR-specific splenocyte secretion of interferon (IFN)-γ, anti-TSHR antibody production and development of hyperthyroidism. B cell depletion 2 weeks after the first immunization, a time-point at which T cells were primed but antibody production was not observed, was still effective at inhibiting antibody production and disease development without inhibiting splenocyte secretion of IFN-γ. By contrast, B cell depletion in hyperthyroid mice was therapeutically ineffective. Together, these data demonstrate that B cells are critical not only as antibody-producing cells but also as antigen-presenting/immune-modulatory cells in the early phase of the induction of experimental Graves' hyperthyroidism and, although therapeutically less effective, B cell depletion is highly efficient for preventing disease development. © 2011 The Authors. Clinical and Experimental Immunology © 2011 British Society for Immunology.

B cell-targeted therapy with anti-CD20 monoclonal antibody in a mouse model of Graves' hyperthyroidism

Science.gov (United States)

Ueki, I; Abiru, N; Kobayashi, M; Nakahara, M; Ichikawa, T; Eguchi, K; Nagayama, Y

2011-01-01

Graves' disease is a B cell-mediated and T cell-dependent autoimmune disease of the thyroid which is characterized by overproduction of thyroid hormones and thyroid enlargement by agonistic anti-thyrotrophin receptor (TSHR) autoantibody. In addition to antibody secretion, B cells have recently been recognized to function as antigen-presenting/immune-modulatory cells. The present study was designed to evaluate the efficacy of B cell depletion by anti-mouse (m) CD20 monoclonal antibody (mAb) on Graves' hyperthyroidism in a mouse model involving repeated injection of adenovirus expressing TSHR A-subunit (Ad-TSHR289). We observe that a single injection of 250 µg/mouse anti-mCD20 mAb eliminated B cells efficiently from the periphery and spleen and to a lesser extent from the peritoneum for more than 3 weeks. B cell depletion before immunization suppressed an increase in serum immunoglobulin (Ig)G levels, TSHR-specific splenocyte secretion of interferon (IFN)-γ, anti-TSHR antibody production and development of hyperthyroidism. B cell depletion 2 weeks after the first immunization, a time-point at which T cells were primed but antibody production was not observed, was still effective at inhibiting antibody production and disease development without inhibiting splenocyte secretion of IFN-γ. By contrast, B cell depletion in hyperthyroid mice was therapeutically ineffective. Together, these data demonstrate that B cells are critical not only as antibody-producing cells but also as antigen-presenting/immune-modulatory cells in the early phase of the induction of experimental Graves' hyperthyroidism and, although therapeutically less effective, B cell depletion is highly efficient for preventing disease development. PMID:21235532

Characterization of a Broadly Reactive Anti-CD40 Agonistic Monoclonal Antibody for Potential Use as an Adjuvant.

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Cameron Martin

Full Text Available Lack of safe and effective adjuvants is a major hindrance to the development of efficacious vaccines. Signaling via CD40 pathway leads to enhanced antigen processing and presentation, nitric oxide expression, pro-inflammatory cytokine expression by antigen presenting cells, and stimulation of B-cells to undergo somatic hypermutation, immunoglobulin class switching, and proliferation. Agonistic anti-CD40 antibodies have shown promising adjuvant qualities in human and mouse vaccine studies. An anti-CD40 monoclonal antibody (mAb, designated 2E4E4, was identified and shown to have strong agonistic effects on primary cells from multiple livestock species. The mAb recognize swine, bovine, caprine, and ovine CD40, and evoked 25-fold or greater proliferation of peripheral blood mononuclear cells (PBMCs from these species relative to cells incubated with an isotype control (p<0.001. In addition, the mAb induced significant nitric oxide (p<0.0001 release by bovine macrophages. Furthermore, the mAb upregulated the expression of MHC-II by PBMCs, and stimulated significant (p<0.0001 IL-1α, IL6, IL-8, and TNF-α expression by PBMCs. These results suggest that the mAb 2E4E4 can target and stimulate cells from multiple livestock species and thus, it is a potential candidate for adjuvant development. This is the first study to report an anti-swine CD40 agonistic mAb that is also broadly reactive against multiple species.

Immunoscintigraphy of adenocarcinomas by means of 111In-labelled F(ab')2 fragments of anti-CEA monoclonal antibody F023C5

International Nuclear Information System (INIS)

Riva, P.; Paganelli, G.; Callegaro, L.

1988-01-01

F(ab') 2 fragments of F023C5, an anti-CEA monoclonal antibody, were conjugated to diethylenetriamine pentaacetic acid (DTPA) and converted into a ready to use reagent for instant 111 In-labelling. The resulting 111 In radiopharmaceutical was administered intravenously and tested for its ability to image (at 48-72 h after administration) 31 primary and 85 metastatic carcinoma lesions in 70 adenocarcinoma patients (26 gastrointestinal, 18 breast and 26 lung tumour patients) whose serum CEA was elevated in 43 cases and normal in the other 27. (author)

A review of human anti-globulin antibody (HAGA, HAMA, HACA, HAHA) responses to monoclonal antibodies. Not four letter words.

Science.gov (United States)

Mirick, G R; Bradt, B M; Denardo, S J; Denardo, G L

2004-12-01

The United States Food and Drug Administration (FDA) has approved unconjugated monoclonal antibodies (MAbs) for immunotherapy (IT) of B-cell lymphoma, breast cancer and acute myeloid leukemia. More recently, approval has been given for conjugated ZevalinTM ((90)yttrium ibritumomab tiuxetan, IDEC-Y2B8, Biogen Idec, Cambridge, MA) and BexxarTM ((131)I-tositumomab, Corixa, Corp., Seattle, WA and GlaxoSmithKline, Philadelphia, PA) anti-CD20 MAbs for use in radioimmunotherapy (RIT) of non-Hodgkin's lymphoma (NHL), thus redefining the standard care of cancer patients. Because of, and despite a lack of basis for concern about allergic reactions due to human antibody responses to these foreign proteins, assays were developed to determine HAGA (human anti-globulin antibody) levels that developed in patient sera following treatment with MAbs. Strategies were also devised to ''humanize'' MAbs and to temporarily block patient immune function with drugs in order to decrease the seroconversion rates, with considerable success. On the other hand, a survival advantage has been observed in some patients who developed a HAGA following treatment. This correlates with development of an anti-idiotype antibody cascade directed toward the MAbs used to treat these patients. What follows is a selective review of HAGA and its effect on cancer treatment over the past 2 decades.

A review of human anti-globulin antibody (HAGA, HAMA, HACA, HAHA) responses to monoclonal antibodies. Not four letter words

International Nuclear Information System (INIS)

Mirick, G. R.; Bradt, B. M.; Denardo, S. J.; Denardo, G. L.

2004-01-01

The United States Food and Drugs Administration (FDA) has approved unconjugated monoclonal antibodies (MAbs) for immunotherapy (IT) of B-cell lymphoma, breast cancer and acute myeloid leukemia. More recently, approval has been given for conjugated ZevalinTM ( 9 0yttrium ibritumomab tiuxetan, IDEC-Y2B8, Biogen Idec, Cambridge, MA) and BexxarTM ( 1 31I-tositumomab, Corixa, Corp., Seattle, WA and GlaxoSmithKline, Philadelphia, PA) antiCD20 MAns for use in radioimmunotherapy (RIT) of non-Hodgikin's lymphoma (NHL), thus redefining the standard care of cancer patients. Because of, and despite a lack of basis for concern about allergic reactions due to human antibody responses to these foreign proteins, essays were developed to determine HAGE (human anti-globulin antibody) levels that developed in patient sera following treatment with MAbs. Strategies were also devised to humanize MAbs and to temporarily block patient immune function with drugs in order to decrease the seroconversion rates, with considerable success. On the other hand, a survival advantage has been observed in some patients who developed a HAGA following treatment. This correlates with development of an anti-idiotype antibody cascade directed toward the MAbs used to treat these patients. What follows is a selective review of HAGA and its effect on cancer treatment over the past 2 decades

Treatment with anti-interferon-δ monoclonal antibodies modifies experimental autoimmune encephalomyelitis in interferon-δ receptor knockout mice

DEFF Research Database (Denmark)

Espejo, C.; Penkowa, Milena; Saez-Torres, I.

2001-01-01

Neuroinflammation, neuronal degeneration, regeneration, monoclonal antibodies, multiple schlerosis......Neuroinflammation, neuronal degeneration, regeneration, monoclonal antibodies, multiple schlerosis...

Development of a monoclonal-based enzyme-linked immunoassay for saxitoxin-induced protein.

Science.gov (United States)

Smith, D S; Kitts, D D

1994-03-01

A monoclonal antibody was generated against saxitoxin-induced protein (SIP) from the small shore crab Hemigrapsus oregenesis. SIP was induced by saxitoxin injection and could be detected in the crude crab extracts with both polyclonal and monoclonal antibody preparations. On Western blots, the polyclonal serum reacted against several bands which were induced by saxitoxin in the crude extracts. These bands represented proteins related to SIP. The monoclonal (4G5), however, was specific for the 79,000 mol. wt subunit of SIP. A triple antibody sandwich ELISA was developed in which polyclonal anti-SIP IgG was used as a trapping layer and monoclonal 4G5 was used as the detection layer. This assay was shown to be more specific and more accurate than a direct bind assay which employed the polyclonal antiserum alone. Although the polyclonal serum was more sensitive than the monoclonal on Western blots, the triple antibody sandwich and direct bind ELISAs were of comparable sensitivity.

Mass-Production and Characterization of Anti-CD20 Monoclonal Antibody in Peritoneum of Balb/c Mice

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Leili Aghebati

2013-02-01

Full Text Available Purpose: Monoclonal antibodies are important tools are used in basic research as well as, in diagnosis, imaging and treatment of immunodeficiency diseases, infections and cancers. The purpose of this study was to produce large scale of monoclonal antibody against CD20 in order to diagnostic application in leukemia and lymphomas disorders. Methods: Hybridoma cells that produce monoclonal antibody against human CD20 were administered into the peritoneum of the Balb/c mice which have previously been primed with 0.5 ml Pristane. After twelve days, approximately 7 ml ascetic fluid was harvested from the peritoneum of each mouse. Evaluation of mAb titration was assessed by ELISA method. In the present study, we describe a protocol for large scale production of MAbs. Results: We prepared monoclonal antibodies (mAbs with high specificity and sensitivity against human CD20 by hybridoma method and characterized them by ELISA. The subclass of antibody was IgG2a and its light chain was kappa. Ascetic fluid was purified by Protein-A Sepharose affinity chromatography and the purified monoclonal antibody was conjugated with FITC and Immunofluorescence was done for confirming the specific binding. Conclusion: The conjugated monoclonal antibody could have application in diagnosis B-cell lymphomas, hairy cell leukemia, B-cell chronic lymphocytic leukemia, and melanoma cancer stem cells.

Mass-Production and Characterization of Anti-CD20 Monoclonal Antibody in Peritoneum of Balb/c Mice

Science.gov (United States)

Sineh sepehr, Koushan; Baradaran, Behzad; Majidi, Jafar; Abdolalizadeh, Jalal; Aghebati, leili; Zare Shahneh, Fatemeh

2013-01-01

Purpose: Monoclonal antibodies are important tools are used in basic research as well as, in diagnosis, imaging and treatment of immunodeficiency diseases, infections and cancers. The purpose of this study was to produce large scale of monoclonal antibody against CD20 in order to diagnostic application in leukemia and lymphomas disorders. Methods: Hybridoma cells that produce monoclonal antibody against human CD20 were administered into the peritoneum of the Balb/c mice which have previously been primed with 0.5 ml Pristane. After twelve days, approximately 7 ml ascetic fluid was harvested from the peritoneum of each mouse. Evaluation of mAb titration was assessed by ELISA method. In the present study, we describe a protocol for large scale production of MAbs. Results: We prepared monoclonal antibodies (mAbs) with high specificity and sensitivity against human CD20 by hybridoma method and characterized them by ELISA. The subclass of antibody was IgG2a and its light chain was kappa. Ascetic fluid was purified by Protein-A Sepharose affinity chromatography and the purified monoclonal antibody was conjugated with FITC and Immunofluorescence was done for confirming the specific binding. Conclusion: The conjugated monoclonal antibody could have application in diagnosis B-cell lymphomas, hairy cell leukemia, B-cell chronic lymphocytic leukemia, and melanoma cancer stem cells. PMID:24312821

Exposure and Tumor Fn14 Expression as Determinants of Pharmacodynamics of the Anti-TWEAK Monoclonal Antibody RG7212 in Patients with Fn14-Positive Solid Tumors

DEFF Research Database (Denmark)

Meulendijks, Didier; Lassen, Ulrik N; Siu, Lillian L

2016-01-01

PURPOSE: The TWEAK-Fn14 pathway represents a novel anticancer target that is being actively investigated. Understanding the relationship between pharmacokinetics of anti-TWEAK therapeutics and tumor pharmacodynamics is critical. We investigated exposure-response relationships of RG7212, an anti...... changes in tumor TWEAK-Fn14 signaling in paired pre- and posttreatment tumor biopsies. The objectives of the analysis were to define exposure-response relationships and the relationship between pretreatment tumor Fn14 expression and pharmacodynamic effect. Associations between changes in TWEAK-Fn14...... longer time on study was observed with high versus low RG7212 exposure. CONCLUSIONS: RG7212 reduced tumor TWEAK-Fn14 signaling in a systemic exposure-dependent manner. In addition to higher exposure, relatively high Fn14 expression might be required for pharmacodynamic effect of anti-TWEAK monoclonal...

[Diagnostic and therapeutic use of human anti-D (Rho) monoclonal antibodies. Evaluation and perspectives].

Science.gov (United States)

Rouger, P; Goossens, D; Champomier, F; Tsikas, G; Liberge, G; Leblanc, J; Richard, C; Bailleul, C; Salmon, C

1985-12-01

Human monoclonal antibodies will be essential in medicine. They are valuable tools for biological diagnosis and therapeutics. Our model, human monoclonal antibodies directed against the Rhesus D antigen can be used for the determination of the Rhesus D phenotype and for the suppression of Rh(D) immunisation in women. These new products require new procedures of preparation, new regulations for the quality controls, which will be discussed in this paper.

Monoclonal antibodies to Herpes Simplex Virus Type 2

International Nuclear Information System (INIS)

McLean-Pieper, C.S.

1982-01-01

In this thesis the production and characterisation of monoclonal antibodies to Herpes Simplex Virus Type 2 is described. The development of a suitable radioimmunoassay for the detection of anti-HSV-2 antibodies, and the selection of an optimal immunisation schedule, is given. Three assay systems are described and their reliability and sensitivity compared. (Auth.)

Bacterial Molecular Signals in the Sinorhizobium fredii-Soybean Symbiosis

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Francisco J. López-Baena

2016-05-01

Full Text Available Sinorhizobium (Ensifer fredii (S. fredii is a rhizobial species exhibiting a remarkably broad nodulation host-range. Thus, S. fredii is able to effectively nodulate dozens of different legumes, including plants forming determinate nodules, such as the important crops soybean and cowpea, and plants forming indeterminate nodules, such as Glycyrrhiza uralensis and pigeon-pea. This capacity of adaptation to different symbioses makes the study of the molecular signals produced by S. fredii strains of increasing interest since it allows the analysis of their symbiotic role in different types of nodule. In this review, we analyze in depth different S. fredii molecules that act as signals in symbiosis, including nodulation factors, different surface polysaccharides (exopolysaccharides, lipopolysaccharides, cyclic glucans, and K-antigen capsular polysaccharides, and effectors delivered to the interior of the host cells through a symbiotic type 3 secretion system.

H2Mab-77 is a Sensitive and Specific Anti-HER2 Monoclonal Antibody Against Breast Cancer.

Science.gov (United States)

Itai, Shunsuke; Fujii, Yuki; Kaneko, Mika K; Yamada, Shinji; Nakamura, Takuro; Yanaka, Miyuki; Saidoh, Noriko; Chang, Yao-Wen; Handa, Saori; Takahashi, Maki; Suzuki, Hiroyoshi; Harada, Hiroyuki; Kato, Yukinari

2017-08-01

Human epidermal growth factor receptor 2 (HER2) plays a critical role in the progression of breast cancers, and HER2 overexpression is associated with poor clinical outcomes. Trastuzumab is an anti-HER2 humanized antibody that leads to significant survival benefits in patients with HER2-positive metastatic breast cancers. In this study, we developed novel anti-HER2 monoclonal antibodies (mAbs) and characterized their efficacy in flow cytometry, Western blot, and immunohistochemical analyses. Initially, we expressed the full length or ectodomain of HER2 in LN229 glioblastoma cells and then immunized mice with ectodomain of HER2 or LN229/HER2, and performed the first screening by enzyme-linked immunosorbent assays using ectodomain of HER2. Subsequently, we selected mAbs according to their efficacy in flow cytometry (second screening), Western blot (third screening), and immunohistochemical analyses (fourth screening). Among 100 mAb clones, only three mAbs reacted with HER2 in Western blot, and clone H 2 Mab-77 (IgG 1 , kappa) was selected. Finally, immunohistochemical analyses with H 2 Mab-77 showed sensitive and specific reactions against breast cancer cells, warranting the use of H 2 Mab-77 to detect HER2 in pathological analyses of breast cancers.

Using anti-VEGF monoclonal antibody and magnetic nanoparticles

International Nuclear Information System (INIS)

Chen Jing; Wuhua; Hang Deyan; Xie Changsheng

2004-01-01

Objective: To study the biodistribution of 131 I-anti-vascular endothelial growth factor (VEGF) monoclonal antibody (Sc-7269)-dextran magnetic nanoparticles (DMN) in nude mice bearing human liver cancer where an external magnetic field was focused on, and to evaluate its therapeutic effects and safety. Methods: Eighteen nude mice bearing human liver cancer where an external magnetic field was focused on, were used for the bio-distribution study after intratumoral injection (n=9) or intravenous injection (n=9) of 131 I-Sc-7269-DMN. Another 25 tumor-bearing nude mice were divided into five groups, four groups of them were treated with 74 MBq/ml 131 I-Sc-7269-DMN, 131 I-Sc-7269, 131 I-DMN and 131 I by a single intratumoral injection, respectively. And an external magnetic field was bound to the tumor of the nude mice that were injected 131 I-Sc-7269-DMN or 131 I-DMN. For control study, the remaining one group was injected with physiological saline. Tumor growth delay (TGD) and tumor inhibition rate were observed as antitumor effects. Peripheral white cell counts and the loss of body weight were tested as indicators of systemic toxicity. Results: The retention percentages of radioactivity (%ID/g) in tumors after intratumoral injection were 104.06, 101.58 and 100.96%ID/g at 4, 24 and 48 h, respectively, while in the case of intravenous injection, the %ID/g values were lower (85.33, 89.67 and 90.00%ID/g, respectively, P 131 I-Sc-7269-DMN [ (13.3 ± 3.3) d] was the longest, and tumor inhibition rate (89.0%)was the highest compared with that in other groups (P 131 I-Sc-7269-DMN-treated mice as monitored by the decrease in peripheral white cell counts and the loss of body weight. Conclusions: The radioimmunotherapy with intratumoral injection of 131 I-Sc-7269-DMN may be safe and efficient for the treatment of liver cancer. Furthermore, the radioimmunotherapy using DMN as a carrier system may be a highly potential approach in targeted treatment of other kinds of tumors

Monoclonal antibody anti-AFB1: scale-up in vitro for biotools development Anticorpo monoclonal antiAFB1: produção in vitro visando desenvolvimento de bioferramentas

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Eiko Nakagawa Itano

2010-12-01

Full Text Available Aflatoxin B1 (AFB1 is a mycotoxin classified as group 1 (human carcinogen by International Agency for Research on Cancer - IARC, causing hazardous contamination in a wide variety of food and feed, where the monitoring depends on precision and accuracy of analytical method. The culture of AFB1 specific monoclonal antibody (mAb secreting hybridoma was performed for further development of immunochemical methods. The growth of hybridoma AF2 was carried out in RPMI medium + 15 % fetal bovine serum (FBS, as well as the same medium gradually amended with H-SFM medium (25, 50, 75 and 100 % H-SFM. The protein concentration in the culture supernatant ranged from 1.80 to 10.88 mg/mL. The culture amended with FBS-free synthetic H-SFM medium reached production of reagent with higher degree of purity and lower risk, in addition to lower protein content (2.29 mg/mL reached with 100 % H-SFM, which approaches the real content of pure mAb. The indirect competitive enzyme-linked immunosorbent assay (ic-ELISA and SDS-polyacrylamide gel electrophoresis (SDS-PAGE showed anti-AFB1 activity and IgG corresponding bands, respectively, indicating feasible application of mAb produced in 100, 75 and 50 % H-SFM for further use in the development of AFB1 detecting biotools. This mAb production can be an initial step that can supply the self-sufficient immune-reagent in the rapid diagnosis at national condition, which is essential in the food quality and safety.Aflatoxina B1 (AFB1 é uma micotoxina classificada pela International Agency for Research on Cancer - IARC no Grupo 1 (carcinógeno ao humano, responsável pelo perigo de contaminação em ampla variedade de alimento e ração, cujo monitoramento depende de metodologia analítica precisa e exata. O trabalho visou cultivo do hibridoma secretor de anticorpo monoclonal (AcM específico para AFB1 visando desenvolvimento de métodos imunoquímicos. Hibridoma AF2 foi cultivado em meio RPMI + 15 % de soro fetal bovino (SFB

Growth inhibition of tumor cells in vitro by using monoclonal antibodies against gonadotropin-releasing hormone receptor.

Science.gov (United States)

Lee, Gregory; Ge, Bixia

2010-07-01

As the continuation of a previous study, synthetic peptides corresponding to the extracellular domains of human gonadotropin-releasing hormone (GnRH) receptor were used to generate additional monoclonal antibodies which were further characterized biochemically and immunologically. Among those identified to recognize GnRH receptor, monoclonal antibodies designated as GHR-103, GHR-106 and GHR-114 were found to exhibit high affinity (Kd L37), when cancer cells were incubated with GnRH or GHR-106. The widespread expressions of GnRH receptor in almost all of the studied human cancer cell lines were also demonstrated by RT-PCR and Western blot assay, as well as indirect immunofluorescence assay with either of these monoclonal antibodies as the primary antibody. In view of the longer half life of antibodies as compared to that of GnRH or its analogs, anti-GnRH receptor monoclonal antibodies in humanized forms could function as GnRH analogs and serve as an ideal candidate of anti-cancer drugs for therapeutic treatments of various cancers in humans as well as for fertility regulations.

Comparative tumour localization properties of radiolabelled monoclonal antibody preparations of defined immunoreactivities

International Nuclear Information System (INIS)

Pimm, M.V.; Baldwin, R.W.

1987-01-01

The immunoreactive fraction of an anti-CEA monoclonal antibody preparation has been progressively decreased by the addition of increasing proportions of impurity in the form of immunologically inert mouse immunoglobulin. Following radioiodination, the immunoreactive fractions of the preparations were determined and their localization in a human tumour xenograft in nude mice was assessed. There was a progressive decline in tumour localization, from tumour to blood ratios of 2:1 with unadulterated antibody to 0.6:1 with preparations only 15% with respect to the initial antibody. These findings demonstrate that the immunoreactive fraction of monoclonal antibody preparations is a major limiting factor in tumour localization and this has implications for experimental and clinical applications of monoclonal antibodies. (orig.)

Septal endocarditis, bone infection and severe leg ischemia detected in Tc-99m labelled monoclonal anti granulocyte scan

International Nuclear Information System (INIS)

Bechelaghem, A.I; Habbeche, M; Benlabgaa, R; Ghedbane, IE; Hanzal, A; Khelifa, A; Mechcken, F; Bourezak, SE; Bouyoucef, SE

2006-01-01

Patient 28 years old has continued to have a persistent fever (39.2 O C), despite ten days treatment by specific antibiotics for bacterial endocarditis associated to a recent claudication of the right lower leg. The persistent fever has motivated a 99mTc-labelled monoclonal anti granulocyte scan which has showed an important uptake in the myocardial septum, and other infection locations in temporal bone and in right tibial arteries. Two days after, a nanocolloids-99mTc WBS showed no uptake in the heart area, a total absence of uptake of the nanocolloids in the bone marrow of right tibia b and cranial SPECT views confirmed the infectious site in the right temporal bone. New antibiotic strategy was adopted successfully associated with surgical amputation of the right lower leg (au)

Monoclonal antibody studies in B(non-T)-cell malignancies.

Science.gov (United States)

Shimoyama, M; Minato, K; Tobinai, K; Nagai, M; Hirose, M

1983-09-01

Tumor cells suspensions prepared from 129 B- or non-T cell malignancies were investigated with a panel of 10 monoclonal antibodies and conventional surface marker techniques. Surface immunoglobulin (sIg) and B1 antigen proved to be the most useful markers for B-cell lineage. Six major subtypes of acute lymphoblastic leukemia (ALL) of non-T cell nature are now recognized by these immunological techniques, including null-ALL, Ia-ALL, lymphoid stem cell ALL, pre-pre-B ALL, pre-B ALL and B-ALL. In cases of chronic leukemias and lymphomas of non-T cell nature, 80% of the tumor was defined by sIg and 88% by B1 antigen as definitely of B-cell lineage. The clonal character was also defined in 68% of the tumor on the basis of the detection of predominant single light chain in sIg. Ia-like antigen was detected in almost all cases (96%). Leukemic cells from all cases of chronic lymphocytic leukemia (CLL), chronic lymphosarcoma cell leukemia (CLsCL) and hairy cell leukemia (HCL) reacted with OKIa1 and anti-B1, and leukemic cells from most of them with anti-pan T monoclonal antibody (10.2). In more than half of CLL and CLsCL, leukemic cells were reactive with J5, OKM1, 9.6 and OKT8, but not with OKT3, OKT4 and OKT6. HCL cells had almost the same reactivity with these monoclonal antibodies as CLL and CLsCL cells except that J5 remained unreactive. These results indicated that Japanese CLL, CLsCL and HCL were different from Western ones at least with respect to surface marker characteristics. In cases of lymphomas, heavy chains of sIg were expressed in polyclonal fashion, especially in follicular lymphoma and diffuse lymphomas of medium sized cell type and large cell type, indicating that lymphomas of these types may originate from follicular center cells of the heavy chain switching stage. Anti-T monoclonals were also reactive with lymphoma cells. In about half of follicular lymphomas and diffuse lymphomas of the medium sized cell type, lymphoma cells reacted with 10.2, and less

Molecular and Therapeutic Characterization of Anti-ectodysplasin A Receptor (EDAR) Agonist Monoclonal Antibodies*

Science.gov (United States)

Kowalczyk, Christine; Dunkel, Nathalie; Willen, Laure; Casal, Margret L.; Mauldin, Elizabeth A.; Gaide, Olivier; Tardivel, Aubry; Badic, Giovanna; Etter, Anne-Lise; Favre, Manuel; Jefferson, Douglas M.; Headon, Denis J.; Demotz, Stéphane; Schneider, Pascal

2011-01-01

The TNF family ligand ectodysplasin A (EDA) and its receptor EDAR are required for proper development of skin appendages such as hair, teeth, and eccrine sweat glands. Loss of function mutations in the Eda gene cause X-linked hypohidrotic ectodermal dysplasia (XLHED), a condition that can be ameliorated in mice and dogs by timely administration of recombinant EDA. In this study, several agonist anti-EDAR monoclonal antibodies were generated that cross-react with the extracellular domains of human, dog, rat, mouse, and chicken EDAR. Their half-life in adult mice was about 11 days. They induced tail hair and sweat gland formation when administered to newborn EDA-deficient Tabby mice, with an EC50 of 0.1 to 0.7 mg/kg. Divalency was necessary and sufficient for this therapeutic activity. Only some antibodies were also agonists in an in vitro surrogate activity assay based on the activation of the apoptotic Fas pathway. Activity in this assay correlated with small dissociation constants. When administered in utero in mice or at birth in dogs, agonist antibodies reverted several ectodermal dysplasia features, including tooth morphology. These antibodies are therefore predicted to efficiently trigger EDAR signaling in many vertebrate species and will be particularly suited for long term treatments. PMID:21730053

Synthetic methyl hexagalacturonate hapten inhibitors of antihomogalacturonan monoclonal antibodies LM7, JIM5 and JIM7

DEFF Research Database (Denmark)

Clausen, Mads Hartvig; Willats, William George Tycho; Knox, J. Paul

2003-01-01

A range of synthetic methyl hexagalacturonates were used as potential hapten inhibitors in competitive-inhibition enzyme-linked immunosorbent assays (ELISAs) with anti-homogalacturonan monoclonal antibodies LM7, JIM5 and JIM7. The selective inhibition of these antibodies by different haptens...... provides insight into the structures of the partially methyl-esterified pectin epitopes of these widely used monoclonal antibodies....

The Effects of Clinorotation on the Host Plant, Medicago truncatula, and Its Microbial Symbionts

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Ariel J.C. Dauzart

2016-02-01

Full Text Available Understanding the outcome of the plant-microbe symbiosis in altered gravity is vital to developing life support systems for long-distance space travel and colonization of other planets. Thus, the aim of this research was to understand mutualistic relationships between plants and endophytic microbes under the influence of altered gravity. This project utilized the model tripartite relationship among Medicago truncatula ¬– Sinorhizobium meliloti – Rhizophagus irregularis. Plants were inoculated with rhizobial bacteria (S. meliloti, arbuscular mycorrhizal fungi (R. irregularis, or both microbes, and placed on a rotating clinostat. Vertical and horizontal static controls were also performed. Clinorotation significantly reduced M. truncatula dry mass and fresh mass compared to the static controls. The addition of rhizobia treatments under clinorotation also altered total root length and root-to-shoot fresh mass ratio. Nodule size decreased under rhizobia + clinorotation treatment, and nodule density was significantly decreased compared to the vertical treatment. However, inoculation with arbuscular mycorrhizal fungi was shown to increase biomass accumulation and nodule size. Thus, clinorotation significantly affected M. truncatula and its symbiotic relationships with S. meliloti and R. irregularis. In the long term, the results observed in this clinostat study on the changes of plant-microbe mutualism need to be investigated in spaceflight experiments. Thus, careful consideration of the symbiotic microbes of plants should be included in the design of bioregenerative life support systems needed for space travel.

Novel Clostridium difficile Anti-Toxin (TcdA and TcdB Humanized Monoclonal Antibodies Demonstrate In Vitro Neutralization across a Broad Spectrum of Clinical Strains and In Vivo Potency in a Hamster Spore Challenge Model.

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Hongyu Qiu

Full Text Available Clostridium difficile (C. difficile infection (CDI is the main cause of nosocomial antibiotic-associated colitis and increased incidence of community-associated diarrhea in industrialized countries. At present, the primary treatment of CDI is antibiotic administration, which is effective but often associated with recurrence, especially in the elderly. Pathogenic strains produce enterotoxin, toxin A (TcdA, and cytotoxin, toxin B (TcdB, which are necessary for C. difficile induced diarrhea and gut pathological changes. Administration of anti-toxin antibodies provides an alternative approach to treat CDI, and has shown promising results in preclinical and clinical studies. In the current study, several humanized anti-TcdA and anti-TcdB monoclonal antibodies were generated and their protective potency was characterized in a hamster infection model. The humanized anti-TcdA (CANmAbA4 and anti-TcdB (CANmAbB4 and CANmAbB1 antibodies showed broad spectrum in vitro neutralization of toxins from clinical strains and neutralization in a mouse toxin challenge model. Moreover, co-administration of humanized antibodies (CANmAbA4 and CANmAbB4 cocktail provided a high level of protection in a dose dependent manner (85% versus 57% survival at day 22 for 50 mg/kg and 20 mg/kg doses, respectively in a hamster gastrointestinal infection (GI model. This study describes the protective effects conferred by novel neutralizing anti-toxin monoclonal antibodies against C. difficile toxins and their potential as therapeutic agents in treating CDI.

Molecular imaging of rheumatoid arthritis by radiolabelled monoclonal antibodies: new imaging strategies to guide molecular therapies

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Malviya, G.; Dierckx, R.A. [Department of Nuclear Medicine and Molecular Imaging, University Medical Centre Groningen, University of Groningen (Netherlands); Conti, F. [Rheumatology Unit, I Faculty of Medicine and Surgery, Sapienza University of Rome (Italy); Chianelli, M. [Department of Nuclear Medicine and Molecular Imaging, University Medical Centre Groningen, University of Groningen (Netherlands); Unit of Nuclear Medicine, Regina apostolorum Hospital, Albano, Rome (Italy); Scopinaro, F. [Nuclear Medicine Department, Sapienza University of Rome, St. Andrea Hospital, Rome (Italy); Signore, A. [Department of Nuclear Medicine and Molecular Imaging, University Medical Centre Groningen, University of Groningen (Netherlands); Nuclear Medicine Department, Sapienza University of Rome, St. Andrea Hospital, Rome (Italy)

2010-02-15

The closing of the last century opened a wide variety of approaches for inflammation imaging and treatment of patients with rheumatoid arthritis (RA). The introduction of biological therapies for the management of RA started a revolution in the therapeutic armamentarium with the development of several novel monoclonal antibodies (mAbs), which can be murine, chimeric, humanised and fully human antibodies. Monoclonal antibodies specifically bind to their target, which could be adhesion molecules, activation markers, antigens or receptors, to interfere with specific inflammation pathways at the molecular level, leading to immune-modulation of the underlying pathogenic process. These new generation of mAbs can also be radiolabelled by using direct or indirect method, with a variety of nuclides, depending upon the specific diagnostic application. For studying rheumatoid arthritis patients, several monoclonal antibodies and their fragments, including anti-TNF-{alpha}, anti-CD20, anti-CD3, anti-CD4 and anti-E-selectin antibody, have been radiolabelled mainly with {sup 99m}Tc or {sup 111}In. Scintigraphy with these radiolabelled antibodies may offer an exciting possibility for the study of RA patients and holds two types of information: (1) it allows better staging of the disease and diagnosis of the state of activity by early detection of inflamed joints that might be difficult to assess; (2) it might provide a possibility to perform 'evidence-based biological therapy' of arthritis with a view to assessing whether an antibody will localise in an inflamed joint before using the same unlabelled antibody therapeutically. This might prove particularly important for the selection of patients to be treated since biological therapies can be associated with severe side-effects and are considerably expensive. This article reviews the use of radiolabelled mAbs in the study of RA with particular emphasis on the use of different radiolabelled monoclonal antibodies for

Molecular imaging of rheumatoid arthritis by radiolabelled monoclonal antibodies: new imaging strategies to guide molecular therapies

International Nuclear Information System (INIS)

Malviya, G.; Dierckx, R.A.; Conti, F.; Chianelli, M.; Scopinaro, F.; Signore, A.

2010-01-01

The closing of the last century opened a wide variety of approaches for inflammation imaging and treatment of patients with rheumatoid arthritis (RA). The introduction of biological therapies for the management of RA started a revolution in the therapeutic armamentarium with the development of several novel monoclonal antibodies (mAbs), which can be murine, chimeric, humanised and fully human antibodies. Monoclonal antibodies specifically bind to their target, which could be adhesion molecules, activation markers, antigens or receptors, to interfere with specific inflammation pathways at the molecular level, leading to immune-modulation of the underlying pathogenic process. These new generation of mAbs can also be radiolabelled by using direct or indirect method, with a variety of nuclides, depending upon the specific diagnostic application. For studying rheumatoid arthritis patients, several monoclonal antibodies and their fragments, including anti-TNF-α, anti-CD20, anti-CD3, anti-CD4 and anti-E-selectin antibody, have been radiolabelled mainly with 99m Tc or 111 In. Scintigraphy with these radiolabelled antibodies may offer an exciting possibility for the study of RA patients and holds two types of information: (1) it allows better staging of the disease and diagnosis of the state of activity by early detection of inflamed joints that might be difficult to assess; (2) it might provide a possibility to perform 'evidence-based biological therapy' of arthritis with a view to assessing whether an antibody will localise in an inflamed joint before using the same unlabelled antibody therapeutically. This might prove particularly important for the selection of patients to be treated since biological therapies can be associated with severe side-effects and are considerably expensive. This article reviews the use of radiolabelled mAbs in the study of RA with particular emphasis on the use of different radiolabelled monoclonal antibodies for therapy decision-making and

Exploring the plant-associated bacterial communities in Medicago sativa L

Directory of Open Access Journals (Sweden)

Pini Francesco

2012-05-01

Full Text Available Abstract Background Plant-associated bacterial communities caught the attention of several investigators which study the relationships between plants and soil and the potential application of selected bacterial species in crop improvement and protection. Medicago sativa L. is a legume crop of high economic importance as forage in temperate areas and one of the most popular model plants for investigations on the symbiosis with nitrogen fixing rhizobia (mainly belonging to the alphaproteobacterial species Sinorhizobium meliloti. However, despite its importance, no studies have been carried out looking at the total bacterial community associated with the plant. In this work we explored for the first time the total bacterial community associated with M. sativa plants grown in mesocosms conditions, looking at a wide taxonomic spectrum, from the class to the single species (S. meliloti level. Results Results, obtained by using Terminal-Restriction Fragment Length Polymorphism (T-RFLP analysis, quantitative PCR and sequencing of 16 S rRNA gene libraries, showed a high taxonomic diversity as well as a dominance by members of the class Alphaproteobacteria in plant tissues. Within Alphaproteobacteria the families Sphingomonadaceae and Methylobacteriaceae were abundant inside plant tissues, while soil Alphaproteobacteria were represented by the families of Hyphomicrobiaceae, Methylocystaceae, Bradyirhizobiaceae and Caulobacteraceae. At the single species level, we were able to detect the presence of S. meliloti populations in aerial tissues, nodules and soil. An analysis of population diversity on nodules and soil showed a relatively low sharing of haplotypes (30-40% between the two environments and between replicate mesocosms, suggesting drift as main force shaping S. meliloti population at least in this system. Conclusions In this work we shed some light on the bacterial communities associated with M. sativa plants, showing that Alphaproteobacteria may

A novel monoclonal anti-CD81 antibody produced by genetic immunization efficiently inhibits Hepatitis C virus cell-cell transmission.

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Isabel Fofana

Full Text Available Hepatitis C virus (HCV infection is a challenge to prevent and treat because of the rapid development of drug resistance and escape. Viral entry is required for initiation, spread, and maintenance of infection, making it an attractive target for antiviral strategies.Using genetic immunization, we produced four monoclonal antibodies (mAbs against the HCV host entry factor CD81. The effects of antibodies on inhibition of HCV infection and dissemination were analyzed in HCV permissive human liver cell lines.The anti-CD81 mAbs efficiently inhibited infection by HCV of different genotypes as well as a HCV escape variant selected during liver transplantation and re-infecting the liver graft. Kinetic studies indicated that anti-CD81 mAbs target a post-binding step during HCV entry. In addition to inhibiting cell-free HCV infection, one antibody was also able to block neutralizing antibody-resistant HCV cell-cell transmission and viral dissemination without displaying any detectable toxicity.A novel anti-CD81 mAb generated by genetic immunization efficiently blocks HCV spread and dissemination. This antibody will be useful to further unravel the role of virus-host interactions during HCV entry and cell-cell transmission. Furthermore, this antibody may be of interest for the development of antivirals for prevention and treatment of HCV infection.

A review of human anti-globulin antibody (HAGA, HAMA, HACA, HAHA) responses to monoclonal antibodies. Not four letter words

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Mirick, G. R.; Bradt, B. M.; Denardo, S. J.; Denardo, G. L. [Calfornia Univ., Sacramento (United States). Davis Medical Center

2004-12-01

The United States Food and Drugs Administration (FDA) has approved unconjugated monoclonal antibodies (MAbs) for immunotherapy (IT) of B-cell lymphoma, breast cancer and acute myeloid leukemia. More recently, approval has been given for conjugated ZevalinTM ({sup 9}0yttrium ibritumomab tiuxetan, IDEC-Y2B8, Biogen Idec, Cambridge, MA) and BexxarTM ({sup 1}31I-tositumomab, Corixa, Corp., Seattle, WA and GlaxoSmithKline, Philadelphia, PA) antiCD20 MAns for use in radioimmunotherapy (RIT) of non-Hodgikin's lymphoma (NHL), thus redefining the standard care of cancer patients. Because of, and despite a lack of basis for concern about allergic reactions due to human antibody responses to these foreign proteins, essays were developed to determine HAGE (human anti-globulin antibody) levels that developed in patient sera following treatment with MAbs. Strategies were also devised to humanize MAbs and to temporarily block patient immune function with drugs in order to decrease the seroconversion rates, with considerable success. On the other hand, a survival advantage has been observed in some patients who developed a HAGA following treatment. This correlates with development of an anti-idiotype antibody cascade directed toward the MAbs used to treat these patients. What follows is a selective review of HAGA and its effect on cancer treatment over the past 2 decades.

Acute neurological worsening after Rituximab treatment in patients with anti-MAG neuropathy.

Science.gov (United States)

Sala, Emilie; Robert-Varvat, Florence; Paul, Stéphane; Camdessanché, Jean-Philippe; Antoine, Jean-Christophe

2014-10-15

Patients with peripheral neuropathy and anti-MAG monoclonal IgM may respond to Rituximab, a humanized monoclonal anti-CD20 antibody. We report on three patients with peripheral neuropathy and anti-MAG monoclonal IgM who deteriorated under Rituximab and reviewed seven previously published cases. Worsening was acute and severe, and occurred during the treatment period. All the patients improved after deterioration but at final evaluation only one was improved comparatively to baseline, five were worsened and four were stabilized. Deterioration was not clearly associated with an increase of the anti-MAG antibody titer. Two patients received Rituximab prior or after the course which induced worsening without adverse reaction. Although rare, acute worsening of the neuropathy can occur after Rituximab. The deterioration is however reversible within some weeks to several months. Copyright © 2014 Elsevier B.V. All rights reserved.

Large Scale Generation and Characterization of Anti-Human CD34 Monoclonal Antibody in Ascetic Fluid of Balb/c Mice

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Koushan Sineh sepehr

2013-02-01

Full Text Available Purpose: Monoclonal antibodies or specific antibodies are now an essential tool of biomedical research and are of great commercial and medical value. The purpose of this study was to produce large scale of monoclonal antibody against CD34 in order to diagnostic application in leukemia and purification of human hematopoietic stem/progenitor cells. Methods: For large scale production of monoclonal antibody, hybridoma cells that produce monoclonal antibody against human CD34 were injected into the peritoneum of the Balb/c mice which have previously been primed with 0.5 ml Pristane. 5 ml ascitic fluid was harvested from each mouse in two times. Evaluation of mAb titration was assessed by ELISA method. The ascitic fluid was examined for class and subclasses by ELISA mouse mAb isotyping Kit. mAb was purified from ascitic fluid by affinity chromatography on Protein A-Sepharose. Purity of monoclonal antibody was monitored by SDS -PAGE and the purified monoclonal antibody was conjugated with FITC. Results: Monoclonal antibodies with high specificity and sensitivity against human CD34 by hybridoma technology were prepared. The subclass of antibody was IgG1 and its light chain was kappa. Conclusion: The conjugated monoclonal antibody could be a useful tool for isolation, purification and characterization of human hematopoietic stem cells.

Large Scale Generation and Characterization of Anti-Human CD34 Monoclonal Antibody in Ascetic Fluid of Balb/c Mice

Science.gov (United States)

Aghebati Maleki, Leili; Majidi, Jafar; Baradaran, Behzad; Abdolalizadeh, Jalal; Kazemi, Tohid; Aghebati Maleki, Ali; Sineh sepehr, Koushan

2013-01-01

Purpose: Monoclonal antibodies or specific antibodies are now an essential tool of biomedical research and are of great commercial and medical value. The purpose of this study was to produce large scale of monoclonal antibody against CD34 in order to diagnostic application in leukemia and purification of human hematopoietic stem/progenitor cells. Methods: For large scale production of monoclonal antibody, hybridoma cells that produce monoclonal antibody against human CD34 were injected into the peritoneum of the Balb/c mice which have previously been primed with 0.5 ml Pristane. 5 ml ascitic fluid was harvested from each mouse in two times. Evaluation of mAb titration was assessed by ELISA method. The ascitic fluid was examined for class and subclasses by ELISA mouse mAb isotyping Kit. mAb was purified from ascitic fluid by affinity chromatography on Protein A-Sepharose. Purity of monoclonal antibody was monitored by SDS -PAGE and the purified monoclonal antibody was conjugated with FITC. Results: Monoclonal antibodies with high specificity and sensitivity against human CD34 by hybridoma technology were prepared. The subclass of antibody was IgG1 and its light chain was kappa. Conclusion: The conjugated monoclonal antibody could be a useful tool for isolation, purification and characterization of human hematopoietic stem cells. PMID:24312838

Large Scale Generation and Characterization of Anti-Human IgA Monoclonal Antibody in Ascitic Fluid of Balb/c Mice

OpenAIRE

Fatemeh Ezzatifar; Jafar Majidi; Behzad Baradaran; Leili Aghebati Maleki; Jalal Abdolalizadeh; Mehdi Yousefi

2015-01-01

Purpose: Monoclonal antibodies are potentially powerful tools used in biomedical research, diagnosis, and treatment of infectious diseases and cancers. The monoclonal antibody against Human IgA can be used as a diagnostic application to detect infectious diseases. The aim of this study was to improve an appropriate protocol for large-scale production of mAbs against IgA. Methods: For large-scale production of the monoclonal antibody, hybridoma cells that produce monoclonal antibodies again...

Large Scale Generation and Characterization of Anti-Human CD34 Monoclonal Antibody in Ascetic Fluid of Balb/c Mice

OpenAIRE

Aghebati Maleki, Leili; Majidi, Jafar; Baradaran, Behzad; Abdolalizadeh, Jalal; Kazemi, Tohid; Aghebati Maleki, Ali; Sineh sepehr, Koushan

2013-01-01

Purpose: Monoclonal antibodies or specific antibodies are now an essential tool of biomedical research and are of great commercial and medical value. The purpose of this study was to produce large scale of monoclonal antibody against CD34 in order to diagnostic application in leukemia and purification of human hematopoietic stem/progenitor cells. Methods: For large scale production of monoclonal antibody, hybridoma cells that produce monoclonal antibody against human CD34 were injected into t...

Combined use of anti-ErbB monoclonal antibodies and erlotinib enhances antibody-dependent cellular cytotoxicity of wild-type erlotinib-sensitive NSCLC cell lines

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Cavazzoni Andrea

2012-12-01

Full Text Available Abstract Background The epidermal growth factor receptor (EGFR is an established target for anti-cancer treatment in different tumour types. Two different strategies have been explored to inhibit this pivotal molecule in epithelial cancer development: small molecules TKIs and monoclonal antibodies. ErbB/HER-targeting by monoclonal antibodies such as cetuximab and trastuzumab or tyrosine-kinase inhibitors as gefitinib or erlotinib has been proven effective in the treatment of advanced NSCLC. Results In this study we explored the potential of combining either erlotinib with cetuximab or trastuzumab to improve the efficacy of EGFR targeted therapy in EGFR wild-type NSCLC cell lines. Erlotinib treatment was observed to increase EGFR and/or HER2 expression at the plasma membrane level only in NSCLC cell lines sensitive to the drug inducing protein stabilization. The combined treatment had marginal effect on cell proliferation but markedly increased antibody-dependent, NK mediated, cytotoxicity in vitro. Moreover, in the Calu-3 xenograft model, the combination significantly inhibited tumour growth when compared with erlotinib and cetuximab alone. Conclusion Our results indicate that erlotinib increases surface expression of EGFR and/or HER2 only in EGFR-TKI sensitive NSCLC cell lines and, in turns, leads to increased susceptibility to ADCC both in vitro and in a xenograft models. The combination of erlotinib with monoclonal antibodies represents a potential strategy to improve the treatment of wild-type EGFR NSCLC patients sensitive to erlotinib.

Phase I trial of anti-GD2 monoclonal antibody hu3F8 plus GM-CSF: Impact of body weight, immunogenicity and anti-GD2 response on pharmacokinetics and survival.

Science.gov (United States)

Cheung, Irene Y; Kushner, Brian H; Modak, Shakeel; Basu, Ellen M; Roberts, Stephen S; Cheung, Nai-Kong V

2017-01-01

Fifty-seven stage 4 patients with refractory/relapsed neuroblastoma were enrolled in a phase I trial (Clinicaltrials.gov NCT01757626) using humanized anti-GD2 monoclonal antibody hu3F8 in combination with granulocyte-macrophage colony-stimulating factor. The influence of body weight and human anti-human antibody (HAHA) on the pharmacokinetics (PK) of hu3F8, and the effect of de novo anti-GD2 response on patient outcome were explored. Serum samples before hu3F8 infusion, and serially up to day 12 during treatment cycle #1, and at 5 min after each hu3F8 infusion for all subsequent cycles were collected. PK was analyzed using non-compartmental modeling. Immunogenicity was assayed by HAHA response, and vaccination effect by induced host anti-GD2 response measured periodically until disease progression or last followup. Progression-free and overall survival was estimated by the Kaplan-Meier method. Despite dosing being based on body weight, smaller patients had consistently lower area-under-the-curve and faster clearance over the 15 dose levels (0.9 to 9.6 mg/kg per treatment cycle) in this trial. Positive HAHA, defined by the upper limit of normal, when measured within 10 days from the last hu3F8 dose received, was associated with significantly lower serum hu3F8. Despite prior sensitization to other anti-GD2 antibody, e.g. mouse 3F8 or ch14.18, 75% of the patients never developed HAHA response even after getting more treatment cycles. Hu3F8 induced a de novo anti-GD2 response in patients, which was prognostic of progression-free survival. We conclude that hu3F8 had low immunogenicity. During treatment, positive HAHA and low body weight affected PK adversely, whereas induced anti-GD2 response was an outcome predictor.

Diversity and numbers of root-nodule bacteria (rhizobia in Polish soils

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Stefan Martyniuk

2011-01-01

Full Text Available Using a sand pouch-plant infection method, populations of several species of root-nodule bacteria (rhizobia were enumerated in eighty soils collected throughout Poland. Rhizobium leguminosarum bv. viciae (symbionts of pea, faba bean, vetch and R. leguminosarum bv. trifolii (symbionts of clover were detected in 77 and 76 soils, respectively. Most of these soils contained moderate and high numbers of these species of the rhizobia. Symbionts of beans, R. leguminosarum bv. phaseoli, were assessed in 76 soils; of this number 15 soils had no detectable populations of bean rhizobia and in 40 soils high or moderate numbers of these bacteria were found. Bradyrhizobium sp. (Lupinus, root-nodule bacteria of lupine and serradella, were absent in 19 soils, out of 80 tested, and 34 soils were colonised by high or moderate populations of bradyrhizobia. Sinorhizobium meliloti, rhizobia nodulating alfalfa, were sparse in the examined soils; with 56 soil containing no detectable numbers of S. meliloti and only 6 soils harbouring high or moderate populations of this species. The estimated numbers of the rhizobia in the studied soils were also related to some physical and chemical properties of these soils.

Monoclonal antibodies against pregnancy-specific β1-glycoprotein (SP1) in immunohistochemistry and radioimmunoassay

International Nuclear Information System (INIS)

Wahlstroem, T.; Heikinheimo, M.

1983-01-01

Monoclonal mouse antibodies against pregnancy-specific beta-1-glycoprotein (SP 1 ) have been studied for their suitability in immunoperoxidase staining and radioimmunoassay methodologies. These antibodies were useful in staining normal placentas, hydatidiform moles, invasive moles and choriocarcinomas. They showed good specificity, with minimal background staining, and will thus be superior to conventional polyclonal antisera in immunohistochemistry. However, the presently tested monoclonal anti-SP 1 antibodies were found not to be suitable for radioimmunoassay. (Auth.)

Large Scale Generation and Characterization of Anti-Human IgA Monoclonal Antibody in Ascitic Fluid of Balb/c Mice

Science.gov (United States)

Ezzatifar, Fatemeh; Majidi, Jafar; Baradaran, Behzad; Aghebati Maleki, Leili; Abdolalizadeh, Jalal; Yousefi, Mehdi

2015-01-01

Purpose: Monoclonal antibodies are potentially powerful tools used in biomedical research, diagnosis, and treatment of infectious diseases and cancers. The monoclonal antibody against Human IgA can be used as a diagnostic application to detect infectious diseases. The aim of this study was to improve an appropriate protocol for large-scale production of mAbs against IgA. Methods: For large-scale production of the monoclonal antibody, hybridoma cells that produce monoclonal antibodies against Human IgA were injected intraperitoneally into Balb/c mice that were previously primed with 0.5 ml Pristane. After ten days, ascitic fluid was harvested from the peritoneum of each mouse. The ELISA method was carried out for evaluation of the titration of produced mAbs. The ascitic fluid was investigated in terms of class and subclass by a mouse mAb isotyping kit. MAb was purified from the ascitic fluid by ion exchange chromatography. The purity of the monoclonal antibody was confirmed by SDS-PAGE, and the purified monoclonal antibody was conjugated with HRP. Results: Monoclonal antibodies with high specificity and sensitivity against Human IgA were prepared by hybridoma technology. The subclass of antibody was IgG1 and its light chain was the kappa type. Conclusion: This conjugated monoclonal antibody could have applications in designing ELISA kits in order to diagnose different infectious diseases such as toxoplasmosis and H. Pylori. PMID:25789225

Large Scale Generation and Characterization of Anti-Human IgA Monoclonal Antibody in Ascitic Fluid of Balb/c Mice

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Fatemeh Ezzatifar

2015-03-01

Full Text Available Purpose: Monoclonal antibodies are potentially powerful tools used in biomedical research, diagnosis, and treatment of infectious diseases and cancers. The monoclonal antibody against Human IgA can be used as a diagnostic application to detect infectious diseases. The aim of this study was to improve an appropriate protocol for large-scale production of mAbs against IgA. Methods: For large-scale production of the monoclonal antibody, hybridoma cells that produce monoclonal antibodies against Human IgA were injected intraperitoneally into Balb/c mice that were previously primed with 0.5 ml Pristane. After ten days, ascitic fluid was harvested from the peritoneum of each mouse. The ELISA method was carried out for evaluation of the titration of produced mAbs. The ascitic fluid was investigated in terms of class and subclass by a mouse mAb isotyping kit. MAb was purified from the ascitic fluid by ion exchange chromatography. The purity of the monoclonal antibody was confirmed by SDS-PAGE, and the purified monoclonal antibody was conjugated with HRP. Results: Monoclonal antibodies with high specificity and sensitivity against Human IgA were prepared by hybridoma technology. The subclass of antibody was IgG1 and its light chain was the kappa type. Conclusion: This conjugated monoclonal antibody could have applications in designing ELISA kits in order to diagnose different infectious diseases such as toxoplasmosis and H. Pylori.

Characterization of a purified nicotinic receptor from rat brain by using idiotypic and anti-idiotypic antibodies

International Nuclear Information System (INIS)

Abood, L.G.; Langone, J.J.; Bjercke, R.; Lu, X.; Banerjee, S.

1987-01-01

The availability of an anti-nicotine monoclonal antibody has made it possible to further establish the nature of the nicotine recognition proteins purified from rat brain by affinity chromatography and to provide a highly sensitive assay for determining [ 3 H]nicotine binding to the purified material. An enantiomeric analogue of nicotine. (-)-6-hydroxymethylnicotine, was used to prepare the affinity column. In addition, with the use of an anti-idiotypic monoclonal antibody, it was confirmed that the recognition site for nicotine resides on a protein complex composed of two components with molecular masses of 62 and 57 kDa. It was also demonstrated that the same two proteins could be purified by immunoaffinity chromatography with the use of an anti-idiotypic monoclonal antibody. With the use of the anti-nicotine antibody to measure [ 3 H]nicotine binding, the purified material was shown to bind 250 pmol/mg of protein. By utilizing a procedure in which the purified receptor protein was conjugated to membranes by disulfide bonds, a binding activity of 80 pmol/mg was obtained. With the availability of sterospecific monoclonal antibodies to (-)-nicotine as well as monoclonal anti-idiotypic antibodies derived when the anti-nicotine antibodies were used as immunogens, additional procedures became available for the further characterization of the purified nicotine receptor and examining its (-)-[ 3 H]nicotine-binding characteristics

Monoclonal antibody

International Nuclear Information System (INIS)

Oyamada, Hiyoshimaru

1987-01-01

Some aspects of monoclonal antibodies are described, centering on studies made by the author and those presented at the Second International Conference on Monoclonal Antibody Immunoconjugates for Cancer held in March this year (1987). The history of immuno-nuclear medicine and procedures for producing monoclonal antibodies are briefly outlined. Monoclonal antibodies are immunoglobulins. Here, the structure of IgG, which is used most frequently, is described. An IgG is composed of two antigen binding fragments (Fab) and one crystallizable fragment (Fc). The end portion of a Fab reacts with an antigen. One of the major applications of immuno-nuclear medicine is the diagnosis of cancer. As label nucleides, 131 I and 111 I were selected in most cases in the past while 123 I and 99m Tc are currently used more often. Advantages and disadvantages of this diagnosis method is discussed citing studies presented at the First (1986) and Second (1987) International Conference on Monoclonal Antibody Immunoconjugates for Cancer. The present status of the application of monoclonal antibodies to treatment of cancer is also described. (Nogami, K.)

Production and characterisation of monoclonal antibodies against native and disassembled human catalase

NARCIS (Netherlands)

Wiemer, E. A.; Ofman, R.; Middelkoop, E.; de Boer, M.; Wanders, R. J.; Tager, J. M.

1992-01-01

Catalase isolated from human erythrocytes was used to immunise mice, in order to generate hybridomas producing specific monoclonal antibodies to the enzyme. Hybridomas secreting anti-(catalase) antibodies were identified by a modified enzyme-linked immunosorbent assay (ELISA) using either

Clinical prospective study with radioiodinated monoclonal antibodies directed against colorectal cancer

International Nuclear Information System (INIS)

Chatal, J.F.; Douillard, J.Y.; Kremer, M.; Curtet, C.; Le Mevel, B.; Saccavini, J.C.; Maurel, C.; Aubry, J.

1985-01-01

The diagnostic application of three monoclonal antibodies are studied: an anti-carcinoembryonic antigen (CEA) antibody designated as 202 and two monoclonal antibodies, designated as 17-1A and 19-9, which recognize different antigens associated with gastrointestinal carcinomas. The complementary specificity of these antibodies was determined by an immuno-histochemical study and the scintigraphic detection parameters by a radiopharmacokinetic study in colic-tumour-bearing nude mice. On the basis of a prospective study, the value of immunoscintigraphy was compared with conventional methods such as ultrasonography and computed tomography for localization of recurrences of colorectal cancers. (UK)

Risk of fatigue in cancer patients receiving anti-EGFR monoclonal antibodies: results from a systematic review and meta-analysis of randomized controlled trial.

Science.gov (United States)

Zhu, Jianhong; Zhao, Wenxia; Liang, Dan; Li, Guocheng; Qiu, Kaifeng; Wu, Junyan; Li, Jianfang

2018-04-01

To evaluate the association between fatigue and anti-epidermal growth factor receptor monoclonal antibodies (anti-EGFR MAbs), we conducted the first meta-analysis to access the incidence and risk of fatigue associated with anti-EGFR MAbs. Electronic databases were searched for randomized controlled trials (RCTs) published up to February 2017. Eligible studies were selected according to PRISMA statement. Incidence rates, risk ratio (RRs), and 95% confidence intervals (CIs) were calculated using fixed-effects or random-effects models. Outcomes of quality were summarized in accordance with the GRADE (Grading of Recommendations Assessment, Development, and Evaluation) methodology. Thirty-five RCTs (including 15,622 patients) were included; median follow-up ranged from 8.1 to 71.4 months, and the fatigue events were recorded and graded according to the Common Toxicity Criteria for Adverse Events version 2.0 or 3.0 in most of the included trials. For patients receiving anti-EGFR MAbs, the overall incidence of all-grade and high-grade fatigue was 54.1% and 10.5%, respectively. Compared with control, anti-EGFR MAbs significantly increased the risk of all-grade fatigue (RR 1.10, 95% CI, 1.05-1.14, moderate-quality evidence) and high-grade fatigue (RR 1.31, 95% CI, 1.19-1.45, moderate-quality evidence). No significant differences among subgroup analyses (anti-EGFR MAbs, tumor type, and median follow-up) on high-grade fatigue were observed. No evidence of publication bias was observed. The present study suggested that anti-EGFR MAbs may increase the risk of fatigue in cancer patients.

Ofatumumab, a human anti-CD20 monoclonal antibody, for treatment of rheumatoid arthritis with an inadequate response to one or more disease-modifying antirheumatic drugs: results of a randomized, double-blind, placebo-controlled, phase I/II study

DEFF Research Database (Denmark)

Østergaard, Mikkel; Baslund, Bo; Rigby, William

2010-01-01

To investigate the safety and efficacy of ofatumumab, a novel human anti-CD20 monoclonal antibody (mAb), in patients with active rheumatoid arthritis (RA) whose disease did not respond to > or = 1 disease-modifying antirheumatic drug....

Anti-idiotypic antibodies directed against anti-HBs among the patients with chronic hepatitis B.

Science.gov (United States)

Kobayashi, K; Suzuki, H; Ueno, Y; Nagatomi, R; Kanno, A; Otsuki, M; Toyota, T

1990-08-01

Anti-idiotypic antibodies (anti-Id) against anti-HBs were found in the sera of patients with chronic hepatitis type B. Anti-idiotypic antibodies were detected by an enzyme-linked immunosorbent assay using horseradish peroxidase conjugated mouse monoclonal anti-HBs. Ten of 72 HBsAg positive sera contained anti-Id (13.9%). The prevalence of anti-Id did not appear to correlate with HBeAg/anti-HBe system. However, HB virus specific DNA polymerase activity was significantly higher in anti-Id positive sera. In the sera obtained from the patients treated with predonisolone before, anti-Id positive rate was higher than that in the patients without a history of predonisolone therapy. These results suggest that anti-Id may be related to the immunoregulatory mechanism of HB virus replication.

Itolizumab – a humanized anti-CD6 monoclonal antibody with better side effects profile for the treatment of psoriasis [Corrigendum

Directory of Open Access Journals (Sweden)

Menon R

2015-06-01

Full Text Available Menon R, David BG. Clinical, Cosmetic and Investigational Dermatology. 2015;8:215–22.On page 215, please note correspondence should have been listed as:   Roshni Menon, D II/17,JIPMER Campus, Dhanvanthri Nagar,Pondicherry, India 605006Tel +91 944 320 8140Email roshnijagdish@gmail.com.On page 215, the first sentence of the Introduction was “Psoriasis is a chronic inflammatory disease of the skin characterized by exacerbations and remissions affecting 1%–3% of the world’s population, and approximately 20% of patients have moderate to severe disease.1,2” however should have been “Psoriasis is a chronic inflammatory disease of the skin characterized by exacerbations and remissions affecting 1%–3% of the world’s population. Approximately 20% of patients have moderate to severe disease.1,2”On page 217, 219, and 221 the running header was “Itolizumab – aCD6 monoclonal antibody for the treatment of psoriasis” however should have been “Itolizumab – a humanized anti CD6 monoclonal antibody for the treatment of psoriasis”.On page 218, Table 1, the second column heading was listed as “Anand et al25 n=40 (moderate–severe psoriasis” however should have been “Anand et al25 n=40/32 weeks (moderate–severe psoriasis”.Read the original article 

IgM but not IgG monoclonal anti-Nocardia brasiliensis antibodies confer protection against experimental actinomycetoma in BALB/c mice.

Science.gov (United States)

Gonzalez-Suarez, Maria L; Salinas-Carmona, Mario C; Pérez-Rivera, Isabel

2009-10-01

Nocardia brasiliensis is a facultative intracellular microorganism that produces a human chronic infection known as actinomycetoma. Human and mouse anti-N. brasiliensis antibody response identify P24, P26 and P61 immunodominant antigens. In this work, we generated immunoglobulin M (IgM) and IgG monoclonal antibodies (mAbs) specific to immunodominant P61 antigen. The monoclonal IgM (NbM1) and IgG2a (NbG1) antibodies were assessed for their in vitro bactericidal activity, in vivo protective effect and ability to block catalase activity. These mAbs specifically recognized P61, but they did not inhibit its enzyme activity. The in vitro bactericidal effect of NbG1 was higher than the killing ability of the IgM mAb. In vivo experiments with a murine model of experimental infection with N. brasiliensis injected into rear footpads was used to test the effect of NbM1 and NbG1. The negative untreated group developed a chronic actinomycetoma within 4 weeks. IgM mAbs conferred protection to BALB/c mice infected with N. brasiliensis. IgG mAb lacked this protective effect. IgM mAb showed a dose-response correlation between antibody concentration and lesion size. These results demonstrate that humoral immune response mediated by antigen-specific IgM antibody protects against an intracellular bacterial infection.

Humanized versus murine anti-human epidermal growth factor receptor monoclonal antibodies for immunoscintigraphic studies

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Morales, Alejo A. Morales; Duconge, Jorge; Alvarez-Ruiz, Daniel; Becquer-Viart, Maria de Los Angeles; Nunez-Gandolff, Gilda; Fernandez, Eduardo; Caballero-Torres, Idania; Iznaga-Escobar, Normando

2000-02-01

The anti-human epidermal growth factor receptor (EGF-R) humanized antibody h-R3 (IgG{sub 1}), which binds to an extracellular domain of EGF-R, was used to evaluate the biodistribution on nude mice xenografted with A431 epidermoid carcinoma cell line. Results are compared with its murine version ior egf/r3 monoclonal antibody (mAb). Twenty-one athymic female 4NMRI nu/nu mice were injected intravenously with 10 {mu}g/100 {mu}Ci of {sup 99m}Tc-labeled mAbs. The mAb ior C5 that recognizes an antigen expressed preferentially on the surface of malignant and cytoplasm of normal colorectal cells was used as negative control. Immunoreactivity of {sup 99m}Tc-labeled mAbs was measured by enzyme linked immunosorbent assay on A431 cell line and the immunoreactive fractions determined by Lindmo method. Among all organs significant accumulation was found in tumor (6.14{+-}2.50 %ID/g, 5.06{+-}2.61 %ID/g for murine and humanized mAbs, respectively) 4 h after injection. The immunoreactive fractions were found to be 0.88 and 0.81 for murine and humanized mAb, respectively. Thus, we expect better results using the humanized mAb h-R3 for diagnostic immunoscintigraphy.

Humanized versus murine anti-human epidermal growth factor receptor monoclonal antibodies for immunoscintigraphic studies

International Nuclear Information System (INIS)

Morales, Alejo A. Morales; Duconge, Jorge; Alvarez-Ruiz, Daniel; Becquer-Viart, Maria de Los Angeles; Nunez-Gandolff, Gilda; Fernandez, Eduardo; Caballero-Torres, Idania; Iznaga-Escobar, Normando

2000-01-01

The anti-human epidermal growth factor receptor (EGF-R) humanized antibody h-R3 (IgG 1 ), which binds to an extracellular domain of EGF-R, was used to evaluate the biodistribution on nude mice xenografted with A431 epidermoid carcinoma cell line. Results are compared with its murine version ior egf/r3 monoclonal antibody (mAb). Twenty-one athymic female 4NMRI nu/nu mice were injected intravenously with 10 μg/100 μCi of 99m Tc-labeled mAbs. The mAb ior C5 that recognizes an antigen expressed preferentially on the surface of malignant and cytoplasm of normal colorectal cells was used as negative control. Immunoreactivity of 99m Tc-labeled mAbs was measured by enzyme linked immunosorbent assay on A431 cell line and the immunoreactive fractions determined by Lindmo method. Among all organs significant accumulation was found in tumor (6.14±2.50 %ID/g, 5.06±2.61 %ID/g for murine and humanized mAbs, respectively) 4 h after injection. The immunoreactive fractions were found to be 0.88 and 0.81 for murine and humanized mAb, respectively. Thus, we expect better results using the humanized mAb h-R3 for diagnostic immunoscintigraphy

Intraperitoneal delivery of monoclonal antibodies: enhanced regional delivery advantage using intravenous unlabeled anti-mouse antibody

International Nuclear Information System (INIS)

Wahl, R.L.; Fisher, S.

1987-01-01

Radiolabeled monoclonal antibodies (MAb) delivered intraperitoneally expose cells in contact with peritoneal fluid to considerably higher levels of MAb than if the MAb dose were given intravenously. This regional delivery advantage for intact MAb is present mainly due to the relatively slow exit of MAb from the peritoneal fluid to the blood. Eventually, following i.p. injection, blood levels of MAb rise resulting in exposure of the animal to high systemic MAb levels and potential toxicity. In this series of experiments, systemic exposure was minimized by the administration of unlabeled goat polyclonal anti-mouse antibody intravenously from 1 1/2 to 6 h following i.p. MAb injection. This maneuver results in the formation of immune complexes with their subsequent clearance and dehalogenation by the reticuloendothelial system, thus minimizing systemic MAb exposure. This approach, of increasing systemic clearance of MAb, did not alter intraperitoneal MAb levels and thus significantly increased the regional delivery advantage to the peritoneal cavity by 70-100%. This approach provides an immunologic rationale for the further enhancement of MAb delivery to i.p. foci of malignant disease and may have diagnostic and therapeutic utility. (author)

Symbiotic Properties of Sinorhizobium Fredii, J-TGS50 an Indonesian Soybean Nodule Forming Bacteria

International Nuclear Information System (INIS)

Setiyo Hadi Waluyo

2004-01-01

Green House experiments were conducted to study symbiotic properties of Sinorhizobium Fredii, J-TGS50. Sinorhizobium Fredii USDA 192, USDA 201, USDA 205, USDA 206, USDA 217 and Bradyrhizobium japonicum USDA 110 were used as references. Yeast extract mannitol broth culture of the bacteria were made and used as inoculation for several local and imported soybean varieties used in this study. Plants were harvested at 20 days after inoculation. Number of nodules were counted, fresh weight of nodules and shoot were determined. S. Fredii J-TGS50 and S. Fredii USDA 192, USDA 201, USDA 205, USDA 206, USDA 217 were found different in their symbiotic properties. S. Fredii J-TGS50 formed nodules on same imported soybean. While there were no nodules obtained from the plant inoculated with S. Fredii USDA 192, USDA 201, USDA 205, USDA 206, USDA 217. S. Fredii J-TGS50 and recommended B. Japonicum USDA 110 formed nodule on several local soybean varieties. There was no differences between those two bacteria either in nodulation efficiency or in the effectiveness of the formed nodules. Results of this study can be concluded that S. Fredii, J-TGS50 is a native to Indonesian soil and it is a promising soybean nodule forming bacteria in Indonesia. Using indigenous bacteria is valuable. Since they are mostly more tolerant and adaptable than the introduced ones. An important aspect for the success of Biological Nitrogen Fixation (BNF) is insight in the structure of indigenous soybean rhizobia populations. Study on the biodiversity of soybean rhizobia was important conducted. (author)

A Monoclonal Antibody against Wnt-1 Induces Apoptosis in Human Cancer Cells

Directory of Open Access Journals (Sweden)

Biao He

2004-01-01

Full Text Available Aberrant activation of the Wingless-type (Wnt/β-catenin signaling pathway is associated with a variety of human cancers. Little is known regarding the role that Wnt ligands play in human carcinogenesis. To test whether a Wnt-1 signal is a survival factor in human cancer cells and thus may serve as a potential cancer therapeutic target, we investigated the effect of inhibition of Wnt-1 signaling in a variety of human cancer cell lines, including non small cell lung cancer, breast cancer, mesothelioma, and sarcoma. Both monoclonal antibody and RNA interference (RNAi were used to inhibit Wnt-1 signaling. We found that incubation of a monoclonal anti-Wnt-1 antibody induced apoptosis and caused downstream protein changes in cancer cells overexpressing Wnt-1. In contrast, apoptosis was not detected in cells lacking or having minimal Wnt-1 expression after the antibody incubation. RNAi targeting of Wnt-1 in cancer cells overexpressing Wnt-1 demonstrated similar downstream protein changes and induction of apoptosis. The antibody also suppressed tumor growth in vivo. Our results indicate that both monoclonal anti-Wnt-1 antibody and Wnt-1 siRNA inhibit Wnt-1 signaling and can induce apoptosis in human cancer cells. These findings hold promise as a novel therapeutic strategy for cancer.

Production of nodulation factors by Rhizobium meliloti: fermentation, purification and characterization of glycolipids.

Science.gov (United States)

Kohring, B; Baier, R; Niehaus, K; Pühler, A; Flaschel, E

1997-12-01

Lipooligosaccharides, synthesized by soil bacteria of the genera Rhizobium, are known to have multifunctional effects on a wide variety of plants as signal substances in symbiosis initiation, cell response elicitation and growth regulation. These so called nodulation (Nod-) factors represent interesting biotechnological products with respect to fundamental studies of symbiotic interactions as well as for potential applications. Therefore, a batch fermentation process on a scale of 30 l has been developed by means of the Rhizobium meliloti strain R.m. 1021 (pEK327) strongly overexpressing the genes for the synthesis of Nod factors. Induction by the flavone luteolin led to growth associated production of the lipooligosaccharides. Ultrafiltration was used for separating the biomass from the filtrate containing the extracellular Nod factors. Simultaneously, ultrafiltration reduced the amount of lipophilic substances, which would otherwise interfere with processes downstream. The second separation step consisted in adsorption on XAD-2, a nonspecific hydrophobic adsorptive resin. Adsorption of Nod factors was carried out by batch operation of a stirred tank. Desorption was performed by elution with methanol in a fixed bed column. A semi-preparative reversed phase HPLC (Polygoprep 100-30 C18) was chosen as the final purification step. The Nod factors were obtained after evaporation and lyophilization. Thus, about 600 mg of Nod factors were produced from 20 l of fermentation broth. The Nod factors produced by Rhizobium meliloti R.m. 1021 (pEK327) were identified by liquid secondary ion mass spectrometry and by reversed-phase HPLC as fluorescent derivatives of 2-aminobenzamide. The biological activity of the products was demonstrated by means of the root hair deformation (HAD-) assay.

Standardization of methodology to derivatization and radiolabeling of the anti-CD20 monoclonal antibody from bifunctional chelator DOTA-NHS-Ester

International Nuclear Information System (INIS)

Massicano, Adriana V.F.; Akanji, Akinkunmi G.; Santos, Josefina S.; Pujatti, Priscilla B.; Couto, Renata M.; Massicano, Felipe; Araujo, Elaine Bortoleti de

2009-01-01

Lymphomas are cancers of the lymphatic system, being the most common the non-Hodgkin lymphoma (NHL). The Radioimmunotherapy (RIT), that increase the cytotoxic effect of monoclonal antibodies (mAb), therefore labeling these Mab with different radioisotopes. RIT combines the specificity of the antibody and the toxicity of the radionuclides. The mAb anti-CD20 is used for treatment of relapse or refractory NHL. The labeling of anti- CD20 with 177 Lu, requires a bifunctional chelating agent that is designed to make a 'connect bridge' between the mAb and the radionuclide. The incorporation of the chelating group in mAb structure is called derivatization. The aim of this work is to study the derivatization of anti-CD20 antibody with DOTA-NHS-ester chelating group and labeling parameters to produce 177 Lu-DOTA-Anti CD20. Five milligrams of anti-CD20 were purified by dialysis against phosphate buffer pH 8.0 and derivatized with DOTA-NHS-ester in 1:250, 1:500 and 1:1000 molar ratios. The reaction was conducted for 1 hour in gently mixing at room temperature and remained under refrigeration for 48 hours. The reaction mixture was purified in gel column Sephadex G-50 ; the aliquots that presented greater protein concentration, were mixed and concentrated. The purified antibody conjugated was added to 111-185MBq (3-5mCi) of 177 LuCl3 diluted in 0.4 M acetate buffer pH 5.5. Radiochemical purity was less than 95% in all the molar ratios, indicating necessity of the purification after the labeling. The mAb derivatized showed stable when stored for to 1 month to 4 deg C and 4 days at -20 deg C. (author)

Anti-VEGF drugs: evidence for effectiveness

OpenAIRE

Evans, Jennifer; Virgili, Gianni

2014-01-01

Anti-vascular endothelial growth factors (anti-VEGF) are targeted biological drugs (e.g. monoclonal antibodies) that prevent the growth of new vessels by inhibiting VEGF. VEGF is a cytokine (cell-signalling protein) that promotes the growth of, and leakage from, new vessels. Currently there are three anti-VEGF drugs licensed for use in eye disease: pegaptanib, aflibercept, ranibizumab and one that is not licensed but is commonly used off-label (bevacizumab).

Anti-influenza M2e antibody

Science.gov (United States)

Bradbury, Andrew M [Santa Fe, NM

2011-12-20

Humanized recombinant and monoclonal antibodies specific for the ectodomain of the influenza virus M2 ion channel protein are disclosed. The antibodies of the invention have anti-viral activity and may be useful as anti-viral therapeutics and/or prophylactic/vaccine agents for inhibiting influenza virus replication and for treating individuals infected with influenza.

Porcine humoral immune responses to multiple injections of murine monoclonal antibodies

DEFF Research Database (Denmark)

Lohse, Louise; Nielsen, Jens; Kamstrup, Søren

2005-01-01

In humans and cattle, multiple injections of murine monoclonal antibodies (m-mAbs) induce anti-mouse antibody responses. The objectives of the present. study were to investigate whether a similar response could be seen when pigs were subjected to m-mAb therapy, and to study the kinetics of such a...

Generation and testing anti-influenza human monoclonal antibodies in a new humanized mouse model (DRAGA: HLA-A2. HLA-DR4. Rag1 KO. IL-2Rγc KO. NOD).

Science.gov (United States)

Mendoza, Mirian; Ballesteros, Angela; Qiu, Qi; Pow Sang, Luis; Shashikumar, Soumya; Casares, Sofia; Brumeanu, Teodor-D

2018-02-01

Pandemic outbreaks of influenza type A viruses have resulted in numerous fatalities around the globe. Since the conventional influenza vaccines (CIV) provide less than 20% protection for individuals with weak immune system, it has been considered that broadly cross-neutralizing antibodies may provide a better protection. Herein, we showed that a recently generated humanized mouse (DRAGA mouse; HLA-A2. HLA-DR4. Rag1KO. IL-2Rgc KO. NOD) that lacks the murine immune system and expresses a functional human immune system can be used to generate cross-reactive, human anti-influenza monoclonal antibodies (hu-mAb). DRAGA mouse was also found to be suitable for influenza virus infection, as it can clear a sub-lethal infection and sustain a lethal infection with PR8/A/34 influenza virus. The hu-mAbs were designed for targeting a human B-cell epitope ( 180 WGIHHPPNSKEQ QNLY 195 ) of hemagglutinin (HA) envelope protein of PR8/A/34 (H1N1) virus with high homology among seven influenza type A viruses. A single administration of HA 180-195 specific hu-mAb in PR8-infected DRAGA mice significantly delayed the lethality by reducing the lung damage. The results demonstrated that DRAGA mouse is a suitable tool to (i) generate heterotype cross-reactive, anti-influenza human monoclonal antibodies, (ii) serve as a humanized mouse model for influenza infection, and (iii) assess the efficacy of anti-influenza antibody-based therapeutics for human use.

Novel monoclonal treatments in severe asthma.

Science.gov (United States)

Meteran, Howraman; Meteran, Hanieh; Porsbjerg, Celeste; Backer, Vibeke

2017-12-01

To provide a general overview of the current biological treatments and discuss their potential anti-asthmatic effects. We reviewed articles in PubMed found using the search words "Asthma/therapy AND antibodies, monoclonal/therapeutic use AND cytokines." Only articles published in English since 2000 were considered. The search identified 29 studies; 8 additional studies were found by hand search, generating 37 studies. Of the 37 studies investigating biological treatments of asthma, 5 were on the effects of anti-IgE (omalizumab); 12 on anti-IL-5; 8 on anti-IL-13; 5 on anti-IL-4R-α; 3 on anti-IL-9; one on TNF-α; one on anti-IL-2R-α; one on TSLP (Thymic Stromal Lymphopoietin); and one on OX40L. Sample sizes ranged from 3 to 943 participants. Studies of therapies targeting IgE, IL-2, IL4R-α, IL-5, and IL-13 showed some efficacy, whereas those targeting TSLP, IL-9, and TNF-α lacked convincing effectiveness. Research on the biological treatment of asthma shows promising results. While anti-IgE (omalizumab) has been used in the treatment of asthma for some years, anti-IL-5 has recently been approved for use. The efficacy of results of other large studies with a longer duration is needed to draw a firm conclusion. Such studies should not only focus on clinical outcomes, but also consider asthma-related quality of life. Knowledge on the asthma phenotypes and identification of biomarkers associated with these will be useful for physicians considering the right treatment for the asthma patient.

Predominant antitumor effects by fully human anti-TRAIL-receptor2 (DR5) monoclonal antibodies in human glioma cells in vitro and in vivo

Science.gov (United States)

Nagane, Motoo; Shimizu, Saki; Mori, Eiji; Kataoka, Shiro; Shiokawa, Yoshiaki

2010-01-01

Tumor necrosis factor–related apoptosis-inducing ligand (TRAIL/Apo2 L) preferentially induces apoptosis in human tumor cells through its cognate death receptors DR4 or DR5, thereby being investigated as a potential agent for cancer therapy. Here, we applied fully human anti-human TRAIL receptor monoclonal antibodies (mAbs) to specifically target one of death receptors for TRAIL in human glioma cells, which could also reduce potential TRAIL-induced toxicity in humans. Twelve human glioma cell lines treated with several fully human anti-human TRAIL receptor mAbs were sensitive to only anti-DR5 mAbs, whereas they were totally insensitive to anti-DR4 mAb. Treatment with anti-DR5 mAbs exerted rapid cytotoxicity and lead to apoptosis induction. The cellular sensitivity was closely associated with cell-surface expression of DR5. Expression of c-FLIPL, Akt, and Cyclin D1 significantly correlated with sensitivity to anti-DR5 mAbs. Primary cultures of glioma cells were also relatively resistant to anti-DR5 mAbs, exhibiting both lower DR5 and higher c-FLIPL expression. Downregulation of c-FLIPL expression resulted in the sensitization of human glioma cells to anti-DR5 mAbs, whereas overexpression of c-FLIPL conferred resistance to anti-DR5 mAb. Treatment of tumor-burden nude mice with the direct agonist anti-DR5 mAb KMTR2 significantly suppressed growth of subcutaneous glioma xenografts leading to complete regression. Similarly, treatment of nude mice bearing intracerebral glioma xenografts with KMTR2 significantly elongated lifespan without tumor recurrence. These results suggest that DR5 is the predominant TRAIL receptor mediating apoptotic signals in human glioma cells, and sensitivity to anti-DR5 mAbs was determined at least in part by the expression level of c-FLIPL and Akt. Specific targeting of death receptor pathway through DR5 using fully human mAbs might provide a novel therapeutic strategy for intractable malignant gliomas. PMID:20511188

Establishment of H2Mab-119, an Anti-Human Epidermal Growth Factor Receptor 2 Monoclonal Antibody, Against Pancreatic Cancer.

Science.gov (United States)

Yamada, Shinji; Itai, Shunsuke; Nakamura, Takuro; Chang, Yao-Wen; Harada, Hiroyuki; Suzuki, Hiroyoshi; Kaneko, Mika K; Kato, Yukinari

2017-12-01

Human epidermal growth factor receptor 2 (HER2) is overexpressed in breast cancer and is associated with poor clinical outcomes. In addition, HER2 expression has been reported in other cancers, such as gastric, colorectal, lung, and pancreatic cancers. An anti-HER2 humanized antibody, trastuzumab, leads to significant survival benefits in patients with HER2-overexpressing breast cancers and gastric cancers. Herein, we established a novel anti-HER2 monoclonal antibody (mAb), H 2 Mab-119 (IgG 1 , kappa), and characterized its efficacy against pancreatic cancers using flow cytometry, Western blot, and immunohistochemical analyses. H 2 Mab-119 reacted with pancreatic cancer cell lines, such as KLM-1, Capan-2, and MIA PaCa-2, but did not react with PANC-1 in flow cytometry analysis. Western blot analysis also revealed a moderate signal for KLM-1 and a weak signal for MIA PaCa-2, although H 2 Mab-119 reacted strongly with LN229/HER2 cells. Finally, immunohistochemical analyses with H 2 Mab-119 revealed sensitive and specific reactions against breast and colon cancers but did not react with pancreatic cancers, indicating that H 2 Mab-119 is useful for detecting HER2 overexpression in pancreatic cancers using flow cytometry and Western blot analyses.

[Anti-angiogenic drugs].

Science.gov (United States)

Sato, Yasufumi

2010-06-01

Angiogenesis or neovascularization, the formation of neo-vessels, is a physiological phenomenon endued in vasculature, but is involved in various pathological conditions. Angiogenesis is required for tumor growth and metastasis, and thus constitutes an important target for the control of tumor progression. Indeed, the recent development of bevacizumab, a neutralizing anti-VEGF monoclonal antibody as the first anti-angiogenic drug, legalized the clinical merit of anti-angiogenesis in cancers. Thereafter, various drugs targeting VEGF-mediated signals have been developed to control tumor angiogenesis. Thus, anti-angiogenic drugs are now recognized in the clinic as a major step forward for the treatment of cancers. This review focuses on the current status of antiangiogenesis treatment in cancers.

Rhizobial symbiosis effect on the growth, metal uptake, and antioxidant responses of Medicago lupulina under copper stress.

Science.gov (United States)

Kong, Zhaoyu; Mohamad, Osama Abdalla; Deng, Zhenshan; Liu, Xiaodong; Glick, Bernard R; Wei, Gehong

2015-08-01

The effects of rhizobial symbiosis on the growth, metal uptake, and antioxidant responses of Medicago lupulina in the presence of 200 mg kg(-1) Cu(2+) throughout different stages of symbiosis development were studied. The symbiosis with Sinorhizobium meliloti CCNWSX0020 induced an increase in plant growth and nitrogen content irrespective of the presence of Cu(2+). The total amount of Cu uptake of inoculated plants significantly increased by 34.0 and 120.4% in shoots and roots, respectively, compared with non-inoculated plants. However, although the rhizobial symbiosis promoted Cu accumulation both in shoots and roots, the increase in roots was much higher than in shoots, thus decreasing the translocation factor and helping Cu phytostabilization. The rate of lipid peroxidation was significantly decreased in both shoots and roots of inoculated vs. non-inoculated plants when measured either 8, 13, or 18 days post-inoculation. In comparison with non-inoculated plants, the activities of superoxide dismutase and ascorbate peroxidase of shoots of inoculated plants exposed to excess Cu were significantly elevated at different stages of symbiosis development; similar increases occurred in the activities of superoxide dismutase, catalase, and glutathione reductase of inoculated roots. The symbiosis with S. meliloti CCNWSX0020 also upregulated the corresponding genes involved in antioxidant responses in the plants treated with excess Cu. The results indicated that the rhizobial symbiosis with S. meliloti CCNWSX0020 not only enhanced plant growth and metal uptake but also improved the responses of plant antioxidant defense to excess Cu stress.

Monoclonal Antibodies for Non-Hodgkin's Lymphoma: State of the Art and Perspectives

Directory of Open Access Journals (Sweden)

Giulia Motta

2010-01-01

Full Text Available Monoclonal antibodies have been the most successful therapeutics ever brought to cancer treatment by immune technologies. The use of monoclonal antibodies in B-cell Non-Hodgkin's lymphomas (NHL represents the greatest example of these advances, as the introduction of the anti-CD20 antibody rituximab has had a dramatic impact on how we treat this group of diseases today. Despite this success, several questions about how to optimize the use of monoclonal antibodies in NHL remain open. The best administration schedules, as well as the optimal duration of rituximab treatment, have yet to be determined. A deeper knowledge of the mechanisms underlying resistance to rituximab is also necessary in order to improve the activity of this and of similar therapeutics. Finally, new antibodies and biological agents are entering the scene and their advantages over rituximab will have to be assessed. We will discuss these issues and present an overview of the most significant clinical studies with monoclonal antibodies for NHL treatment carried out to date.

Biodistribution and pharmacokinetics of monoclonal antibody T1h and variant anti-CD6 murine 10D12 in healthy animals and in experimental arthritis model

International Nuclear Information System (INIS)

León, M; Hernández, I; Aldana, L; Ayra, F; Castro, Y; Leyva, R; García, L; Pérez, S.; Casaco, A.

2016-01-01

Biodistribution and pharmacokinetic of two radio labeled monoclonal antibodies was performed with the help of imaging techniques. Isotopic labeling was carried out by means of standardized methods. Pharmacokinetic evaluation was performed using the population approach and sparse data design. Introduction: Targeted therapy with monoclonal antibodies (MAb) is an efficient option for the treatment of rheumatoid arthritis. Th1 is a MAb anti human CD6 developed for the treatment of autoimmune disease and 10D12 is its counterpart anti murine CD6 developed as a pharmacological tool to get deep into the response mechanisms in animals models of rheumatoid arthritis.To investigate the behavior of both antibodies in the assay system, molecules were labeled with 125I to evaluate pharmacokinetic in healthy animals and with 99mTc to evaluate the antibody uptake in inflamed area of induced arthritis. Materials and methods: Antibodies were supplied by the Center of Molecular immunology. Iodination was performed by the iodogen method and technetium labeling was carried out directly by Schwarz method. Female C57BL6 from CENPALAB were used for experiments. Biodistribution and pharmacokinetic was performed by a sparse data design using the population approach. Uptake in region of inflammation was quantified by gammagraphy at the same time points of blood sampling. A compartmental model was build to quantify uptake kinetic. Pharmacokinetic profiles were analyzed using MONOLIX software version 4.2. Results: Minor pharmacokinetic differences were found between monoclonal antibodies labeled with 125I and 99mTc. As a humanized antibody, T1h shows a faster clearance than 10D12 and a biodistribution pattern reflecting preference for excretion mechanisms. The arthritis accumulation was not consistent with a targeted mediated uptake. On the other hand, radio labeled 10D12 shows an accumulation profile in arthritis with two peaks of maximum concentration representing an initial transit to

Therapeutic effects of anti-CD115 monoclonal antibody in mouse cancer models through dual inhibition of tumor-associated macrophages and osteoclasts.

Directory of Open Access Journals (Sweden)

Laetitia Fend

Full Text Available Tumor progression is promoted by Tumor-Associated Macrophages (TAMs and metastasis-induced bone destruction by osteoclasts. Both myeloid cell types depend on the CD115-CSF-1 pathway for their differentiation and function. We used 3 different mouse cancer models to study the effects of targeting cancer host myeloid cells with a monoclonal antibody (mAb capable of blocking CSF-1 binding to murine CD115. In mice bearing sub-cutaneous EL4 tumors, which are CD115-negative, the anti-CD115 mAb depleted F4/80(+ CD163(+ M2-type TAMs and reduced tumor growth, resulting in prolonged survival. In the MMTV-PyMT mouse model, the spontaneous appearance of palpable mammary tumors was delayed when the anti-CD115 mAb was administered before malignant transition and tumors became palpable only after termination of the immunotherapy. When administered to mice already bearing established PyMT tumors, anti-CD115 treatment prolonged their survival and potentiated the effect of chemotherapy with Paclitaxel. As shown by immunohistochemistry, this therapeutic effect correlated with the depletion of F4/80(+CD163(+ M2-polarized TAMs. In a breast cancer model of bone metastasis, the anti-CD115 mAb potently blocked the differentiation of osteoclasts and their bone destruction activity. This resulted in the inhibition of cancer-induced weight loss. CD115 thus represents a promising target for cancer immunotherapy, since a specific blocking antibody may not only inhibit the growth of a primary tumor through TAM depletion, but also metastasis-induced bone destruction through osteoclast inhibition.

Radioimmunodetection of human melanoma tumor xenografts with human monoclonal antibodies

International Nuclear Information System (INIS)

Gomibuchi, Makoto; Saxton, R.E.; Lake, R.R.; Katano, Mitsuo; Irie, R.F.

1986-01-01

A human IgM monoclonal antibody has been established that defines a tumor-associated membrane antigen expressed on human melanoma cells. The antigen has been identified as the ganglioside GD2. In this paper, the authors describe the potential usefulness of the human monoclonal antibody for radioimaging. Nude mice bearing tumors derived from a human melanoma cell line were used as a model. Antibody activity was degradated significantly after labeling with 131 I by the use of a modified chloramine-T method. After testing various concentrations, labeled antibody of a specific activity of 2.8μCi/μg produced the best results. Balb/c nude mice bearing a GD2-positive M14 melanoma cell line were injected with 10-30μg of labeled antibody, and its radiolocalization in different organs and in the whole body were evaluated. The best tumor image was obtained on Day 6. The labeled antibody uptake ratio between tumor and muscle was 9.2:1; the ratio between tumor and liver was 1.4:1. These studies represent the first report of experimental tumor imaging with human monoclonal antibody. Human monoclonals will probably prove to be superior reagents for tumor imaging in melanoma patients if the problem of anti-body radiolysis is resolved. (author)

Anti-tick monoclonal antibody applied by artificial capillary feeding in Rhipicephalus (Boophilus) microplus females.

Science.gov (United States)

Gonsioroski, Andressa Varella; Bezerra, Isis Abel; Utiumi, Kiyoko Uemura; Driemeier, David; Farias, Sandra Estrazulas; da Silva Vaz, Itabajara; Masuda, Aoi

2012-04-01

The tick Rhipicephalus microplus is an ectoparasite harmful to livestock, a vector of disease agents that affects meat and milk production. However, resistance to acaricides reflects the need for alternative tick control methods, among which vaccines have gained increasing relevance. In this scenario, monoclonal antibodies can be used to identify and characterize antigens that can be used as vaccine immunogens. Capillary tube artificial feeding of partially engorged R. microplus females with monoclonal antibodies against proteins from the gut of tick were used to test the effects of immunoglobulins in the physiology of the parasite. The results of artificial feeding showed that female ticks over 25mg and under 60 mg in weight performed better in the artificial feeding process, with a 94-168% weight increase after 24h of feeding. Results showed that artificial feeding of ticks proved to be a viable technique to study the effects of antibodies or drugs in the physiology of the parasite. One monoclonal antibody (BrBm2) induced decreased oviposition. Moreover, the antigen recognized by BrBm2 was identified as a 27-kDa protein and immunolabeled on digestive vesicles membranes of digestive cells of partially and fully engorged females. Copyright © 2012 Elsevier Inc. All rights reserved.

Rituximab for the treatment of refractory simultaneous anti-glomerular basement membrane (anti-GBM) and membranous nephropathy.

Science.gov (United States)

Bandak, Ghassan; Jones, Bruce A; Li, Jian; Yee, Jerry; Umanath, Kausik

2014-02-01

Antibody-mediated anti-glomerular basement membrane (anti-GBM) disease occurs rarely in the presence of another B-cell disorder, membranous nephropathy. The coexistence of these two autoimmune disorders would be anticipated to require differing, specific therapies targeted to each disease process. We describe a case of concomitant membranous nephropathy and anti-GBM disease in which conventional therapy, including steroids, plasmapheresis and cyclophosphamide, failed to attenuate the anti-GBM disease, yet responded to an alternative treatment of rituximab. This B-cell directed, monoclonal, chimeric antibody treatment substantially reduced anti-GBM antibody titers and led to discontinuation of plasmapheresis, while maintaining the remission of membranous nephropathy and anti-GBM disease.

Purification of bovine thyroid-stimulating hormone by a monoclonal antibody

International Nuclear Information System (INIS)

Lock, A.J.; van Denderen, J.; Aarden, L.A.

1988-01-01

A monoclonal antibody directed against bovine TSH was obtained by hybridoma technology. This antibody was specific for TSH and did not react with bovine LH and FSH. Affinity chromatography of crude TSH was performed on anti-TSH Sepharose. Bovine TSH was purified in a single step to near homogeneity by this technique, as shown by cation exchange chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified TSH. The biological activity of the hormone was not affected during the purification, as determined by [ 3 H]thymidine incorporation of the TSH-dependent FRTL5 cell line. The results indicate that affinity purification of TSH by means of a monoclonal antibody is a simple one-step procedure for the production of biologically active, highly purified TSH

Molecular and genetic characterization of the rhizopine catabolism (mocABRC) genes of Rhizobium meliloti L5-30.

Science.gov (United States)

Rossbach, S; Kulpa, D A; Rossbach, U; de Bruijn, F J

1994-10-17

Rhizopine (L-3-O-methyl-scyllo-inosamine, 3-O-MSI) is a symbiosis-specific compound, which is synthesized in nitrogen-fixing nodules of Medicago sativa induced by Rhizobium meliloti strain L5-30. 3-O-MSI is thought to function as an unusual growth substrate for R. meliloti L5-30, which carries a locus (mos) responsible for its synthesis closely linked to a locus (moc) responsible for its degradation. Here, the essential moc genes were delimited by Tn5 mutagenesis and shown to be organized into two regions, separated by 3 kb of DNA. The DNA sequence of a 9-kb fragment spanning the two moc regions was determined, and four genes were identified that play an essential role in rhizopine catabolism (mocABC and mocR). The analysis of the DNA sequence and the amino acid sequence of the deduced protein products revealed that MocA resembles NADH-dependent dehydrogenases. MocB exhibits characteristic features of periplasmic-binding proteins that are components of high-affinity transport systems. MocC does not share significant homology with any protein in the database. MocR shows homology with the GntR class of bacterial regulator proteins. These results suggest that the mocABC genes are involved in the uptake and subsequent degradation of rhizopine, whereas mocR is likely to play a regulatory role.

Monoclonal antibodies to Nocardia asteroides and Nocardia brasiliensis antigens.

OpenAIRE

Jiménez, T; Díaz, A M; Zlotnik, H

1990-01-01

Nocardia asteroides and Nocardia brasiliensis whole-cell extracts were used as antigens to generate monoclonal antibodies (MAbs). Six stable hybrid cell lines secreting anti-Nocardia spp. MAbs were obtained. These were characterized by enzyme-linked immunosorbent assay, Western blot (immunoblot), and immunofluorescence assay. Although all the MAbs exhibited different degrees of cross-reactivity with N. asteroides and N. brasiliensis antigens as well as with culture-filtrate antigens from Myco...

The development and applications of polyclonal and monoclonal antibodies for the detection of illicit drugs in saliva samples

OpenAIRE

Fanning, Lorna M.

2002-01-01

Anti-tetrahydrocannabinol (THC), anti-cocaine and anti-morphine polyclonal antibodies were produced. These antibodies were successfully applied to an ELISA format for the detection of THC, cocaine, and morphine in saliva samples. Monoclonal antibodies against amphetamine and its derivatives were produced using two different conjugates, amphetamine-bovine serum albumin and methamphetaminebovine serum albumin. Two successful clones were produced, and the antibodies were applied to an ELISA ...

Radiolabeled monoclonal antibodies for imaging and therapy: Potential, problems, and prospects: Scientific highlights

International Nuclear Information System (INIS)

Srivastava, S.C.; Buraggi, G.L.

1986-01-01

This meeting focused on areas of research on radiolabeled monoclonal antibodies. Topics covered included the production, purification, and fragmentation of monoclonal antibodies and immunochemistry of hybridomas; the production and the chemistry of radionuclides; the radiohalogenation and radiometal labeling techniques; the in-vivo pharmacokinetics of radiolabeled antibodies; the considerations of immunoreactivity of radiolabeled preparations; the instrumentation and imaging techniques as applied to radioimmunodetection; the radiation dosimetry in diagnostic and therapeutic use of labeled antibodies; the radioimmunoscintigraphy and radioimmunotherapy studies; and perspectives and directions for future research. Tutorial as well as scientific lectures describing the latest research data on the above topics were presented. Three workshop panels were convened on ''Methods for Determining Immunoreactivity of Radiolabeled Monoclonal Antibodies - Problems and Pitfalls,'' Radiobiological and Dosimetric Considerations for Immunotherapy with Labeled Antibodies,'' and ''The Human Anti-Mouse Antibody Response in Patients.''

Oxidized LDL-induced angiogenesis involves sphingosine 1-phosphate: prevention by anti-S1P antibody.

Science.gov (United States)

Camaré, Caroline; Trayssac, Magali; Garmy-Susini, Barbara; Mucher, Elodie; Sabbadini, Roger; Salvayre, Robert; Negre-Salvayre, Anne

2015-01-01

Neovascularization occurring in atherosclerotic lesions may promote plaque expansion, intraplaque haemorrhage and rupture. Oxidized LDL (oxLDL) are atherogenic, but their angiogenic effect is controversial; both angiogenic and anti-angiogenic effects have been reported. The angiogenic mechanism of oxLDL is partly understood, but the role of the angiogenic sphingolipid, sphingosine 1-phosphate (S1P), in this process is not known. Thus, we investigated whether S1P is involved in the oxLDL-induced angiogenesis and whether an anti-S1P monoclonal antibody can prevent this effect. Angiogenesis was assessed by capillary tube formation by human microvascular endothelial cells (HMEC-1) cultured on Matrigel and in vivo by the Matrigel plug assay in C57BL/6 mice. Human oxLDL exhibited a biphasic angiogenic effect on HMEC-1; low concentrations were angiogenic, higher concentrations were cytotoxic. The angiogenic response to oxLDL was blocked by the sphingosine kinase (SPHK) inhibitor, dimethylsphingosine, by SPHK1-siRNA and by an anti-S1P monoclonal antibody. Moreover, inhibition of oxLDL uptake and subsequent redox signalling by anti-CD36 and anti-LOX-1 receptor antibodies and by N-acetylcysteine, respectively, blocked SPHK1 activation and tube formation. In vivo, in the Matrigel plug assay, low concentrations of human oxLDL or murine oxVLDL also triggered angiogenesis, which was prevented by i.p. injection of the anti-S1P antibody. These data highlight the role of S1P in angiogenesis induced by oxLDL both in HMEC-1 cultured on Matrigel and in vivo in the Matrigel plug model in mice, and demonstrate that the anti-S1P antibody effectively blocks the angiogenic effect of oxLDL. © 2014 The British Pharmacological Society.

ADCC responses and blocking of EGFR-mediated signaling and cell growth by combining the anti-EGFR antibodies imgatuzumab and cetuximab in NSCLC cells

NARCIS (Netherlands)

Kol, Arjan; Terwisscha van Scheltinga, Anton; Pool, Martin; Gerdes, Christian; de Vries, Elisabeth; de Jong, Steven

2017-01-01

Imgatuzumab is a novel glycoengineered anti-epidermal growth factor receptor (EGFR) monoclonal antibody optimized to induce both antibody-dependent cellular cytotoxicity (ADCC) and EGFR signal transduction inhibition. We investigated antiEGFR monoclonal antibodies imgatuzumab and cetuximab-induced

Host-secreted antimicrobial peptide enforces symbiotic selectivity in Medicago truncatula.

Science.gov (United States)

Wang, Qi; Yang, Shengming; Liu, Jinge; Terecskei, Kata; Ábrahám, Edit; Gombár, Anikó; Domonkos, Ágota; Szűcs, Attila; Körmöczi, Péter; Wang, Ting; Fodor, Lili; Mao, Linyong; Fei, Zhangjun; Kondorosi, Éva; Kaló, Péter; Kereszt, Attila; Zhu, Hongyan

2017-06-27

Legumes engage in root nodule symbioses with nitrogen-fixing soil bacteria known as rhizobia. In nodule cells, bacteria are enclosed in membrane-bound vesicles called symbiosomes and differentiate into bacteroids that are capable of converting atmospheric nitrogen into ammonia. Bacteroid differentiation and prolonged intracellular survival are essential for development of functional nodules. However, in the Medicago truncatula - Sinorhizobium meliloti symbiosis, incompatibility between symbiotic partners frequently occurs, leading to the formation of infected nodules defective in nitrogen fixation (Fix - ). Here, we report the identification and cloning of the M. truncatula NFS2 gene that regulates this type of specificity pertaining to S. meliloti strain Rm41. We demonstrate that NFS2 encodes a nodule-specific cysteine-rich (NCR) peptide that acts to promote bacterial lysis after differentiation. The negative role of NFS2 in symbiosis is contingent on host genetic background and can be counteracted by other genes encoded by the host. This work extends the paradigm of NCR function to include the negative regulation of symbiotic persistence in host-strain interactions. Our data suggest that NCR peptides are host determinants of symbiotic specificity in M. truncatula and possibly in closely related legumes that form indeterminate nodules in which bacterial symbionts undergo terminal differentiation.

Minimal contribution of cell-bound antibodies to the immunoscintigraphy of inflamed joints with 99mTc-anti-CD4 monoclonal antibodies

International Nuclear Information System (INIS)

Kinne, R.W.; Palombo-Kinne, E.; Wolski, A.; Wolf, F.; Emmrich, F.; Becker, W.

2002-01-01

Aim: The cellular joint infiltrate in rheumatoid arthritis patients is rich in CD4-positive T-helper lymphocytes and macrophages, rendering anti-CD4 monoclonal antibodies (mAbs) suitable for specific immunoscintigraphy of human/experimental arthritis. Following intravenous injection, however, mAbs are present both in the free form and bound to CD4-positive, circulating monocytes and T-cells. Thus, the present study aimed at analyzing the relative contribution of the free and the cell-bound component to the imaging of inflamed joints in experimental adjuvant arthritis (AA). Methods: AA rat pertioneal macrophages or lymph node T-cells were incubated in vitro with saturating amounts of 99mTc-anti-CD4 mAb (W3/25) and injected i.v. into rats with AA. Results: In vitro release of 99m Tc-anti-CD4 mAb from the cells was limited (on average 1.57%/h for macrophages and 0.84%/h for T-cells). Following i.v. injection, whole body/joints scans and tissue measurements showed only negligible accumulation of radioactivity in inflamed ankle joints (tissue: 0.22 and 0.34% of the injected activity, respectively), whereas the radioactivity was concentrated in liver (tissue: 79% and 71%, respectively), kidney, and urinary bladder. Unlike macrophages, however, anti-CD4 mAb-coated T-cells significantly accumulated in lymphoid organs, the inflamed synovial membrane of the ankle joints, as well as in elbow and knee joints. Conclusion: While the overall contribution of cell-bound mAbs to the imaging of arthritic joints with anti-CD4 mAbs is minimal, differential accumulation of macrophages and T-cells in lymphoid organs and the inflamed synovial membrane indicates preferential migration patterns of these 2 cell populations in arthritic rats. Although only validated for 99m Tc-anti-CD4 mAbs, extrapolation of the results to other anticellular mAbs with similar affinity for their antigen may be possible. (orig.) [de

Radiobromination of humanized anti-HER2 monoclonal antibody trastuzumab using N-succinimidyl 5-bromo-3-pyridinecarboxylate, a potential label for immunoPET

Energy Technology Data Exchange (ETDEWEB)

Mume, Eskender [Organic Chemistry, Department of Chemistry, Uppsala University, S-751 24 Uppsala (Sweden); Orlova, Anna [Affibody AB, S-161 02 Bromma (Sweden); Malmstroem, Per-Uno [Division of Urology, Department of Surgical Sciences, Uppsala University, S-751 85 Uppsala (Sweden); Lundqvist, Hans [Unit of Biomedical Radiation Sciences, Rudbeck Laboratory, Uppsala University, S-751 85 Uppsala (Sweden); Sjoeberg, Stefan [Organic Chemistry, Department of Chemistry, Uppsala University, S-751 24 Uppsala (Sweden); Tolmachev, Vladimir [Unit of Biomedical Radiation Sciences, Rudbeck Laboratory, Uppsala University, S-751 85 Uppsala (Sweden)]. E-mail: vladimir.tolmachev@bms.uu.se

2005-08-01

Combining the specificity of radioimmunoscintigraphy and the high sensitivity of PET in an in vivo detection technique could improve the quality of nuclear diagnostics. Positron-emitting nuclide {sup 76}Br (T {sub 1/2}=16.2 h) might be a possible candidate for labeling monoclonal antibodies (mAbs) and their fragments, provided that the appropriate labeling chemistry has been established. For internalizing antibodies, such as the humanized anti-HER2 monoclonal antibody, trastuzumab, radiobromine label should be residualizing, i.e., ensuring that radiocatabolites are trapped intracellularly after the proteolytic degradation of antibody. This study evaluated the chemistry of indirect radiobromination of trastuzumab using N-succinimidyl 5-(tributylstannyl)-3-pyridinecarboxylate. Literature data indicated that the use of this method provided residualizing properties for iodine and astatine labels on some antibodies. An optimized 'one-pot' procedure produced an overall labeling efficiency of 45.5{+-}1.2% over 15 min. The bromine label was stable under physiological and denaturing conditions. The labeled trastuzumab retained its capacity to bind specifically to HER2-expressing SKOV-3 ovarian carcinoma cells in vitro (immunoreactivity more than 75%). However, in vitro cell test did not demonstrate that the radiobromination of trastuzumab using N-succinimidyl 5-bromo-3-pyridinecarboxylate improves cellular retention of radioactivity in comparison with the use of N-succinimidyl 4-bromobenzoate.

Application of four anti-human interferon-alpha monoclonal antibodies for immunoassay and comparative analysis of natural interferon-alpha mixtures

International Nuclear Information System (INIS)

Andersson, G.; Lundgren, E.; Ekre, H.P.

1991-01-01

Four different mouse monoclonal antibodies to human interferon-alpha (IFN-alpha) were evaluated for application in quantitative and comparative analysis of natural IFN-alpha mixtures. Binding to IFN-alpha subtypes in solution revealed individual reactivity patterns. These patterns changed if the IFN-alpha molecules were immobilized either passively to a surface or bound by another antibody. Also, substitution of a single amino acid in IFN-alpha 2 affected the binding, apparently by altering the conformation. Isoelectric focusing of three natural IFN-alpha preparations from different sources, followed by immunoblotting, resulted in individual patterns with each of the four mAbs and also demonstrated variation in the composition of the IFN-alpha preparations. None of the mAbs was subtype specific, but by combining the different mAbs, and also applying polyclonal anti-human IFN-alpha antibodies, it was possible to design sensitive sandwich ELISAs with broad or more limited IFN-alpha subtype specificity

Monoclonal Antibody Fragments for Targeting Therapeutics to Growth Plate Cartilage | NCI Technology Transfer Center | TTC

Science.gov (United States)

Researchers at The Eunice Kennedy Shriver National Institute on Child Health and Human Development (NICHD) have discovered monoclonal antibodies that bind to matrilin-3, a protein specifically expressed in cartilage tissue, that could be used for treating or inhibiting growth plate disorders, such as a skeletal dysplasia or short stature. The monoclonal antibodies can also be used to target therapeutic agents, such as anti-arthritis agents, to cartilage tissue. NICHD seeks statements of capability or interest from parties interested in collaborative research to co-develop, evaluate, or commercialize treatment of skeletal disorders using targeting antibodies.

Radiolabeled Humanized Anti-CD3 Monoclonal Antibody Visilizumab for Imaging Human T-Lymphocytes

NARCIS (Netherlands)

Malviya, Gaurav; D'Alessandria, Calogero; Bonanno, Elena; Vexler, Vladimir; Massari, Roberto; Trotta, Carlo; Scopinaro, Francesco; Dierckx, Rudi; Signore, Alberto

2009-01-01

Visilizumab is an IgG(2) humanized monoclonal antibody (mAb) characterized by non-Fc gamma R binding and specific to the CD3 antigen, expressed on more than 95% of circulating resting T-lymphocytes and on activated T-lymphocytes homing in inflamed tissues. We hypothesized that the use of a

Generation and Characterization of Anti-CD34 Monoclonal Antibodies that React with Hematopoietic Stem Cells

Science.gov (United States)

Aghebati Maleki, Leili; Majidi, Jafar; Baradaran, Behzad; Movassaghpour, Aliakbar; Abdolalizadeh, Jalal

2014-01-01

CD34 is a type I membrane protein with a molecular mass of approximately 110 kDa. This antigen is associated with human hematopoietic progenitor cells and is a differentiation stage-specific leukocyte antigen. In this study we have generated and characterized monoclonal antibodies (mAbs) directed against a CD34 marker. Mice were immunized with two keyhole lympet hemocyanin (KLH)-conjugated CD34 peptides. Fused cells were grown in hypoxanthine, aminopterine and thymidine (HAT) selective medium and cloned by the limiting dilution (L.D) method. Several monoclones were isolated by three rounds of limited dilutions. From these, we chose stable clones that presented sustained antibody production for subsequent characterization. Antibodies were tested for their reactivity and specificity to recognize the CD34 peptides and further screened by enzyme-linked immunosorbent assay (ELISA) and Western blotting analyses. One of the mAbs (3D5) was strongly reactive against the CD34 peptide and with native CD34 from human umbilical cord blood cells (UCB) in ELISA and Western blotting analyses. The results have shown that this antibody is highly specific and functional in biomedical applications such as ELISA and Western blot assays. This monoclonal antibodies (mAb) can be a useful tool for isolation and purification of human hematopoietic stem cells (HSCs). PMID:24611141

DETECTION AND PURIFICATION OF TYROSINE-SULFATED PROTEINS USING A NOVEL ANTI-SULFOTYROSINE MONOCLONAL ANTIBODY*

Science.gov (United States)

Hoffhines, Adam J.; Damoc, Eugen; Bridges, Kristie G.; Leary, Julie A.; Moore, Kevin L.

2006-01-01

Protein-tyrosine O-sulfation is a post-translational modification mediated by one of two Golgi tyrosylprotein sulfotransferases (TPST-1 & TPST-2) that catalyze the transfer of sulfate to tyrosine residues in secreted and transmembrane proteins. Tyrosine sulfation plays a role in protein-protein interactions in several well-defined systems. Although dozens of tyrosine-sulfated proteins are known, many more are likely to exist and await description. Advancing our understanding of the importance of tyrosine sulfation in biological systems requires the development of new tools for the detection and study of tyrosine-sulfated proteins. We have developed a novel anti-sulfotyrosine monoclonal antibody, called PSG2, that binds with high affinity and exquisite specificity to sulfotyrosine residues in peptides and proteins independent of sequence context. We show that it can detect tyrosinesulfated proteins in complex biological samples and can be used as a probe to assess the role of tyrosine sulfation in protein function. We also demonstrate the utility of PSG2 in the purification of tyrosine-sulfated proteins from crude tissue samples. Finally, Western blot analysis using PSG2 indicates that certain sperm/epididymal proteins are undersulfated in Tpst2−/− mice. This indicates that TPST-1 and TPST-2 have distinct macromolecular substrate specificities and provides clues as to the molecular mechanism of the infertility of Tpst2−/− males. PSG2 should be widely applicable for identification of tyrosine-sulfated proteins in other systems and organisms. PMID:17046811

Heterogeneity of Polyneuropathy Associated with Anti-MAG Antibodies

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Laurent Magy

2015-01-01

Full Text Available Polyneuropathy associated with IgM monoclonal gammopathy and anti-myelin associated glycoprotein (MAG antibodies is an immune-mediated demyelinating neuropathy. The pathophysiology of this condition is likely to involve anti-MAG antibody deposition on myelin sheaths of the peripheral nerves and it is supposed to be distinct from chronic inflammatory demyelinating neuropathy (CIDP, another immune-mediated demyelinating peripheral neuropathy. In this series, we have retrospectively reviewed clinical and laboratory findings from 60 patients with polyneuropathy, IgM gammopathy, and anti-MAG antibodies. We found that the clinical picture in these patients is highly variable suggesting a direct link between the monoclonal gammopathy and the neuropathy. Conversely, one-third of patients had a CIDP-like phenotype on electrodiagnostic testing and this was correlated with a low titer of anti-MAG antibodies and the absence of widening of myelin lamellae. Our data suggest that polyneuropathy associated with anti-MAG antibodies is less homogeneous than previously said and that the pathophysiology of the condition is likely to be heterogeneous as well with the self-antigen being MAG in most of the patients but possibly being another component of myelin in the others.

Effects of in vivo injection of anti-chicken CD25 monoclonal antibody on regulatory T cell depletion and CD4+CD25- T cell properties in chickens.

Science.gov (United States)

Shanmugasundaram, Revathi; Selvaraj, Ramesh K

2012-03-01

Regulatory T cells (Tregs) are defined as CD4(+)CD25(+) cells in chickens. This study examined the effects of an anti-chicken CD25 monoclonal antibody injection (0.5 mg/bird) on in vivo depletion of Tregs and the properties of CD4(+)CD25(-) cells in Treg-depleted birds. The CD4(+)CD25(+) cell percentage in the blood was lower at 8 d post injection than at 0 d. Anti-CD25-mediated CD4(+)CD25(+) cell depletion in blood was maximum at 12 d post injection. The anti-CD25 antibody injection depleted CD4(+)CD25(+) cells in the spleen and cecal tonsils, but not in the thymus, at 12 d post antibody injection. CD4(+)CD25(-) cells from the spleen and cecal tonsils of birds injected with the anti-chicken CD25 antibody had higher proliferation and higher IL-2 and IFNγ mRNA amounts than the controls at 12 d post injection. At 20 d post injection, CD4(+)CD25(+) cell percentages in the blood, spleen and thymus were comparable to that of the 0 d post injection. It could be concluded that anti-chicken CD25 injection temporarily depleted Treg population and increased and IL-2 and IFNγ mRNA amounts in CD4(+)CD25(-) cells at 12d post injection. Copyright © 2011 Elsevier Ltd. All rights reserved.

Inhibition of the alternative complement activation pathway in traumatic brain injury by a monoclonal anti-factor B antibody: a randomized placebo-controlled study in mice

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Holers V Michael

2007-05-01

Full Text Available Abstract Background The posttraumatic response to traumatic brain injury (TBI is characterized, in part, by activation of the innate immune response, including the complement system. We have recently shown that mice devoid of a functional alternative pathway of complement activation (factor B-/- mice are protected from complement-mediated neuroinflammation and neuropathology after TBI. In the present study, we extrapolated this knowledge from studies in genetically engineered mice to a pharmacological approach using a monoclonal anti-factor B antibody. This neutralizing antibody represents a specific and potent inhibitor of the alternative complement pathway in mice. Methods A focal trauma was applied to the left hemisphere of C57BL/6 mice (n = 89 using a standardized electric weight-drop model. Animals were randomly assigned to two treatment groups: (1 Systemic injection of 1 mg monoclonal anti-factor B antibody (mAb 1379 in 400 μl phosphate-buffered saline (PBS at 1 hour and 24 hours after trauma; (2 Systemic injection of vehicle only (400 μl PBS, as placebo control, at identical time-points after trauma. Sham-operated and untreated mice served as additional negative controls. Evaluation of neurological scores and analysis of brain tissue specimens and serum samples was performed at defined time-points for up to 1 week. Complement activation in serum was assessed by zymosan assay and by murine C5a ELISA. Brain samples were analyzed by immunohistochemistry, terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL histochemistry, and real-time RT-PCR. Results The mAb 1379 leads to a significant inhibition of alternative pathway complement activity and to significantly attenuated C5a levels in serum, as compared to head-injured placebo-treated control mice. TBI induced histomorphological signs of neuroinflammation and neuronal apoptosis in the injured brain hemisphere of placebo-treated control mice for up to 7 days. In contrast, the

Toxic effects of arsenic on Sinorhizobium-Medicago sativa symbiotic interaction

Energy Technology Data Exchange (ETDEWEB)

Pajuelo, Eloisa [Department of Microbiology and Parasitology, Faculty of Pharmacy, University of Seville, 41012 Seville (Spain); Rodriguez-Llorente, Ignacio D. [Department of Microbiology and Parasitology, Faculty of Pharmacy, University of Seville, 41012 Seville (Spain)], E-mail: irodri@us.es; Dary, Mohammed; Palomares, Antonio J. [Department of Microbiology and Parasitology, Faculty of Pharmacy, University of Seville, 41012 Seville (Spain)

2008-07-15

Recently, the Rhizobium-legume symbiotic interaction has been proposed as an interesting tool in bioremediation. However, little is known about the effect of most common contaminants on this process. The phytotoxic effects of arsenic on nodulation of Medicago sativa have been examined in vitro using the highly arsenic resistant and symbiotically effective Sinorhizobium sp. strain MA11. The bacteria were able to grow on plates containing As concentrations as high as 10 mM. Nevertheless, as little as 25-35 {mu}M arsenite produced a 75% decrease in the total number of nodules, due to a 90% reduction in the number of rhizobial infections, as could be determined using the strain MA11 carrying a lacZ reporter gene. This effect was associated to root hair damage and a shorter infective root zone. However, once nodulation was established nodule development seemed to continue normally, although earlier senescence could be observed in nodules of arsenic-grown plants. - First steps of nodulation of alfalfa, in particular infection thread formation, are more sensitive to As than nitrogen fixation due to plant effects.

Nuclear medicine: Monoclonal antibodies

International Nuclear Information System (INIS)

Endo, K.; Sakahara, H.; Koizumi, M.; Kawamura, Y.; Torizuka, K.; Yokoyama, A.

1986-01-01

Antitumor monoclonal antibody was successfully labeled with Tc-99m by using dithiosemicarbazone (DTS) as a bifunctional chelating agent. In the first step, DTS was coupled to antibody without loss of immunoreactivity; the compound then efficiently formed a neutral 1:1 chelate with pentavalent or tetravalent Tc-99m. Imaging with Tc-99m-labeled monoclonal antibody to human osteosarcoma (OST-7) clearly displayed a small tumor in nude mice at 6 and 24 hours after intravenous administration. The tumor-to-blood ratio of the Tc-99m-labeled monoclonal antibody was higher than that of a radioiodinated antibody and similar to that of an In-111-labeled antibody. Thus, conjugation of DTS to monoclonal antibody followed by radiometalation is a simple and efficient method of preparing Tc-99m-labeled monoclonal antibody

Three phylogenetic groups of nodA and nifH genes in Sinorhizobium and Mesorhizobium isolates from leguminous trees growing in Africa and Latin America.

Science.gov (United States)

Haukka, K; Lindström, K; Young, J P

1998-02-01

The diversity and phylogeny of nodA and nifH genes were studied by using 52 rhizobial isolates from Acacia senegal, Prosopis chilensis, and related leguminous trees growing in Africa and Latin America. All of the strains had similar host ranges and belonged to the genera Sinorhizobium and Mesorhizobium, as previously determined by 16S rRNA gene sequence analysis. The restriction patterns and a sequence analysis of the nodA and nifH genes divided the strains into the following three distinct groups: sinorhizobia from Africa, sinorhizobia from Latin America, and mesorhizobia from both regions. In a phylogenetic tree also containing previously published sequences, the nodA genes of our rhizobia formed a branch of their own, but within the branch no correlation between symbiotic genes and host trees was apparent. Within the large group of African sinorhizobia, similar symbiotic gene types were found in different chromosomal backgrounds, suggesting that transfer of symbiotic genes has occurred across species boundaries. Most strains had plasmids, and the presence of plasmid-borne nifH was demonstrated by hybridization for some examples. The nodA and nifH genes of Sinorhizobium teranga ORS1009T grouped with the nodA and nifH genes of the other African sinorhizobia, but Sinorhizobium saheli ORS609T had a totally different nodA sequence, although it was closely related based on the 16S rRNA gene and nifH data. This might be because this S. saheli strain was originally isolated from Sesbania sp., which belongs to a different cross-nodulation group than Acacia and Prosopis spp. The factors that appear to have influenced the evolution of rhizobial symbiotic genes vary in importance at different taxonomic levels.

The development of glioblastoma multiforme reactive monoclonal antibodies and their use in drug targeting

International Nuclear Information System (INIS)

Klaich, G.M.

1989-01-01

The objectives of this project were to develop monoclonal antibodies reactive with the tumor glioblastoma multiforme and to use them to study and develop new treatment modalities for this disease. A tumor antigen enriched immunogen, prepared by immunoaffinity chromatography, was compared to a whole tumor homogenate immunogen with the difference in the yield of tumor reactive, normal brain unreactive monoclonal antibodies proving to be significant. Monoclonal antibody A7, reactive with tumor tissue but unreactive with normal tissue, was isotyped to be an IgG2a immunoglobulin and could be purified to electrophoretic homogeneity by using serum-free culture conditions and protein A sepharose chromatography. Monoclonal antibody A7 is noncytotoxic as measured by the 3 H-nicotinamide release assay and binds to a 138 kd membrane antigen which is not internalized. Localization studies using 14 C-labeled monoclonal antibody A7 and the U-87 MG nude mouse xenograft model resulted in a tumor:serum ratio of 1.25:1.0 as compared to 0.29:1.0 for the negative control. A monoclonal antibody A7-doxorubicin immunoconjugate proved to be more cytotoxic than free doxorubicin in vitro while lethality studies using Swiss mice demonstrated the lack of toxicity of the immunoconjugate as compared to free doxorubicin. In vivo chemotherapy studies using the U-87 MG nude mouse xenograft failed to demonstrate any immunoconjugate anti-tumor activity which may be attributable to the route of administration

Binding-site analysis of opioid receptors using monoclonal anti-idiotypic antibodies

International Nuclear Information System (INIS)

Conroy, W.G.

1988-01-01

Structural relatedness between the variable region of anti-ligand antibodies and opioid binding sites allowed the generation of anti-idiotypic antibodies which recognized opioid receptors. The IgG 3 k antibodies which bound to opioid receptors were obtained when an anti-morphine antiserum was the idiotype. Both antibodies bound to opioid receptors, but only one of these blocked the binding of [ 3 H]naloxone. The antibody which did not inhibit the binding of [ 3 H]naloxone was itself displaced from the receptor by opioid ligands. The unique binding properties displayed by this antibody indicated that anti-idiotypic antibodies are not always a perfect image of the original ligand, and therefore may be more useful than typical ligands as probes for the receptor. An auto-anti-idiotypic technique was successfully used to obtain anti-opioid receptor antibodies. Another IgG 3 k antibody that blocked the binding of [ 3 H]naloxone to rat brain opioid receptors was obtained when a mouse was immunized with naloxone conjugated to bovine serum albumin. These data confirmed that an idiotype-anti-idiotype network which can generate an anti-receptor antibody normally functions when an opioid ligand is introduced into an animal in an immunogenic form

Obtenção e caracterização de anticorpo monoclonal murino anti-fator VIII da coagulação sangüínea Attainment and characterization of murine monoclonal anti-factor VIII antibodies

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Rosana Rossi-Ferreira

2006-06-01

Full Text Available Entre os avanços da engenharia celular e biotecnologia nas últimas décadas, destaca-se a produção de anticorpos monoclonais murinos (AcMm utilizados no aprimoramento diagnóstico nas rotinas laboratoriais. A produção de fator VIII de alta pureza sempre foi o desejo e a preocupação das indústrias de hemoderivados para tratamento de pacientes portadores de hemofilia A, porém este produto inexiste no Brasil, sendo necessária sua obtenção no mercado internacional a custos elevados. O trabalho tem por objetivo a produção de AcMm anti-fator VIII humano (FVIII H através da expansão dos clones e caracterização imunoquímica do anticorpo. Camundongos Balb/c foram imunizados com FVIII H purificado como também proveniente de crioprecipitado e as células esplênicas dos animais foram fusionadas com células mielomatosas murinas segundo o método descrito por Kohler e Milstein para produção de híbridos em cultura. Foram testados 1.983 híbridos dos quais 105 foram submetidos à clonagem. Destes, 39 obtiveram monoclonalidade e 7 destes clones foram caracterizados através de técnicas de immunoblotting. Foram submetidas à purificação por cromatografia três imunoglobulinas de diferentes classes pertencentes aos clones LAMB1-10A1A4, LAMB1-17A1A1 e LAMB1-24A2A1. A imunoglobulina purificada pertencente ao clone LAMB1-10A1A4 foi adsorvida em coluna de imunoafinidade para purificação de concentrado de FVIII proveniente de crioprecipitado plasmático.Among the advances in cellular engineering and biotechnology over the last decades, the production of murine monoclonal antibodies (AcMm, used to improve laboratory diagnoses, stands out. The production of very pure factor VIII has always been a concern of suppliers of blood products to treat patients with hemophilia A and this product is still not produced in Brazil. Hence, it can only be attained on the international market at a high cost. The aim of this work was to produce AcMm anti

Development and characterization of a TAPIR-like mouse monoclonal antibody to amyloid-beta.

Science.gov (United States)

Wang, Jun; Hara, Hideo; Makifuchi, Takao; Tabira, Takeshi

2008-06-01

Tissue amyloid plaque immuno-reactive (TAPIR) antibody was better related to the effect of immunotherapy in Alzheimer's disease (AD) than ELISA antibody. Here we used a hybridoma technique to develop a TAPIR-like anti-human amyloid-beta (Abeta) mouse monoclonal antibody. The obtained monoclonal antibody, 3.4A10, was an IgG2b isotype and recognized N-terminal portion of Abeta1-42 without binding denatured or native amyloid-beta protein precursor. It had higher affinity to Abeta1-42 than to Abeta1-40 by Biacore affinity analysis and stained preferably the peripheral part of senile plaques and recognized the plaque core less than 4G8. It inhibited the Abeta1-42 fibril formation as well as degraded pre-aggregated Abeta1-42 peptide in a thioflavin T fluorescence spectrophotometry assay. The in vivo studies showed that 3.4A10 treatment decreased amyloid burden compared to the control group and significantly reduced Abeta42 levels rather than Abeta40 levels in brain lysates as well as the Abeta*56 oligomer (12mer) in TBS fraction of the brain lysates. 3.4A10 entered brain and decorated some plaques, which is surrounded by more Iba1-positive microglia. 3.4A10 therapy did not induce lymphocytic infiltration and obvious increase in microhemorrhage. We conclude that 3.4A10 is a TAPIR-like anti-human amyloid monoclonal antibody, and has a potential of therapeutic application for AD.

Novel rabies virus-neutralizing epitope recognized by human monoclonal antibody: Fine mapping and escape mutant analysis

NARCIS (Netherlands)

Marissen, W.E.; Kramer, R.A.; Rice, A.; Weldon, W.C.; Niezgoda, M.; Faber, M.; Slootstra, J.W.; Meloen, R.H.; Clijsters-van der Horst, M.; Visser, T.J.; Jongeneelen, M.; Thijsse, S.; Throsby, M.; Kruif, de J.; Rupprecht, C.E.; Dietzschold, B.; Goudsmit, J.; Bakker, A.B.H.

2005-01-01

Anti-rabies virus immunoglobulin combined with rabies vaccine protects humans from lethal rabies infections. For cost and safety reasons, replacement of the human or equine polyclonal immunoglobulin is advocated, and the use of rabies virus-specific monoclonal antibodies (MAbs) is recommended. We

Novel rabies virus-neutralizing epitope recognized by human monoclonal antibody: fine mapping and escape mutant analysis

NARCIS (Netherlands)

Marissen, Wilfred E.; Kramer, R. Arjen; Rice, Amy; Weldon, William C.; Niezgoda, Michael; Faber, Milosz; Slootstra, Jerry W.; Meloen, Rob H.; Clijsters-van der Horst, Marieke; Visser, Therese J.; Jongeneelen, Mandy; Thijsse, Sandra; Throsby, Mark; de Kruif, John; Rupprecht, Charles E.; Dietzschold, Bernhard; Goudsmit, Jaap; Bakker, Alexander B. H.

2005-01-01

Anti-rabies virus immunoglobulin combined with rabies vaccine protects humans from lethal rabies infections. For cost and safety reasons, replacement of the human or equine polyclonal immunoglobulin is advocated, and the use of rabies virus-specific monoclonal antibodies (MAbs) is recommended. We

Use of monoclonal antibodies against Hendra and Nipah viruses in an antigen capture ELISA

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Spiropoulou Christina F

2010-06-01

Full Text Available Abstract Background Outbreaks of Hendra (HeV and Nipah (NiV viruses have been reported starting in 1994 and 1998, respectively. Both viruses are capable of causing fatal disease in humans and effecting great economical loss in the livestock industry. Results Through screening of hybridomas derived from mice immunized with γ-irradiated Nipah virus, we identified two secreted antibodies; one reactive with the nucleocapsid (N protein and the other, the phosphoprotein (P of henipaviruses. Epitope mapping and protein sequence alignments between NiV and HeV suggest the last 14 amino acids of the carboxyl terminus of the N protein is the target of the anti-N antibody. The anti-P antibody recognizes an epitope in the amino-terminal half of P protein. These monoclonal antibodies were used to develop two antigen capture ELISAs, one for virus detection and the other for differentiation between NiV and HeV. The lower limit of detection of the capture assay with both monoclonal antibodies was 400 pfu. The anti-N antibody was used to successfully detect NiV in a lung tissue suspension from an infected pig. Conclusion The antigen capture ELISA developed is potentially affordable tool to provide rapid detection and differentiation between the henipaviruses.

Epitope Sequences in Dengue Virus NS1 Protein Identified by Monoclonal Antibodies

Directory of Open Access Journals (Sweden)

Leticia Barboza Rocha

2017-10-01

Full Text Available Dengue nonstructural protein 1 (NS1 is a multi-functional glycoprotein with essential functions both in viral replication and modulation of host innate immune responses. NS1 has been established as a good surrogate marker for infection. In the present study, we generated four anti-NS1 monoclonal antibodies against recombinant NS1 protein from dengue virus serotype 2 (DENV2, which were used to map three NS1 epitopes. The sequence 193AVHADMGYWIESALNDT209 was recognized by monoclonal antibodies 2H5 and 4H1BC, which also cross-reacted with Zika virus (ZIKV protein. On the other hand, the sequence 25VHTWTEQYKFQPES38 was recognized by mAb 4F6 that did not cross react with ZIKV. Lastly, a previously unidentified DENV2 NS1-specific epitope, represented by the sequence 127ELHNQTFLIDGPETAEC143, is described in the present study after reaction with mAb 4H2, which also did not cross react with ZIKV. The selection and characterization of the epitope, specificity of anti-NS1 mAbs, may contribute to the development of diagnostic tools able to differentiate DENV and ZIKV infections.

A mathematical model of a recombinant humanized anti-cocaine monoclonal antibody's effects on cocaine pharmacokinetics in mice.

Science.gov (United States)

Wetzel, Hanna N; Zhang, Tongli; Norman, Andrew B

2017-09-01

A recombinant humanized anti-cocaine monoclonal antibody (mAb), h2E2, is at an advanced stage of pre-clinical development as an immunotherapy for cocaine abuse. It is hypothesized that h2E2 binds to and sequesters cocaine in the blood. A three-compartment model of the effects of h2E2 on cocaine's distribution was constructed. The model assumes that h2E2 binds to cocaine and that the h2E2-cocaine complex does not enter the brain but distributes between the central and peripheral compartments. Free cocaine is eliminated from both the central and peripheral compartments, and h2E2 and the h2E2-cocaine complex are eliminated from the central compartment only. This model was tested against a new dataset measuring cocaine concentrations in the brain and plasma over 1h in the presence and absence of h2E2. The mAb significantly increased plasma cocaine concentrations with a concomitant significant decrease in brain concentration. Plasma concentrations declined over the 1-hour sampling period in both groups. With a set of parameters within reasonable physiological ranges, the three-compartment model was able to qualitatively and quantitatively simulate the increased plasma concentration in the presence of the antibody and the decreased peak brain concentration in the presence of antibody. Importantly, the model explained the decline in plasma concentrations over time as distribution of the cocaine-h2E2 complex into a peripheral compartment. This model will facilitate the targeting of ideal mAb PK/PD properties thus accelerating the identification of lead candidate anti-drug mAbs. Copyright © 2017 Elsevier Inc. All rights reserved.

Characterization of Palytoxin Binding to HaCaT Cells Using a Monoclonal Anti-Palytoxin Antibody

Directory of Open Access Journals (Sweden)

Chiara Florio

2013-02-01

Full Text Available Palytoxin (PLTX is the reference compound for a group of potent marine biotoxins, for which the molecular target is Na+/K+-ATPase. Indeed, ouabain (OUA, a potent blocker of the pump, is used to inhibit some PLTX effects in vitro. However, in an effort to explain incomplete inhibition of PLTX cytotoxicity, some studies suggest the possibility of two different binding sites on Na+/K+-ATPase. Hence, this study was performed to characterize PLTX binding to intact HaCaT keratinocytes and to investigate the ability of OUA to compete for this binding. PLTX binding to HaCaT cells was demonstrated by immunocytochemical analysis after 10 min exposure. An anti-PLTX monoclonal antibody-based ELISA showed that the binding was saturable and reversible, with a Kd of 3 × 10−10 M. However, kinetic experiments revealed that PLTX binding dissociation was incomplete, suggesting an additional, OUA-insensitive, PLTX binding site. Competitive experiments suggested that OUA acts as a negative allosteric modulator against high PLTX concentrations (0.3–1.0 × 10−7 M and possibly as a non-competitive antagonist against low PLTX concentrations (0.1–3.0 × 10−9 M. Antagonism was supported by PLTX cytotoxicity inhibition at OUA concentrations that displaced PLTX binding (1 × 10−5 M. However, this inhibition was incomplete, supporting the existence of both OUA-sensitive and -insensitive PLTX binding sites.

Increased T cell proliferative responses to islet antigens identify clinical responders to anti-CD20 monoclonal antibody (rituximab) therapy in type 1 diabetes.

Science.gov (United States)

Herold, Kevan C; Pescovitz, Mark D; McGee, Paula; Krause-Steinrauf, Heidi; Spain, Lisa M; Bourcier, Kasia; Asare, Adam; Liu, Zhugong; Lachin, John M; Dosch, H Michael

2011-08-15

Type 1 diabetes mellitus is believed to be due to the autoimmune destruction of β-cells by T lymphocytes, but a single course of rituximab, a monoclonal anti-CD20 B lymphocyte Ab, can attenuate C-peptide loss over the first year of disease. The effects of B cell depletion on disease-associated T cell responses have not been studied. We compare changes in lymphocyte subsets, T cell proliferative responses to disease-associated target Ags, and C-peptide levels of participants who did (responders) or did not (nonresponders) show signs of β-cell preservation 1 y after rituximab therapy in a placebo-controlled TrialNet trial. Rituximab decreased B lymphocyte levels after four weekly doses of mAb. T cell proliferative responses to diabetes-associated Ags were present at baseline in 75% of anti-CD20- and 82% of placebo-treated subjects and were not different over time. However, in rituximab-treated subjects with significant C-peptide preservation at 6 mo (58%), the proliferative responses to diabetes-associated total (p = 0.032), islet-specific (p = 0.048), and neuronal autoantigens (p = 0.005) increased over the 12-mo observation period. This relationship was not seen in placebo-treated patients. We conclude that in patients with type 1 diabetes mellitus, anti-B cell mAb causes increased proliferative responses to diabetes Ags and attenuated β-cell loss. The way in which these responses affect the disease course remains unknown.

Molecular mechanism for the action of the anti-CD44 monoclonal antibody MEM-85

Czech Academy of Sciences Publication Activity Database

Škerlová, Jana; Král, V.; Kachala, M.; Fábry, M.; Bumba, L.; Svergun, D. I.; Tošner, Z.; Veverka, Václav; Řezáčová, Pavlína

2015-01-01

Roč. 191, č. 2 (2015), s. 214-223 ISSN 1047-8477 R&D Projects: GA MŠk(CZ) LK11205; GA MŠk(CZ) LO1304 Institutional support: RVO:61388963 Keywords : CD44 * epitope mapping * monoclonal antibody * MEM-85 * NMR * SAXS Subject RIV: CE - Biochemistry Impact factor: 2.570, year: 2015

Library of monoclonal antibodies against brush border membrane epithelial antigens

International Nuclear Information System (INIS)

Behar, M.; Katz, A.; Silverman, M.

1986-01-01

A purified fraction of proximal tubule brush border membranes (BBM) was prepared from dog kidney and used to immunize mice. The standard technique of hybridoma production was followed as described by Kohler and Milstein. Production of antibodies was detected by indirect immunofluorescence on dog kidney slices and by immunodot against the purified fraction on nitrocellulose. Five hybrids exhibited anti BBM activity. These were cloned twice and yielded stable cell lines producing IgG type monoclonal antibodies against BBM. They were designated A 1 , C 7 , D 3 , D 7 and H 4 . As a family these five monoclonals have broad tissue specificity, i.e. positive staining of the surface mucosa of intestinal kidney proximal tubules. D 3 exhibits even broader specificity for epithelium reacting with bile canaliculi and choroid plexus. The authors have verified that at least 4/5 antibodies are directed against BBM protein as revealed by immunoprecipitation of solubilized BBM and detected by Coomassie blue staining or autoradiography of lactoperoxidase labelled BBM. Most interestingly all antibodies bind to the surface of LL CPK 1 cells, a continuous pig kidney cell line of undefined origin but exhibiting many characteristics of proximal tubule cells. The library of monoclonal antibodies obtained provide important probes with which to study membrane biogenesis and polarization in epithelial cells

Production and characterization of monoclonal antibodies against cathepsin B and cathepsin B-Like proteins of Naegleria fowleri.

Science.gov (United States)

Seong, Gi-Sang; Sohn, Hae-Jin; Kang, Heekyoung; Seo, Ga-Eun; Kim, Jong-Hyun; Shin, Ho-Joon

2017-12-01

Naegleria fowleri causes fatal primary amoebic meningoencephalitis (PAM) in humans and experimental animals. In previous studies, cathepsin B (nfcpb) and cathepsin B-like (nfcpb-L) genes of N. fowleri were cloned, and it was suggested that refolding rNfCPB and rNfCPB-L proteins could play important roles in host tissue invasion, immune response evasion and nutrient uptake. In this study, we produced anti-NfCPB and anti-NfCPB-L monoclonal antibodies (McAb) using a cell fusion technique, and observed their immunological characteristics. Seven hybridoma cells secreting rNfCPB McAbs and three hybridoma cells secreting rNfCPB-L McAbs were produced. Among these, 2C9 (monoclone for rNfCPB) and 1C8 (monoclone for rNfCPB-L) McAb showed high antibody titres and were finally selected for use. As determined by western blotting, 2C9 McAb bound to N. fowleri lysates, specifically the rNfCPB protein, which had bands of 28 kDa and 38.4 kDa. 1C8 McAb reacted with N. fowleri lysates, specifically the rNfCPB-L protein, which had bands of 24 kDa and 34 kDa. 2C9 and 1C8 monoclonal antibodies did not bind to lysates of other amoebae, such as N. gruberi, Acanthamoeba castellanii and A. polyphaga in western blot analyses. Immuno-cytochemistry analysis detected NfCPB and NfCPB-L proteins in the cytoplasm of N. fowleri trophozoites, particularly in the pseudopodia and food-cup. These results suggest that monoclonal antibodies produced against rNfCPB and rNfCPB-L proteins may be useful for further immunological study of PAM. Copyright © 2017 Elsevier Inc. All rights reserved.

High-affinity monoclonal antibodies specific for deoxynucleosides structurally modified by alkylating agents: Applications for immunoanalysis

International Nuclear Information System (INIS)

Adamkiewicz, J.; Ahrens, O.; Rajewsky, M.F.

1984-01-01

So far the results of attempts to use monoclonal antibodies for the demonstration of carcinogen-DNA adducts in cells by immunostaining have been promising. Thus the authors have established a standardized procedure for the quantitation of specific alkyl-deoxynucleosides in the nuclear DNA of individual cells by direct immunofluorescence, using tetramethylrhodamine isothiocyanate-labeled monoclonal antibodies and a computer-based image analysis of electronically intensified fluorescence signals. With a fluorescent anti-(O/sup 6/-EtdGuo) monoclonal antibody, the present detection limit for O/sup 6/-Etd-Guo in the nuclei of individual cells previously exposed to an ethylating N-nitroso compound (e.g., N-ethyl-N-nitrosourea) is -- 700 O/sup 6/-EtdGuo molecules per diploid genome, i.e., similar to the detection limit for the same ethylation product in a hydrolysate of (O/sup 6/-EtdGuo)-containing DNA analyzed by competitive RIA

Potency of a human monoclonal antibody to diphtheria toxin relative to equine diphtheria anti-toxin in a guinea pig intoxication model.

Science.gov (United States)

Smith, Heidi L; Cheslock, Peter; Leney, Mark; Barton, Bruce; Molrine, Deborah C

2016-08-17

Prompt administration of anti-toxin reduces mortality following Corynebacterium diphtheriae infection. Current treatment relies upon equine diphtheria anti-toxin (DAT), with a 10% risk of serum sickness and rarely anaphylaxis. The global DAT supply is extremely limited; most manufacturers have ceased production. S315 is a neutralizing human IgG1 monoclonal antibody to diphtheria toxin that may provide a safe and effective alternative to equine DAT and address critical supply issues. To guide dose selection for IND-enabling pharmacology and toxicology studies, we dose-ranged S315 and DAT in a guinea pig model of diphtheria intoxication based on the NIH Minimum Requirements potency assay. Animals received a single injection of antibody premixed with toxin, were monitored for 30 days, and assigned a numeric score for clinical signs of disease. Animals receiving ≥ 27.5 µg of S315 or ≥ 1.75 IU of DAT survived whereas animals receiving ≤ 22.5 µg of S315 or ≤ 1.25 IU of DAT died, yielding a potency estimate of 17 µg S315/IU DAT (95% CI 16-21) for an endpoint of survival. Because some surviving animals exhibited transient limb weakness, likely a systemic sign of toxicity, DAT and S315 doses required to prevent hind limb paralysis were also determined, yielding a relative potency of 48 µg/IU (95% CI 38-59) for this alternate endpoint. To support advancement of S315 into clinical trials, potency estimates will be used to evaluate the efficacy of S315 versus DAT in an animal model with antibody administration after toxin exposure, more closely modeling anti-toxin therapy in humans.

Localization of radiolabeled anti-DNA monoclonal antibodies in murine systemic lupus erythematosus (SLE)

International Nuclear Information System (INIS)

Wahl, R.; Hahn, B.; Ebling, F.

1984-01-01

The diagnosis of SLE can be extremely difficult. This multi-system disease is characterized by the deposition of DNA-anti-DNA antibody (Ab) complexes in many tissues, producing glomerulonephritis and systemic vasculitis. This study evaluates an IGG monoclonal (Mo) Ab directe3d against DNA (MrSSl) for potential radioimmunodiagnosis of SLE. Six 15 wk. old F-1 female hybrids of NZB+NZW mice (an animal SLE model that develops vasculitis and nephritis) were injected with 50 μCl of I-131 MrSSl and 15 μCl of I-125 isotype-matched control mouse myeloma (LPC-1) (non-reactive with DNA). Imaging and tissue distribution were studied. Two animals were also imaged using I-131 LPC Ab. Images at 2 and 9 days showed no clear differences in scan patterns using MrSSl or LPC-1 Ab. Tissue distribution studies at six days, however, showed a significantly higher accumulation of MrSSl in the kidneys vs. control Ab (2.7% vs. 1.8% of injected dose) (p < .04). Similarly, higher levels of MrSS were also seen in the spleen, liver and lungs (p < .03). Blood levels tended to be higher with the specific antibody as well. These differences were not apparent at 3 days post injection. The increased concentration of MrSSl present at 9 days in several organs may be secondary to MrSSl binding to DNA containing immune complexes present in diseased tissues. Blocked clearance by immune complexes or DNA, or differences in electrical charges of the antibodies could be contributing to the higher MrSSl levels seen. Images did not suggest deiodination as responsible. Further studies are necessary to determine if the amount of MrSSl retained by diseased animals is indicative of SLE disease activity

Monoclonal antibodies and cancer

International Nuclear Information System (INIS)

Haisma, H.J.

1987-01-01

The usefulness of radiolabeled monoclonal antibodies for imaging and treatment of human (ovarian) cancer was investigated. A review of tumor imaging with monoclonal antibodies is presented. Special attention is given to factors that influence the localization of the antibodies in tumors, isotope choice and methods of radiolabeling of the monoclonal antibodies. Two monoclonal antibodies, OC125 and OV-TL3, with high specificity for human epithelial ovarian cancer are characterized. A simple radio-iodination technique was developed for clinical application of the monoclonal antibodies. The behavior of monoclonal antibodies in human tumor xenograft systems and in man are described. Imaging of tumors is complicated because of high background levels of radioactivity in other sites than the tumor, especially in the bloodpool. A technique was developed to improve imaging of human tumor xenographs in nude mice, using subtraction of a specific and a non-specific antibody, radiolabeled with 111 In, 67 Ga and 131 I. To investigate the capability of the two monoclonal antibodies, to specifically localize in human ovarian carcinomas, distribution studies in mice bearing human ovarian carcinoma xenografts were performed. One of the antibodies, OC125, was used for distribution studies in ovarian cancer patients. OC125 was used because of availability and approval to use this antibody in patients. The same antibody was used to investigate the usefulness of radioimmunoimaging in ovarian cancer patients. The interaction of injected radiolabeled antibody OC125 with circulating antigen and an assay to measure the antibody response in ovarian cancer patients after injection of the antibody is described. 265 refs.; 30 figs.; 19 tabs

The Effects of Clinorotation on the Host Plant, Medicago truncatula, and Its Microbial Symbionts

Energy Technology Data Exchange (ETDEWEB)

Dauzart, Ariel J. C.; Vandenbrink, Joshua P.; Kiss, John Z., E-mail: jzkiss@olemiss.edu [Department of Biology, Graduate School, University of Mississippi, University, MS (United States)

2016-02-26

Understanding the outcome of the plant-microbe symbiosis in reduced or altered is vital to developing life support systems for long-distance space travel and colonization of other planets. Thus, the aim of this research was to understand mutualistic relationships between plants and endophytic microbes under the influence of altered gravity. This project utilized the model tripartite relationship among Medicago truncatula—Sinorhizobium meliloti—Rhizophagus irregularis. Plants were inoculated with rhizobial bacteria (S. meliloti), arbuscular mycorrhizal fungi (R. irregularis), or both microbes, and placed on a rotating clinostat. Vertical and horizontal static controls were also performed. Clinorotation significantly reduced M. truncatula dry mass and fresh mass compared to the static controls. The addition of rhizobia treatments under clinorotation also altered total root length and root-to-shoot fresh mass ratio. Nodule size decreased under rhizobia + clinorotation treatment, and nodule density was significantly decreased compared to the vertical treatment. However, inoculation with arbuscular mycorrhizal fungi was shown to increase biomass accumulation and nodule size. Thus, clinorotation significantly affected M. truncatula and its symbiotic relationships with S. meliloti and R. irregularis. In the long term, the results observed in this clinostat study on the changes of plant-microbe mutualism need to be investigated in spaceflight experiments. Thus, careful consideration of the symbiotic microbes of plants should be included in the design of bioregenerative life support systems needed for space travel.

Co-inoculation of arbusculr mycorrhizae and nitrogen fixing bacteria enhance alfalfa yield under saline conditions

International Nuclear Information System (INIS)

Zhu, R.; Tang, F.; Liu, F.; Chen, J.

2016-01-01

The study was to investigate the effects of combined inoculation of Glomus mosseae (arbusculr mycorrhizae fungi, AMF) and Sinorhizobium meliloti (nitrogen-fixing bacteria, i.e., an Rhizobium meliloti, RM) on yield, nutrient contents, nodulation and mycorrhizal colonization of different alfalfa cultivars under saline conditions. An experiment was conducted to test the efficacy of AMF and RM inoculation in development of salt tolerance in alfalfa cultivars (Zhaodong, Nongjing and Longmu) under different salinity levels (0, 60, 120 and 180 mM NaCl). We found that under non stress condition, double inoculation of alfalfa with rhizobium and AM increased the alfalfa yield, nodule weight and number, as well as shoot proline contents, the most when plants were double inoculated followed by AM and rhizobium inoculation, respectively. Whereas under salinity condition, double inoculation of alfalfa with rhizobium and AM increased alfalfa yield, mycorrhizal infection, nodule weight and number as well as increased in shoot proline content, the most followed by AM and rhizobium inoculation, respectively. The Results suggest that growth of alfalfa may be improved by combined inoculation of alfalfa with AM and rhizobium under salt and non-stress conditions. Alleviation of alfalfa growth under saline condition was perhaps due to an increase in mycorrhizal infection and nodule weight and number as well as an increased in shoot proline content by dual inoculation. (author)

The Effects of Clinorotation on the Host Plant, Medicago truncatula, and Its Microbial Symbionts

International Nuclear Information System (INIS)

Dauzart, Ariel J. C.; Vandenbrink, Joshua P.; Kiss, John Z.

2016-01-01

Understanding the outcome of the plant-microbe symbiosis in reduced or altered is vital to developing life support systems for long-distance space travel and colonization of other planets. Thus, the aim of this research was to understand mutualistic relationships between plants and endophytic microbes under the influence of altered gravity. This project utilized the model tripartite relationship among Medicago truncatula—Sinorhizobium meliloti—Rhizophagus irregularis. Plants were inoculated with rhizobial bacteria (S. meliloti), arbuscular mycorrhizal fungi (R. irregularis), or both microbes, and placed on a rotating clinostat. Vertical and horizontal static controls were also performed. Clinorotation significantly reduced M. truncatula dry mass and fresh mass compared to the static controls. The addition of rhizobia treatments under clinorotation also altered total root length and root-to-shoot fresh mass ratio. Nodule size decreased under rhizobia + clinorotation treatment, and nodule density was significantly decreased compared to the vertical treatment. However, inoculation with arbuscular mycorrhizal fungi was shown to increase biomass accumulation and nodule size. Thus, clinorotation significantly affected M. truncatula and its symbiotic relationships with S. meliloti and R. irregularis. In the long term, the results observed in this clinostat study on the changes of plant-microbe mutualism need to be investigated in spaceflight experiments. Thus, careful consideration of the symbiotic microbes of plants should be included in the design of bioregenerative life support systems needed for space travel.

Anti-CD40-mediated cancer immunotherapy

DEFF Research Database (Denmark)

Hassan, Sufia Butt; Sørensen, Jesper Freddie; Olsen, Barbara Nicola

2014-01-01

activation and thus enhancement of immune responses. Treatment with anti-CD40 monoclonal antibodies has been exploited in several cancer immunotherapy studies in mice and led to the development of anti-CD40 antibodies for clinical use. Here, Dacetuzumab and Lucatumumab are in the most advanced stage...... with other cancer immunotherapies, in particular interleukin (IL)-2. An in-depth analysis of this immunotherapy is provided elsewhere. In the present review, we provide an update of the most recent clinical trials with anti-CD40 antibodies. We present and discuss recent and ongoing clinical trials...... in this field, including clinical studies which combine anti-CD40 treatment with other cancer-treatments, such as Rituximab and Tremelimumab....

A monoclonal antibody to pestviruses in bovine and ovine sera

International Nuclear Information System (INIS)

Mweene, A.S.

1991-01-01

An enzyme-linked immunoabsorbent assay (ELISA) has been developed to defeat antibodies to pestviruses in bovine and ovine sera. Single sera from 211 cattle and 22 sheep from 7 different farms were tested using ELISA and Serum Neutralisation Test (SNT). 17 Monoclonal antibodies (Mabs) directed against P80, gp48 and gp53 were tested for ability to coat ELISA plates and capture the bovine viral diarrhea antigen. 5 mabs(WB 103, WB, 105, WB 112 against P80 kDa protein, WB 210 and WB 214 directed against gp48 and gp 53 kDa protein. Specific antibody to BVDV was detected by rabbit anti-bovine and anti-ovine IgG antisera. The quantitative correlation between two tests was good

Monoclonal antibodies in pediatric allergy

Directory of Open Access Journals (Sweden)

Amelia Licari

2015-10-01

Full Text Available Production of monoclonal antibodies (mAbs involving human-mouse hybrid cells was first described in 1970s, but these biologics are now used for a variety of diseases including cancers, autoimmune disorders and allergic diseases. The aim of this article is to review current and future applications of mAbs, in particular focusing on anti-IgE therapy, in the field of pediatric allergy. Proceedings of the 11th International Workshop on Neonatology and Satellite Meetings · Cagliari (Italy · October 26th-31st, 2015 · From the womb to the adultGuest Editors: Vassilios Fanos (Cagliari, Italy, Michele Mussap (Genoa, Italy, Antonio Del Vecchio (Bari, Italy, Bo Sun (Shanghai, China, Dorret I. Boomsma (Amsterdam, the Netherlands, Gavino Faa (Cagliari, Italy, Antonio Giordano (Philadelphia, USA

Molecular mechanism for the action of the anti-CD44 monoclonal antibody MEM-85

Czech Academy of Sciences Publication Activity Database

Škerlová, Jana; Král, Vlastimil; Kachala, M.; Fábry, Milan; Bumba, Ladislav; Svergun, D.I.; Tosner, Z.; Veverka, V.; Řezáčová, Pavlína

2015-01-01

Roč. 191, č. 2 (2015), s. 214-223 ISSN 1047-8477 R&D Projects: GA ČR(CZ) GA15-11851S EU Projects: European Commission 264257 Institutional support: RVO:68378050 ; RVO:61388971 Keywords : CD44 * Epitope mapping * Monoclonal antibody * MEM-85 * SAXS * NMR Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.570, year: 2015

Antibodies to poliovirus detected by immunoradiometric assay with a monoclonal antibody

International Nuclear Information System (INIS)

Spitz, M.; Fossati, C.A.; Schild, G.C.; Spitz, L.; Brasher, M.

1982-01-01

An immunoradiometric assay (IRMA) for the assay of antibodies to poliovirus antigens is described. Dilutions of the test sera or whole (finger prick) blood samples were incubated with the poliovirus antigen bound to a solid phase and the specific antibody was detected by the addition of a mouse anti-human IgG monoclonal antibody (McAb), which was itself revealed by iodinated sheep IgG antimouse F(ab). The authors have shown that this technique is suitable for the estimation of IgG anti-poliovirus antibodies induced in children following polio vaccine. The present study shows that SPRIA provides a simple and inexpensive method for serological studies with poliovirus particularly for use in large-scale surveys. (Auth.)

Antibodies to poliovirus detected by immunoradiometric assay with a monoclonal antibody

Energy Technology Data Exchange (ETDEWEB)

Spitz, M.; Fossati, C.A.; Schild, G.C.; Spitz, L.; Brasher, M. (National Inst. for Biological Standards and Control, London (UK))

1982-10-01

An immunoradiometric assay (IRMA) for the assay of antibodies to poliovirus antigens is described. Dilutions of the test sera or whole (finger prick) blood samples were incubated with the poliovirus antigen bound to a solid phase and the specific antibody was detected by the addition of a mouse anti-human IgG monoclonal antibody (McAb), which was itself revealed by iodinated sheep IgG antimouse F(ab). The authors have shown that this technique is suitable for the estimation of IgG anti-poliovirus antibodies induced in children following polio vaccine. The present study shows that SPRIA provides a simple and inexpensive method for serological studies with poliovirus particularly for use in large-scale surveys.

In vivo imaging and quantitation of renal transplant rejection using indium-111 labelled anti-lymphocyte and anti-MHC class I and II monoclonal antibodies in a rat model

International Nuclear Information System (INIS)

Loutfi, I.; Batchelor, J.R.; Lavender, J.P.

1992-01-01

It has been described in this report, non-invasive and specific method for imaging and assessment of acute kidney transplant rejection in rat model. This model can serve as a basis for application in man using a cocktail of monoclonal antibodies with different specificities starting with monoclonal antibodies labelled with indium-111 which have been used in this technique. 3 refs., 1 tab., 2 figs

The alternative Medicago truncatula defense proteome of ROS – defective transgenic roots during early microbial infection

Directory of Open Access Journals (Sweden)

Leonard Muriithi Kiirika

2014-07-01

Full Text Available ROP-type GTPases of plants function as molecular switches within elementary signal transduction pathways such as the regulation of ROS synthesis via activation of NADPH oxidases (RBOH-respiratory burst oxidase homologue in plants. Previously, we reported that silencing of the Medicago truncatula GTPase MtROP9 led to reduced ROS production and suppressed induction of ROS-related enzymes in transgenic roots (MtROP9i infected with pathogenic (Aphanomyces euteiches and symbiotic microorganisms (Glomus intraradices, Sinorhizobium meliloti. While fungal infections were enhanced, S. meliloti infection was drastically impaired. In this study, we investigate the temporal proteome response of M. truncatula MtROP9i transgenic roots during the same microbial interactions under conditions of deprived potential to synthesize ROS. In comparison with control roots (Mtvector, we present a comprehensive proteomic analysis using sensitive MS protein identification. For four early infection time-points (1, 3, 5, 24 hpi, 733 spots were found to be different in abundance: 213 spots comprising 984 proteins (607 unique were identified after S. meliloti infection, 230 spots comprising 796 proteins (580 unique after G. intraradices infection, and 290 spots comprising 1240 proteins (828 unique after A. euteiches infection. Data evaluation by GelMap in combination with a heatmap tool allowed recognition of key proteome changes during microbial interactions under conditions of hampered ROS synthesis. Overall, the number of induced proteins in MtROP9i was low as compared with controls, indicating a dual function of ROS in defense signaling as well as alternative response patterns activated during microbial infection. Qualitative analysis of induced proteins showed that enzymes linked to ROS production and scavenging were highly induced in control roots, while in MtROP9i the majority of proteins were involved in alternative defense pathways such as cell wall and protein

Effectivenes of inoculation in alfalfa breeding in ecological conditions of the Bjelovar and Bilogora county

Directory of Open Access Journals (Sweden)

Darko Uher

2012-09-01

Full Text Available Development and basic existence of animal production as well as production of high quality milk depends upon possibility of sufficient production of quality and protein sufficient forage. Forage crop that satisfies these demands is alfalfa which is one of the most important perennial forage crop legumes. The aim of this study was to enhance alfalfa production on acid soil by liming and alfalfa seed inoculation with efficient Sinorhizobium meliloti strains in order to reduce the use of mineral nitrogen fertilization and enable qualitative and cost effective production of forage on the dairy farms. Field trial was established at family farm in the area of Bjelovar and Bilogora county. During two years experimental period statistically significant influence of inoculation and liming on forage and dry matteryield was determined. Significantly the lowest yields were determined on untreated plots without liming material. In all untreated plots, significantly lower yields were determined, but significant differences in yields were also obtained by inoculation with different S. meliloti strains, emphasizing the importance of strains selection used for alfalfa inoculation. In both experimental years total forage yield were ranging from 34 t/ha (untreated plots without liming up to 60 t/ha on plots inoculated with strain 2011 and without liming. Values of total dry matter yield for both experimental years ranged from 6.5 t/ha (untreated plots without liming up to 15,7 t/ha on plots inoculated with strain 2011 without liming. Results of this study showed that application of liming materials for acidity removal had positive effect on alfalfa yields in both experimental years and significantly improved alfalfa production on acid soils. The results of this study clearly showed that inoculation with selected S. meliloti strains may improve alfalfa production on acid soils and may contribute to more efficient forage production for dairy farms under particular

Analyses of the peripheral immunome following multiple administrations of avelumab, a human IgG1 anti-PD-L1 monoclonal antibody.

Science.gov (United States)

Donahue, Renee N; Lepone, Lauren M; Grenga, Italia; Jochems, Caroline; Fantini, Massimo; Madan, Ravi A; Heery, Christopher R; Gulley, James L; Schlom, Jeffrey

2017-01-01

Multiple anti-PD-L1/PD-1 checkpoint monoclonal antibodies (MAb) have shown clear evidence of clinical benefit. All except one have been designed or engineered to omit the possibility to mediate antibody-dependent cell-mediated cytotoxicity (ADCC) as a second potential mode of anti-tumor activity; the reason for this is the concern of lysis of PD-L1 positive immune cells. Avelumab is a fully human IgG1 MAb which has been shown in prior in vitro studies to mediate ADCC versus a range of human tumor cells, and clinical studies have demonstrated anti-tumor activity versus a range of human cancers. This study was designed to investigate the effect on immune cell subsets in the peripheral blood of cancer patients prior to and following multiple administrations of avelumab. One hundred twenty-three distinct immune cell subsets in the peripheral blood of cancer patients ( n  = 28) in a phase I trial were analyzed by flow cytometry prior to and following one, three, and nine cycles of avelumab. Changes in soluble (s) CD27 and sCD40L in plasma were also evaluated. In vitro studies were also performed to determine if avelumab would mediate ADCC of PBMC. No statistically significant changes in any of the 123 immune cell subsets analyzed were observed at any dose level, or number of doses, of avelumab. Increases in the ratio of sCD27:sCD40L were observed, suggesting potential immune activation. Controlled in vitro studies also showed lysis of tumor cells by avelumab versus no lysis of PBMC from five donors. These studies demonstrate the lack of any significant effect on multiple immune cell subsets, even those expressing PD-L1, following multiple cycles of avelumab. These results complement prior studies showing anti-tumor effects of avelumab and comparable levels of adverse events with avelumab versus other anti-PD-1/PD-L1 MAbs. These studies provide the rationale to further exploit the potential ADCC mechanism of action of avelumab as well as other human IgG1 checkpoint

Dashboard systems: Pharmacokinetic/pharmacodynamic mediated dose optimization for monoclonal antibodies.

Science.gov (United States)

Mould, Diane R; Dubinsky, Marla C

2015-03-01

Many marketed drugs exhibit high variability in exposure and response. While these drugs are efficacious in their approved indications, finding appropriate dose regimens for individual patients is not straightforward. Similar dose adjustment problems are also seen with drugs that have a complex relationship between exposure and response and/or a narrow therapeutic window. This is particularly true for monoclonal antibodies, where prolonged dosing at a sub-therapeutic dose can also elicit anti-drug antibodies which will further compromise safety and efficacy. Thus, finding appropriate doses quickly would represent a substantial improvement in healthcare. Dashboard systems, which are decision-support tools, offer an improved, convenient means of tailoring treatment for individual patients. This article reviews the clinical need for this approach, particularly with monoclonal antibodies, the design, development, and testing of such systems, and the likely benefits of dashboard systems in clinical practice. We focus on infliximab for reference. © 2015, The American College of Clinical Pharmacology.

Golimumab and certolizumab: The two new anti-tumor necrosis factor kids on the block

Directory of Open Access Journals (Sweden)

Mittal Mohit

2010-01-01

Full Text Available Anti-tumor necrosis factor (anti-TNF agents have revolutionized treatment of psoriasis and many other inflammatory diseases of autoimmune origin. They have considerable advantages over the existing immunomodulators. Anti-TNF agents are designed to target a very specific component of the immune-mediated inflammatory cascades. Thus, they have lower risks of systemic side-effects. In a brief period of 10 years, a growing number of biological therapies are entering the clinical arena while many more biologicals remain on the horizon. With time, the long-term side-effects and efficacies of these individual agents will become clearer and help to determine which ones are the most suitable for long-term care. Golimumab (a human monoclonal anti-TNF-α antibody and Certolizumab (a PEGylated Fab fragment of humanized monoclonal TNF-α antibody are the two latest additions to the anti-TNF regimen. Here, we are providing a brief description about these two drugs and their uses.

Cloning of the first human anti-JCPyV/VP1 neutralizing monoclonal antibody: epitope definition and implications in risk stratification of patients under natalizumab therapy.

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Diotti, Roberta Antonia; Mancini, Nicasio; Clementi, Nicola; Sautto, Giuseppe; Moreno, Guisella Janett; Criscuolo, Elena; Cappelletti, Francesca; Man, Petr; Forest, Eric; Remy, Louise; Giannecchini, Simone; Clementi, Massimo; Burioni, Roberto

2014-08-01

JC virus (JCPyV) has gained novel clinical importance as cause of progressive multifocal leukoencephalopathy (PML), a rare demyelinating disease recently associated to immunomodulatory drugs, such as natalizumab used in multiple sclerosis (MS) cases. Little is known about the mechanisms leading to PML, and this makes the need of PML risk stratification among natalizumab-treated patients very compelling. Clinical and laboratory-based risk-stratification markers have been proposed, one of these is represented by the JCPyV-seropositive status, which includes about 54% of MS patients. We recently proposed to investigate the possible protective role of neutralizing humoral immune response in preventing JCPyV reactivation. In this proof-of-concept study, by cloning the first human monoclonal antibody (GRE1) directed against a neutralizing epitope on JCPyV/VP1, we optimized a robust anti-JCPyV neutralization assay. This allowed us to evaluate the neutralizing activity in JCPyV-positive sera from MS patients, demonstrating the lack of correlation between the level of anti-JCPyV antibody and anti-JCPyV neutralizing activity. Relevant consequences may derive from future clinical studies induced by these findings; indeed the study of the serum anti-JCPyV neutralizing activity could allow not only a better risk stratification of the patients during natalizumab treatment, but also a better understanding of the pathophysiological mechanisms leading to PML, highlighting the contribution of peripheral versus central nervous system JCPyV reactivation. Noteworthy, the availability of GRE1 could allow the design of novel immunoprophylactic strategies during the immunomodulatory treatment. Copyright © 2014 Elsevier B.V. All rights reserved.

The challenge of treating hepatitis C virus-associated cryoglobulinemic vasculitis in the era of anti-CD20 monoclonal antibodies and direct antiviral agents.

Science.gov (United States)

Roccatello, Dario; Sciascia, Savino; Rossi, Daniela; Solfietti, Laura; Fenoglio, Roberta; Menegatti, Elisa; Baldovino, Simone

2017-06-20

Mixed cryoglobulinemia syndrome (MC) is a systemic vasculitis involving kidneys, joints, skin, and peripheral nerves. While many autoimmune, lymphoproliferative, and neoplastic disorders have been associated with this disorder, hepatitis C virus (HCV) is known to be the etiologic agent in the majority of patients. Therefore, clinical research has focused on anti-viral drugs and, more recently, on the new, highly potent Direct-acting Antiviral Agents (DAAs). These drugs assure sustained virologic response (SVR) rates >90%. Nevertheless, data on their efficacy in patients with HCV-associated cryoglobulinemic vasculitis are disappointing, possibly due to the inability of the drugs to suppress the immune-mediated process once it has been triggered.Despite the potential risk of exacerbation of the infection, immunosuppression has traditionally been regarded as the first-line intervention in cryoglobulinemic vasculitis, especially if renal involvement is severe. Biologic agents have raised hopes for more manageable therapeutic approaches, and Rituximab (RTX), an anti CD20 monoclonal antibody, is the most widely used biologic drug. It has proved to be safer than conventional immunosuppressants, thus substantially changing the natural history of HCV-associated cryoglobulinemic vasculitis by providing long-term remission, especially with intensive regimens.The present review focuses on the new therapeutic opportunities offered by the combination of biological drugs, mainly Rituximab, with DAAs.

Mammalian Cell Culture Clarification: A Case Study Using Chimeric Anti-CEA Monoclonal Antibodies

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Mohamed Ali Abol Hassan

2011-12-01

Full Text Available The extracellular expression of monoclonal antibodies (mAbs in mammalian cell culture provides both opportunities and restrictions for the design of robust harvest and clarification operations. With advances in cell culture media and cell lines, it is now possible to achieve high titers of over 5 g/l for mAbs. However, Mammalian cells are sensitive to breakage due to shear stress that can result in release of proteases and other host cell proteins (HCPs which eventually affects product stability and purity. There is larger number of mAbs undergoing clinical development and it has placed significant importance on platform technologies of process development. Generally, Centrifugation and microfiltration are the primary harvest techniques used in the industry and depth filtration is also used as a step operation on clarification. This study compares the unit operations; centrifugation, microfiltration and depth filtration for maximum recovery of monoclonal antibodies. The results have shown that the depth filtration as more suitable operation for mammalian cell culture clarification since it gives 96% recovery of mAbs in comparison to centrifugation and microfiltration. ABSTRAK: Pengungkapan luar sel dari antibodi monoklon (monoclonal antibodies ((mAbs dalam kultur sel mamalia memberi ruang dan batasan terhadap reka bentuk penuaian yang cekap dan penerangan operasi. Dengan kemajuan dalam media sel kultur dan cell lines (produk yang berupa sel kekal yang digunakan untuk tujuan kajian biologi, kini adalah berkemungkinan untuk memperolehi titer tinggi melebihi 5g/l untuk mAbs [2]. Walaupun begitu, sel mamalia sensitif terhadap retakan disebabkan tegasan ricih yang menyebabkan pengeluaran protease dan hos sel protein yang lain, (host cell proteins (HCPs akhirnya mempengaruhi kestabilan dan keaslian produk. Terdapat mAbs dalam jumlah besar yang masih menjalani pembangunan klinikal dan sesungguhnya ini penting sebagai satu landasan teknologi dalam

Systems approach for the selection of micro-RNAs as therapeutic biomarkers of anti-EGFR monoclonal antibody treatment in colorectal cancer

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Deyati, Avisek; Bagewadi, Shweta; Senger, Philipp; Hofmann-Apitius, Martin; Novac, Natalia

2015-01-01

miRNA plays an important role in tumourgenesis by regulating expression of oncogenes and tumour suppressors. Thus affects cell proliferation and differentiation, apoptosis, invasion and angiogenesis. miRNAs are potential biomarkers for diagnosis, prognosis and therapies of different forms of cancer. However, relationship between response of cancer patients towards targeted therapy and the resulting modifications of the miRNA transcriptome in the context of pathway regulation is poorly understood. With ever-increasing pathways and miRNA-mRNA interaction databases, freely available mRNA and miRNA expression data in multiple cancer therapy have produced an unprecedented opportunity to decipher the role of miRNAs in early prediction of therapeutic efficacy in diseases. Efficient translation of -omics data and accumulated knowledge to clinical decision-making are of paramount scientific and public health interest. Well-structured translational algorithms are needed to bridge the gap from databases to decisions. Herein, we present a novel SMARTmiR algorithm to prospectively predict the role of miRNA as therapeutic biomarker for an anti-EGFR monoclonal antibody i.e. cetuximab treatment in colorectal cancer.

Serological blind spots for variants of human IgG3 and IgG4 by a commonly used anti-immunoglobulin reagent.

Science.gov (United States)

Howie, Heather L; Delaney, Meghan; Wang, Xiaohong; Er, Lay See; Vidarsson, Gestur; Stegmann, Tamara C; Kapp, Linda; Lebedev, Jenna N; Wu, Yanyun; AuBuchon, James P; Zimring, James C

2016-12-01

Human immunoglobulin G (IgG) includes four different subtypes (IgG1, IgG2, IgG3, and IgG4), and it is also now appreciated that there are genetic variations within IgG subtypes (called isoallotypes). Twenty-nine different isoallotypes have been described, with 7, 4, 15, and 3 isoallotypes described for IgG1, IgG2, IgG3, and IgG4, respectively. The reactivity of anti-IgG with different isoallotypes has not been characterized. A novel monoclonal anti-K antibody (PugetSound Monoclonal Antibody 1 [PUMA1]) was isolated and sequenced, and a panel of PUMA1 variants was expressed, consisting of the 29 known IgG isoallotypes. The resulting panel of antibodies was preincubated with K-positive red blood cells (RBCs) and then subjected to testing with currently approved anti-IgG by flow cytometry, solid phase systems, gel cards, and tube testing. A US Food and Drug Administration (FDA)-approved monoclonal anti-IgG (gamma-clone) failed to recognize 2 of 15 IgG3 isoallotypes (IgG3-03 and IgG3-13) and 3 of 3 IgG4 isoallotypes (IgG4-01, IgG4-02, and IgG4-03). In contrast, an FDA-approved rabbit polyclonal anti-IgG recognized each of the known human IgG isoallotypes. These findings demonstrate "blind spots" in isoalloantibody detection by a monoclonal anti-IgG. If a patient has anti-RBC antibodies predominantly of an IgG3 subtype (the IgG3-03 and/or IgG3-13 variety), then it is possible that a clinically significant alloantibody would be missed. IgG-03 and IgG-13 have an estimated frequency of 1% to 3% in Caucasian populations and 20% to 30% in certain African populations. Nonreactivity with IgG4 is a known characteristic of this monoclonal anti-IgG, but IgG4 isoallotypes have not been previously reported. © 2016 AABB.

In vivo localization of radiolabeled monoclonal antibody to carcinoembryonic antigen (CEA) in a CEA-producing tumor

International Nuclear Information System (INIS)

Kamei, Tetsuya; Seto, Hikaru; Taki, Kuniyasu; Soya, Toshio; Kakishita, Masao; Maeda, Masatoshi; Honda, Takashi; Koshimura, Saburou.

1987-01-01

To compare accumulation of the 125 I-labeled antibodies(anti-carcinoembryonic antigen(CEA) monoclonal antibody and polyclonal antibody) to a CEA-producing tumor (SC-2-JCK), an in vivo localization study was performed in nude mice. The tumor-to-blood ratio at 120 hours after injection rose to 4.6 for the monoclonal antibody, but remained at 1.3 for the polyclonal antibody. However, no differences were noted between the antibodies up to 72 hours after injection. In autoradiograms, selective accumulation of the tracer was noted in the tumor for both antibodies. However, no superiority or inferiority of imaging for either of the antibodies could be definitely determined. (author)

Canakinumab: un anticuerpo monoclonal prometedor en el tratamiento de enfermedades cardiovasculares Canakinumab: a promising monoclonal antibody in the treatment of cardiovascular diseases

Directory of Open Access Journals (Sweden)

Fernando Manzur

2013-02-01

Full Text Available El canakinumab es un anticuerpo monoclonal anti-IL-1β totalmente humano desarrollado por Novartis, cuyo mecanismo de acción se basa en la neutralización de la señalización IL-1β, lo cual conduce a la supresión de la inflamación en pacientes con trastornos de origen autoinmune. La IL-1β actúa como un mediador de la respuesta inmune periférica durante la infección y la inflamación. Mediante la unión antígeno-anticuerpo el canakinumab inhibe la acción de la IL1-β evitando sus efectos pro-inflamatorios. En la actualidad, está en evaluación como un nuevo posible agente dirigido frente a la IL-1β, con el objetivo de reducir la tasa de eventos cardiovasculares y la diabetes de aparición reciente (estudio CANTOS.Canakinumab is a totally human monoclonal antibody anti-IL-1β developed by Novartis, whose mode of action is based on the neutralization of IL-1β signaling, which leads to suppression of inflammation in patients with autoimmune disorders. The IL-1β acts as a mediator of the peripheral immune response during infection and inflammation. By the antigen-antibody binding, canakinumab inhibits the action of IL1-β avoiding its pro-inflammatory effects. Currently, it is being evaluated as a new possible agent directed against IL-1β, with the goal of reducing the rate of cardiovascular events and new onset diabetes (study CANTOS.

Preparation of anti-ciguatoxin monoclonal antibodies using synthetic haptens: sandwich ELISA detection of ciguatoxins.

Science.gov (United States)

Tsumuraya, Takeshi; Fujii, Ikuo; Hirama, Masahiro

2014-01-01

Ciguatera fish poisoning (CFP) is a form of food poisoning caused by the consumption of fish that have accumulated a type of sodium channel activator toxin called ciguatoxins (CTXs), which are produced by dinoflagellates of the genus Gambierdiscus through the food chain. CFP affects more than 50000 people each year. The extremely low level of CTXs in tainted fish has hampered the development of antibodies for the detection of these toxins. Monoclonal antibodies (mAbs) specific against major congeners of CTX3C, 51-hydroxyCTX3C, CTX1B, and 54-deoxyCTX1B were prepared by immunization of mice with protein conjugates of rationally designed synthetic haptens in place of the natural toxins. We found that haptenic groups possessing a surface area larger than 400 angstroms2 were required to produce mAbs that can bind strongly to CTXs. Direct sandwich ELISA utilizing two different monoclonal antibodies that bind specifically to one of the two wings of a CTX were established to detect CTXs. No cross-reactivity was observed against the other marine toxins tested, including brevetoxin A, brevetoxin B, okadaic acid, and maitotoxin.

Regulation of levels of serum antibodies to ryegrass pollen allergen Lol pIV by an internal image anti-idiotypic monoclonal antibody.

Science.gov (United States)

Zhou, E M; Kisil, F T

1995-03-01

A murine monoclonal anti-idiotypic antibody (anti-Id), designated B1/1, was produced against an idiotope of a murine antibody (mAb91), which recognizes the epitope, site A, of allergen Lol pIV, one of the major groups of allergens in ryegrass (Lolium perenne) pollen. The ability of B1/1 to modulate the antibody responses to Lol pIV was investigated in murine model systems. In the first system, B1/1-keyhole limpet haemocyanin (KLH) conjugate was administered to treat three different strains of mice (C57BL/6, BALB/c and C3H). In the second and third model systems, a solution of B1/1 in phosphate-buffered saline (PBS) was used to treat syngeneic BALB/c mice at various doses and time intervals, respectively. The treatment with either form of B1/1, administered at doses ranging from 100 ng to 100 micrograms mouse, resulted in a reduction of the levels of the antibodies to Lol pIV. In particular, the level of IgE antibodies to Lol pIV was greatly reduced. The administration of a single intravenous (i.v.) injection of a solution of B1/1 8 weeks prior to the challenge with Lol pIV was still effective in reducing the level of antibodies to the allergen. Moreover, the level of antibodies to Lol pIV that expressed the idiotope mAb91 was also markedly decreased. By contrast, it was observed that the level of antibodies to Lol pIV in mice pretreated with B1/1 in PBS at a dose of 10 ng/mouse increased (albeit slightly) compared to that in mice treated with control mAb. These experimental models lend themselves for investigating the mechanism(s) by which an anti-Id modulates antibody responses to a grass pollen allergen.

Location of Primary Tumor and Benefit From Anti-Epidermal Growth Factor Receptor Monoclonal Antibodies in Patients With RAS and BRAF Wild-Type Metastatic Colorectal Cancer

Science.gov (United States)

Moretto, Roberto; Cremolini, Chiara; Rossini, Daniele; Pietrantonio, Filippo; Battaglin, Francesca; Mennitto, Alessia; Bergamo, Francesca; Loupakis, Fotios; Marmorino, Federica; Berenato, Rosa; Marsico, Valentina Angela; Caporale, Marta; Antoniotti, Carlotta; Masi, Gianluca; Salvatore, Lisa; Borelli, Beatrice; Fontanini, Gabriella; Lonardi, Sara; De Braud, Filippo

2016-01-01

Introduction. Right- and left-sided colorectal cancers (CRCs) differ in clinical and molecular characteristics. Some retrospective analyses suggested that patients with right-sided tumors derive less benefit from anti-epidermal growth factor receptor (EGFR) antibodies; however, molecular selection in those studies was not extensive. Patients and Methods. Patients with RAS and BRAF wild-type metastatic CRC (mCRC) who were treated with single-agent anti-EGFRs or with cetuximab-irinotecan (if refractory to previous irinotecan) were included in the study. Differences in outcome between patients with right- and left-sided tumors were investigated. Results. Of 75 patients, 14 and 61 had right- and left-sided tumors, respectively. None of the right-sided tumors responded according to RECIST, compared with 24 left-sided tumors (overall response rate: 0% vs. 41%; p = .0032), and only 2 patients with right-sided tumors (15%) versus 47 patients with left-sided tumors (80%) achieved disease control (p < .0001). The median duration of progression-free survival was 2.3 and 6.6 months in patients with right-sided and left-sided tumors, respectively (hazard ratio: 3.97; 95% confidence interval: 2.09–7.53; p < .0001). Conclusion. Patients with right-sided RAS and BRAF wild-type mCRC seemed to derive no benefit from single-agent anti-EGFRs. Implications for Practice: Right- and left-sided colorectal tumors have peculiar epidemiological and clinicopathological characteristics, distinct gene expression profiles and genetic alterations, and different prognoses. This study assessed the potential predictive impact of primary tumor site with regard to anti-epidermal growth factor receptor (EGFR) monoclonal antibody treatment in patients with RAS and BRAF wild-type metastatic colorectal cancer. The results demonstrated the lack of activity of anti-EGFRs in RAS and BRAF wild-type, right-sided tumors, thus suggesting a potential role for primary tumor location in driving treatment choices

The application of anti-ESAT-6 monoclonal antibody fluorescent probe in ex vivo near-infrared fluorescence imaging in mice with pulmonary tuberculosis.

Science.gov (United States)

Feng, Feng; Zhang, Haoling; Zhu, Zhaoqin; Li, Cong; Shi, Yuxin; Zhang, Zhiyong

2014-09-01

Here, we aimed to assess the feasibility of anti-ESAT-6 monoclonal antibody (mAb) coupling with IR783 and rhodamine fluorescent probe in the detection of ESAT-6 expression in tuberculosis tissue of mice using near-infrared fluorescence imaging. IR783 and rhodamine were conjugated to the anti-ESAT-6 mAb or IgG. Mice in the experimental group were injected with fluorescence-labeled mAb probe, and mice in the control group were injected with fluorescence-labeled non-specific IgG antibody. Twenty-four hours later, the lung tissue of mice was examined using ex vivo near-infrared fluorescence imaging. In addition, the contrast-to-noise ratio (CNR) was calculated by measuring the signal intensities of the pulmonary lesions, normal lung tissue and background noise. The frozen lung tissue section was examined under fluorescence microscopy and compared with hemoxylin and eosin (HE) staining. The ex vivo near-infrared fluorescence imaging showed that the fluorescence signal in the lung tuberculosis lesions in the experimental group was significantly enhanced, whereas there was only a weak fluorescence signal or even no fluorescence signal in the control group. CNR values were 64.40 ± 7.02 (n = 6) and 8.75 ± 3.87 (n = 6), respectively (t = 17.01, p fluorescence accumulation distribution detected under fluorescence microscopy was consistent with HE staining of the tuberculosis region. In conclusion, anti-ESAT-6 mAb fluorescent probe could target and be applied in specific ex vivo imaging of mice tuberculosis, and may be of further use in tuberculosis in living mice. Copyright © 2013 John Wiley & Sons, Ltd.

Targeting connective tissue growth factor (CTGF) in acute lymphoblastic leukemia preclinical models: anti-CTGF monoclonal antibody attenuates leukemia growth.

Science.gov (United States)

Lu, Hongbo; Kojima, Kensuke; Battula, Venkata Lokesh; Korchin, Borys; Shi, Yuexi; Chen, Ye; Spong, Suzanne; Thomas, Deborah A; Kantarjian, Hagop; Lock, Richard B; Andreeff, Michael; Konopleva, Marina

2014-03-01

Connective tissue growth factor (CTGF/CCN2) is involved in extracellular matrix production, tumor cell proliferation, adhesion, migration, and metastasis. Recent studies have shown that CTGF expression is elevated in precursor B-acute lymphoblastic leukemia (ALL) and that increased expression of CTGF is associated with inferior outcome in B-ALL. In this study, we characterized the functional role and downstream signaling pathways of CTGF in ALL cells. First, we utilized lentiviral shRNA to knockdown CTGF in RS4;11 and REH ALL cells expressing high levels of CTGF mRNA. Silencing of CTGF resulted in significant suppression of leukemia cell growth compared to control vector, which was associated with AKT/mTOR inactivation and increased levels of cyclin-dependent kinase inhibitor p27. CTGF knockdown sensitized ALL cells to vincristine and methotrexate. Treatment with an anti-CTGF monoclonal antibody, FG-3019, significantly prolonged survival of mice injected with primary xenograft B-ALL cells when co-treated with conventional chemotherapy (vincristine, L-asparaginase and dexamethasone). Data suggest that CTGF represents a targetable molecular aberration in B-ALL, and blocking CTGF signaling in conjunction with administration of chemotherapy may represent a novel therapeutic approach for ALL patients.

Uses of monoclonal antibody 8H9

Science.gov (United States)

Cheung, Nai-Kong V.

2013-04-09

This invention provides a composition comprising an effective amount of monoclonal antibody 8H9 or a derivative thereof and a suitable carrier. This invention provides a pharmaceutical composition comprising an effective amount of monoclonal antibody 8H9 or a derivative thereof and a pharmaceutically acceptable carrier. This invention also provides an antibody other than the monoclonal antibody 8H9 comprising the complementary determining regions of monoclonal antibody 8H9 or a derivative thereof, capable of binding to the same antigen as the monoclonal antibody 8H9. This invention provides a substance capable of competitively inhibiting the binding of monoclonal antibody 8H9. This invention also provides an isolated scFv of monoclonal antibody 8H9 or a derivative thereof. This invention also provides the 8H9 antigen. This invention also provides different uses of the monoclonal antibody 8H9 or its derivative.

An Anti-C1s Monoclonal, TNT003, Inhibits Complement Activation Induced by Antibodies Against HLA.

Science.gov (United States)

Thomas, K A; Valenzuela, N M; Gjertson, D; Mulder, A; Fishbein, M C; Parry, G C; Panicker, S; Reed, E F

2015-08-01

Antibody-mediated rejection (AMR) of solid organ transplants (SOT) is characterized by damage triggered by donor-specific antibodies (DSA) binding donor Class I and II HLA (HLA-I and HLA-II) expressed on endothelial cells. While F(ab')2 portions of DSA cause cellular activation and proliferation, Fc regions activate the classical complement cascade, resulting in complement deposition and leukocyte recruitment, both hallmark features of AMR. We characterized the ability of an anti-C1s monoclonal antibody, TNT003, to inhibit HLA antibody (HLA-Ab)-induced complement activation. Complement deposition induced by HLA-Ab was evaluated using novel cell- and bead-based assays. Human aortic endothelial cells (HAEC) were cultured with HLA-Ab and human complement; production of activated complement proteins was measured by flow cytometry. Additionally, C3d deposition was measured on single antigen beads (SAB) mixed with HLA-Ab and human complement. TNT003 inhibited HLA-Ab mediated complement deposition on HAEC in a concentration-dependent manner; C3a, C4a and C5a anaphylatoxin production was also diminished by TNT003. Finally, TNT003 blocked C3d deposition induced by Class I (HLAI-Ab)- and Class II (HLAII-Ab)-specific antibodies on SAB. These data suggest TNT003 may be useful for modulating the effects of DSA, as TNT003 inhibits complement deposition and split product formation generated by HLA-I/II-Ab in vitro. © 2015 The Authors. American Journal of Transplantation Published by Wiley Periodicals, Inc.

Antigenic modulation limits the effector cell mechanisms employed by type I anti-CD20 monoclonal antibodies.

Science.gov (United States)

Tipton, Thomas R W; Roghanian, Ali; Oldham, Robert J; Carter, Matthew J; Cox, Kerry L; Mockridge, C Ian; French, Ruth R; Dahal, Lekh N; Duriez, Patrick J; Hargreaves, Philip G; Cragg, Mark S; Beers, Stephen A

2015-03-19

Following the success of rituximab, 2 other anti-CD20 monoclonal antibodies (mAbs), ofatumumab and obinutuzumab, have entered clinical use. Ofatumumab has enhanced capacity for complement-dependent cytotoxicity, whereas obinutuzumab, a type II mAb, lacks the ability to redistribute into lipid rafts and is glycoengineered for augmented antibody-dependent cellular cytotoxicity (ADCC). We previously showed that type I mAbs such as rituximab have a propensity to undergo enhanced antigenic modulation compared with type II. Here we assessed the key effector mechanisms affected, comparing type I and II antibodies of various isotypes in ADCC and antibody-dependent cellular-phagocytosis (ADCP) assays. Rituximab and ofatumumab depleted both normal and leukemic human CD20-expressing B cells in the mouse less effectively than glycoengineered and wild-type forms of obinutuzumab, particularly when human immunoglobulin G1 (hIgG1) mAbs were compared. In contrast to mouse IgG2a, hIgG1 mAbs were ineffective in ADCC assays with murine natural killer cells as effectors, whereas ADCP was equivalent for mouse IgG2a and hIgG1. However, rituximab's ability to elicit both ADCC and ADCP was reduced by antigenic modulation, whereas type II antibodies remained unaffected. These data demonstrate that ADCP and ADCC are impaired by antigenic modulation and that ADCP is the main effector function employed in vivo. © 2015 by The American Society of Hematology.

Diagnosis of colorectal carcinomas and recurrence with 99m Tc labeled monoclonal anti-CEA-antibody (BW 431/26)

International Nuclear Information System (INIS)

Lind, P.; Langsteger, W.; Koeltringer, P.; Eber, O.; Beham, A.

1989-01-01

With the introduction of 99m Tc labeled monoclonal antibodies against CEA, a clinically relevant extension can be expected in the diagnosis of colorectal tumors by immunoscintigraphy (IS). This study comprises a total of 31 patients (primary tumors, occult neoplasms with elevated CEA serum level, suspicious recurrences). In primary tumors (n = 14), all coloscopically diagnosed carcinomas were confirmed and correctly localised by IS (n = 8). In 4 cases IS was true negative, in one case false positive; in one patient a stomach adenocarcinoma could be demonstrated. In the diagnosis of recurrences (n = 17) IS revealed an uptake in TCT (transmission computed tomography) and coloscopically suspicious areas in 10 cases. In 6 cases IS was negative (5 true negative findings in scar or granulation tissue, 1 false negative finding in paraaortal lymphnodes). In one patient the raised CEA level was due to multiple liver metastases, a local recurrence could not be detected. Elevated serum CEA-levels were found only in 10 of 19 patients with true positive IS. In postoperative cancer care IS with 99m Tc-labeled anti-CEA antibody (MAK BW 431/26) plays a preeminent role in the exclusion or diagnosis of kolorectal recurrences in case of ambiguous TCT or endoscopic findings. (Author)

Prognosis in monoclonal proteinaemia

NARCIS (Netherlands)

Schaar, Cornelis Gerardus

2006-01-01

Monoclonal proteinaemia (M-proteinemia) is associated with multiple myeloma (MM) or other hematological malignancies. In the absence of these diseases the term MGUS (Monoclonal Gammopathy of Undetermined Significance) is used. During 1991-1993 1464 patients with newly diagnosed M-proteinemia in the

Uses of monoclonal antibody 8H9

Energy Technology Data Exchange (ETDEWEB)

Cheung, Nai-Kong V.

2018-04-10

This invention provides a composition comprising an effective amount of monoclonal antibody 8H9 or a derivative thereof and a suitable carrier. This invention provides a pharmaceutical composition comprising an effective amount of monoclonal antibody 8H9 or a derivative thereof and a pharmaceutically acceptable carrier. This invention also provides an antibody other than the monoclonal antibody 8H9 comprising the complementary determining regions of monoclonal antibody 8H9 or a derivative thereof, capable of binding to the same antigen as the monoclonal antibody 8H9. This invention provides a substance capable of competitively inhibiting the binding of monoclonal antibody 8H9. This invention also provides an isolated scFv of monoclonal antibody 8H9 or a derivative thereof. This invention also provides the 8H9 antigen. This invention also provides a method of inhibiting the growth of tumor cells comprising contacting said tumor cells with an appropriate amount of monoclonal antibody 8H9 or a derivative thereof.

Anti-B cell antibody therapies for inflammatory rheumatic diseases

DEFF Research Database (Denmark)

Faurschou, Mikkel; Jayne, David R W

2014-01-01

Several monoclonal antibodies targeting B cells have been tested as therapeutics for inflammatory rheumatic diseases. We review important observations from randomized clinical trials regarding the efficacy and safety of anti-B cell antibody-based therapies for rheumatoid arthritis, systemic lupus...... and functions in rheumatic disorders. Future studies should also evaluate how to maintain disease control by means of conventional and/or biologic immunosuppressants after remission-induction with anti-B cell antibodies....

Analysis of cell kinetics after gamma ray irradiation using anti-BrdU monoclonal antibody

International Nuclear Information System (INIS)

Akagi, Kiyoshi; Tanaka, Yoshimasa

1989-01-01

The cell cycle was analyzed using anti-BrdU monoclonal antibody, and changes in cell kinetics after gamma ray irradiation as evaluated by this BrdU-PI double staining were compared with those evaluated by the DNA histogram method based on PI staining. The effect of irradiation on the cell kinetics has been studied according primarily to the number of G2 blocked cells. By the present BrdU method, rapid transition of the G1-S phase was observed within 2 hours of irradiation, and then G1 block was observed. Cells in the S phase progressed to the G2 + M cells returned to the G1 phase after 18 or more hours. These initial G1 blocked cells induced by irradiation were confirmed for the fist time by the present BrdU-PI double staining. By the conventional method based on the DNA histogram, accurate determination of S cell fraction was difficult due to overlapping of the DNA contents of G1 cells and early S cells and those of late S cells and G2 cells. On the other hand, BrdU-PI double staining allowed direct differentiation of G1, S, and G2 + M cells, especially between G1-S and S-G2 + M cells. The analysis of cell kinetics using BrdU is advantageous over the conventional autoradiographic methods in that it allowed more rapid assay with very high sensitivity. In addition, BrdU is alrady used clinically as an enhancement agent in radiation therapy for cancer. The present method is considered to be indispensable for evaluation of the percentage of S cells in the tumor tissue and analysis of cell kinetics after irradiation and chemotherapy against cancer. (author)

Rapid High-Level Production of Functional HIV Broadly Neutralizing Monoclonal Antibodies in Transient Plant Expression Systems

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Rosenberg, Yvonne; Sack, Markus; Montefiori, David; Forthal, Donald; Mao, Lingjun; -Abanto, Segundo Hernandez; Urban, Lori; Landucci, Gary; Fischer, Rainer; Jiang, Xiaoming

2013-01-01

Passive immunotherapy using anti-HIV broadly neutralizing monoclonal antibodies (mAbs) has shown promise as an HIV treatment, reducing mother-to-child-transmission (MTCT) of simian/human immunodeficiency virus (SHIV) in non-human primates and decreasing viral rebound in patients who ceased receiving anti-viral drugs. In addition, a cocktail of potent mAbs may be useful as mucosal microbicides and provide an effective therapy for post-exposure prophylaxis. However, even highly neutralizing HIV mAbs used today may lose their effectiveness if resistance occurs, requiring the rapid production of new or engineered mAbs on an ongoing basis in order to counteract the viral resistance or the spread of a certain HIV-1 clade in a particular region or patient. Plant-based expression systems are fast, inexpensive and scalable and are becoming increasingly popular for the production of proteins and monoclonal antibodies. In the present study, Agrobacterium-mediated transient transfection of plants, utilizing two species of Nicotiana, have been tested to rapidly produce high levels of an HIV 89.6PΔ140env and several well-studied anti-HIV neutralizing monoclonal antibodies (b12, 2G12, 2F5, 4E10, m43, VRC01) or a single chain antibody construct (m9), for evaluation in cell-based viral inhibition assays. The protein-A purified plant-derived antibodies were intact, efficiently bound HIV envelope, and were equivalent to, or in one case better than, their counterparts produced in mammalian CHO or HEK-293 cells in both neutralization and antibody dependent viral inhibition assays. These data indicate that transient plant-based transient expression systems are very adaptable and could rapidly generate high levels of newly identified functional recombinant HIV neutralizing antibodies when required. In addition, they warrant detailed cost-benefit analysis of prolonged incubation in plants to further increase mAb production. PMID:23533588

Rapid high-level production of functional HIV broadly neutralizing monoclonal antibodies in transient plant expression systems.

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Yvonne Rosenberg

Full Text Available Passive immunotherapy using anti-HIV broadly neutralizing monoclonal antibodies (mAbs has shown promise as an HIV treatment, reducing mother-to-child-transmission (MTCT of simian/human immunodeficiency virus (SHIV in non-human primates and decreasing viral rebound in patients who ceased receiving anti-viral drugs. In addition, a cocktail of potent mAbs may be useful as mucosal microbicides and provide an effective therapy for post-exposure prophylaxis. However, even highly neutralizing HIV mAbs used today may lose their effectiveness if resistance occurs, requiring the rapid production of new or engineered mAbs on an ongoing basis in order to counteract the viral resistance or the spread of a certain HIV-1 clade in a particular region or patient. Plant-based expression systems are fast, inexpensive and scalable and are becoming increasingly popular for the production of proteins and monoclonal antibodies. In the present study, Agrobacterium-mediated transient transfection of plants, utilizing two species of Nicotiana, have been tested to rapidly produce high levels of an HIV 89.6PΔ140env and several well-studied anti-HIV neutralizing monoclonal antibodies (b12, 2G12, 2F5, 4E10, m43, VRC01 or a single chain antibody construct (m9, for evaluation in cell-based viral inhibition assays. The protein-A purified plant-derived antibodies were intact, efficiently bound HIV envelope, and were equivalent to, or in one case better than, their counterparts produced in mammalian CHO or HEK-293 cells in both neutralization and antibody dependent viral inhibition assays. These data indicate that transient plant-based transient expression systems are very adaptable and could rapidly generate high levels of newly identified functional recombinant HIV neutralizing antibodies when required. In addition, they warrant detailed cost-benefit analysis of prolonged incubation in plants to further increase mAb production.

NOSOTROPIC SUBSTANTIATION OF ANTI IgE ANIBODY THERAPY

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Yu.G. Levina

2008-01-01

Full Text Available This article studies the specifics of allergic diseases pathogenesis. Such common allergic diseases as bronchial asthma, allergic rhinitis and atopic dermatitis are conditioned by processes based on increase of immunoglobulin e synthesis. Omalizunab, which is a recombinant humanized monoclone anti-Ige antibody, prevents Ige fixing to membrane receptors of mast cells and significantly reduces the level of immunoglobulin e circulating in blood.Key words: anti-Ige antibody, omalizumab, allergic diseases, Bronchial asthma, children.

Monoclonal antibodies against human angiotensinogen, their characterization and use in an angiotensinogen enzyme linked immunosorbent assay.

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Rubin, I; Lykkegaard, S; Olsen, A A; Selmer, J; Ballegaard, M

1988-01-01

Monoclonal antibodies were produced against human angiotensinogen. An enzyme linked immunosorbent assay (ELISA) was developed using a high affinity monoclonal antibody as catching antibody and a polyclonal rabbit anti human angiotensinogen antibody as detecting antibody in a "sandwich" ELISA. Linear range of the ELISA was 15-450 pmol/l of human angiotensinogen. Intra- and inter- assay variation coefficients were in the range of 2% to 8%. A correlation coefficient, r = 0.97, (n = 20), with values obtained by radioimmunoassay. This correlation coefficient, obtained by using both normal and pregnant sera, confirmed that the ELISA fulfill the requirements for clinical useful assay. Characterization of the antibodies were performed with respect to affinity constant and epitopes.

Pro region engineering of nerve growth factor by deep mutational scanning enables a yeast platform for conformational epitope mapping of anti-NGF monoclonal antibodies.

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Medina-Cucurella, Angélica V; Zhu, Yaqi; Bowen, Scott J; Bergeron, Lisa M; Whitehead, Timothy A

2018-04-12

Nerve growth factor (NGF) plays a central role in multiple chronic pain conditions. As such, anti-NGF monoclonal antibodies (mAbs) that function by antagonizing NGF downstream signaling are leading drug candidates for non-opioid pain relief. To evaluate anti-canine NGF (cNGF) mAbs we sought a yeast surface display platform of cNGF. Both mature cNGF and pro-cNGF displayed on the yeast surface but bound conformationally sensitive mAbs at most 2.5-fold in mean fluorescence intensity above background, suggesting that cNGF was mostly misfolded. To improve the amount of folded, displayed cNGF, we used comprehensive mutagenesis, FACS, and deep sequencing to identify point mutants in the pro-region of canine NGF that properly enhance the folded protein displayed on the yeast surface. Out of 1,737 tested single point mutants in the pro region, 49 increased the amount of NGF recognized by conformationally sensitive mAbs. These gain-of-function mutations cluster around residues A-61-P-26. Gain-of-function mutants were additive, and a construct containing three mutations increased amount of folded cNGF to 23- fold above background. Using this new cNGF construct, fine conformational epitopes for tanezumab and three anti-cNGF mAbs were evaluated. The epitope revealed by the yeast experiments largely overlapped with the tanezumab epitope previously determined by X-ray crystallography. The other mAbs showed site-specific differences with tanezumab. As the number of binding epitopes of functionally neutralizing anti-NGF mAbs on NGF are limited, subtle differences in the individual interacting residues on NGF that bind each mAb contribute to the understanding of each antibody and variations in its neutralizing activity. These results demonstrate the potential of deep sequencing-guided protein engineering to improve the production of folded surface-displayed protein, and the resulting cNGF construct provides a platform to map conformational epitopes for other anti-neurotrophin m

A MONOCLONAL ANTIBODY-BETA-GLUCURONIDASE CONJUGATE AS ACTIVATOR OF THE PRODRUG EPIRUBICIN-GLUCURONIDE FOR SPECIFIC TREATMENT OF CANCER

NARCIS (Netherlands)

Haisma, Hidde; BOVEN, E; VANMUIJEN, M; DEJONG, J; VANDERVIJGH, WJF; PINEDO, HM

The anti-pan carcinoma monoclonal antibody (MAb) 323/A3, linked to E. coli-derived beta-glucuronidase (GUS) was used to study the tumour-site-selective activation of the prodrug Epirubicin-glucuronide (Epi-glu). Epi-glu was isolated from the urine of patients treated with Epirubicin (Epi) by

Therapeutic Recombinant Monoclonal Antibodies

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Bakhtiar, Ray

2012-01-01

During the last two decades, the rapid growth of biotechnology-derived techniques has led to a myriad of therapeutic recombinant monoclonal antibodies with significant clinical benefits. Recombinant monoclonal antibodies can be obtained from a number of natural sources such as animal cell cultures using recombinant DNA engineering. In contrast to…

Long-term measurement of anti-adalimumab using pH-shift-anti-idiotype antigen binding test shows predictive value and transient antibody formation

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van Schouwenburg, Pauline A.; Krieckaert, Charlotte L.; Rispens, Theo; Aarden, Lucien; Wolbink, Gerrit Jan; Wouters, Diana

2013-01-01

Therapeutic monoclonal antibodies are effective drugs for many different diseases. However, the formation of anti-drug antibodies (ADA) against a biological can result in reduced clinical response in some patients. Measurement of ADA in the presence of (high) drug levels is difficult due to drug

Mapping of cat albumin using monoclonal antibodies: identification of determinants common to cat and dog.

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Boutin, Y; Hébert, J; Vrancken, E R; Mourad, W

1989-01-01

Cat and dog albumins from commercial extracts were used to produce monoclonal antibodies (MoAb). Anti-cat albumin MoAb recognized both cat and dog albumin equally, as did anti-dog albumin MoAb; this confirms cross-reactivity between cat and dog. The MoAb were separated into two groups according to their epitopic specificity; they recognized two overlapping epitopes of cat albumin. Furthermore, by competitive inhibition of radio-allergosorbent test (RAST), it was shown that one MoAb group inhibited significantly the binding of human IgE antibodies (from a pool of 13 patients allergic to both cats and dogs) to insolubilized cat or dog extracts. These observations suggest that murine anti-cat or anti-dog MoAb and human IgE antibodies recognize identical or closely related determinants on cat and dog albumin. Images Fig. 1 Fig. 2 PMID:2478325

A four-step sandwich radioimmunoassay for direct selection of monoclonal antibodies to allergen molecules

International Nuclear Information System (INIS)

Ley, V.; Corbi, A.L.; Sanchez-Madrid, F.; Carreira, J.C.

1985-01-01

A 4-step radioimmunoassay has been devised for direct identification of monoclonal antibodies (MAb) directed to IgE-binding molecules. Polyvinyl chloride wells coated with purified anti-mouse kappa chain MAb (187-1) were successively incubated with: (1) MAb-containing hybridoma supernatants, (2) allergen extract, (3) allergic patients' serum pool, and (4) 125 I-labeled anti-human IgE antiserum, to detect MAb-allergen-IgE complexes. MAb to allergens from Parietaria judaica pollen and Dermatophagoides mites have been selected with this screening procedure. The affinity-purified allergen molecules competed the binding of IgE to allergen extracts coated to paper discs in a RAST inhibition assay, confirming the anti-allergen specificity of the selected MAb. This screening method is sensitive enough to allow detection of MAb directed to poorly represented allergens. (Auth.)

Pharmacokinetics, biodistribution and dosimetry of 99mTc-labeled anti-human epidermal growth factor receptor humanized monoclonal antibody R3 in rats

International Nuclear Information System (INIS)

Escobar, Normando Iznaga; Morales, Alejo Morales; Duconge, Jorge; Torres, Idania Caballero; Fernandez, Eduardo; Gomez, Jose A.

1998-01-01

The pharmacokinetics, biodistribution and dosimetry of 99m Tc-labeled anti-human epidermal growth factor receptor (anti-hEGF-r) humanized monoclonal antibody (MAb) R3 was investigated following intravenous injection in normal Wistar rats. Serum disappearance curves were best fit by a two-compartment model having a mean distribution half-life (t (1(2α)) ) of 0.250 h and a mean elimination (t (1(2β)) ) of 13.89 h. Among the various organs, a little accumulation of the radiolabeled antibody was found only in kidneys. Biodistribution and dosimetry studies in humans were performed by extrapolation of the animal data to humans. Absorbed dose to normal organs and the remainder of the whole body were estimated using the medical internal radiation dose formula, and dose contributions from radioactivity in transit through the gastrointestinal tract were estimated using a compartment model. Extrapolated values of radiation absorbed dose to normal organs in rads per millicurie administered were whole body, 0.0085; lower large intestine wall, 0.0898; small intestine, 0.0530; upper large intestine wall, 0.0731; and kidneys, 0.0455. The effective dose equivalent predicted was 0.0162 rem/mCi and the effective dose was found to be 0.015 rem/mCi. On the basis of the pharmacokinetics, biodistribution and internal radiation dosimetry information obtained in this study, a diagnostic phase I clinical trial with 99m Tc-labeled humanized MAb R3 conjugate in patients should be supported

MAXIMIZATION OF DNA DAMAGE TO MGMT(+ EGFR(+ GBM CELLS USING OPTIMAL COMBINATION OF TEMOZOLOMIDE-ANTI EGFR MONOCLONAL ANTIBODY NIMOTUZUMAB

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M. A. M. Inggas

2015-09-01

Full Text Available Background: Glioblastoma multiforme (GBM is the most aggressive primary brain tumor in adultswith dismal prognosis due to the unavailability of an effective therapy. Up to now, there had been no definitive studies published on EGFR inhibition therapy as a chemosensitizer for GBM therapy using Temozolomide (TMZ. This study aims to reveal the most effective method and timing to administer TMZ-anti EGFR targeted therapy which causes maximal DNA damage on GBM cells.Methods: Various regimens of anti EGFR monoclonal antibody Nimotuzumab (NMZ was administered in different combinations with TMZ, performed on U87MG MGMT(+ EGFR(+ cells. The effectiveness of the combinations were evaluated by measuring yH2AX levels which reflects the degree of DNA damage. One-way Anova and LSD tests were performed to determine the effects of each treatment with p<0.05. Results and discussion: the mean SD of yH2AX of each treatment was: 11,90±1,25 for the control group; 29.33±1.91 for NMZ alone; 28.13±1.58 for TMZ alone; 41.53±3.51 for concurrent use; 35.67 ±2.65 for NMZ after 24 hours TMZ; 31.87±2.94 for NMZ after 48 hours TMZ; 39.57±4.2 for TMZ after 24 hours NMZ; and 35.93 ±3.56 for TMZ after 48 hours NMZ. The administration of TMZ concurrent with or after 24 hours NMZ gives the highest amount of DNA damage to GBM cells. Conclusion: The administration of Nimotuzumab targeted therapy up to 24 hours before Temozolomide chemotherapy has been proven to be effective in maximizing the amount of DNA damage done to GBM cells in vitro. 

Overview of the trastuzumab (Herceptin) anti-HER2 monoclonal antibody clinical program in HER2-overexpressing metastatic breast cancer. Herceptin Multinational Investigator Study Group.

Science.gov (United States)

Shak, S

1999-08-01

The recombinant humanized anti-HER2 monoclonal antibody trastuzumab (Herceptin; Genentech, San Francisco, CA) was evaluated in human clinical trials for treatment of women with metastatic breast cancer who have tumors that overexpress HER2. The trastuzumab clinical program consisted of a series of phase I, phase II, and phase III clinical trials. Clinical experience with this novel biologic has been obtained in more than 1,000 women with HER2-overexpressing metastatic breast cancer. Two pivotal trials were performed to evaluate trastuzumab efficacy and safety: (1) trastuzumab in combination with chemotherapy as first-line therapy and (2) trastuzumab as a single agent in second- and third-line chemotherapy. Preliminary results of the pivotal clinical trials that have been presented at national meetings are summarized below. The data suggest that trastuzumab will be an important new treatment option for women with HER2-overexpressing metastatic breast cancer.

Identification of a second T-cell antigen receptor in human and mouse by an anti-peptide γ-chain-specific monoclonal antibody

International Nuclear Information System (INIS)

Ioannides, C.G.; Itoh, K.; Fox, F.E.; Pahwa, R.; Good, R.A.; Platsoucas, C.D.

1987-01-01

The authors developed a monoclonal antibody (mAb) (9D7) against a synthetic peptide (P13K) selected from the deduced amino acid sequence of the constant region of the λ chain of the murine T-cell antigen receptor (TCR) (amino acids 118-130). Using this mAb, they identified a putative second TCR expressed on peripheral blood lymphocytes from a patient with severe combined immunodeficiency (SCID) that were propagated in culture with recombinant interleukin 2 (rIL-2) and Con A. This mAb immunoprecipitated two polypeptide chains of 40 and 58 kDa under nonreducing conditions and of 40 and 56 kDa under reducing conditions from 125 I-labeled denatured lysates of T3 + WT31 - lymphocytes expanded in culture from a SCID patient. Chemical crosslinking of 125 I-labeled cells followed by immunoprecipitation with anti-Leu-4 mAb under nonreducing or reducing conditions revealed that the 40- and 56-kDa polypeptide chains were associated with the T3 differentiation antigen. These experiments were done with polyclonal cell populations. Cloned T3 + WT31 - cell populations are required to determine whether the TCR contains two λ polypeptide chains. Using the same 9D7 anti-P18K mAb and immunoblotting analysis, they identified a 35 kDa γ-chain polypeptide under reducing conditions expressed on purified L3T4 - Lyt2 - BALB/c mouse thymocytes. This γ-chain TCR is disulfide linked and has a molecular mass of 80 kDa under nonreducing conditions

Production and characterization of human anti-V3 monoclonal antibodies from the cells of HIV-1 infected Indian donors

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Andrabi Raiees

2012-09-01

Full Text Available Abstract Background Analysis of human monoclonal antibodies (mAbs developed from HIV-1 infected donors have enormously contributed to the identification of neutralization sensitive epitopes on the HIV-1 envelope glycoprotein. The third variable region (V3 is a crucial target on gp120, primarily due to its involvement in co-receptor (CXCR4 or CCR5 binding and presence of epitopes recognized by broadly neutralizing antibodies. Methods Thirty-three HIV-1 seropositive drug naive patients (18 males and 15 females within the age range of 20–57 years (median = 33 years were recruited in this study for mAb production. The mAbs were selected from EBV transformed cultures with conformationally constrained Cholera-toxin-B containing V3C (V3C-CTB fusion protein. We tested the mAbs for their binding with HIV-1 derived proteins and peptides by ELISA and for neutralization against HIV-1 viruses by TZM-bl assays. Results We isolated three anti-V3 mAbs, 277, 903 and 904 from the cells of different individuals. The ELISA binding revealed a subtype-C and subtype-A specific binding of antibody 277 and 903 while mAb 904 exhibited cross reactivity also with subtype-B V3. Epitope mapping of mAbs with overlapping V3 peptides showed exclusive binding to V3 crown. The antibodies displayed high and low neutralizing activity against 2/5 tier 1 and 1/6 tier 2 viruses respectively. Overall, we observed a resistance of the tier 2 viruses to neutralization by the anti-V3 mAbs, despite the exposure of the epitopes recognized by these antibodies on two representative native viruses (Du156.12 and JRFL, suggesting that the affinity of mAb might equally be crucial for neutralization, as the epitope recognition. Conclusions Our study suggests that the anti-V3 antibodies derived from subtype-C infected Indian patients display neutralization potential against tier 1 viruses while such activity may be limited against more resistant tier 2 viruses. Defining the fine epitope

Enavatuzumab, a Humanized Anti-TWEAK Receptor Monoclonal Antibody, Exerts Antitumor Activity through Attracting and Activating Innate Immune Effector Cells

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Shiming Ye

2017-01-01

Full Text Available Enavatuzumab is a humanized IgG1 anti-TWEAK receptor monoclonal antibody that was evaluated in a phase I clinical study for the treatment of solid malignancies. The current study was to determine whether and how myeloid effector cells were involved in postulated mechanisms for its potent antitumor activity in xenograft models. The initial evidence for a role of effector cells was obtained in a subset of tumor xenograft mouse models whose response to enavatuzumab relied on the binding of Fc of the antibody to Fcγ receptor. The involvement of effector cells was further confirmed by immunohistochemistry, which revealed strong infiltration of CD45+ effector cells into tumor xenografts in responding models, but minimal infiltration in nonresponders. Consistent with the xenograft studies, human effector cells preferentially migrated toward in vivo-responsive tumor cells treated by enavatuzumab in vitro, with the majority of migratory cells being monocytes. Conditioned media from enavatuzumab-treated tumor cells contained elevated levels of chemokines, which might be responsible for enavatuzumab-triggered effector cell migration. These preclinical studies demonstrate that enavatuzumab can exert its potent antitumor activity by actively recruiting and activating myeloid effectors to kill tumor cells. Enavatuzumab-induced chemokines warrant further evaluation in clinical studies as potential biomarkers for such activity.

NPR1 protein regulates pathogenic and symbiotic interactions between Rhizobium and legumes and non-legumes.

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Smadar Peleg-Grossman

Full Text Available BACKGROUND: Legumes are unique in their ability to establish symbiotic interaction with rhizobacteria from Rhizobium genus, which provide them with available nitrogen. Nodulation factors (NFs produced by Rhizobium initiate legume root hair deformation and curling that entrap the bacteria, and allow it to grow inside the plant. In contrast, legumes and non-legumes activate defense responses when inoculated with pathogenic bacteria. One major defense pathway is mediated by salicylic acid (SA. SA is sensed and transduced to downstream defense components by a redox-regulated protein called NPR1. METHODOLOGY/PRINCIPAL FINDINGS: We used Arabidopsis mutants in SA defense pathway to test the role of NPR1 in symbiotic interactions. Inoculation of Sinorhizobium meliloti or purified NF on Medicago truncatula or nim1/npr1 A. thaliana mutants induced root hair deformation and transcription of early and late nodulins. Application of S. meliloti or NF on M. truncatula or A. thaliana roots also induced a strong oxidative burst that lasted much longer than in plants inoculated with pathogenic or mutualistic bacteria. Transient overexpression of NPR1 in M. truncatula suppressed root hair curling, while inhibition of NPR1 expression by RNAi accelerated curling. CONCLUSIONS/SIGNIFICANCE: We show that, while NPR1 has a positive effect on pathogen resistance, it has a negative effect on symbiotic interactions, by inhibiting root hair deformation and nodulin expression. Our results also show that basic plant responses to Rhizobium inoculation are conserved in legumes and non-legumes.

Thermodynamic and kinetic approaches for evaluation of monoclonal antibody - Lipoprotein interactions.

Science.gov (United States)

Multia, Evgen; Sirén, Heli; Andersson, Karl; Samuelsson, Jörgen; Forssén, Patrik; Fornstedt, Torgny; Öörni, Katariina; Jauhiainen, Matti; Riekkola, Marja-Liisa

2017-02-01

Two complementary instrumental techniques were used, and the data generated was processed with advanced numerical tools to investigate the interactions between anti-human apoB-100 monoclonal antibody (anti-apoB-100 Mab) and apoB-100 containing lipoproteins. Partial Filling Affinity Capillary Electrophoresis (PF-ACE) combined with Adsorption Energy Distribution (AED) calculations provided information on the heterogeneity of the interactions without any a priori model assumptions. The AED calculations evidenced a homogenous binding site distribution for the interactions. Quartz Crystal Microbalance (QCM) studies were used to evaluate thermodynamics and kinetics of the Low-Density Lipoprotein (LDL) and anti-apoB-100 Mab interactions. High affinity and selectivity were observed, and the emerging data sets were analysed with so called Interaction Maps. In thermodynamic studies, the interaction between LDL and anti-apoB-100 Mab was found to be predominantly enthalpy driven. Both techniques were also used to study antibody interactions with Intermediate-Density (IDL) and Very Low-Density (VLDL) Lipoproteins. By screening affinity constants for IDL-VLDL sample in a single injection we were able to distinguish affinity constants for both subpopulations using the numerical Interaction Map tool. Copyright © 2016 Elsevier Inc. All rights reserved.

Anti-PD-L1 Treatment Induced Central Diabetes Insipidus.

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Zhao, Chen; Tella, Sri Harsha; Del Rivero, Jaydira; Kommalapati, Anuhya; Ebenuwa, Ifechukwude; Gulley, James; Strauss, Julius; Brownell, Isaac

2018-02-01

Immune checkpoint inhibitors, including anti-programmed cell death protein 1 (PD-1), anti-programmed cell death protein ligand 1 (PD-L1), and anti-cytotoxic T-lymphocyte antigen 4 (anti-CTLA4) monoclonal antibodies, have been widely used in cancer treatment. They are known to cause immune-related adverse events (irAEs), which resemble autoimmune diseases. Anterior pituitary hypophysitis with secondary hypopituitarism is a frequently reported irAE, especially in patients receiving anti-CTLA4 treatment. In contrast, posterior pituitary involvement, such as central diabetes insipidus (DI), is relatively rare and is unreported in patients undergoing PD-1/PD-L1 blockade. We describe a case of a 73-year-old man with Merkel cell carcinoma who received the anti-PD-L1 monoclonal antibody avelumab and achieved partial response. The patient developed nocturia, polydipsia, and polyuria 3 months after starting avelumab. Further laboratory testing revealed central DI. Avelumab was held and he received desmopressin for the management of central DI. Within 6 weeks after discontinuation of avelumab, the patient's symptoms resolved and he was eventually taken off desmopressin. The patient remained off avelumab and there were no signs or symptoms of DI 2 months after the discontinuation of desmopressin. To our knowledge, this is the first report of central DI associated with anti-PD-L1 immunotherapy. The patient's endocrinopathy was successfully managed by holding treatment with the immune checkpoint inhibitor. This case highlights the importance of early screening and appropriate management of hormonal irAEs in subjects undergoing treatment with immune checkpoint inhibitors to minimize morbidity and mortality. Copyright © 2017 Endocrine Society

Pharmacokinetics of internally labeled monoclonal antibodies as a gold standard: comparison of biodistribution of 75Se-, 111In-, and 125I-labeled monoclonal antibodies in osteogenic sarcoma xenografts in nude mice

International Nuclear Information System (INIS)

Koizumi, M.; Endo, K.; Watanabe, Y.; Saga, T.; Sakahara, H.; Konishi, J.; Yamamuro, T.; Toyama, S.

1989-01-01

In order to know the true biodistribution of anti-tumor monoclonal antibodies, three monoclonal antibodies (OST6, OST7, and OST15) against human osteosarcoma and control antibody were internally labeled with 75Se by incubating [75Se]methionine and hybridoma cells. 75Se-labeled monoclonal antibodies were evaluated both in vitro and in vivo using the human osteogenic sarcoma cell line KT005, and the results were compared with those of 125I- and 111In-labeled antibodies. 75Se-, 125I- and 111In-labeled monoclonal antibodies had identical binding activities to KT005 cells, and the immunoreactivity was in the decreasing order of OST6, OST7, and OST15. On the contrary, in vivo tumor uptake (% injected dose/g) of 75Se- and 125I-labeled antibodies assessed using nude mice bearing human osteosarcoma KT005 was in the order of OST7, OST6, and OST15. In the case of 111In, the order was OST6, OST7, and OST15. High liver uptake was similarly seen with 75Se- and 111In-labeled antibodies, whereas 125I-labeled antibodies showed the lowest tumor and liver uptake. These data indicate that tumor targeting of antibody conjugates are not always predictable from cell binding studies due to the difference of blood clearance of labeled antibodies. Furthermore, biodistribution of both 111In- and 125I-labeled antibodies are not identical with internally labeled antibody. Admitting that internally labeled antibody is a ''gold standard'' of biodistribution of monoclonal antibody, high liver uptake of 111In-radiolabeled antibodies may be inherent to antibodies. Little, if any, increase in tumor-to-normal tissue ratios of antibody conjugates will be expected compared to those of 111In-labeled antibodies if stably coupled conjugates are administered i.v

Anti-PrPC monoclonal antibody infusion as a novel treatment for cognitive deficits in an alzheimer's disease model mouse

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Strittmatter Stephen M

2010-10-01

Full Text Available Abstract Background Alzheimer's Disease (AD is the most common of the conformational neurodegenerative disorders characterized by the conversion of a normal biological protein into a β-sheet-rich pathological isoform. In AD the normal soluble Aβ (sAβ forms oligomers and fibrils which assemble into neuritic plaques. The most toxic form of Aβ is thought to be oligomeric. A recent study reveals the cellular prion protein, PrPC, to be a receptor for Aβ oligomers. Aβ oligomers suppress LTP signal in murine hippocampal slices but activity remains when pretreated with the PrP monoclonal anti-PrP antibody, 6D11. We hypothesized that targeting of PrPC to prevent Aβ oligomer-related cognitive deficits is a potentially novel therapeutic approach. APP/PS1 transgenic mice aged 8 months were intraperitoneally (i.p. injected with 1 mg 6D11 for 5 days/week for 2 weeks. Two wild-type control groups were given either the same 6D11 injections or vehicle solution. Additional groups of APP/PS1 transgenic mice were given either i.p. injections of vehicle solution or the same dose of mouse IgG over the same period. The mice were then subjected to cognitive behavioral testing using a radial arm maze, over a period of 10 days. At the conclusion of behavioral testing, animals were sacrificed and brain tissue was analyzed biochemically or immunohistochemically for the levels of amyloid plaques, PrPC, synaptophysin, Aβ40/42 and Aβ oligomers. Results Behavioral testing showed a marked decrease in errors in 6D11 treated APP/PS1 Tg mice compared with the non-6D11 treated Tg groups (p C or Aβ oligomer levels. 6D11 treated APP/PS1 Tg mice had significantly greater synaptophysin immunoreactivity in the dentate gyrus molecular layer of the hippocampus compared to vehicle treated APP/PS1 Tg mice (p Conclusions Even short term treatment with monoclonal antibodies such as 6D11 or other compounds which block the binding of Aβ oligomers to PrPC can be used to treat

Use of monoclonal antibodies as an effective strategy for treatment of ciguatera poisoning.

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Inoue, Masayuki; Lee, Nayoung; Tsumuraya, Takeshi; Fujii, Ikuo; Hirama, Masahiro

2009-06-01

Ciguatera is a global food poisoning caused by the consumption of fish that have accumulated sodium channel activator toxins, ciguatoxins. At present, most diagnosed cases of ciguatera are treated with symptomatic and supportive remedies, and no specific therapy has been devised. Here we report that ciguatoxin CTX3C can be effectively neutralized in vitro and in vivo by simultaneous use of two anti-ciguatoxin monoclonal antibodies, providing the first rational approach toward directly preventing and treating ciguatera.

Detection of the argonaute protein Ago2 and microRNAs in the RNA induced silencing complex (RISC) using a monoclonal antibody.

Science.gov (United States)

Ikeda, Keigo; Satoh, Minoru; Pauley, Kaleb M; Fritzler, Marvin J; Reeves, Westley H; Chan, Edward K L

2006-12-20

MicroRNAs (miRNAs) are short RNA molecules responsible for post-transcriptional gene silencing by the degradation or translational inhibition of their target messenger RNAs (mRNAs). This process of gene silencing, known as RNA interference (RNAi), is mediated by highly conserved Argonaute (Ago) proteins which are the key components of the RNA induced silencing complex (RISC). In humans, Ago2 is responsible for the endonuclease cleavage of targeted mRNA and it interacts with the mRNA-binding protein GW182, which is a marker for cytoplasmic foci referred to as GW bodies (GWBs). We demonstrated that the anti-Ago2 monoclonal antibody 4F9 recognized GWBs in a cell cycle dependent manner and was capable of capturing miRNAs associated with Ago2. Since Ago2 protein is the effector protein of RNAi, anti-Ago2 monoclonal antibody may be useful in capturing functional miRNAs.

Radiolabelled monoclonal antibodies against alpha-fetoprotein for in vivo localization of human hepatocellular carcinoma by immunotomoscintigraphy

International Nuclear Information System (INIS)

Bergmann, J.F.; Lumbroso, J.D.; Manil, L.; Saccavini, J.C.; Rougier, P.; Assicot, M.; Mathieu, A.; Bellet, D.; Bohuon, C.

1987-01-01

Two high affinity monoclonal antibodies, designated AF01 and AF04, directed against distinct epitopes of human alpha-fetoprotein (AFP) and the Fab fragments of one of them, were labelled with 131 I and injected into 18 patients with AFP producing hepatocellular carcinoma (HCC) in order to carry out imaging studies by tomoscintigraphy. Twelve patients were injected with whole antibody, only three of seven patients injected with AF01 and two of five patients injected with AF04 had a positive scan. In contrast, five out of six patients injected with labelled Fab fragments of AF04 had positive imaging. These results confirm that tumour imaging of HCC using 131 I labelled monoclonal antibody against AFP is feasible. Moreover, utilization of tomoscintigraphy in place of linear scintigraphy and Fab fragments instead of whole immunoglobulin may improve the sensitivity of radioimmunolocalization. This technique provides useful information on the in vivo distribution of monoclonal antibodies directed against AFP and on the practicability of the eventual therapeutic use of anti-AFP antibodies in HCC. (orig.)

Monoclonal antibodies to Pneumocystis carinii

DEFF Research Database (Denmark)

Kovacs, J A; Halpern, J L; Lundgren, B

1989-01-01

To increase understanding of the antigenic structure of Pneumocystis carinii, we developed monoclonal antibodies to rat and human P. carinii. The specificity of the antibodies was demonstrated by immunofluorescence and immunoblot studies. Only one of five monoclonal antibodies to rat P. carinii r...

Analysis of Tn antigenicity with a panel of new IgM and IgG1 monoclonal antibodies raised against leukemic cells

DEFF Research Database (Denmark)

Blixt, Klas Ola; Lavrova, Olga I; Mazurov, Dmitriy V

2012-01-01

antigenicity remain poorly understood. As a consequence, a broad variety of anti-Tn monoclonal antibodies (mAbs) have been generated. To further investigate the nature and complexity of the Tn antigen, we generated seven different anti-Tn mAbs of IgM and IgG classes raised against human Jurkat T cells, which...... are Tn-positive due to the low activity of T-synthase and mutation in specific chaperone Cosmc. The binding analysis of anti-Tn mAbs with the array of synthetic saccharides, glycopeptides and O-glycoproteins revealed unexpected differences in specificities of anti-Tn mAbs. IgM mAbs bound the terminal Gal...

Development and characterization of anti-glycopeptide monoclonal antibodies against human podoplanin, using glycan-deficient cell lines generated by CRISPR/Cas9 and TALEN.

Science.gov (United States)

Kaneko, Mika K; Nakamura, Takuro; Honma, Ryusuke; Ogasawara, Satoshi; Fujii, Yuki; Abe, Shinji; Takagi, Michiaki; Harada, Hiroyuki; Suzuki, Hiroyoshi; Nishioka, Yasuhiko; Kato, Yukinari

2017-02-01

Human podoplanin (hPDPN), which binds to C-type lectin-like receptor-2 (CLEC-2), is involved in platelet aggregation and cancer metastasis. The expression of hPDPN in cancer cells or cancer-associated fibroblasts indicates poor prognosis. Human lymphatic endothelial cells, lung-type I alveolar cells, and renal glomerular epithelial cells express hPDPN. Although numerous monoclonal antibodies (mAbs) against hPDPN are available, they recognize peptide epitopes of hPDPN. Here, we generated a novel anti-hPDPN mAb, LpMab-21. To characterize the hPDPN epitope recognized by the LpMab-21, we established glycan-deficient CHO-S and HEK-293T cell lines, using the CRISPR/Cas9 or TALEN. Flow cytometric analysis revealed that the minimum hPDPN epitope, in which sialic acid is linked to Thr76, recognized by LpMab-21 is Thr76-Arg79. LpMab-21 detected hPDPN expression in glioblastoma, oral squamous carcinoma, and seminoma cells as well as in normal lymphatic endothelial cells. However, LpMab-21 did not react with renal glomerular epithelial cells or lung type I alveolar cells, indicating that sialylation of hPDPN Thr76 is cell-type-specific. LpMab-21 combined with other anti-hPDPN antibodies that recognize different epitopes may therefore be useful for determining the physiological function of sialylated hPDPN. © 2016 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

Radioimmunoscintigraphy of experimental arterial and venous thrombi in animals with 99Tcm labelled monoclonal antibody against thrombus elements

International Nuclear Information System (INIS)

Ji Shundong; Liu Xiaojian; Zhang Rongjun; Wan Weixing; Jin Jian; Yuan Changgeng

2000-01-01

Object: To evaluate the use of 99 Tc m labelled anti P-Selection monoclonal antibody (McAb)SZ-51 and anti-fibrin McAb SZ-63 in detection of experimental thrombi in rabbits and dogs. Method: The McAb SZ-51 and SZ-63 were labelled by using the method of 2-imino-thiolane modification and 99 Tc m -glucoheptonate (GH) trans-chelation. The experimental femoral arterial and venous thrombosis were prepared, then 99 Tc m -McAb was injected into ear-edge vein, finally imaged by SPECT. 99 Tc m -labelled murine IgG was used as a negative control. Results: The fresh arterial and venous thrombi in dogs were clearly imaged 0.5 to 2 h and 2 to 4 h after injection of 99 Tc m -SZ-51/63 and 99 Tc m -SZ-51, respectively. The old arterial and venous thrombi in rabbits were clearly imaged 2 to 4 h after injection of 99 Tc m -SZ-63. Conclusion: the monoclonal antibody SZ-51 and SZ-63 would be a potential agent for imaging diagnosis of thrombotic disease

At least two Fc Neu5Gc residues of monoclonal antibodies are required for binding to anti-Neu5Gc antibody.

Science.gov (United States)

Yu, Chuanfei; Gao, Kai; Zhu, Lei; Wang, Wenbo; Wang, Lan; Zhang, Feng; Liu, Chunyu; Li, Meng; Wormald, Mark R; Rudd, Pauline M; Wang, Junzhi

2016-01-29

Two non-human glycan epitopes, galactose-α-1,3-galactose (α-gal) and Neu5Gc-α-2-6-galactose (Neu5Gc) have been shown to be antigenic when attached to Fab oligosaccharides of monoclonal antibodies (mAbs) , while α-gal attached to Fc glycans was not. However, the antigenicity of Neu5Gc on the Fc glycans remains unclear in the context that most mAbs carry only Fc glycans. After studying two clinical mAbs carrying significant amounts of Fc Neu5Gc, we show that their binding activity with anti-Neu5Gc antibody resided in a small subset of mAbs carrying two or more Fc Neu5Gc, while mAbs harboring only one Neu5Gc showed no reactivity. Since most Neu5Gc epitopes were distributed singly on the Fc of mAbs, our results suggest that the potential antigenicity of Fc Neu5Gc is low. Our study could be referenced in the process design and optimization of mAb production in murine myeloma cells and in the quality control of mAbs for industries and regulatory authorities.

Screening for epitope specificity directly on culture supernatants in the early phase of monoclonal antibody production by an ELISA with biotin-labeled antigen.

Science.gov (United States)

Andersen, Ditte C; Jensen, Charlotte H; Gregersen, Annemette; Brandt, Jette; Kliem, Anette; Skjødt, Karsten; Koch, Claus; Teisner, Børge

2004-01-01

This report describes an assay for comparison of epitope specificity in groups of monoclonal antibodies against a given antigen. The only prerequisite is the biotin-labeled antigen. One of the monoclonal antibodies is captured onto a plastic surface via a rabbit anti-mouse Ig, and the other preincubated with biotinylated antigen. When the two antibodies react with the same epitope subsequent binding of the biotin-labeled antigen is abolished (inhibition). In the cases where no inhibition was observed, the two antibodies were considered to react with distinct, independent epitopes. The obvious advantages using this assay, are that it can be performed directly on culture supernatants in the early phase of monoclonal antibody production, and also works for antigens with repetitive epitopes. Moreover, the bonus effect, i.e., a signal in excess of the reference signal when sets of monoclonal antibodies with different epitope specificity are compared, gives a relative measure of affinity.

Effects of anti-vascular endothelial growth factor monoclonal antibody (bevacizumab on lens epithelial cells

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Jun JH

2016-06-01

Full Text Available Jong Hwa Jun,1 Wern-Joo Sohn,2 Youngkyun Lee,2 Jae-Young Kim21Department of Ophthalmology, School of Medicine, Dongsan Medical Center, Keimyung University, 2Department of Oral Biochemistry, School of Dentistry, IHBR, Kyungpook National University, Daegu, South KoreaAbstract: The molecular and cellular effects of anti-vascular endothelial growth factor monoclonal antibody (bevacizumab on lens epithelial cells (LECs were examined using both an immortalized human lens epithelial cell line and a porcine capsular bag model. After treatment with various concentrations of bevacizumab, cell viability and proliferation patterns were evaluated using the water-soluble tetrazolium salt assay and 5-bromo-2'-deoxyuridine enzyme-linked immunosorbent assay, respectively. The scratch assay and Western blot analysis were employed to validate the cell migration pattern and altered expression levels of signaling molecules related to the epithelial–mesenchymal transition (EMT. Application of bevacizumab induced a range of altered cellular events in a concentration-dependent manner. A 0.1–2 mg/mL concentration demonstrated dose-dependent increase in proliferation and viability of LECs. However, 4 mg/mL decreased cell proliferation and viability. Cell migrations displayed dose-dependent retardation from 0.1 mg/mL bevacizumab treatment. Transforming growth factor-β2 expression was markedly increased in a dose-dependent manner, and α-smooth muscle actin, matrix metalloproteinase-9, and vimentin expression levels showed dose-dependent changes in a B3 cell line. Microscopic observation of porcine capsular bag revealed changes in cellular morphology and a decline in cell density compared to the control after 2 mg/mL treatment. The central aspect of posterior capsule showed delayed confluence, and the factors related to EMT revealed similar expression patterns to those identified in the cell line. Based on these results, bevacizumab modulates the proliferation

Screening for epitope specificity directly on culture supernatants in the early phase of monoclonal antibody production by an ELISA with biotin-labeled antigen

DEFF Research Database (Denmark)

Andersen, Ditte C; Jensen, Charlotte H; Gregersen, Annemette

2004-01-01

This report describes an assay for comparison of epitope specificity in groups of monoclonal antibodies against a given antigen. The only prerequisite is the biotin-labeled antigen. One of the monoclonal antibodies is captured onto a plastic surface via a rabbit anti-mouse Ig, and the other...... preincubated with biotinylated antigen. When the two antibodies react with the same epitope subsequent binding of the biotin-labeled antigen is abolished (inhibition). In the cases where no inhibition was observed, the two antibodies were considered to react with distinct, independent epitopes. The obvious...

An ultra-sensitive monoclonal antibody-based fluorescent microsphere immunochromatographic test strip assay for detecting aflatoxin M1 in milk

Science.gov (United States)

A rapid lateral flow fluorescent microspheres immunochromatography test strip (FMs-ICTS) has been developed for the detection of aflatoxin M1 (AFM1) residues in milk. For this purpose, an ultra-sensitive anti-AFM1 monoclonal antibody (MAb) 1D3 was prepared and identified. The IC50 value of the MA...

Quantitative assessment of antibody internalization with novel monoclonal antibodies against Alexa fluorophores.

Science.gov (United States)

Liao-Chan, Sindy; Daine-Matsuoka, Barbara; Heald, Nathan; Wong, Tiffany; Lin, Tracey; Cai, Allen G; Lai, Michelle; D'Alessio, Joseph A; Theunissen, Jan-Willem

2015-01-01

Antibodies against cell surface antigens may be internalized through their specific interactions with these proteins and in some cases may induce or perturb antigen internalization. The anti-cancer efficacy of antibody-drug conjugates is thought to rely on their uptake by cancer cells expressing the surface antigen. Numerous techniques, including microscopy and flow cytometry, have been used to identify antibodies with desired cellular uptake rates. To enable quantitative measurements of internalization of labeled antibodies, an assay based on internalized and quenched fluorescence was developed. For this approach, we generated novel anti-Alexa Fluor monoclonal antibodies (mAbs) that effectively and specifically quench cell surface-bound Alexa Fluor 488 or Alexa Fluor 594 fluorescence. Utilizing Alexa Fluor-labeled mAbs against the EphA2 receptor tyrosine kinase, we showed that the anti-Alexa Fluor reagents could be used to monitor internalization quantitatively over time. The anti-Alexa Fluor mAbs were also validated in a proof of concept dual-label internalization assay with simultaneous exposure of cells to two different mAbs. Importantly, the unique anti-Alexa Fluor mAbs described here may also enable other single- and dual-label experiments, including label detection and signal enhancement in macromolecules, trafficking of proteins and microorganisms, and cell migration and morphology.

Quantitative assessment of antibody internalization with novel monoclonal antibodies against Alexa fluorophores.

Directory of Open Access Journals (Sweden)

Sindy Liao-Chan

Full Text Available Antibodies against cell surface antigens may be internalized through their specific interactions with these proteins and in some cases may induce or perturb antigen internalization. The anti-cancer efficacy of antibody-drug conjugates is thought to rely on their uptake by cancer cells expressing the surface antigen. Numerous techniques, including microscopy and flow cytometry, have been used to identify antibodies with desired cellular uptake rates. To enable quantitative measurements of internalization of labeled antibodies, an assay based on internalized and quenched fluorescence was developed. For this approach, we generated novel anti-Alexa Fluor monoclonal antibodies (mAbs that effectively and specifically quench cell surface-bound Alexa Fluor 488 or Alexa Fluor 594 fluorescence. Utilizing Alexa Fluor-labeled mAbs against the EphA2 receptor tyrosine kinase, we showed that the anti-Alexa Fluor reagents could be used to monitor internalization quantitatively over time. The anti-Alexa Fluor mAbs were also validated in a proof of concept dual-label internalization assay with simultaneous exposure of cells to two different mAbs. Importantly, the unique anti-Alexa Fluor mAbs described here may also enable other single- and dual-label experiments, including label detection and signal enhancement in macromolecules, trafficking of proteins and microorganisms, and cell migration and morphology.

High resolution imaging of surface patterns of single bacterial cells

International Nuclear Information System (INIS)

Greif, Dominik; Wesner, Daniel; Regtmeier, Jan; Anselmetti, Dario

2010-01-01

We systematically studied the origin of surface patterns observed on single Sinorhizobium meliloti bacterial cells by comparing the complementary techniques atomic force microscopy (AFM) and scanning electron microscopy (SEM). Conditions ranged from living bacteria in liquid to fixed bacteria in high vacuum. Stepwise, we applied different sample modifications (fixation, drying, metal coating, etc.) and characterized the observed surface patterns. A detailed analysis revealed that the surface structure with wrinkled protrusions in SEM images were not generated de novo but most likely evolved from similar and naturally present structures on the surface of living bacteria. The influence of osmotic stress to the surface structure of living cells was evaluated and also the contribution of exopolysaccharide and lipopolysaccharide (LPS) by imaging two mutant strains of the bacterium under native conditions. AFM images of living bacteria in culture medium exhibited surface structures of the size of single proteins emphasizing the usefulness of AFM for high resolution cell imaging.

High-quality forage production under salinity by using a salt-tolerant AtNXH1-expressing transgenic alfalfa combined with a natural stress-resistant nitrogen-fixing bacterium.

Science.gov (United States)

Stritzler, Margarita; Elba, Pagano; Berini, Carolina; Gomez, Cristina; Ayub, Nicolás; Soto, Gabriela

2018-06-20

Alfalfa, usually known as the "Queen of Forages", is the main source of vegetable protein to meat and milk production systems worldwide. This legume is extremely rich in proteins due to its highly efficient symbiotic association with nitrogen-fixing strains. In the last years, alfalfa culture has been displaced to saline environments by other important crops, including major cereals, a fact that has reduced its biomass production and symbiotic nitrogen fixation. In this short communication, we report the high forage production and nutrient quality of alfalfa under saline conditions by alfalfa transformation with the AtNHX1 Na + /H + antiporter and inoculation with the stress-resistant nitrogen-fixing strain Sinorhizobium meliloti B401. Therefore, the incorporation of transgenic traits into salt-sensitive legumes in association with the inoculation with natural stress-resistant isolates could be a robust approach to improve the productivity and quality of these important nitrogen-fixing crops. Copyright © 2018. Published by Elsevier B.V.

Anti-S100A4 antibody suppresses metastasis formation by blocking stroma cell invasion

DEFF Research Database (Denmark)

Klingelhöfer, Jörg; Grum-Schwensen, Birgitte; Beck, Mette K

2012-01-01

microenvironment, making it an attractive target for anti-cancer therapy. In this study, we produced a function-blocking anti-S100A4 monoclonal antibody with metastasis-suppressing activity. Antibody treatment significantly reduced metastatic burden in the lungs of experimental animals by blocking the recruitment...... of T cells to the site of the primary tumor. In vitro studies demonstrated that this antibody efficiently reduced the invasion of T cells in a fibroblast monolayer. Moreover, it was capable of suppressing the invasive growth of human and mouse fibroblasts. We presume therefore that the antibody exerts...... its activity by suppressing stroma cell recruitment to the site of the growing tumor. Our epitope mapping studies suggested that the antibody recognition site overlaps with the target binding interface of human S100A4. We conclude here that this antibody could serve as a solid basis for development...

Monoclonal antibodies in oncology

International Nuclear Information System (INIS)

Chan, S.Y.T.; Sikora, K.

1986-01-01

Monoclonal antibodies (MCAs) can be used to differentiate between normal and neoplastic cells and thus exploited for diagnostic and, ultimately, therapeutic gain. The evidence for the existence of human tumour antigens is reviewed. Several areas of diagnosis are already benefiting from the application of the monoclonal technology. Immunohistology can help the pathologist with difficult diagnostic problems. New classifications of lymphoma and leukaemia can be based on specific surface molecules. Similarly, the detection of shed tumour antigens is already established as part of the routine assessment of many patients with common solid tumours. Isotopically labeled monoclonal antibodies have been used to localise primary and metastatic tumours. The use of antibodies in this way is not only a promising diagnostic tool but also the first step in studying the possibility of arming antibodies to provide therapeutic agents. Such trials are currently in progress. (Auth.)

Monoclonal Antibodies Against Fusicoccin with Binding Characteristics Similar to the Putative Fusicoccin Receptor of Higher Plants 1

Science.gov (United States)

Feyerabend, Martin; Weiler, Elmar W.

1987-01-01

Monoclonal antibodies were raised against fusicoccin. The toxin, linked to bovine serum albumin through its t-pentenyl moiety, served as immunogen. Hybridomas secreting anti-fusicoccin antibodies were screened by radioimmunoassay employing a novel radioactive derivative, [3H]-nor-fusicoccin-alcohol of high specific activity (1.5 × 1014Bq/mole). The two monoclonal antibodies reported here are of high apparent affinity for fusicoccin (0.71 × 10−9 molar and 1.85 × 10−9 molar). This is comparable to the apparent affinity of rabbit antiserum raised against the same type of conjugate (9.3 × 10−9 molar). A method for the single step purification of the monoclonal antibodies from ascites fluid is reported. A solid-phase immunoassay, using alkaline phosphatase as enzyme, exhibits a measuring range from 0.1 to 1.5 picomoles (about 70 picograms to 1 nanogram) of fusicoccin. The displacement of [3H]-nor-fusicoccin-alcohol from the antibodies by compounds structurally related to fusicoccin exhibits similar selectivity as a microsomal binding assay with the same tracer as radiolabeled probe. Images Fig. 2 PMID:16665786

Cloning and molecular characterization of the cDNAs encoding the variable regions of an anti-CD20 monoclonal antibody.

Science.gov (United States)

Shanehbandi, Dariush; Majidi, Jafar; Kazemi, Tohid; Baradaran, Behzad; Aghebati-Maleki, Leili

2017-01-01

CD20-based targeting of B-cells in hematologic malignancies and autoimmune disorders is associated with outstanding clinical outcomes. Isolation and characterization of VH and VL cDNAs encoding the variable regions of the heavy and light chains of monoclonal antibodies (MAb) is necessary to produce next generation MAbs and their derivatives such as bispecific antibodies (bsAb) and single-chain variable fragments (scFv). This study was aimed at cloning and characterization of the VH and VL cDNAs from a hybridoma cell line producing an anti-CD20 MAb. VH and VL fragments were amplified, cloned and characterized. Furthermore, amino acid sequences of VH, VL and corresponding complementarity-determining regions (CDR) were determined and compared with those of four approved MAbs including Rituximab (RTX), Ibritumomab tiuxetan, Ofatumumab and GA101. The cloned VH and VL cDNAs were found to be functional and follow a consensus pattern. Amino acid sequences corresponding to the VH and VL fragments also indicated noticeable homologies to those of RTX and Ibritumomab. Furthermore, amino acid sequences of the relating CDRs had remarkable similarities to their counterparts in RTX and Ibritumomab. Successful recovery of VH and VL fragments encourages the development of novel CD20 targeting bsAbs, scFvs, antibody conjugates and T-cells armed with chimeric antigen receptors.

Safety, Pharmacokinetics, Immunogenicity, and Biodistribution of (186)Re-Labeled Humanized Monoclonal Antibody BIWA 4 (Bivatuzumab( in Patients with Early-Stage Breast Cancer.

NARCIS (Netherlands)

Koppe, M.; Schaijk, F. van; Roos, J.C.; Leeuwen, P.; Heider, K.H.; Kuthan, H.; Bleichrodt, R.P.

2004-01-01

The aim of this prospective study was to evaluate the safety, pharmacokinetics, immunogenicity, and biodistribution of (186)Re-labeled humanized anti-CD44v6 monoclonal antibody (MAb( BIWA 4 (Bivatuzumab( in 9 patients with early-stage breast cancer. Radioimmunoscintigraphy (RIS( was performed within

An Autopsy Case of Amyotrophic Lateral Sclerosis with Waldenström Macroglobulinemia and Anti-MAG Gammopathy

Directory of Open Access Journals (Sweden)

Snejana Jurici

2011-12-01

Full Text Available We report the case of a 71-year-old woman with typical signs of bulbar amyotrophic lateral sclerosis (ALS associated with immunoglobulin M (IgM monoclonal gammopathy and anti-MAG (myelin-associated glycoprotein antibodies. This unusual association between ALS and anti-MAG antibodies has previously been reported in a single case. Our present case, at neuropathological examination, demonstrated no causative link between anti-MAG antibodies and ALS.

Antibodies and Selection of Monoclonal Antibodies.

Science.gov (United States)

Hanack, Katja; Messerschmidt, Katrin; Listek, Martin

Monoclonal antibodies are universal binding molecules with a high specificity for their target and are indispensable tools in research, diagnostics and therapy. The biotechnological generation of monoclonal antibodies was enabled by the hybridoma technology published in 1975 by Köhler and Milstein. Today monoclonal antibodies are used in a variety of applications as flow cytometry, magnetic cell sorting, immunoassays or therapeutic approaches. First step of the generation process is the immunization of the organism with appropriate antigen. After a positive immune response the spleen cells are isolated and fused with myeloma cells in order to generate stable, long-living antibody-producing cell lines - hybridoma cells. In the subsequent identification step the culture supernatants of all hybridoma cells are screened weekly for the production of the antibody of interest. Hybridoma cells producing the antibody of interest are cloned by limited dilution till a monoclonal hybridoma is found. This is a very time-consuming and laborious process and therefore different selection strategies were developed since 1975 in order to facilitate the generation of monoclonal antibodies. Apart from common automation of pipetting processes and ELISA testing there are some promising approaches to select the right monoclonal antibody very early in the process to reduce time and effort of the generation. In this chapter different selection strategies for antibody-producing hybridoma cells are presented and analysed regarding to their benefits compared to conventional limited dilution technology.

Development of EMab-51, a Sensitive and Specific Anti-Epidermal Growth Factor Receptor Monoclonal Antibody in Flow Cytometry, Western Blot, and Immunohistochemistry.

Science.gov (United States)

Itai, Shunsuke; Kaneko, Mika K; Fujii, Yuki; Yamada, Shinji; Nakamura, Takuro; Yanaka, Miyuki; Saidoh, Noriko; Handa, Saori; Chang, Yao-Wen; Suzuki, Hiroyoshi; Harada, Hiroyuki; Kato, Yukinari

2017-10-01

The epidermal growth factor receptor (EGFR) is a member of the human epidermal growth factor receptor (HER) family of receptor tyrosine kinases and is involved in cell growth and differentiation. EGFR homodimers or heterodimers with other HER members, such as HER2 and HER3, activate downstream signaling cascades in many cancers. In this study, we developed novel anti-EGFR monoclonal antibodies (mAbs) and characterized their efficacy in flow cytometry, Western blot, and immunohistochemical analyses. First, we expressed the full-length or ectodomain of EGFR in LN229 glioblastoma cells and then immunized mice with LN229/EGFR or ectodomain of EGFR, and performed the first screening using enzyme-linked immunosorbent assays. Subsequently, we selected mAbs according to their efficacy in flow cytometry (second screening), Western blot (third screening), and immunohistochemical (fourth screening) analyses. Among 100 mAbs, only one clone EMab-51 (IgG 1 , kappa) reacted with EGFR in Western blot analysis. Finally, immunohistochemical analyses with EMab-51 showed sensitive and specific reactions against oral cancer cells, warranting the use of EMab-51 to detect EGFR in pathological analyses of EGFR-expressing cancers.

Use of Re-188 labelled anti-EGFr humanized monoclonal antibody h-R3 for radioimmunotherapy of gliomas

International Nuclear Information System (INIS)

Perera, A.; Torres, L.A.; Lopez, G.; Casaco, A.; Batista, J.F.; Pena, Y.; Coca, M.A.; Leyva, R.; Garcia, I.

2007-01-01

MBq of 188Re. In patients included in the group I transitory worsening of p re-existing neurological symptoms were observed. In this group: 1 patient with glioblastoma multiforme (GBM) died in progression after 6 months of treatment, 2 patients (one with GBM and one with anaplastic astrocitoma) developed stable disease during 3 months. One GBM patient had partial response for more than 1 year and 2 patients (one with GBM and one with anaplastic astrocitoma) were asymptomatic and in complete response after 3 years of treatment. No patient developed a HAMA response. Conclusions: Proposed procedure allowed the stable efficient labelling of h-R3 with 188Re. Preliminary results of this study strongly suggest that loco-regional radioimmunotherapy of high grade glioma using the anti-EGFr humanized monoclonal antibody h-R3 labelled with 188Re may be safe and constitute a promising therapeutic approach for these patients. (author)

Pharmacokinetics, biodistribution and dosimetry of {sup 99m}Tc-labeled anti-human epidermal growth factor receptor humanized monoclonal antibody R3 in rats

Energy Technology Data Exchange (ETDEWEB)

Escobar, Normando Iznaga; Morales, Alejo Morales; Duconge, Jorge; Torres, Idania Caballero; Fernandez, Eduardo; Gomez, Jose A

1998-01-01

The pharmacokinetics, biodistribution and dosimetry of {sup 99m}Tc-labeled anti-human epidermal growth factor receptor (anti-hEGF-r) humanized monoclonal antibody (MAb) R3 was investigated following intravenous injection in normal Wistar rats. Serum disappearance curves were best fit by a two-compartment model having a mean distribution half-life (t{sub (1(2{alpha}}{sub ))}) of 0.250 h and a mean elimination (t{sub (1(2{beta}}{sub ))}) of 13.89 h. Among the various organs, a little accumulation of the radiolabeled antibody was found only in kidneys. Biodistribution and dosimetry studies in humans were performed by extrapolation of the animal data to humans. Absorbed dose to normal organs and the remainder of the whole body were estimated using the medical internal radiation dose formula, and dose contributions from radioactivity in transit through the gastrointestinal tract were estimated using a compartment model. Extrapolated values of radiation absorbed dose to normal organs in rads per millicurie administered were whole body, 0.0085; lower large intestine wall, 0.0898; small intestine, 0.0530; upper large intestine wall, 0.0731; and kidneys, 0.0455. The effective dose equivalent predicted was 0.0162 rem/mCi and the effective dose was found to be 0.015 rem/mCi. On the basis of the pharmacokinetics, biodistribution and internal radiation dosimetry information obtained in this study, a diagnostic phase I clinical trial with {sup 99m}Tc-labeled humanized MAb R3 conjugate in patients should be supported.

Chimeric antigen receptor (CAR-specific monoclonal antibody to detect CD19-specific T cells in clinical trials.

Directory of Open Access Journals (Sweden)

Bipulendu Jena

Full Text Available Clinical trials targeting CD19 on B-cell malignancies are underway with encouraging anti-tumor responses. Most infuse T cells genetically modified to express a chimeric antigen receptor (CAR with specificity derived from the scFv region of a CD19-specific mouse monoclonal antibody (mAb, clone FMC63. We describe a novel anti-idiotype monoclonal antibody (mAb to detect CD19-specific CAR(+ T cells before and after their adoptive transfer. This mouse mAb was generated by immunizing with a cellular vaccine expressing the antigen-recognition domain of FMC63. The specificity of the mAb (clone no. 136.20.1 was confined to the scFv region of the CAR as validated by inhibiting CAR-dependent lysis of CD19(+ tumor targets. This clone can be used to detect CD19-specific CAR(+ T cells in peripheral blood mononuclear cells at a sensitivity of 1∶1,000. In clinical settings the mAb is used to inform on the immunophenotype and persistence of administered CD19-specific T cells. Thus, our CD19-specific CAR mAb (clone no. 136.20.1 will be useful to investigators implementing CD19-specific CAR(+ T cells to treat B-lineage malignancies. The methodology described to develop a CAR-specific anti-idiotypic mAb could be extended to other gene therapy trials targeting different tumor associated antigens in the context of CAR-based adoptive T-cell therapy.

Preparation of human cardiac anti-myosin: a review

International Nuclear Information System (INIS)

Okada, H.; Souza, I.T.T.

1990-01-01

The present communication is a review of the physicochemical characterization and immunological properties of myosin isolated from the cardiac muscle, the production of monoclonal antibody anti-myosin, the radiolabeling of this antibody and its applications as radiopharmaceuticals to imaging myocardial infarcts. The classical example of radioimmunologic diagnosis of non malignant tissues is the detection of myocardial infarction by radiolabeled antibodies to myosin. (author)

Contribution of tryptophan residues to the combining site of a monoclonal anti dinitrophenyl spin-label antibody

International Nuclear Information System (INIS)

Anglister, J.; Bond, M.W.; Frey, T.; Leahy, D.; Levitt, M.; McConnell, H.M.; Rule, G.S.; Tomasello, J.; Whittaker, M.

1987-01-01

Two Fab fragments of the monoclonal anti dinitrophenyl (DNP) spin-label antibody AN02 were prepared by recombination of specifically deuterated heavy and light chains. In the recombinant H(I)L(II) all the tyrosines and phenylalanines were perdeuterated as were the tryptophan residues of the heavy chain. In the recombinant H(II)L(I) all the tyrosines and phenylalanines were perdeuterated as were the tryptophan residues of the light chain. Saturation of three resonances of H(I)L(II), assigned to tryptophan protons of the light chain, resulted in magnetization transfer to the aromatic proton at position 6 of the DNP ring and to the CH2 protons of the glycines linked to the DNP in a diamagnetic hapten (DNP-DG). Saturation of three resonances of H(II)L(I) assigned to tryptophan protons of the heavy chain resulted in magnetization transfer to the CH2 protons of the glycines in DNP-DG. From the dependence of the magnetization transfer on the irradiation time, the cross relaxation rates between the involved protons were estimated. The inferred distances between these protons of the hapten and certain tryptophan protons are 3-4 A. It is concluded that in the combining site of AN02 there is one tryptophan from the light chain and one tryptophan from the heavy chain that are very near the hapten. When all tyrosines and phenylalanines were perdeuterated and all tryptophan aromatic protons were deuterated except for the protons at positions 2 and 5, titration of the Fab fragments with variable amounts of paramagnetic hapten showed that one proton from the light chain tryptophan is near (less than 7 A) the unpaired electron and that three other protons are significantly closer than 15 A

Contribution of tryptophan residues to the combining site of a monoclonal anti dinitrophenyl spin-label antibody

Energy Technology Data Exchange (ETDEWEB)

Anglister, J.; Bond, M.W.; Frey, T.; Leahy, D.; Levitt, M.; McConnell, H.M.; Rule, G.S.; Tomasello, J.; Whittaker, M.

1987-09-22

Two Fab fragments of the monoclonal anti dinitrophenyl (DNP) spin-label antibody AN02 were prepared by recombination of specifically deuterated heavy and light chains. In the recombinant H(I)L(II) all the tyrosines and phenylalanines were perdeuterated as were the tryptophan residues of the heavy chain. In the recombinant H(II)L(I) all the tyrosines and phenylalanines were perdeuterated as were the tryptophan residues of the light chain. Saturation of three resonances of H(I)L(II), assigned to tryptophan protons of the light chain, resulted in magnetization transfer to the aromatic proton at position 6 of the DNP ring and to the CH2 protons of the glycines linked to the DNP in a diamagnetic hapten (DNP-DG). Saturation of three resonances of H(II)L(I) assigned to tryptophan protons of the heavy chain resulted in magnetization transfer to the CH2 protons of the glycines in DNP-DG. From the dependence of the magnetization transfer on the irradiation time, the cross relaxation rates between the involved protons were estimated. The inferred distances between these protons of the hapten and certain tryptophan protons are 3-4 A. It is concluded that in the combining site of AN02 there is one tryptophan from the light chain and one tryptophan from the heavy chain that are very near the hapten. When all tyrosines and phenylalanines were perdeuterated and all tryptophan aromatic protons were deuterated except for the protons at positions 2 and 5, titration of the Fab fragments with variable amounts of paramagnetic hapten showed that one proton from the light chain tryptophan is near (less than 7 A) the unpaired electron and that three other protons are significantly closer than 15 A.

Monoclonal gammopathy: a diagnosis for to keep in mind; Gammapatia monoclonal: un diagnostico a tener en cuenta

Energy Technology Data Exchange (ETDEWEB)

Howland Alvarez, Ivon; Figueredo Peguero, Yrving; Luna Conde, Clara, E-mail: ihowlanda@infomed.sld.cu [Centro de Investigaciones Medico Quirurgicas, La Habana (Cuba); others, and

2011-07-01

How to identify monoclonal gammopathies at risk for progression has been studied for the last year. 40 patients were studied in which a monoclonal band had been detected, in some of the cases de novo. The electrophoresis was performed in the Hydrasys system. Of the total of electrophoresis carried out, the 14% was monoclonal gammopathy. In 36% a diagnostic assumption was not stated. Most frequent diagnosis in the group of patients with a diagnosis was multiple myeloma. Average age of patients was 61.5 years and there were differences among percentages for sex.

Quantification in mass units of group 1 grass allergens by a monoclonal antibody-based sandwich ELISA.

Science.gov (United States)

Arilla, M C; Ibarrola, I; Eraso, E; Aguirre, M; Martínez, A; Asturias, J A

2001-08-01

Grass pollen extracts currently used for allergy diagnosis and immunotherapy are a complex mixture of proteins of which only a few have allergenic activity. Lol p 1 is one of the most important allergens in grass pollen extracts. To develop a two-site enzyme-linked immunosorbent assay for the quantification of Lol p 1 and other group 1 allergens from grass species, and to assess its suitability for quantifying this group of allergens. Balb/c mice immunized with recombinant Lol p 1 were used for the production of monoclonal antibodies. Screening of hybridomas was performed by direct ELISA, and selected monoclonal antibodies were immobilized on ELISA plates and incubated with samples containing group 1 allergens. Bound allergens were detected by a combination of biotinylated Lol p 1-specific monoclonal antibody and peroxidase-streptavidin conjugate. The assay is based on three Lol p 1-specific monoclonal antibodies with different epitope specificities. The optimized ELISA measured Lol p 1 concentrations ranging from 125 to 1000 ng/mL and could quantify group 1 allergen from grass species belonging to the Pooidea subfamily. The assay does not depend on anti-sera production or availability of human sera and thus reactives can be produced in unlimited amounts. This sensitive and specific Lol p 1 assay will be helpful both for quantifying the group 1 allergen content of Pooideae pollen extracts intended for clinical use and for studying cross-reactivities among pollen extracts.

Tumor imaging with monoclonal antibodies

International Nuclear Information System (INIS)

Haisma, H.; Hilgers, J.

1987-01-01

Many monoclonal antibodies directed against tumor-associated antigens have been identified, but so far none of these are tumor specific. Polyclonal and monoclonal antibodies have been used for imaging of a wide variety of tumors with success. Radiolabeling of antibody is usually done with iodine isotopes of which 123 I is the best candidate for radioimmunodetection purposes. The labeling of antibodies through chelates makes it possible to use metal radioisotopes like 111 In, which is the best radioisotope for imaging with monoclonal antibodies due to its favorable half-life of 2.5 days. Usually imaging cannot be performed within 24 h after injection, but clearance of antibody can be increased by using F(ab) 2 of Fab. Another approach is to clear non-bound antibody by a second antibody, directed against the first. The detection limit of immunoimaging is about 2 cm, but will be improved by tomography or SPECT. There is still a high false positive and false negative rate, which makes it impossible to use radioimmunodetection as the only technique for diagnosis of tumors. In combination with other detection techniques, tumor imaging with monoclonal antibodies can improve diagnosis. 44 refs.; 3 tabs

Bacterial PerO Permeases Transport Sulfate and Related Oxyanions.

Science.gov (United States)

Hoffmann, Marie-Christine; Pfänder, Yvonne; Tintel, Marc; Masepohl, Bernd

2017-07-15

Rhodobacter capsulatus synthesizes the high-affinity ABC transporters CysTWA and ModABC to specifically import the chemically related oxyanions sulfate and molybdate, respectively. In addition, R. capsulatus has the low-affinity permease PerO acting as a general oxyanion transporter, whose elimination increases tolerance to molybdate and tungstate. Although PerO-like permeases are widespread in bacteria, their function has not been examined in any other species to date. Here, we present evidence that PerO permeases from the alphaproteobacteria Agrobacterium tumefaciens , Dinoroseobacter shibae , Rhodobacter sphaeroides , and Sinorhizobium meliloti and the gammaproteobacterium Pseudomonas stutzeri functionally substitute for R. capsulatus PerO in sulfate uptake and sulfate-dependent growth, as shown by assimilation of radioactively labeled sulfate and heterologous complementation. Disruption of perO genes in A. tumefaciens , R. sphaeroides , and S. meliloti increased tolerance to tungstate and, in the case of R. sphaeroides , to molybdate, suggesting that heterometal oxyanions are common substrates of PerO permeases. This study supports the view that bacterial PerO permeases typically transport sulfate and related oxyanions and, hence, form a functionally conserved permease family. IMPORTANCE Despite the widespread distribution of PerO-like permeases in bacteria, our knowledge about PerO function until now was limited to one species, Rhodobacter capsulatus In this study, we showed that PerO proteins from diverse bacteria are functionally similar to the R. capsulatus prototype, suggesting that PerO permeases form a conserved family whose members transport sulfate and related oxyanions. Copyright © 2017 American Society for Microbiology.

Epitope and functional specificity of monoclonal antibodies to mouse gamma interferon: the synthetic peptide approach

International Nuclear Information System (INIS)

Russell, J.K.; Hayes, M.P.; Carter, J.M.; Torres, B.A.; Dunn, B.M.; Johnson, H.M.

1986-01-01

Four anti-recombinant mouse gamma interferon (α-IFNγ) monoclonal antibodies were generated using hamster spleen cells. Binding of 125 I-IFNγ by these protein A-bound antibodies was specifically blocked by cold IFNγ. Binding by three of these antibodies was also blocked by a synthetic peptide corresponding to the N-terminal 1-39 amino acids of IFNγ, while a corresponding C-terminal (95-133) peptide had no effect on binding. One of the N-terminal specific monoclonal antibodies inhibited both the antiviral and macrophage priming (for tumor cell killing) activities of IFNγ, while the other two had no effect on either biological function. Blocking experiments with cold IFNγ and N-terminal peptide suggest that the epitope specificities of the monoclonal antibodies could be determined by the conformational or topographic structure of IFNγ. Polyclonal antibodies to either the N-terminal or C-terminal peptides also inhibited both the antiviral and macrophage priming activities of IFNγ. All of the antibodies that inhibited IFNγ function also blocked binding of IFNγ to membrane receptor on cells, while antibodies that did not inhibit function also did not block binding. The data suggest that both the N-terminal and C-terminal domains of IFNγ play an important role in its antiviral and macrophage priming functions, possibly in a cooperative manner

Production of Monoclonal Antibodies specific for Progesterone

OpenAIRE

YÜCEL, Fatıma

2014-01-01

Progesterone levels in milk and serum are indicators of pregnancy in cattle. The progesterone level reaches a peak on the 21 st and 22 nd days of pregnancy. Monoclonal antibodies specific to progesterone could be used for the immunodetection of milk and serum progesterone levels. We report here the development of hybrid cells prdoducing monoclonal antibodies specific for progesterone using hybridoma technology. Hybridoma cells secreting monoclonal antibodies against progesterone (MAM 2H1...

Vascular targeted therapy with anti-prostate-specific membrane antigen monoclonal antibody J591 in advanced solid tumors.

Science.gov (United States)

Milowsky, Matthew I; Nanus, David M; Kostakoglu, Lale; Sheehan, Christine E; Vallabhajosula, Shankar; Goldsmith, Stanley J; Ross, Jeffrey S; Bander, Neil H

2007-02-10

Based on prostate-specific membrane antigen (PSMA) expression on the vasculature of solid tumors, we performed a phase I trial of antibody J591, targeting the extracellular domain of PSMA, in patients with advanced solid tumor malignancies. This was a proof-of-principle evaluation of PSMA as a potential neovascular target. The primary end points were targeting,toxicity, maximum-tolerated dose, pharmacokinetics (PK), and human antihuman antibody (HAHA) response. Patients had advanced solid tumors previously shown to express PSMA on the neovasculature. They received 111Indium (111ln)-J591 for scintigraphy and PK, followed 2 weeks later by J591 with a reduced amount of 111In for additional PK measurements. J591 dose levels were 5, 10, 20, 40, and 80 mg. The protocol was amended for six weekly administrations of unchelated J591. Patients with a response or stable disease were eligible for re-treatment. Immunohistochemistry assessed PSMA expression in tumor tissues. Twenty-seven patients received monoclonal antibody (mAb) J591. Treatment was well tolerated. Twenty (74%) of 27 patients had at least one area of known metastatic disease targeted by 111In-J591, with positive imaging seen in patients with kidney, bladder, lung, breast, colorectal, and pancreatic cancers, and melanoma. Seven of 10 patient specimens available for immunohistochemical assessment of PSMA expression in tumor-associated vasculature demonstrated PSMA staining. No HAHA response was seen. Three patients of 27 with stable disease received re-treatment. Acceptable toxicity and excellent targeting of known sites of metastases were demonstrated in patients with multiple solid tumor types, highlighting a potential role for the anti-PSMA antibody J591 as a vascular-targeting agent.

Levels of house dust mite-specific serum immunoglobulin E (IgE) in different cat populations using a monoclonal based anti-IgE enzyme-linked immunosorbent assay.

Science.gov (United States)

Bexley, Jennifer; Hogg, Janice E; Hammerberg, Bruce; Halliwell, Richard E W

2009-10-01

Levels of serum immunoglobulin E (IgE) specific for the house dust mites (HDMs) Dermatophagoides farinae (DF) and Dermatophagoides pteronyssinus (DP) in 58 cats with clinical signs suggestive of atopic dermatitis (allergic dermatitis cats), 52 cats with no history of allergic or immunological disease (nonallergic cats) and 26 specific pathogen-free (SPF) cats were measured using a monoclonal anti-IgE enzyme-linked immunosorbent assay. Reactivity to both native and reduced HDM allergens was compared. SPF cats had significantly lower levels of HDM-specific serum IgE than cats with allergic dermatitis and nonallergic cats. The difference in levels of HDM-specific IgE in the serum of cats with allergic dermatitis and nonallergic cats was significant for native DF allergen, but not for native DP allergen or reduced HDM allergens. The results suggest that DF in its native form may be a significant allergen in cats with allergic dermatitis. The clinical relevance of these reactions, however, remains to be proven.

Application of monoclonal antibodies for diagnosis and treatment of human digestive cancer

International Nuclear Information System (INIS)

Otsuji, Eigo

2007-01-01

Radioimmunoscintigraphic applications of monoclonal antibodies (Mabs) for noninvasive detection and visualization of target tumors have grown immensely, and it suggests that Mabs can reach specifically to the targeted tumors in the human body. Radionuclides, cytotoxic drugs and anti-cancer drugs can be coupled to these specific MAbs to detect the extent of disease and/or to treat the tumors. Many of such immunoconjugates were studied for targeting therapy for cancer in animal experiments and some of them have applied to human. In this paper, we described the existing status of application of Mabs for diagnosis and immunotargeting therapy of digestive cancers. (author)

Generation and characterization of monoclonal antibodies against Giardia muris trophozoites.

Science.gov (United States)

Heyworth, M F; Ho, K E; Pappo, J

1989-11-01

Mouse monoclonal antibodies (mAb) were produced against Giardia muris trophozoite surface antigens. To generate B-cell hybridomas, P3/NS1/1-Ag4-1 myeloma cells were fused with splenic lymphocytes from BALB/c mice that had been immunized parenterally with G. muris trophozoites. Hybridoma culture supernatants were screened for mAb by flow cytometry of G. muris trophozoites incubated with culture supernatant followed by fluorescein-conjugated anti-mouse IgG and IgM. Flow cytometry showed three types of trophozoite staining by mAb: (i) bright staining of greater than 90% of trophozoites, with aggregation of the organisms; (ii) bright staining of approximately 90% of trophozoites, with little or no aggregation; (iii) dull staining of approximately 20% of trophozoites, without aggregation. Western blotting of mAb on G. muris trophozoite antigens separated by polyacrylamide gel electrophoresis showed that a mAb exhibiting the third of these flow cytometry staining patterns recognized trophozoite antigens of MW approximately 31,000 and 35,000. Immunoprecipitation studies indicated that the same mAb specifically precipitated two 125I-labelled trophozoite surface antigens of MW approximately 30,000. Monoclonal antibodies generated in this study may facilitate the purification and biochemical characterization of trophozoite antigens that are targets for protective intestinal antibody in G. muris-infected mice.

Efecto de la inoculación con rizobios procedentes de Alberta, Canadá, en sorgo (Sorghum bicolor L. Moench, en condiciones de campo Effect of the inoculation with rhizobia from Alberta, Canada, in sorghum (Sorghum bicolor L. Moench, under field conditions

Directory of Open Access Journals (Sweden)

C. J Bécquer

2011-09-01

Full Text Available Se realizó un experimento de campo con el objetivo de medir el efecto de cepas de rizobio en las variables agronómicas del sorgo, en las condiciones ambientales de Sancti Spíritus, Cuba. Se utilizaron 10 cepas de Sinorhizobium meliloti, procedentes de ecosistemas ganaderos de Alberta, Canadá; así como cuatro cepas de referencia pertenecientes a diferentes géneros y especies de rizobio, que procedían de la colección de Agriculture and AgriFood Canada. La confección de los inóculos y la inoculación de las semillas se realizaron por métodos estándar. El diseño experimental fue de bloques al azar, con 16 tratamientos y cuatro réplicas. Se evaluó el peso seco aéreo, la longitud del tallo y la longitud de la panoja; además, se calculó el incremento del peso seco aéreo en los tratamientos inoculados con relación al control absoluto. Los resultados demostraron la capacidad de las cepas estudiadas de influir en las variables agronómicas, ya que los tratamientos seleccionados igualaron sus valores a los del control fertilizado y presentaron un incremento de más del 100% del peso seco aéreo, comparado con el control absoluto.A field trial was conducted with the objective of measuring the effect of rhizobium strains on the agronomic variables of sorghum under the environmental conditions of Sancti Spiritus, Cuba. Ten Sinorhizobium meliloti strains, from livestock production ecosystems of Alberta, Canada, were used; as well as four reference strains belonging to different rhizobium genera and strains, which were from the collection of Agriculture and AgriFood Canada. The inoculi confection and seed inoculation were made by standard methods. The experimental design was randomized blocks, with 16 treatments and four replications. The dry aerial weight, stem length and ear length were evaluated; in addition, the increase of aerial dry weight was calculated in the inoculated treatments as compared to the absolute control. The results

Scintigraphy of infection and inflammation with autologous leukocytes and murine monoclonal antibodies

International Nuclear Information System (INIS)

Becker, W.

1992-01-01

Scintigraphy of infection and inflammation with autologous leukocytes (In-111- oxin; Tc-99m-HMPAO) and murine monoclonal antibodies (Tc-99m-anti- NCA-95; BW250/183; I-123-anti-NCA-95; AK-47) has been evaluated in different diseases and revealed comparable results. The use of one of these radiopharmaceuticals is dependant both from the diagnostic accuracy in different diseases and stages of disease and from its ready availability and ease of preparation. Tc-99m- HMPAO should be prefered when autologous leucocytes are labeled, except of differential diagnosis of circumscript inflammatory bowel disease from abdominal abscesses and of chronic osteomyelitis. In these cases In-111-oxin is superior. Immunoscintigraphic techniques are superior regarding the ease of preparation and the unnecessity of handling patients blood. Disadvantageous are the possible human antimouse antibodies, especially regarding the development of human antimouse antibodies. (orig.) [de

Generation of anti-idiotype scFv for pharmacokinetic measurement in lymphoma patients treated with chimera anti-CD22 antibody SM03.

Directory of Open Access Journals (Sweden)

Qi Zhao

Full Text Available Pre-clinical and clinical studies of therapeutic antibodies require highly specific reagents to examine their immune responses, bio-distributions, immunogenicity, and pharmacodynamics in patients. Selective antigen-mimicking anti-idiotype antibody facilitates the assessment of therapeutic antibody in the detection, quantitation and characterization of antibody immune responses. Using mouse specific degenerate primer pairs and splenocytic RNA, we generated an idiotype antibody-immunized phage-displayed scFv library in which an anti-idiotype antibody against the therapeutic chimera anti-CD22 antibody SM03 was isolated. The anti-idiotype scFv recognized the idiotype of anti-CD22 antibody and inhibited binding of SM03 to CD22 on Raji cell surface. The anti-idiotype scFv was subsequently classified as Ab2γ type. Moreover, our results also demonstrated firstly that the anti-idiotype scFv could be used for pharmacokinetic measurement of circulating residual antibody in lymphoma patients treated with chimera anti-CD22 monoclonal antibody SM03. Of important, the present approach could be easily adopted to generate anti-idiotype antibodies for therapeutic antibodies targeting membrane proteins, saving the cost and time for producing a soluble antigen.

A novel anti-EMMPRIN function-blocking antibody reduces T cell proliferation and neurotoxicity: relevance to multiple sclerosis.

Science.gov (United States)

Agrawal, Smriti M; Silva, Claudia; Wang, Janet; Tong, Jade Pui-Wai; Yong, V Wee

2012-04-05

Extracellular matrix metalloproteinase inducer (EMMPRIN; CD147, basigin) is an inducer of the expression of several matrix metalloproteinases (MMPs). We reported previously that blocking EMMPRIN activity reduced neuroinflammation and severity of disease in an animal model of multiple sclerosis (MS), experimental autoimmune encephalomyelitis (EAE). To improve upon EMMPRIN blockade, and to help unravel the biological functions of EMMPRIN in inflammatory disorders, we have developed several anti-EMMPRIN monoclonal antibodies. Of these monoclonal antibodies, a particular one, clone 10, was efficient in binding mouse and human cells using several methods of detection. The specificity of clone 10 was demonstrated by its lack of staining of EMMPRIN-null embryos compared to heterozygous and wild-type mouse samples. Functionally, human T cells activated with anti-CD3 and anti-CD28 elevated their expression of EMMPRIN and the treatment of these T cells with clone 10 resulted in decreased proliferation and matrix metalloproteinase- 9 (MMP-9) production. Activated human T cells were toxic to human neurons in culture and clone 10 pretreatment reduced T cell cytotoxicity correspondent with decrease of granzyme B levels within T cells. In vivo, EAE mice treated with clone 10 had a markedly reduced disease score compared to mice treated with IgM isotype control. We have produced a novel anti-EMMPRIN monoclonal antibody that blocks several aspects of T cell activity, thus highlighting the multiple roles of EMMPRIN in T cell biology. Moreover, clone 10 reduces EAE scores in mice compared to controls, and has activity on human cells, potentially allowing for the testing of anti-EMMPRIN treatment not only in EAE, but conceivably also in MS.

A novel anti-EMMPRIN function-blocking antibody reduces T cell proliferation and neurotoxicity: relevance to multiple sclerosis

Directory of Open Access Journals (Sweden)

Agrawal Smriti M

2012-04-01

Full Text Available Abstract Background Extracellular matrix metalloproteinase inducer (EMMPRIN; CD147, basigin is an inducer of the expression of several matrix metalloproteinases (MMPs. We reported previously that blocking EMMPRIN activity reduced neuroinflammation and severity of disease in an animal model of multiple sclerosis (MS, experimental autoimmune encephalomyelitis (EAE. Methods To improve upon EMMPRIN blockade, and to help unravel the biological functions of EMMPRIN in inflammatory disorders, we have developed several anti-EMMPRIN monoclonal antibodies. Results Of these monoclonal antibodies, a particular one, clone 10, was efficient in binding mouse and human cells using several methods of detection. The specificity of clone 10 was demonstrated by its lack of staining of EMMPRIN-null embryos compared to heterozygous and wild-type mouse samples. Functionally, human T cells activated with anti-CD3 and anti-CD28 elevated their expression of EMMPRIN and the treatment of these T cells with clone 10 resulted in decreased proliferation and matrix metalloproteinase- 9 (MMP-9 production. Activated human T cells were toxic to human neurons in culture and clone 10 pretreatment reduced T cell cytotoxicity correspondent with decrease of granzyme B levels within T cells. In vivo, EAE mice treated with clone 10 had a markedly reduced disease score compared to mice treated with IgM isotype control. Conclusions We have produced a novel anti-EMMPRIN monoclonal antibody that blocks several aspects of T cell activity, thus highlighting the multiple roles of EMMPRIN in T cell biology. Moreover, clone 10 reduces EAE scores in mice compared to controls, and has activity on human cells, potentially allowing for the testing of anti-EMMPRIN treatment not only in EAE, but conceivably also in MS.

Anti-GD2 mAb and Vorinostat synergize in the treatment of neuroblastoma

NARCIS (Netherlands)

Kroesen, M.; Bull, C.; Gielen, P.R.; Brok, I.C.; Armandari, I.; Wassink, M.; Looman, M.W.G.; Boon, L.; Brok, M.H.M.G.M. den; Hoogerbrugge, P.M.; Adema, G.J.

2016-01-01

Neuroblastoma (NBL) is a childhood malignancy of the sympathetic nervous system. For high-risk NBL patients, the mortality rate is still over 50%, despite intensive multimodal treatment. Anti-GD2 monoclonal antibody (mAB) in combination with systemic cytokine immunotherapy has shown clinical

Magnetic nanoparticle based purification and enzyme-linked immunosorbent assay using monoclonal antibody against enrofloxacin

Science.gov (United States)

Kim, Nam-Gun; Kim, Myeong-Ae; Park, Young-Il; Jung, Tae-Sung; Son, Seong-Wan; So, ByungJae

2015-01-01

Monoclonal anti-enrofloxacin antibody was prepared for a direct competitive enzyme-linked immunosorbent assay (ELISA) and purification system using monoclonal antibody (mAb) coupled magnetic nanoparticles (MNPs). The IC50 values of the developed mAb for enrofloxacin (ENR), ciprofloxacin, difloxacin, sarafloxacin, pefloxacin, and norfloxacin were 5.0, 8.3, 9.7, 21.7, 36.0, and 63.7 ng/mL, respectively. The lowest detectable level of ENR was 0.7 ng/mL in the prepared ELISA system. To validate the developed ELISA in the food matrix, known amounts of ENR were spiked in meat and egg samples at 10, 20 and 30 ng/mL. Recoveries for ENR ranged from 72.9 to 113.16% with a coefficient of variation (CV) of 2.42 to 10.11%. The applicability of the mAb-MNP system was verified by testing the recoveries for ENR residue in three different matrices. Recoveries for ENR ranged from 75.16 to 86.36%, while the CV ranged from 5.08 to 11.53%. Overall, ENR-specific monoclonal antibody was prepared and developed for use in competitive to ELISAs for the detection of ENR in animal meat samples. Furthermore, we suggest that a purification system for ENR using mAb-coupled MNPs could be useful for determination of ENR residue in food. PMID:26040610

[{sup 177}Lu]DOTA-anti-CD20: Labeling and pre-clinical studies

Energy Technology Data Exchange (ETDEWEB)

Audicio, Paola F., E-mail: paudicio@cin.edu.u [Departamento de Radiofarmacia, Centro de Investigaciones Nucleares, Facultad de Ciencias, Universidad de la Republica, Mataojo 2055, 11400 Montevideo (Uruguay); Castellano, Gustavo, E-mail: gcas@famaf.unc.edu.a [FaMAF, Universidad Nacional de Cordoba, Ciudad Universitaria, 5016 Cordoba (Argentina); Tassano, Marcos R.; Rezzano, Maria E.; Fernandez, Marcelo [Departamento de Radiofarmacia, Centro de Investigaciones Nucleares, Facultad de Ciencias, Universidad de la Republica, Mataojo 2055, 11400 Montevideo (Uruguay); Riva, Eloisa [Clinica Hematologica ' Prof. Dra. L. Diaz' , Hospital de Clinicas. Av. Italia. sn, Montevideo (Uruguay); Robles, Ana; Cabral, Pablo; Balter, Henia; Oliver, Patricia [Departamento de Radiofarmacia, Centro de Investigaciones Nucleares, Facultad de Ciencias, Universidad de la Republica, Mataojo 2055, 11400 Montevideo (Uruguay)

2011-07-15

Anti-CD20 (Rituximab), a specific chimeric monoclonal antibody used in CD20-positive Non-Hodgkin's Lymphoma, was conjugated to a bifunctional quelate (DOTA) and radiolabeled with {sup 177}Lu through a simple method. [{sup 177}Lu]-DOTA-anti-CD20 was obtained with a radiochemical purity higher than 97%, and showed good chemical and biological stability, maintaining its biospecificity to CD20 antigens. Monte Carlo simulation showed high doses deposited on a spheroid tumor mass model. This method seems to be an appropriate alternative for the production of [{sup 177}Lu]-DOTA-anti-CD20 as therapeutic radiopharmaceutical.

Radiometric immunosorbent assay for the detection of anti-hormone-binding protein antibodies

International Nuclear Information System (INIS)

Pierce, E.A.; Dame, M.C.; DeLuca, H.F.

1986-01-01

A radiometric immunosorbent assay (RISA) for the detection of monoclonal antibodies to hormone-binding proteins has been developed. The assay involves incubating hybridoma supernatants in microtiter wells that have been coated with goat anti-mouse IgG antibodies. Any mouse IgG in the test supernatant is thus specifically retained in the wells. Radioactive ligand-binding protein complexes are then incubated in the wells. The presence of anti-binding protein antibodies in the supernatant is indicated by specific retention of radioactive ligand-binding protein complexes in the wells. Crude antigen preparations, such as tissue homogenates, can be used to detect antibodies. The assay is capable of detecting antibody at concentrations 20 ng/ml (approx. 100 pM IgG). The RISA has been used successfully to screen for monoclonal antibodies to the intracellular receptor for 1,25-dihydroxyvitamin D 3 and should be useful for the detection of antibodies to ligand-binding proteins in general

Preclinical evaluation of racotumomab, an anti-idiotype monoclonal antibody to N-glycolyl-containing gangliosides, with or without chemotherapy in a mouse model of non-small cell lung cancer

International Nuclear Information System (INIS)

Segatori, Valeria I.; Vazquez, Ana M.; Gomez, Daniel E.; Gabri, Mariano R.; Alonso, Daniel F.

2012-01-01

N-glycolylneuraminic acid (NeuGc) is a sialic acid molecule usually found in mammalian cells as terminal constituents of different membrane glycoconjugates such as gangliosides. The NeuGcGM3 ganglioside has been described as a tumor antigen for non-small cell lung cancer (NSCLC) in humans. Racotumomab is an anti-NeuGc-containing gangliosides anti-idiotype monoclonal antibody (mAb) (formerly known as 1E10) that has received attention as a potential active immunotherapy for advanced lung cancer in clinical trials. In this work, we have examined the antitumor activity of racotumomab in combination or not with chemotherapy, using the 3LL Lewis lung carcinoma as a preclinical model of NSCLC in C57BL/6 mice. Vaccination with biweekly doses of racotumomab at 50–200 μg/dose formulated in aluminum hydroxide (racotumomab-alum vaccine) demonstrated a significant antitumor effect against the progression of lung tumor nodules. Racotumomab-alum vaccination exerted a comparable effect on lung disease to that of pemetrexed-based chemotherapy (100 mg/kg weekly). Interestingly, chemo-immunotherapy was highly effective against lung nodules and well-tolerated, although no significant synergistic effect was observed as compared to each treatment alone in the present model. We also obtained evidence on the role of the exogenous incorporation of NeuGc in the metastatic potential of 3LL cells. Our preclinical data provide support for the combination of chemotherapy with the anti-idiotype mAb racotumomab, and also reinforce the biological significance of NeuGc in lung cancer.

Preclinical evaluation of racotumomab, an anti-idiotype monoclonal antibody to N-glycolyl-containing gangliosides, with or without chemotherapy in a mouse model of non-small cell lung cancer

Energy Technology Data Exchange (ETDEWEB)

Segatori, Valeria I. [Laboratory of Molecular Oncology, Department of Science and Technology, Quilmes National University, Buenos Aires (Argentina); Vazquez, Ana M. [Center of Molecular Immunology, Innovation Managing Direction, La Habana (Cuba); Gomez, Daniel E.; Gabri, Mariano R.; Alonso, Daniel F., E-mail: dfalonso@unq.edu.ar [Laboratory of Molecular Oncology, Department of Science and Technology, Quilmes National University, Buenos Aires (Argentina)

2012-11-08

N-glycolylneuraminic acid (NeuGc) is a sialic acid molecule usually found in mammalian cells as terminal constituents of different membrane glycoconjugates such as gangliosides. The NeuGcGM3 ganglioside has been described as a tumor antigen for non-small cell lung cancer (NSCLC) in humans. Racotumomab is an anti-NeuGc-containing gangliosides anti-idiotype monoclonal antibody (mAb) (formerly known as 1E10) that has received attention as a potential active immunotherapy for advanced lung cancer in clinical trials. In this work, we have examined the antitumor activity of racotumomab in combination or not with chemotherapy, using the 3LL Lewis lung carcinoma as a preclinical model of NSCLC in C57BL/6 mice. Vaccination with biweekly doses of racotumomab at 50–200 μg/dose formulated in aluminum hydroxide (racotumomab-alum vaccine) demonstrated a significant antitumor effect against the progression of lung tumor nodules. Racotumomab-alum vaccination exerted a comparable effect on lung disease to that of pemetrexed-based chemotherapy (100 mg/kg weekly). Interestingly, chemo-immunotherapy was highly effective against lung nodules and well-tolerated, although no significant synergistic effect was observed as compared to each treatment alone in the present model. We also obtained evidence on the role of the exogenous incorporation of NeuGc in the metastatic potential of 3LL cells. Our preclinical data provide support for the combination of chemotherapy with the anti-idiotype mAb racotumomab, and also reinforce the biological significance of NeuGc in lung cancer.

Prevention and reversal of experimental autoimmune thyroiditis (EAT) in mice by administration of anti-L3T4 monoclonal antibody at different stages of disease development.

Science.gov (United States)

Stull, S J; Kyriakos, M; Sharp, G C; Braley-Mullen, H

1988-11-01

Experimental autoimmune thyroiditis (EAT) can be induced in CBA/J mice following the transfer of spleen cells from mouse thyroglobulin (MTg)-sensitized donors that have been activated in vitro with MTg. Since L3T4+ T cells are required to transfer EAT in this model, the present study was undertaken to assess the effectiveness of the anti-L3T4 monoclonal antibody (mAb) GK1.5 in preventing or arresting the development of EAT. Spleen cells from mice given mAb GK1.5 prior to sensitization with MTg and adjuvant could not transfer EAT to normal recipients and cells from these mice did not proliferate in vitro to MTg. Donor mice given GK1.5 before immunization did not develop anti-MTg autoantibody and recipients of cells from such mice also produced little anti-MTg. GK1.5 could also prevent the proliferation and activation of sensitized effector cell precursors when added to in vitro cultures. When a single injection of mAb GK1.5 was given to recipients of in vitro-activated spleen cells, EAT was reduced whether the mAb was given prior to cell transfer or as late as 19 days after cell transfer. Whereas the incidence and severity of EAT was consistently reduced by injecting recipient mice with GK1.5, the same mice generally had no reduction in anti-MTg autoantibody. Since EAT is consistently induced in control recipients by 14-19 days after cell transfer, the ability of mAb GK1.5 to inhibit EAT when injected 14 or 19 days after cell transfer indicates that a single injection of the mAb GK1.5 can cause reversal of the histopathologic lesions of EAT in mice. These studies further establish the important role of L3T4+ T cells in the pathogenesis of EAT in mice and also suggest that therapy with an appropriate mAb may be an effective treatment for certain autoimmune diseases even when the therapy is initiated late in the course of the disease.

Radiolabeled monoclonal antibodies: a review

International Nuclear Information System (INIS)

Toledo e Souza, I.T. de; Okada, H.

1990-05-01

Since the description by Kohler and Milstein 1975 of their technique for producing monoclonal antibodies of predefined specificity, it has become a mainstay in most laboratories that utilize immunochemical techniques to study problems in basic, applied or clinical research. Paradoxically, the very success of monoclonal antibodies has generated a literature which is now so vast and scattered that it has become difficult to obtain a perspective. This brief review represents the distillation of many publications relating to the production and use of monoclonaal antibodies as radiopharmaceuticals. Significant advances were made possible in the last few years by combined developments in the fields of tumor-associated antigens and of monoclonal antibodies. In fact monoclonal antibodies against some well defined tumor-associated antigens, has led to significantly greater practical possibilities for producing highly specific radiolabeled antibodies as radiopharmaceuticals for diagnosis and therapy of human tumors. One of the main requirements of this methodology is the availability of stable radiopharmaceutical reagents which after labeling in vivo injection retain the capacity of specific interaction with the defined antigen and their molecular integrity. Since injection into human is the objetive of this kind of study all the specifications of radiopharmaceutical have to be fulfilled e.g. sterility, apirogenicity and absence of toxicity. (author) [pt

[Monoclonal antibodies for the treatment of multiple sclerosis].

Science.gov (United States)

Sánchez-Seco, Victoria Galán; Casanova Peño, Ignacio; Arroyo González, Rafael

2014-12-01

Until the mid 1990s, with the appearance of interferon beta and glatiramer acetate, there was no treatment for multiple sclerosis (MS). However, due to their moderate therapeutic potential in some patients, a broad search was continued to find new and more effective treatment strategies, largely concentrated on monoclonal antibodies (MOAB). Natalizumab, the first MOAB for the treatment of MS, was approved at the end of 2004, representing a major advance in the field of neuroimmunology. Today, there is broad experience with natalizumab and other MOAB (alemtuzumab, daclizumab, rituximab, ocrelizumab, ofatumumab and anti-lingo-1) that are pending commercialization or are under phase II or III of development with promising results. The present review analyzes the efficacy and safety results of all these drugs. Copyright © 2014 Elsevier España, S.L.U. All rights reserved.

Radioimmunodetection of human leukemia with anti-interleukin-2 receptor antibody in severe combined immunodeficiency mice

International Nuclear Information System (INIS)

Hosono, Makoto; Takaori-Kondo, Akifumi; Zheng-Sheng, Yao; Kobayashi, Hisataka; Hosono, Masako N.; Sakahara, Harumi; Imada, Kazunori; Okuma, Minoru; Uchiyama, Takashi; Konishi, Junji

1995-01-01

Anti-Tac monoclonal antibody recognizes human interleukin-2 receptor, which is overexpressed in leukemic cells of most adult T-cell leukemia (ATL) patients. To examine the potency of anti-Tac for targeting of ATL, biodistributions of intravenously administered 125 I- and 111 In-labeled anti-Tac were examined in severe combined immunodeficiency (SCID) mice inoculated with ATL cells. Significant amounts of radiolabeled anti-Tac were found in the spleen and thymus. The trafficking of ATL cells in SCID mice was detected using 111 In-oxine-labeled ATL cells. These results were coincident with the histologically confirmed infiltration of ATL cells. The radiolabeled anti-Tac seemed potent for targeting of ATL

Customizing monoclonal antibodies for the treatment of methamphetamine abuse: current and future applications.

Science.gov (United States)

Peterson, Eric C; Gentry, W Brooks; Owens, S Michael

2014-01-01

Monoclonal antibody-based medications designed to bind (+)-methamphetamine (METH) with high affinity are among the newest approaches to the treatment of METH abuse and the associated medical complications. The potential clinical indications for these medications include treatment of overdose, reduction of drug dependence, and protection of vulnerable populations from METH-related complications. Research designed to discover and conduct preclinical and clinical testing of these antibodies suggests a scientific vision for how intact monoclonal antibody (mAb) (singular and plural) or small antigen-binding fragments of mAb could be engineered to optimize the proteins for specific therapeutic applications. In this review, we discuss keys to success in this development process including choosing predictors of specificity, efficacy, duration of action, and safety of the medications in disease models of acute and chronic drug abuse. We consider important aspects of METH-like hapten design and how hapten structural features influence specificity and affinity, with an example of a high-resolution X-ray crystal structure of a high-affinity antibody to demonstrate this structural relationship. Additionally, several prototype anti-METH mAb forms such as antigen-binding fragments and single-chain variable fragments are under development. Unique, customizable aspects of these fragments are presented with specific possible clinical indications. Finally, we discuss clinical trial progress of the first in kind anti-METH mAb, for which METH is the disease target instead of vulnerable central nervous system networks of receptors, binding sites, and neuronal connections. © 2014 Elsevier Inc. All rights reserved.

Comparison of an anti-rabies human monoclonal antibody combination with human polyclonal anti-rabies immune globulin

NARCIS (Netherlands)

Goudsmit, Jaap; Marissen, Wilfred E.; Weldon, William C.; Niezgoda, Michael; Hanlon, Cathleen A.; Rice, Amy B.; Kruif, John de; Dietzschold, Bernhard; Bakker, Alexander B. H.; Rupprecht, Charles E.

2006-01-01

The World Health Organization estimates human mortality from endemic canine rabies to be 55,000 deaths/year. Limited supply hampers the accessibility of appropriate lifesaving treatment, particularly in areas where rabies is endemic. Anti-rabies antibodies are key to protection against lethal

Detección de actividad pectolítica en el cultivo de la cepa GR4 de rhyzobíum meliloti

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P. Martínez M.

2010-07-01

Full Text Available Se ensayaron varios métodos para la obtención y purificación parcial de pectinasas a partir de sobrenadante del cultivo de la cepa GR4 de Rhizobium meliloti. Se describe el método con el cual se obtuvo el sobrenadante en el que se logró detectar la presencia de actividad pectolítica. Empleando una muestra comercial de enzimas pécticas (Pectinex, Novo se estudió la estabilidad de la actividad enzimática durante el proceso de purificación parcial establecido; se observó una pérdida gradual de la actividad en función del tiempo de duración del proceso.

Antigen-specific cytotoxic T cell and antigen-specific proliferating T cell clones can be induced to cytolytic activity by monoclonal antibodies against T3

NARCIS (Netherlands)

Spits, H.; Yssel, H.; Leeuwenberg, J.; de Vries, J. E.

1985-01-01

T3 is a human differentiation antigen expressed exclusively on mature T cells. In this study it is shown that anti-T3 monoclonal antibodies, in addition to their capacity to induce T cells to proliferate, are able to induce antigen-specific cytotoxic T lymphocyte clones to mediate antigen

Clinical observation in nasopharyngeal carcinoma (NPC) treated with the anti-EGFR monoclonal antibody followed by helical tomotherapy

International Nuclear Information System (INIS)

Hou Jun; Feng Linchun; Cai Boning; Lu Na; Du Lei; Ma Lin; Xu Shouping; Xie Chuanbin

2011-01-01

Objective: To evaluate the clinical outcome and the acute toxicity in nasopharyngeal carcinoma (NPC) treated with tomotherapy followed by the anti-EGFR monoclonal antibody. Methods: Between March 2008 and November 2009, 34 newly diagnosed NPC patients were treated with helical tomotherapy combined with nimotuzumab or cetuximab. All the patients underwent tomotherapy at the dose of 70 Gy/33F for the gross tumor volume (pGTV ns ) and positive lymphnodes (GTV nd ), and 60 Gy/33F for the high risk clinical target volume (PTV 1 ), and 56 Gy/33 F for the low risk clinical target volume (PTV 2 ), respectively. 17 patients in group N were given weekly injection of 200 mg for 6-7 times and 17 patients in group C were given initial dosage 400 mg/m 2 followed by subsequent weekly dosage of 250 mg/m 2 for 6-7 times. Acute lesions were evaluated with the RTOG/EORTC criteria. Result: The median follow-up time was 22 months. The effective rates (CR + PR) in 3, 6 and 12 months were 14/17, 12/17, 12/17 in group N and 15/17, 14/17, 14/17 in group C. The 1 year survival rate was 15/17 in group Nand 17/17 in group C. Nimotuzumab had less acute mucositis reaction (u=2.25, P< 0.05), weight loss (t=2.56, P=0.02) and rash (u=4.36, P<0.01) compared with cetuximab. Conclusions: Helical tomotherapy combined with nimotuzumab or cetuximab was effective and made no difference in the short-term efficacy and 1 year survival rate for the patients with NPC. Nimotuzumab has less acute reaction than cetuximab. More studies should be done to prove long-term effects. (authors)

Development of monoclonal antibodies and quantitative ELISAs targeting insulin-degrading enzyme

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Dickson Dennis W

2009-10-01

Full Text Available Abstract Background Insulin-degrading enzyme (IDE is a widely studied zinc-metalloprotease implicated in the pathogenesis of type 2 diabetes mellitus, Alzheimer disease (AD and varicella zoster virus infection. Despite more than six decades of research on IDE, progress has been hampered by the lack of well-characterized reagents targeting this biomedically important protease. To address this important need, we generated and characterized new mouse monoclonal antibodies (mAbs targeting natively folded human and rodent IDE. Results Eight monoclonal hybridoma cell lines were derived in house from mice immunized with full-length, natively folded, recombinant human IDE. The mAbs derived from these lines were shown to detect IDE selectively and sensitively by a wide range of methods. Two mAbs in particular—designated 6A1 and 6H9—proved especially selective for IDE in immunocytochemical and immunohistochemical applications. Using a variety of methods, we show that 6A1 selectively detects both human and rodent IDE, while 6H9 selectively detects human, but not rodent, IDE, with both mAbs showing essentially no cross reactivity with other proteins in these applications. Using these novel anti-IDE mAbs, we also developed sensitive and quantitative sandwich ELISAs capable of quantifying IDE levels present in human brain extracts. Conclusion We succeeded in developing novel mAbs that selectively detect rodent and/or human IDE, which we have shown to be suitable for a wide range of applications, including western blotting, immunoprecipitation, immunocytochemistry, immunohistochemistry, and quantitative sandwich ELISAs. These novel anti-IDE mAbs and the assays derived from them constitute important new tools for addressing many unresolved questions about the basic biology of IDE and its role in multiple highly prevalent human diseases.

Novel immunoradiometric assay of thyroglobulin in serum with use of monoclonal antibodies selected for lack of cross-reactivity with autoantibodies

International Nuclear Information System (INIS)

Piechaczyk, M.; Baldet, L.; Pau, B.; Bastide, J.M.

1989-01-01

A multisite immunoradiometric assay for measurement of serum thyroglobulin (Tg), designated Magnogel-IRMA-Tg, has been developed, involving magnetic microbeads (Magnogel). This assay is based on the use of five anti-Tg monoclonal antibodies (MAbs) directed against three antigenic regions on the Tg molecule that are not recognized by anti-Tg autoantibodies (aAbs). Four of these MAbs, directed against two antigenic domains, were coupled to the magnetic beads and were used to trap the serum antigen. Another MAb, directed against the third region, was iodinated and served as the labeled second antibody. The Magnogel-IRMA-Tg technique is reproducible, rapid, and sensitive (lower detection limit, 3 micrograms/L). The assay reliably measures serum Tg in the presence of anti-Tg aAbs

Enhancement of retroviral infection in vitro by anti-Le(y) IgG: reversal by humanization of monoclonal mouse antibody

DEFF Research Database (Denmark)

Hansen, J E; Sørensen, A M; Arendrup, M

1993-01-01

Monoclonal mouse IgG3 antibody (ABL 364) against the carbohydrate Le(y) antigen enhanced infection in vitro with HTLV-1 and with HIV-1 when propagated in both transformed and normal lymphocytes. Enhancement was independent of complement, occurred with both lymphocytes and monocytes as target cells...

Re-engineering of the PAM1 phage display monoclonal antibody to produce a soluble, versatile anti-homogalacturonan scFv

DEFF Research Database (Denmark)

Manfield, I. W.; Bernal Giraldo, Adriana Jimena; Møller, I.

2006-01-01

Antibody phage display is an increasingly important alternative method for the production of monoclonal antibodies (mAbs) and involves the expression of antibody fragments (scFvs) at the surface of bacteriophage particles. We have previously used this technique to generate a phage mAb (PAM1phage...

Anti-Mycobacterium leprae monoclonal antibodies cross-react with human skin: an alternative explanation for the immune responses in leprosy

NARCIS (Netherlands)

Naafs, B.; Kolk, A. H.; Chin A Lien, R. A.; Faber, W. R.; van Dijk, G.; Kuijper, S.; Stolz, E.; van Joost, T.

1990-01-01

A panel of 17 mouse monoclonal antibodies (MoAb) raised against Mycobacterium leprae (M. leprae) antigens was used to detect antigenic determinants in normal human skin. An indirect immunoperoxidase technique was used. Eight of the MoAb detected epidermal antigens similar to patterns well known for

Biodistribution of an anti-interleukin 2 receptor monoclonal antibody in rat recipients of a heart allograft, and its use as a rejection marker in gamma scintigraphy

International Nuclear Information System (INIS)

Thedrez, P.; Paineau, J.; Jacques, Y.; Chatal, J.F.; Pelegrin, A.; Bouchaud, C.; Soulillou, J.P.

1989-01-01

Anti-interleukin-2 receptor monoclonal antibodies have been shown to prevent allograft rejection. This paper reports on the biodistribution of a mouse MoAb directed at the 55 Kd alpha chain of rat interleukin-2 receptor (IL2-R) during allograft rejection. Only a low percentage (approximately 1%) of intact 125I-labeled MoAb was detected in the rejected graft, and irrelevant control IgG1 was found at a similar level. This suggests that most of the injected intact MoAb bound to graft tissue via its monomorphic Fc segment. In contrast, OX39 F(ab')2 fragments showed a preferential localization in the rejected allograft and did not bind to the LEW-to-LEW syngeneic heart graft. Irrelevant F(ab')2 did not concentrate in the allogeneic graft. Accordingly, F(ab')2 fragments from OX39 or irrelevant MoAb were used for gamma-scintigraphy on allograft recipients together with biodistribution studies. Results show that scintigraphy was able to detect allograft accumulation of 131I OX39 F(ab')2, whereas no imaging was obtained when OX39 F(ab')2 was used in the syngeneic combination or when irrelevant 131-IgG1 F(ab')2 was given to allograft recipients. This method, applied to the clinical situation, could be of interest for detection of early graft rejection episodes by immunoscintigraphy using reagents specific for activation determinants on lymphocyte membranes, such as anti-interleukin-2 receptor MoAb

Radioimmunoscintigraphy with I-131-labelled anti-CEA monclonal antibody in colorectal cancer patients

International Nuclear Information System (INIS)

Xie Tianhao

1988-01-01

Twenty three colorectal carcinoma and two benign polyposis patients with operatively and histologically proven were studied by radioimmunoscintigraphy, using anti-CEA monoclonal antibodies (C14-17 and C50) radiolabeled with I-131 . Computer assisted processing for the subtraction of Tc-99m background radioactivity was used to enhance the detection and localization of tumor which is visualized by immune scintigraphy. The size of tumor and the ratios of tumor to nontumor (T/NT) are two very important factors for the external immunoscintigraphy. The antibody uptake and retention in tumor are likely to depend on the degree of vascularity and diffusion into the viable tumor mass. Based upon the obtained results, the sensitivity of the method (true-poditive) was 91%, its specificity (true-negative) was 100%. This study thus indicates that radioimmunoscintigraphy of cancer with radioactive anti-CEA monoclonal antibody is very uaeful in the diagnoses of patients with CEA-containing neoplasms

Multicompartmental analysis of the kinetics of monoclonal antibody in cancer patients

International Nuclear Information System (INIS)

Koizumi, K.; De Nardo, G.L.; De Nardo, S.J.; Peng, J.S.; Macey, D.J.; Hisada, K.; Tonami, N.

1985-01-01

Multicompartmental models were applied for analysis of kinetics of iodide labeled monoclonal antibody in cancer patients. About 14 compartments such as intravascular antibody pool, interstitial antibody pool, antibody processors, tumor antigen site, intravascular immune complex pool, intravascular iodide pool, and urine iodide pool were assumed. This model accounts for three molecular species, the antibody, and antibody complex, and free iodide or iodinated peptides. Patients were injected with I-123-Lym-1 IgG2a (anti B cell lymphoma antibody). After injection, blood and urine samples were sequentially collected. Plasma and urine were separated by HPLC into fractions of intact antibody, immune complex, and free iodide. This information was used for input data in the theoretical model. SAAM computer program was used to solve these compartmental models. Published linear rate constants for human serum albumin and human non-immune IgG were initially used. However, data calculated from the model differed from observed curves in several respects. The kinetics of mouse monoclonal antibody, a foreign protein in a patient, were significantly different from those reported for human IgG. When a nonlinear, saturable hepatic processor was incorporated in the model, calculated data fit the observed data better. This kinetic model provides a basis for calculating radiation doses for radioiodinated antibodies

Establishment of monoclonal HCC cell lines with organ site-specific tropisms

International Nuclear Information System (INIS)

Wan, Jinliang; Wen, Duo; Dong, Lili; Tang, Jun; Liu, Dongli; Liu, Yang; Tao, Zhonghua; Gao, Dongmei; Sun, Huichuan; Cao, Ya; Fan, Jia; Wu, Weizhong

2015-01-01

Organ site-specific metastasis is an ominous feature for most poor-prognostic hepatocellular carcinoma (HCC) patients. Cancer cell lines and animal models are indispensable for investigating the molecular mechanisms of organ specific tropism. However, till now, little is known about the drivers in HCC metastatic tropism, and also no effective way has been developed to block the process of tropistic metastasis. In this study, we established several monoclonal HCC cell lines from HCCLM3-RFP together with their xenograft models, and then analyzed their metastatic potentials and tropisms using in-vitro and in-vivo assays, and finally elucidated the driving forces of HCC tropistic metastases. Six monoclonal cell lines with different organ site-specific tropism were established successfully. SPARC, VCAM1 and ANGPTL4 were found positively correlated with the potentials of lung metastasis, while ITGA1 had a positive relation to lymph node metastasis of enterocoelia. By our powerful platforms, HCC metastatic tropisms in clinic could be easily mimicked and recapitulated for exploring the bilateral interactions between tumor and its microenvironment, elucidating the drivers of HCC metastatic tropisms, and testing anti-cancer effects of newly developed agent in pre-clinical stage. The online version of this article (doi:10.1186/s12885-015-1692-0) contains supplementary material, which is available to authorized users

Influence of type I IFN signaling on anti-MOG antibody-mediated demyelination

DEFF Research Database (Denmark)

Berg, Carsten Tue; Khorooshi, Reza M. H.; Asgari, Nasrin

2017-01-01

Background Antibodies with specificity for myelin oligodendrocyte glycoprotein (MOG) are implicated in multiple sclerosis and related diseases. The pathogenic importance of anti-MOG antibody in primary demyelinating pathology remains poorly characterized. Objective The objective of this study...... is to investigate whether administration of anti-MOG antibody would be sufficient for demyelination and to determine if type I interferon (IFN) signaling plays a similar role in anti-MOG antibody-mediated pathology, as has been shown for neuromyelitis optica-like pathology. Methods Purified IgG2a monoclonal anti...... demyelination in wild-type and IFNAR1-KO mice. Conclusions Anti-MOG antibody and complement was sufficient to induce callosal demyelination, and pathology was dependent on type I IFN. Induction of EAE in IFNAR1-KO mice overcame the dependence on type I IFN for anti-MOG and complement-mediated demyelination....

Produksi dan Karakterisasi Antibodi Monoklonal Anti-Cysticercus cellulosae (PRODUCTION AND CHRACTERIZATION OF MONOCLONAL ANTIBODIES AGAINST CYSTICERCUS CELLULOSAE

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Ida Bagus Ngurah Swacita

2015-10-01

Full Text Available The purpose of this study is to make a monoclonal antibody against- Cysticercus cellulosae and itscharacterization. Samples antigen prepared from T. solium larvae (C. cellulosae was then used to immunizeBalb/c. The immune response of mice assessed by ELISA test, then the lymphocytes of mice used for theproduction of monoclonal antibodies (MoAb. Origin lymphocytes of mice that produce antibodies againstC. cellulosae antigen, fused with myeloma cells (NS1. Results fusion of two cells produces hybrid cellscalled hybridomas; cells are then screened by ELISA test. Hybridoma cells that produce only MoAb, usedto produce large quantities in vitro. Characterization of MoAb against-C.cellulosae was tested by usingELISA and Western blotting. Mice were immunized with C.cellulosae antigen showed an immune responseproducing antibodies to C.cellulosae. Based on the results of fusion, produced a total of 51 hybridoma cellclones and after being screened, only three clones of hybridoma cells that produced MoAb against–C.cellulosae. MoAb produced, named after the hole where the growth of the ELISA micro plate, the BE6,BE7, and EE9. Characteristics of this MoAb capable of tracking cellulosae of fluid larvae and recognizeantigen protein bands with molecular weight 78kDa.

Anti-Taenia solium monoclonal antibodies for the detection of parasite antigens in body fluids from patients with neurocysticercosis.

Science.gov (United States)

Paredes, Adriana; Sáenz, Patricia; Marzal, Miguel W; Orrego, Miguel A; Castillo, Yesenia; Rivera, Andrea; Mahanty, Siddhartha; Guerra-Giraldez, Cristina; García, Hector H; Nash, Theodore E

2016-07-01

Neurocysticercosis (NCC), an infection of the brain by Taenia solium (Ts) cysts, is the most common cause of adult-onset epilepsy in developing countries. Serological testing consists primarily of varying methods to detect antibodies in body fluids and more recently antigen (Ag) detection assays to identify individuals or animals with viable parasites. Antigen assays currently in use employ monoclonal antibodies (mAbs) raised against T. saginata, which have known cross reactivity to animal cestodes but are highly specific in human samples. We produced, characterized and tested 21 mAbs raised against T. solium whole cyst antigens, vesicular fluid or excretory secretory products. Reactivity of the TsmAbs against specific cyst structures was determined using immunofluorescence and immunohistochemistry on histological sections of Ts muscle cysts. Four TsmAbs reacted to vesicular space alone, 9 to the neck and cyst wall, one to the neck and vesicular space and 7 to the neck, cyst wall and vesicular space. An in-house ELISA assay to detect circulating Ts antigen, using the TsmAbs as capture antibodies and a rabbit polyclonal anti-Ts whole cyst antibody as a detector antibody demonstrated that eight of the 21 TsmAbs detected antigens in known NCC-positive human sera and three of these also in urine samples. Reactivity was expressed as normalized ratios of optical densities (OD positive control/OD negative control). Three TsmAbs had ratios >10 and five between 2 and 10. The TsmAbs have potential utility for the diagnosis and post-treatment monitoring of patients with viable NCC infections. Copyright © 2016 Elsevier Inc. All rights reserved.

Generation of a Monoclonal Antibody against Mycoplasma spp. following Accidental Contamination during Production of a Monoclonal Antibody against Lawsonia intracellularis

OpenAIRE

Hwang, Jeong-Min; Lee, Ji-Hye; Yeh, Jung-Yong

2012-01-01

This report describes Mycoplasma contamination of Lawsonia intracellularis cultures that led to the unintended acquisition of a monoclonal antibody against Mycoplasma spp. during the attempted generation of a monoclonal antibody against L. intracellularis.

A phase I monotherapy study of RG7212, a first-in-class monoclonal antibody targeting TWEAK signaling in patients with advanced cancers

DEFF Research Database (Denmark)

Lassen, Ulrik N; Meulendijks, Didier; Siu, Lilian L

2015-01-01

activation. A phase I study of RG7212, a humanized anti-TWEAK IgG1κ monoclonal antibody, was conducted in patients with advanced solid tumors expressing Fn14. EXPERIMENTAL DESIGN: Dose escalations, over a 200- to 7,200-mg range, were performed with patients enrolled in weekly (QW), bi-weekly (Q2W), or every...

Establishment of EMab-134, a Sensitive and Specific Anti-Epidermal Growth Factor Receptor Monoclonal Antibody for Detecting Squamous Cell Carcinoma Cells of the Oral Cavity.

Science.gov (United States)

Itai, Shunsuke; Yamada, Shinji; Kaneko, Mika K; Chang, Yao-Wen; Harada, Hiroyuki; Kato, Yukinari

2017-12-01

Epidermal growth factor receptor (EGFR), a receptor tyrosine kinase, activates downstream signaling cascades in many tumors. In this study, we established novel anti-EGFR monoclonal antibodies (mAbs) and characterized their efficacy in flow cytometry, Western blot, and immunohistochemical analyses. We immunized mice with a combination of the extracellular domain of EGFR and EGFR-overexpressing LN229 glioblastoma cells (LN229/EGFR) and performed the first screening using enzyme-linked immunosorbent assay. Next, we selected mAbs using flow cytometry. Among 156 established clones, two mAbs, EMab-51 (IgG 1 , kappa) and EMab-134 (IgG 1 , kappa), reacted with EGFR in Western blot analysis; EMab-134 showed a much higher sensitivity compared with EMab-51. We compared the binding affinities of EMab-51 and EMab-134 using flow cytometry; the calculated K D values for EMab-51 and EMab-134 against SAS cells/HSC-2 cells were 9.2 × 10 -9 M/9.9 × 10 -9 M and 2.6 × 10 -9 M/8.3 × 10 -9 M, respectively, indicating that EMab-134 has a higher affinity to EGFR-expressing cells. Immunohistochemical analysis of EMab-51 and EMab-134 showed sensitive and specific reactions against oral cancer cells; EMab-134 demonstrated a much higher sensitivity (36/38 cases; 94.7%) to oral squamous cell carcinomas compared with EMab-51 (6/38 cases; 15.8%). This novel anti-EGFR mAb, EMab-134, could be advantageous for detecting EGFR in the pathological analysis of EGFR-expressing cancers.

Radioimmunotherapy of Nude Mice Bearing Human Colon Carcinoma with I-131 Labeled Anti-carcinoembryonic Antigen

International Nuclear Information System (INIS)

Kim, Byung Tae; Lee, Kyung Han; Kim, Sang Eun; Choi, Yong; Chi, Dae Yoon; Chung, June Key; Lee, Myung Chul; Koh, Chang Soon; Chung, Hong Keun

1995-01-01

This study was designed to evaluate the effects of various factors on the therapeutic effect of the I-l3l labeled anti-carcinoembryonic antigen monoclonal antibody(anti-CEA antibody). Tetrazolium-based colorimetric assay (MTT) was used to compare in vitro cytotoxicity of 3 Korean colon cancer cell lines (SNU-C2A, SNU-C4, SNU-C5) for selection of proper 2 cell lines in this study. The changes of the size of tumor which was xenografted to nude mice (balb/c nu/nu) were compared in 4 groups (group treated I-131 labeled anti-CEA antibody, group treated with non-radiolabeled anti-CEA antibody, group treated with I-131. labeled anti-human chorionic gonadotropin monoclonal antibody (anti-hCG antibody) as nonspecific antibody, and group injected with normal saline as a control). Immunohistochemical staining and in vivo autoradiography were performed after excision of the xenografted tumor. The results were as below mentioned. The in vitro cytotoxic effect of I-131 labeled anti-CEA antibody is most prominent in SNU-C5 cell line between 3 cancer cell lines. The changes of xenografted tumor size in both SNU-C4 and SNU-C5 cell tumors at the thirteenth day after injection of the antibodies were smallest in the group treated with I-131 labeled anti-CEA antibody (SNU-C4/SNU-C5; 324/342%) comparing with other groups, group treated with anti-CEA antibody (622/660%), group treated with I-131 anti-hCG antibody (538/546%), and control group(1030/724%) (p<0.02 in SNU-C4 and p<0.1in SNU-C5 at the 13th day after injection of antibodies). On the thirteenth day after injection of the antibodies nude mice were sacreficed to count the radiouptake of tumor and to check the changes of tumor size. Correlations between radiouptake and change of tumor size were calculated in each groups and significant negative correlation was only obtained in the group treated with I-131 anti-CEA antibody (p<0.05). There were no correlations between antigenic expression of carcinoembryonic antigen and

Increased half-life and enhanced potency of Fc-modified human PCSK9 monoclonal antibodies in primates.

Directory of Open Access Journals (Sweden)

Yijun Shen

Full Text Available Blocking proprotein convertase subtilisin kexin type 9 (PCSK9 binding to low-density lipoprotein receptor (LDLR can profoundly lower plasma LDL levels. Two anti-PCKS9 monoclonal antibodies (mAbs, alirocumab and evolocumab, were approved by the FDA in 2015. The recommended dose is 75 mg to 150 mg every two weeks for alirocumab and 140mg every two weeks or 420 mg once a month for evolocumab. This study attempted to improve the pharmacokinetic properties of F0016A, an IgG1 anti-PCKS9 mAb, to generate biologically superior molecules. We engineered several variants with two or three amino acid substitutions in the Fc fragment based on prior knowledge. The Fc-modified mAbs exhibited increased binding to FcRn, resulting in prolonged serum half-life and enhanced efficacy in vivo. These results demonstrate that Fc-modified anti-PCKS9 antibodies may enable less frequent or lower dosing of antibodies by improved recycling into the blood.

Ta1, a novel 105 KD human T cell activation antigen defined by a monoclonal antibody.

Science.gov (United States)

Fox, D A; Hussey, R E; Fitzgerald, K A; Acuto, O; Poole, C; Palley, L; Daley, J F; Schlossman, S F; Reinherz, E L

1984-09-01

By using a murine monoclonal antibody produced against an IL 2-dependent human T cell line, we defined a T lineage-specific molecule, termed Ta1, that is expressed strongly on activated T lymphocytes of both the T4 and T8 subsets, as well as on T cell lines and clones, but only weakly on a fraction of resting T cells. SDS-PAGE analysis of immunoprecipitates from 125I-labeled, activated T cells demonstrates a single major band of apparent m.w. 105 KD under both reducing and nonreducing conditions. Unlike anti-IL 2 receptor antibodies, anti-Ta1 does not inhibit T cell proliferative responses to mitogen, antigen, or IL 2-containing medium. Moreover, anti-Ta1 has no effect on T cell-mediated cytotoxicity. Ta1 appears to be a novel human T cell-specific activation antigen that may serve as a useful marker of T cell activation in human disease.

IgM MGUS associated with anti-MAG neuropathy: a single institution experience.

Science.gov (United States)

Talamo, Giampaolo; Mir, Muhammad A; Pandey, Manoj K; Sivik, Jeffrey K; Raheja, Divisha

2015-06-01

Anti-MAG neuropathy is a very rare form of acquired polyneuropathy associated with IgM monoclonal gammopathy of undetermined significance (MGUS). We conducted a retrospective review of 194 consecutive MGUS patients seen at the Penn State Hershey Cancer Institute. We identified six patients among 37 (16 %) with IgM MGUS with anti-MAG neuropathy. Interestingly, an additional patient had anti-MAG neuropathy without MGUS. Common clinical manifestations were numbness and paresthesias of the extremities and gait imbalance. All four patients treated with rituximab and none of the three untreated ones had a subjective improvement of their symptoms. We conclude that all patients with IgM MGUS and neuropathy should be screened for anti-MAG antibodies and, if positive, they should be offered treatment with rituximab.

Radiobromination of anti-HER2/neu/ErbB-2 monoclonal antibody using the p-isothiocyanatobenzene derivative of the [76Br]undecahydro-bromo-7,8-dicarba-nido-undecaborate(1-) ion

International Nuclear Information System (INIS)

Winberg, Karl Johan; Persson, Mikael; Malmstroem, Per-Uno; Sjoeberg, Stefan; Tolmachev, Vladimir

2004-01-01

The monoclonal humanized anti-HER2 antibody trastuzumab was radiolabeled with the positron emitter 76 Br (T ((1)/(2)) =16.2 h). Indirect labeling was performed using the p-isothiocyanatobenzene derivative of the [ 76 Br]undecahydro-bromo-7,8-dicarba-nido-undecaborate(1-) ( 76 Br-NBI) as a precursor molecule. 76 Br-NBI was prepared by bromination of the 7-(p-isothiocyanato-phenyl)dodecahydro-7,8-dicarba-nido-undecaborate(1-) ion (NBI) with a yield of 93-95% using Chloramine-T (CAT) as an oxidant. Coupling of radiobrominated NBI to antibody was performed without intermediate purification, in an ''one pot'' reaction. An overall labeling yield of 55.7 ± 4.8% (mean ± maximum error) was achieved when 300 μg of antibody was labeled. The label was stable in vitro in physiological and denaturing conditions. In a cell binding test, trastuzumab remained immunoreactive after labeling

Molecular characterization of the pSinB plasmid of the arsenite oxidizing, metallotolerant Sinorhizobium sp. M14 - insight into the heavy metal resistome of sinorhizobial extrachromosomal replicons.

Science.gov (United States)

Romaniuk, Krzysztof; Dziewit, Lukasz; Decewicz, Przemyslaw; Mielnicki, Sebastian; Radlinska, Monika; Drewniak, Lukasz

2017-01-01

Sinorhizobium sp. M14 is an As(III)-oxidizing, psychrotolerant strain, capable of growth in the presence of extremely high concentrations of arsenic and many other heavy metals. Metallotolerant abilities of the M14 strain depend upon the presence of two extrachromosomal replicons: pSinA (∼ 109 kb) and pSinB (∼ 300 kb). The latter was subjected to complex analysis. The performed analysis demonstrated that the plasmid pSinB is a narrow-host-range repABC-type replicon, which is fully stabilized by the phd-vapC-like toxin-antitoxin stabilizing system. In silico analysis showed that among the phenotypic gene clusters of the plasmid pSinB, eight modules are potentially involved in heavy metals resistance (HMR). These modules carry genes encoding efflux pumps, permeases, transporters and copper oxidases, which provide resistance to arsenic, cadmium, cobalt, copper, iron, mercury, nickel, silver and zinc. The functional analysis revealed that the HMR modules are active and have an effect on the minimal inhibitory concentration (MIC) values observed for the heterological host cells. The phenotype was manifested by an increase or decrease of the MICs of heavy metals and it was strain specific. The analysis of distribution of the heavy metal resistance genes, i.e. resistome, in Sinorhizobium spp. plasmids, revealed that the HMR modules are common in these replicons. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Development and Characterization of Canine Distemper Virus Monoclonal Antibodies.

Science.gov (United States)

Liu, Yuxiu; Hao, Liying; Li, Xiangdong; Wang, Linxiao; Zhang, Jianpo; Deng, Junhua; Tian, Kegong

2017-06-01

Five canine distemper virus monoclonal antibodies were developed by immunizing BALB/c mice with a traditional vaccine strain Snyder Hill. Among these monoclonal antibodies, four antibodies recognized both field and vaccine strains of canine distemper virus without neutralizing ability. One monoclonal antibody, 1A4, against hemagglutinin protein of canine distemper virus was found to react only with vaccine strain virus but not field isolates, and showed neutralizing activity to vaccine strain virus. These monoclonal antibodies could be very useful tools in the study of the pathogenesis of canine distemper virus and the development of diagnostic reagents.

Diagnostic of tumours of epithelial origin with the monoclonal antibody IOR EGF/R3 murino

International Nuclear Information System (INIS)

Ramos, M.

1997-01-01

Despite of the advantages on anti tumoral therapy, the cancer of epithelial origin constitutes one of the first causes of death worldwide. That kind of tumors have a 10-30-fold overexpression of the epidermal growth factor receptor (EGFr). Monoclonal antibody ior egf/r3 is a lgG2a, which recognizes the epidermal growth factor receptor. The aim of the present work was the evaluate the diagnostic efficacy of the 99m Tc-labelled ior egf/r3 for the detection of epithelial derived tumors, its metastasis and its recurrences

Report of the ECCO pathogenesis workshop on anti-TNF therapy failures in inflammatory bowel diseases: definitions, frequency and pharmacological aspects

DEFF Research Database (Denmark)

Allez, Matthieu; Karmiris, Konstantinos; Louis, Edouard

2010-01-01

The first ECCO pathogenesis workshop focused on anti-TNF therapy failures in inflammatory bowel diseases (IBDs). The overall objective was to better understand and explore primary non response and loss of response to anti-TNF agents in IBD. The outcome of this workshop is presented into two parts....... This first section addresses definitions, frequency and pharmacological aspects of anti-TNF therapy failure, including pharmacokinetics of anti-TNF monoclonal antibodies and immune and non-immune mediated clearance of anti-TNF mAbs. The second section concerns the biological roles of TNF and TNF antagonists...

A Unique Report: Development of Super Anti-Human IgG Monoclone with Optical Density Over Than 3

OpenAIRE

Aghebati Maleki, Leili; Baradaran, Behzad; Abdolalizadeh, Jalal; Ezzatifar, Fatemeh; Majidi, Jafar

2013-01-01

Purpose: Monoclonal antibodies and related conjugates are key reagents used in biomedical researches as well as, in treatment, purification and diagnosis of infectious and non- infectious diseases. Methods: Balb/c mice were immunized with purified human IgG. Spleen cells of the most immune mouse were fused with SP2/0 in the presence of Poly Ethylene Glycol (PEG). Supernatant of hybridoma cells was screened for detection of antibody by ELISA. Then, the sample was assessed for cross-reactivity ...

Mouse monoclonal antibodies against human c-Mpl and characterization for flow cytometry applications.

Science.gov (United States)

Abbott, Christina; Huang, Guo; Ellison, Aaron R; Chen, Ching; Arora, Taruna; Szilvassy, Stephen J; Wei, Ping

2010-04-01

Mouse monoclonal antibodies (MAbs) against human c-Mpl, the cognate receptor for thrombopoietin (TPO), were generated using hybridoma technology and characterized by various assays to demonstrate their specificity and affinity. Two such MAbs, 1.6 and 1.75, were determined to be superior for flow cytometry studies and exhibited double-digit picomolar (pM) affinities to soluble human c-Mpl protein. Both MAbs specifically bound to cells engineered to overexpress human c-Mpl protein, immortalized human hematopoietic cell lines that express endogenous c-Mpl, primary human bone marrow and peripheral blood-derived CD34(+) cells, and purified human platelets. No binding was detected on cell lines that did not express c-Mpl. Receptor competition and siRNA knock-down studies further confirmed the specificity of antibodies 1.6 and 1.75 for human c-Mpl. In contrast to these newly generated MAbs, none of eight commercially available anti-c-Mpl antibodies tested were found to bind specifically to human c-Mpl and were thus shown to be unsuitable for flow cytometry studies. Monoclonal antibodies 1.6 and 1.75 will therefore be useful flow cytometry reagents to detect cell surface c-Mpl expression.

/sup 99m/Tc radiolabelling and quality control tests of anti-melanoma monoclonal antibodies and F(ab')/sub 2/ fragments for immunoscintigraphy

International Nuclear Information System (INIS)

Callegaro, L.; Deleide, G.; Dovis, M.; Cecconato, E.; Scassellati, G.A.

1986-01-01

Tumour radioimmunodetection was first developed by using radiolabelled polyclonal antibodies, raised in goats against tumour associated antigens (TAA). The availability of monoclonal antibodies to TAA has definitely contributed to more extensive in vivo use of radiolabelled antibodies. However, many factors are involved in tumour radioimmunolocalization, related either to the antibody and radioisotope features or to the natural history of the tumour itself. The experimental protocol developed by the authors allows a full evaluation of the properties of a particular MoAb.This paper illustrates the work done with on a particular set of monoclonal antibodies, raised against human melanoma associated antigens, with the aim of visualizing primary and metastatic lesions in melanoma patients

Monoclonal antibody PAL-E specific for endothelium

NARCIS (Netherlands)

Schlingemann, R. O.; Dingjan, G. M.; Emeis, J. J.; Blok, J.; Warnaar, S. O.; Ruiter, D. J.

1985-01-01

A monoclonal antibody, PAL-E, is described that is specific for endothelial cells. The monoclonal antibody, an IgG2a, markedly stains endothelium of capillaries, medium-sized and small veins, and venules in frozen sections of human and some animal tissues tested. It reacts not at all or only weakly

Lymphocyte targeting with /sup 111/In-labelled monoclonal antibodies

Energy Technology Data Exchange (ETDEWEB)

Loutfi, I.; Batchelor, J.R.; Lavender, J.P.; Epenetos, A.A.

1988-01-01

In vitro tests were conducted using human T and B cell lines, as well as whole blood, to establish the usefulness of 2 murine monoclonal antibodies (MAbs), an anti-CD5 (Pan T) and a Pan B, for potential radioimmunolocalization and therapy. Both MAbs showed specificity for the cell line in question as tested by indirect immunofluorescence and radioimmunoassay. Assays carried out on whole blood showed 40-70% of the added activity of /sup 111/In-labelled Pan B antibody binding to B cells and 20-24% of /sup 111/In-Pan T antibody binding to T cells. The amount of internalised /sup 111/In-labelled Pan B was 6% of total amount at 24 hr indicating a slow internalization process. These results should allow for in vivo targeting of normal and neoplastic B and T cells.

Monoclonal for cancer detection and therapy

International Nuclear Information System (INIS)

Baldwin, R.W.; Byers, V.S.

1985-01-01

This book contains 18 chapters. Some of the chapter titles are: Monoclonal Antibodies to Breast Cancer and Their Application; Clinical Applications of Radioimmunolocalisation; Localisation of Cancer of the Ovary and Metastases Using 123 I-labelled Monoclonal Antibody HMFG-2 Compared to Surgical Findings; Interest of Globotriaosylceramide Membrane Antigen as Target for Immunotoxins; and Analysis, Results and Future Prospective of the Therapeutic Use of Radiolabeled Antibody in Cancer Therapy

Novel anti-c-Mpl monoclonal antibodies identified multiple differentially glycosylated human c-Mpl proteins in megakaryocytic cells but not in human solid tumors.

Science.gov (United States)

Zhan, Jinghui; Felder, Barbara; Ellison, Aaron R; Winters, Aaron; Salimi-Moosavi, Hossein; Scully, Sheila; Turk, James R; Wei, Ping

2013-06-01

Thrombopoietin and its cognate receptor, c-Mpl, are the primary molecular regulators of megakaryocytopoiesis and platelet production. To date the pattern of c-Mpl expression in human solid tumors and the distribution and biochemical properties of c-Mpl proteins in hematopoietic tissues are largely unknown. We have recently developed highly specific mouse monoclonal antibodies (MAb) against human c-Mpl. In this study we used these antibodies to demonstrate the presence of full-length and truncated human c-Mpl proteins in various megakaryocytic cell types, and their absence in over 100 solid tumor cell lines and in the 12 most common primary human tumor types. Quantitative assays showed a cell context-dependent distribution of full-length and truncated c-Mpl proteins. All forms of human c-Mpl protein were found to be modified with extensive N-linked glycosylation but different degrees of sialylation and O-linked glycosylation. Of note, different variants of full-length c-Mpl protein exhibiting differential glycosylation were expressed in erythromegakaryocytic leukemic cell lines and in platelets from healthy human donors. This work provides a comprehensive analysis of human c-Mpl mRNA and protein expression on normal and malignant hematopoietic and non-hematopoietic cells and demonstrates the multiple applications of several novel anti-c-Mpl antibodies.

An International Standard for specifying the minimum potency of anti-D blood-grouping reagents: evaluation of a candidate preparation in an international collaborative study

NARCIS (Netherlands)

Thorpe, S. J.; Fox, B.; Heath, A. B.; Scott, M.; de Haas, M.; Kochman, S.; Padilla, A.

2006-01-01

The aim of this study was to evaluate a lyophilized monoclonal immunoglobulin M (IgM) anti-D preparation for use as an International Standard to specify a recommended minimum acceptable potency of anti-D blood-grouping reagents. The candidate International Standard (99/836) for specifying the

A model for the catabolism of rhizopine in Rhizobium leguminosarum involves a ferredoxin oxygenase complex and the inositol degradative pathway.

Science.gov (United States)

Bahar, M; de Majnik, J; Wexler, M; Fry, J; Poole, P S; Murphy, P J

1998-11-01

Rhizopines are nodule-specific compounds that confer an intraspecies competitive nodulation advantage to strains that can catabolize them. The rhizopine (3-O-methyl-scyllo-inosamine, 3-O-MSI) catabolic moc gene cluster mocCABRDE(F) in Rhizobium leguminosarum bv. viciae strain 1a is located on the Sym plasmid. MocCABR are homologous to the mocCABR gene products from Sinorhizobium meliloti. MocD and MocE contain motifs corresponding to a TOL-like oxygenase and a [2Fe-2S] Rieske-like ferredoxin, respectively. The mocF gene encodes a ferredoxin reductase that would complete the oxygenase system, but is not essential for rhizopine catabolism. We propose a rhizopine catabolic model whereby MocB transports rhizopine into the cell and MocDE and MocF (or a similar protein elsewhere in the genome), under the regulation of MocR, act in concert to form a ferredoxin oxygenase system that demethylates 3-O-MSI to form scyllo-inosamine (SI). MocA, an NAD(H)-dependent dehydrogenase, and MocC continue the catabolic process. Compounds formed then enter the inositol catabolic pathway.

Characterization of Crude Oil Degrading Bacteria Isolated from Contaminated Soils Surrounding Gas Stations.

Science.gov (United States)

Abou-Shanab, Reda A I; Eraky, Mohamed; Haddad, Ahmed M; Abdel-Gaffar, Abdel-Rahman B; Salem, Ahmed M

2016-11-01

A total of twenty bacterial cultures were isolated from hydrocarbon contaminated soil. Of the 20 isolates, RAM03, RAM06, RAM13, and RAM17 were specifically chosen based on their relatively higher growth on salt medium amended with 4 % crude oil, emulsion index, surface tension, and degradation percentage. These bacterial cultures had 16S rRNA gene sequences that were most similar to Ochrobactrum cytisi (RAM03), Ochrobactrum anthropi (RAM06 and RAM17), and Sinorhizobium meliloti (RAM13) with 96 %, 100 % and 99 %, and 99 % similarity. The tested strains revealed a promising potential for bioremediation of petroleum oil contamination as they could degrade >93 % and 54 % of total petroleum hydrocarbons (TPHs) in a liquid medium and soil amended with 4 % crude oil, respectively, after 30 day incubation. These bacteria could effectively remove both aliphatic and aromatic petroleum hydrocarbons. In conclusion, these strains could be considered as good prospects for their application in bioremediation of hydrocarbon contaminated environment.

Genomic analyses of metal resistance genes in three plant growth promoting bacteria of legume plants in Northwest mine tailings, China.

Science.gov (United States)

Xie, Pin; Hao, Xiuli; Herzberg, Martin; Luo, Yantao; Nies, Dietrich H; Wei, Gehong

2015-01-01

To better understand the diversity of metal resistance genetic determinant from microbes that survived at metal tailings in northwest of China, a highly elevated level of heavy metal containing region, genomic analyses was conducted using genome sequence of three native metal-resistant plant growth promoting bacteria (PGPB). It shows that: Mesorhizobium amorphae CCNWGS0123 contains metal transporters from P-type ATPase, CDF (Cation Diffusion Facilitator), HupE/UreJ and CHR (chromate ion transporter) family involved in copper, zinc, nickel as well as chromate resistance and homeostasis. Meanwhile, the putative CopA/CueO system is expected to mediate copper resistance in Sinorhizobium meliloti CCNWSX0020 while ZntA transporter, assisted with putative CzcD, determines zinc tolerance in Agrobacterium tumefaciens CCNWGS0286. The greenhouse experiment provides the consistent evidence of the plant growth promoting effects of these microbes on their hosts by nitrogen fixation and/or indoleacetic acid (IAA) secretion, indicating a potential in-site phytoremediation usage in the mining tailing regions of China. Copyright © 2014. Published by Elsevier B.V.

[The value of serum free light chain in differential diagnosis of monoclonal gammopathy of renal significance].

Science.gov (United States)

Li, C; Wen, Y B; Li, H; Su, W; Li, J; Cai, J F; Chen, L M; Li, X M; Li, X W

2017-08-08

Objective: To investigate the value of serum free light chain (FLC) in differential diagnosis of monoclonal gammopathy of renal significance (MGRS). Methods: Forty-nine hospitalized patients who underwent renal biopsy in Peking Union Medical College Hospital between January 2013 and December 2015 were included. Monoclonal gammopathy was detected by serum protein electrophoresis (SPE), serum immunofixation electrophoresis (IFE), urine IFE and serum FLC. All patients were classified as MGRS ( n =32) and monoclonal gammopathy of undetermined significance (MGUS) ( n =17). Results: Renal lesions in MGRS subgroup included light chain amyloidosis ( n =24, 75.0%), light chain deposition disease ( n =7, 21.9%), and fibrillary glomerulopathy ( n =1, 3.1%). Renal diseases in MGUS subgroup included membranous nephropathy ( n =10), focal segmental glomerulosclerosi (FSGS) ( n =3), diabetic glomerulopathy ( n =1), Henoch-Schonlein purpura nephritis ( n =1), anti-GBM disease concurrent with membranous nephropathy ( n =1) and glomerulomegaly ( n =1). Positive number of SPE, serum IFE, urine IFE and abnormal number of serum FLC ratio in MGRS subgroup were 12, 16, 23 and 30, respectively. Positive number of SPE, serum IFE, urine IFE and abnormal number of serum FLC ratio in MGUS subgroup were 11, 17, 6 and 3, respectively. MGRS and MGUS subgroups differed significantly in positive rate of serum IFE ( P value for MGRS, which was helpful for differential diagnosis of patients who had contraindication to renal biopsy.

Método fosfatasa alcalina anti-fosfatasa alcalina para el diagnóstico de inmunodeficiencias celulares Alkaline phosphatase-anti-alkaline phosphatase method for the diagnosis of cell immunodeficiencies

Directory of Open Access Journals (Sweden)

Berta B Socarrás Ferrer

2001-12-01

Full Text Available Para el estudio de 25 pacientes con infecciones recurrentes y un grupo control de 25 individuos supuestamente sanos, se aplicó, en nuestro laboratorio, el método inmunocitoquímico de fosfatasa alcalina anti-fosfatasa alcalina, para la cuantificación de las principales subpoblaciones de linfocitos T identificados con los anticuerpos monoclonales: anti-CD3, anti-CD4 y anti-CD8. Se obtuvieron diferencias significativas para las subpoblaciones TCD3 y CD4 positivos (p For the study of 25 patients with recurrent infections and a control group of 25 supposedly healthy individuals, our Laboratory applied the immunocytochemical method of alkaline phosphatase - anti-alkaline phosphatase for the quantitation of the main T-lymphocyte subgroups identified with monoclonal antibodies:antiCD3, anti-CD4 and anti-CD8. There were significant differences in positive TCD3 and CD4 subsets (p<0,05. Because this is a low cost and quick method, it may be applied by other immunodiagnosis labs throughout the country

Identification of the rctA Gene, Which Is Required for Repression of Conjugative Transfer of Rhizobial Symbiotic Megaplasmids†

Science.gov (United States)

Pérez-Mendoza, Daniel; Sepúlveda, Edgardo; Pando, Victoria; Muñoz, Socorro; Nogales, Joaquina; Olivares, José; Soto, Maria J.; Herrera-Cervera, José A.; Romero, David; Brom, Susana; Sanjuán, Juan

2005-01-01

An analysis of the conjugative transfer of pRetCFN42d, the symbiotic plasmid (pSym) of Rhizobium etli, has revealed a novel gene, rctA, as an essential element of a regulatory system for silencing the conjugative transfer of R. etli pSym by repressing the transcription of conjugal transfer genes in standard laboratory media. The rctA gene product lacks sequence conservation with other proteins of known function but may belong to the winged-helix DNA-binding subfamily of transcriptional regulators. Similar to that of many transcriptional repressors, rctA transcription seems to be positively autoregulated. rctA expression is greatly reduced upon overexpression of another gene, rctB, previously identified as a putative activator of R. etli pSym conjugal transfer. Thus, rctB seems to counteract the repressive action of rctA. rctA homologs are present in at least three other bacterial genomes within the order Rhizobiales, where they are invariably located adjacent to and divergently transcribed from putative virB-like operons. We show that similar to that of R. etli pSym, conjugative transfer of the 1.35-Mb symbiotic megaplasmid A of Sinorhizobium meliloti is also subjected to the inhibitory action of rctA. Our data provide strong evidence that the R. etli and S. meliloti pSym plasmids are indeed self-conjugative plasmids and that this property would only be expressed under optimal, as yet unknown conditions that entail inactivation of the rctA function. The rctA gene seems to represent novel but probably widespread regulatory systems controlling the transfer of conjugative elements within the order Rhizobiales. PMID:16237017

An antimicrobial peptide essential for bacterial survival in the nitrogen-fixing symbiosis.

Science.gov (United States)

Kim, Minsoo; Chen, Yuhui; Xi, Jiejun; Waters, Christopher; Chen, Rujin; Wang, Dong

2015-12-08

In the nitrogen-fixing symbiosis between legume hosts and rhizobia, the bacteria are engulfed by a plant cell membrane to become intracellular organelles. In the model legume Medicago truncatula, internalization and differentiation of Sinorhizobium (also known as Ensifer) meliloti is a prerequisite for nitrogen fixation. The host mechanisms that ensure the long-term survival of differentiating intracellular bacteria (bacteroids) in this unusual association are unclear. The M. truncatula defective nitrogen fixation4 (dnf4) mutant is unable to form a productive symbiosis, even though late symbiotic marker genes are expressed in mutant nodules. We discovered that in the dnf4 mutant, bacteroids can apparently differentiate, but they fail to persist within host cells in the process. We found that the DNF4 gene encodes NCR211, a member of the family of nodule-specific cysteine-rich (NCR) peptides. The phenotype of dnf4 suggests that NCR211 acts to promote the intracellular survival of differentiating bacteroids. The greatest expression of DNF4 was observed in the nodule interzone II-III, where bacteroids undergo differentiation. A translational fusion of DNF4 with GFP localizes to the peribacteroid space, and synthetic NCR211 prevents free-living S. meliloti from forming colonies, in contrast to mock controls, suggesting that DNF4 may interact with bacteroids directly or indirectly for its function. Our findings indicate that a successful symbiosis requires host effectors that not only induce bacterial differentiation, but also that maintain intracellular bacteroids during the host-symbiont interaction. The discovery of NCR211 peptides that maintain bacterial survival inside host cells has important implications for improving legume crops.

Epitope Mapping of Monoclonal Antibody PMab-48 Against Dog Podoplanin.

Science.gov (United States)

Yamada, Shinji; Kaneko, Mika K; Itai, Shunsuke; Chang, Yao-Wen; Nakamura, Takuro; Yanaka, Miyuki; Ogasawara, Satoshi; Murata, Takeshi; Uchida, Hiroaki; Tahara, Hideaki; Harada, Hiroyuki; Kato, Yukinari

2018-04-02

Podoplanin (PDPN), a type I transmembrane sialoglycoprotein, is expressed on normal renal podocytes, pulmonary type I alveolar cells, and lymphatic endothelial cells. Increased expression of PDPN in cancers is associated with poor prognosis and hematogenous metastasis through interactions with C-type lectin-like receptor 2 (CLEC-2) on platelets. We previously reported a novel PMab-48 antibody, which is an anti-dog PDPN (dPDPN) monoclonal antibody (mAb) recognizing PDPN expressed in lymphatic endothelial cells. However, the binding epitope of PMab-48 is yet to be clarified. In this study, an enzyme-linked immunosorbent assay and flow cytometry were used to investigate epitopes of PMab-48. The results revealed that the critical epitope of PMab-48 comprises Asp29, Asp30, Ile31, Ile32, and Pro33 of dPDPN.

A phase I study of PRO131921, a novel anti-CD20 monoclonal antibody in patients with relapsed/refractory CD20+ indolent NHL: correlation between clinical responses and AUC pharmacokinetics.

Science.gov (United States)

Casulo, Carla; Vose, Julie M; Ho, William Y; Kahl, Brad; Brunvand, Mark; Goy, Andre; Kasamon, Yvette; Cheson, Bruce; Friedberg, Jonathan W

2014-09-01

PRO131921 is a third-generation, humanized anti-CD20 monoclonal antibody with increased antibody-dependent cytotoxicity and complement-dependent cytotoxicity compared to rituximab. In this phase I study, PRO131921 was administered as a single agent to patients with CD20+, relapsed or refractory, indolent non-Hodgkin lymphoma (NHL) who had been treated with a prior rituximab-containing regimen. The primary aim of this study was safety and tolerability of PRO131921. The secondary aim of the study, and focus of this report, was to determine the pharmacokinetics (PK) profile of PRO131921 and establish a correlation between drug exposure and clinical efficacy. Patients were treated with PRO131921 by intravenous infusion weekly for 4 weeks and the dose was escalated based on safety in a 3+3 design. Twenty-four patients were treated with PRO131921 at doses from 25mg/m(2) to 800 mg/m(2). Analysis of PK data demonstrated a correlation between higher normalized drug exposure (normalized AUC) and tumor shrinkage (p = .0035). Also, normalized AUC levels were higher among responders and subjects displaying tumor shrinkage versus subjects progressing or showing no regression (p = 0.030). In conclusion, PRO131921 demonstrated clinical activity in rituximab-relapsed and refractory indolent NHL patients. The observation that higher normalized AUC may be associated with improved clinical responses has potential implications in future trials of monoclonal antibody-based therapies, and emphasizes the importance of early PK studies to optimize antibody efficacy. Copyright © 2014 Elsevier Inc. All rights reserved.

uPAR as anti-cancer target

DEFF Research Database (Denmark)

Lund, Ida K; Illemann, Martin; Thurison, Tine

2011-01-01

, and a potential diagnostic and predictive impact of the different uPAR forms has been reported. Hence, pericellular proteolysis seems to be a suitable target for anti-cancer therapy and numerous approaches have been pursued. Targeting of this process may be achieved by preventing the binding of uPA to u...... using mouse monoclonal antibodies (mAbs) against mouse uPA or uPAR. These reagents will target uPA and uPAR in both stromal cells and cancer cells, and their therapeutic potential can now be assessed in syngenic mouse cancer models....

Optimization of IGF-1R SPECT/CT Imaging Using In-111-Labeled F(ab ')(2) and Fab Fragments of Article the Monoclonal Antibody R1507

NARCIS (Netherlands)

Heskamp, Sandra; van Laarhoven, Hanneke W. M.; Molkenboer-Kuenen, Janneke D. M.; Bouwman, Wilbert H.; van der Graaf, Winette T. A.; Oyen, Wim J. G.; Boerman, Otto C.

2012-01-01

The insulin-like growth factor 1 receptor (IGF-1R) is a potential new target for the treatment of breast cancer. Patients with breast cancer lesions that express IGF-1R may benefit from treatment with anti-IGF-IR antibodies. IGF-1R expression can be visualized using radiolabeled R1507, a monoclonal