Different Antibodies (different + antibody)

Distribution by Scientific Domains


Selected Abstracts


Vasoactive intestinal polypeptide immunoreactivity in the human cerebellum: qualitative and quantitative analyses

JOURNAL OF ANATOMY, Issue 3 2009
Vincenzo Benagiano
Abstract Although autoradiographic, reverse transcription-polymerase chain reaction and immunohistochemical studies have demonstrated receptors for vasoactive intestinal polypeptide (VIP) in the cerebellum of various species, immunohistochemistry has never shown immunoreactivity for VIP within cerebellar neuronal bodies and processes. The present study aimed to ascertain whether VIP immunoreactivity really does exist in the human cerebellum by making a systematic analysis of samples removed post-mortem from all of the cerebellar lobes. The study was carried out using light microscopy immunohistochemical techniques based on a set of four different antibodies (three polyclonal and one monoclonal) against VIP, carefully selected on the basis of control tests performed on human colon. All of the antibodies used showed VIP-immunoreactive neuronal bodies and processes distributed in the cerebellar cortex and subjacent white matter of all of the cerebellum lobes, having similar qualitative patterns of distribution. Immunoreactive neurons included subpopulations of the main neuron types of the cortex. Statistical analysis of the quantitative data on the VIP immunoreactivity revealed by the different antibodies in the different cerebellar lobes did not demonstrate any significant differences. In conclusion, using four different anti-VIP antibodies, the first evidence of VIP immunoreactivity is herein supplied in the human post-mortem cerebellum, with similar qualitative/quantitative patterns of distribution among the different cerebellum lobes. Owing to the function performed by VIP as a neurotransmitter/neuromodulator, it is a candidate for a role in intrinsic and extrinsic (projective) circuits of the cerebellum, in agreement with previous demonstrations of receptors for VIP in the cerebellar cortex and nuclei. As VIP signalling pathways are implicated in the regulation of cognitive and psychic functions, cerebral blood flow and metabolism, processes of histomorphogenesis, differentiation and outgrowth of nervous tissues, the results of this study could be applied to clinical neurology and psychiatry, opening new perspectives for the interpretation of neurodevelopment disorders and development of new therapeutic strategies in cerebellar diseases. [source]


Distribution of mucins and antimicrobial substances lysozyme and lactoferrin in the laryngeal subglottic region

JOURNAL OF ANATOMY, Issue 4 2008
Hannes Kutta
Abstract The subglottic region of the larynx is of high clinical relevance with regard to infections and malignancies. Little is known about the distribution of mucins and antimicrobial substances in this area. In this study, we have investigated the mucin distribution in the normal subglottis of the larynx. Moreover, we analysed the expression of lysozyme and lactoferrin in this area. Therefore, the subglottic region of 34 larynges was investigated immunohistochemically with different antibodies to mucins and antimicrobial substances. The epithelium reacted positive with antibodies to mucins MUC1 (34/34), 5AC (26/34), 5B (10/34), 7 (8/34), 8 (10/34) and 16 (19/34); submucosal glands were positive to mucins MUC1 (34/34), 5B (10/34), 7 (8/34), and 16 (19/34); high columnar epithelial cells and serous parts of subepithelial seromucous glands were also positive for lysozyme (34/34) and lactoferrin (34/34). The results show that human subglottic epithelium and subepithelial submucosal glands produce a broad spectrum of mucins that is almost comparable with that in other areas of the respiratory tract. We hypothesize that the mucin diversity of the subglottis has an impact on positive functional consequences during vocal production and antimicrobial defence. This antimicrobial defence is supported by synthesis and secretion of antimicrobial substances such as lysozyme and lactoferrin. Moreover, knowledge of the observed distribution pattern of mucins in the subglottis can be a useful tool for a classification of subglottic laryngeal carcinomas. [source]


Phosphorylation-dependent dimerization and subcellular localization of islet-brain 1/c-Jun N-terminal kinase-interacting protein 1

JOURNAL OF NEUROSCIENCE RESEARCH, Issue 16 2007
T. Borsello
Abstract Islet-brain 1 [IB1; also termed c-Jun N-terminal kinase (JNK)-interacting protein 1 (JIP-1] is involved in the apoptotic signaling cascade of JNK and functions as a scaffold protein. It organizes several MAP kinases and the microtubule-transport motor protein kinesin and relates to other signal-transducing molecules such as the amyloid precursor protein. Here we have identified IB1/JIP-1 using different antibodies that reacted with either a monomeric or a dimeric form of IB1/JIP-1. By immunoelectron microscopy, differences in the subcellular localization were observed. The monomeric form was found in the cytoplasmic compartment and is associated with the cytoskeleton and with membranes, whereas the dimeric form was found in addition in nuclei. After treatment of mouse brain homogenates with alkaline phosphatase, the dimeric form disappeared and the monomeric form decreased its molecular weight, suggesting that an IB1/JIP-1 dimerization is phosphorylation dependent and that IB1 exists in several phospho- forms. N-methyl-D-aspartate receptor activation induced a dephosphorylation of IB1/JIP-1 in primary cultures of cortical neurons and reduced homodimerization. In conclusion, these data suggest that IB1/JIP-1 monomers and dimers may differ in compartmental localization and thus function as a scaffold protein of the JNK signaling cascade in the cytoplasm or as a transcription factor in nuclei. © 2007 Wiley-Liss, Inc. [source]


LRRK2 is a component of granular alpha-synuclein pathology in the brainstem of Parkinson's disease

NEUROPATHOLOGY & APPLIED NEUROBIOLOGY, Issue 3 2008
J. Alegre-Abarrategui
Classical Parkinson's disease (PD) is characterized by the appearance of Lewy bodies (LBs) in affected brain regions, showing mostly compact alpha-synuclein deposition, in contrast with punctate or granular deposition, hypothesized to represent early stages of aggregation. Leucine-rich repeat kinase 2 (LRRK2) is the commonest mutated gene in inherited and idiopathic PD. LRRK2 mutation carriers display a diverse neuropathology, including alpha-synuclein and tau inclusions, suggesting an upstream role for LRRK2 in protein aggregation. We studied LRRK2 expression throughout the normal human brain with three different antibodies. We also examined the pattern of LRRK2 expression in relation to alpha-synuclein aggregation and LB formation in the brainstem of sporadic LB disease. Physiological LRRK2 expression was not restricted to regions preferentially affected in PD and LRRK2 often localized to the nuclear envelope in addition to the known cytoplasmic expression. In PD, we were able to consistently detect LRRK2 in the halo of a minority (approximately 10%) of nigral LBs using three different antibodies. Only one antibody detected LRRK2 in the core of approximately 80% of classic LBs. In the lower brainstem, most notably in the dorsal motor nucleus of the vagus, we found previously unrecognized LRRK2 labelling of complex globular lesions, filled with LB-like matter showing a punctate or granular staining for alpha-synuclein. This was often accompanied by strong LRRK2 expression within dystrophic neurites. Our findings confirm widespread physiological LRRK2 expression in the human brain and suggest an association of LRRK2 with possible early-stage alpha-synuclein pathology in the brainstem of PD. [source]


Production of antibodies in plants: status after twenty years

PLANT BIOTECHNOLOGY JOURNAL, Issue 5 2010
Benoit De Muynck
Summary Thanks to their potential to bind virtually all types of molecules; monoclonal antibodies are in increasing demand as therapeutics and diagnostics. To overcome the overloading of current production facilities, alternative expression systems have been developed, of which plants appear the most promising. In this review, we focus on the expression of monoclonal IgG or IgM in plant species. We analyse the data for 32 different antibodies expressed in various ways, differing in DNA construction, transformation method, signal peptide source, presence or absence of an endoplasmic reticulum retention sequence, host species and the organs tested, together resulting in 98 reported combinations. A large heterogeneity is found in the quantity and quality of the antibody produced. We discuss in more detail the strategy used to express both chains, the nature of the transcription promoters, subcellular localization and unintended proteolysis, when encountered. [source]


Development and optimization of a process for automated recovery of single cells identified by microengraving

BIOTECHNOLOGY PROGRESS, Issue 3 2010
Jae Hyeok Choi
Abstract Microfabricated devices are useful tools for manipulating and interrogating large numbers of single cells in a rapid and cost-effective manner, but connecting these systems to the existing platforms used in routine high-throughput screening of libraries of cells remains challenging. Methods to sort individual cells of interest from custom microscale devices to standardized culture dishes in an efficient and automated manner without affecting the viability of the cells are critical. Combining a commercially available instrument for colony picking (CellCelector, AVISO GmbH) and a customized software module, we have established an optimized process for the automated retrieval of individual antibody-producing cells, secreting desirable antibodies, from dense arrays of subnanoliter containers. The selection of cells for retrieval is guided by data obtained from a high-throughput, single-cell screening method called microengraving. Using this system, 100 clones from a mixed population of two cell lines secreting different antibodies (12CA5 and HYB099-01) were sorted with 100% accuracy (50 clones of each) in ,2 h, and the cells retained viability. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010 [source]


The effect of low intensity ultraviolet-C light on monoclonal antibodies

BIOTECHNOLOGY PROGRESS, Issue 2 2009
Christopher M. Lorenz
Abstract As part of an investigation to identify potential new viral reduction strategies, ultraviolet-C (UV-C) light was examined. Although this technology has been known for decades to possess excellent virus inactivation capabilities, UV-C light can also introduce significant unwanted damage to proteins. To study the effect on monoclonal antibodies, three different antibodies were subjected to varying levels of UV-C light using a novel dosing device from Bayer Technology Services GmbH. The range of fluencies (or doses) covered was between 0 and 300 J/m2 at a wavelength of 254 nm. Product quality data generated from the processed pools showed only minimal damage done to the antibodies. Aggregate formation was low for two of the three antibodies tested. Acidic and basic variants increased for all three antibodies, with the basic species increasing more than the acidic species. Peptide maps made for the three sets of pools showed no damage to two of the three antibody backbones, whereas the third antibody had very low levels of methionine oxidation evident. Samples held at 2,8°C for 33 days showed no increase in aggregates or charge variants, indicating that the proteins did not degrade and were not damaged further by reactive or catalytic species that may have been created on exposure to UV-C light. Overall, UV-C light was shown to induce very little damage to monoclonal antibodies at lower fluencies and appears to be a viable option for viral inactivation in biotechnology applications. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009 [source]


N-linked glycosylation is an important parameter for optimal selection of cell lines producing biopharmaceutical human IgG

BIOTECHNOLOGY PROGRESS, Issue 1 2009
Patrick H. C. van Berkel
Abstract We studied the variations in N-linked glycosylation of human IgG molecules derived from 105 different stable cell lines each expressing one of the six different antibodies. Antibody expression was based on glutamine synthetase selection technology in suspension growing CHO-K1SV cells. The glycans detected on the Fc fragment were mainly of the core-fucosylated complex type containing zero or one galactose and little to no sialic acid. The glycosylation was highly consistent for the same cell line when grown multiple times, indicating the robustness of the production and glycan analysis procedure. However, a twofold to threefold difference was observed in the level of galactosylation and/or non-core-fucosylation between the 105 different cell lines, suggesting clone-to-clone variation. These differences may change the Fc-mediated effector functions by such antibodies. Large variation was also observed in the oligomannose-5 glycan content, which, when present, may lead to undesired rapid clearance of the antibody in vivo. Statistically significant differences were noticed between the various glycan parameters for the six different antibodies, indicating that the variable domains and/or light chain isotype influence Fc glycosylation. The glycosylation altered when batch production in shaker was changed to fed-batch production in bioreactor, but was consistent again when the process was scaled from 400 to 5,000 L. Taken together, the observed clone-to-clone glycosylation variation but batch-to-batch consistency provides a rationale for selection of optimal production cell lines for large-scale manufacturing of biopharmaceutical human IgG. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009 [source]


Improved Fermentation Processes for NS0 Cell Lines Expressing Human Antibodies and Glutamine Synthetase

BIOTECHNOLOGY PROGRESS, Issue 1 2003
Jonathan Dempsey
To meet the increasing requirement for therapeutic antibodies to conduct clinical trials, an enhanced culture medium and fed-batch process was developed for GS-NS0 cell lines. This process was shown to produce high concentrations of monoclonal antibodies for several cell lines expressing different antibodies. Cells were adapted to growth in a glutamine- and serum-free medium containing bovine serum albumin (BSA), cholesterol, and transferrin. A number of amino acids were found to be depleted during cell culture. The concentrations of these amino acids were increased, and further cell culture analyses were performed. This process of cell growth and analysis was repeated over multiple cycles until no depletion was detected. This resulted in an amino acid supplement that was shown to be generic and enhanced antibody productivity up to 5-fold for the three cell lines tested. Transferrin was replaced using tropolone, a lipophilic iron chelator and ferric ammonium citrate. Cell growth was equivalent to that in transferrin-containing medium over the wide ranges tested. A concentrated feed solution, based on the amino acid supplement and the components of the serum-and protein-free supplements, was formulated. Addition of this feed in response to metabolic requirements resulted in a harvest titer a further 2-fold higher than the enhanced culture medium. Harvest antibody titers of up to 600 mg/L were achieved for three cell lines expressing different antibodies, representing an increase of 10-fold over the starting concentrations. [source]


Antikörper-Nachweis bei invasiver Aspergillose

MYCOSES, Issue 2004
R. Kappe
Aspergillus; aspergillosis; invasive infection; antibody detection Zusammenfassung Der Stellenwert von Aspergillus -Antikörpertesten zur Diagnostik der invasiven Aspergillose (IA) ist unklar. In zwei Studien wurden drei verschiedene Antikörperteste an Patienten mit gesicherter IA evaluiert: (i) kommerzieller Hämagglutinationstest (HAT), (ii) kommerzieller Enzymimmunoassay (EIA) IgG, IgM, IgA und (iii) ein experimenteller Mitogillin EIA IgG, IgM, IgA. In der ersten Studie wurden 99 Serumproben von 26 Patienten mit IA und 22 Serumproben von 22 Kontrollpatienten mit allen drei Tests untersucht. 10 von 26 Patienten (38%) waren in mindestens einem Antikörpertest positiv. Die höchste Sensitivität wies der IgG-Nachweis in beiden EIA-Tests auf (22 bzw. 21%), der HAT erreichte eine Sensitivität von 8%. IgM-Antikörper wurden nur bei zwei Patienten, IgA-Antikörper bei keinem Patienten nachgewiesen. Die Spezifität des HAT betrug 85%, des IgG-EIA 72%. Bei zwei Patienten mit gesicherter bzw. wahrscheinlicher IA war der Antikörpernachweis der einzig positive Labortest. In der zweiten Studie wurden retrospektiv die Ergebnisse von 60 Patienten mit gesicherter IA untersucht. 14 Patienten (23%) wiesen einen positiven Antikörpernachweis im EIA und/oder HAT auf. Die Untersuchung des zeitlichen Verlaufs der Antikörper zeigte, daß die IgG-Antikörperbildung bei immunsupprimierten Patienten durchschnittlich 10,8 Tage nach der Diagnosestellung der IA einsetzte. Schlußfolgerungen Aspergillus -Antikörper waren bei ca. 23% der Patienten mit invasiver Aspergillose nachweisbar. Die Antikörperbildung setzte bei erfolgreich therapierten immunsupprimierten Patienten durchschnittlich 10,8 Tage nach Beginn der Infektion ein. Insbesondere der IgG-Antikörpernachweis im EIA-Format kann hilfreich für die Diagnose-Sicherung und Verlaufskontrolle sein. Summary The clinical significance of Aspergillus antibody assays for the diagnosis of invasive aspergillosis (IA) is unclear. In two studies, three different antibody assays were evaluated with patients suffering from proven IA: (i) a commercial haemagglutination test (HAT), (ii) a commercial enzyme immunoassay (EIA) for IgG, IgM, and IgA, and (iii) an experimental mitogillin enzyme immunoassay for IgG, IgM, and IgA. In the first study, 99 serum samples from 26 patients with IA and 22 serum samples from 22 control patients were tested with all the three tests. Ten of the 26 patients (38%) reacted positively in at least one antibody assay. The highest sensitivity was generated by the detection of IgG using the EIA formats (22 and 21%, respectively), the HAT had a sensitivity of 8%. IgM type antibodies were detected in only two patients; no IgA type antibodies were detected. The specificities of the IgG EIA and the HAT were 72 and 85%, respectively. Antibody detection was the single positive laboratory test in two patients with proven and probable IA. In the second study, antibody test results of 60 patients with proven IA were retrospectively evaluated. Fourteen patients (23%) tested positive in the EIA and/or in the HAT. Investigations of the antibody levels in individual immunocompromised patients over time revealed that IgG production started after a mean of 10.8 days after diagnosis of IA. To conclude, antibodies against Aspergillus were detected in 23% of patients with IA. The antibody production started in successfully treated immunosuppressed patients after a mean of 10.8 days after the onset of infection. In particular, the detection of IgG-antibodies with an EIA can be useful for the confirmation of the diagnosis of IA and for the monitoring of the treatment of IA. [source]