Abstract
Serum antibodies from myriad species, particularly birds, can provide key information regarding the transmission and the expansion of the territory of emerging pathogens. Expedient antibody analysis is constrained by a lack of species-specific reagents, a deficiency potentially highlighted by the recent swine-origin influenza A virus (H1N1) outbreak. Available methodologies present difficulties that discourage thorough serologic monitoring of potential disease vectors or hosts. Rapid high-throughput procedures that combined serum amine labeling via biotinylation, contaminant removal, and microsphere-based immunoassays for antibodies to three arboviruses were developed. Agent-specific adaptations of this simple format should facilitate expanded surveillance and diagnostic capabilities regarding pathogens of human and veterinary importance.
Drug Metab Dispos. 38:84-91 (2010)
Impact of molecular processing in the hinge region of therapeutic IgG4 antibodies on disposition profiles in cynomolgus monkeys.
Stubenrauch K, Wessels U, Regula JT, Kettenberger H, Schleypen J, Kohnert U.
Departments of Bioanalytics, Pharma Research Penzberg, Roche Diagnostics, Nonnenwald 2, 82377 Penzberg, Germany. kay-gunnar.stubenrauch@roche.com
Abstract
The IgG4 isotype antibody is a potential candidate for immunotherapy when reduced effector functions are desirable. However, antigen binding fragment (Fab) arm exchange leads to functional monovalency with potentially reduced therapeutic efficacy. Mutagenesis studies suggested that the CH3 domain and not the core hinge is dominantly involved in in vivo molecular processing. This work investigated whether stabilization of the core hinge of a therapeutic IgG4 antibody by mutation of Ser228 to Pro (S228P) would be sufficient to prevent in vivo Fab arm exchange. In vitro experiments evaluated the influence of different levels of oxidation-reduction conditions in buffer and serum on Fab arm exchange (swapping) of wild-type (WT) IgG4 and IgG1 and of IgG4 S228P, which included a sterically neutral second mutation (Leu235 replaced by Glu). The objective of single-dose pharmacokinetic experiments in cynomolgus monkeys was to determine whether the mutation reduced IgG4 swapping in vivo. The results indicated that S228P mutation did not completely prevent Fab arm exchange in vitro in buffer under reducing conditions relative to IgG4 WT. The immunoassay findings were confirmed by mass spectrometry measurements. Results of the in vivo studies suggested that the therapeutic IgG4 WT antibody exchanged Fab arms with endogenous cynomolgus monkey IgG4, resulting in bispecific IgG4 antibodies with monovalency for the therapeutic target. In contrast, serum from cynomolgus monkeys dosed with the IgG4 mutant was virtually free of swapped IgG4. In conclusion, the results indicated that IgG4 swapping in vivo was markedly attenuated by S228P mutation.
Plant Cell 22:468-80 (2010)
A novel role for Arabidopsis mitochondrial ABC transporter ATM3 in molybdenum cofactor biosynthesis.
Teschner J, Lachmann N, Schulze J, Geisler M, Selbach K, Santamaria-Araujo J, Balk J, Mendel RR, Bittner F.
Institut für Pflanzenbiologie, Technische Universität Braunschweig, 38023 Braunschweig, Germany.
Abstract
The molybdenum cofactor (Moco) is a prosthetic group required by a number of enzymes, such as nitrate reductase, sulfite oxidase, xanthine dehydrogenase, and aldehyde oxidase. Its biosynthesis in eukaryotes can be divided into four steps, of which the last three are proposed to occur in the cytosol. Here, we report that the mitochondrial ABC transporter ATM3, previously implicated in the maturation of extramitochondrial iron-sulfur proteins, has a crucial role also in Moco biosynthesis. In ATM3 insertion mutants of Arabidopsis thaliana, the activities of nitrate reductase and sulfite oxidase were decreased to approximately 50%, whereas the activities of xanthine dehydrogenase and aldehyde oxidase, whose activities also depend on iron-sulfur clusters, were virtually undetectable. Moreover, atm3 mutants accumulated cyclic pyranopterin monophosphate, the first intermediate of Moco biosynthesis, but showed decreased amounts of Moco. Specific antibodies against the Moco biosynthesis proteins CNX2 and CNX3 showed that the first step of Moco biosynthesis is localized in the mitochondrial matrix. Together with the observation that cyclic pyranopterin monophosphate accumulated in purified mitochondria, particularly in atm3 mutants, our data suggest that mitochondria and the ABC transporter ATM3 have a novel role in the biosynthesis of Moco.
Placenta. 30:1078-82 (2009)
Inhibition of RET and JAK2 signals and upregultation of VEGFR3 phosphorylation in vitro by galectin-1 in trophoblast tumor cells BeW0
Fischer I, Schulze S, Kuhn C, Friese K, Walzel H, Markert UR, Jeschke U.
Ludwig Maximilians University of Munich, Department of Obstetrics and Gynecology, Munich, Germany.
Abstract
BACKGROUND: Galectin-1 (gal-1), a member of the mammalian beta-galactoside-binding proteins, binds to cell surface glycoproteins (Mucin-1) on trophoblast cells. Although it has been demonstrated that gal-1 induces cell differentiation processes in these cells, no information on its signal transduction processes is available so far. As tyrosine phosphorylation is a major mechanism that controls multiple biological processes including cell differentiation, survival and proliferation, the aim of this study was to examine which human receptor tyrosine kinases (RTKs) were phosphorylated in trophoblast cells by gal-1.
MATERIALS AND METHODS: BeWo choriocarcinoma cells were incubated for 24h in the absence (controls) and presence of 60microg/ml galectin-1. With the RayBio Human RTK Phosphorylation Antibody Array 1, the relative levels of phosphorylation of different human RTKs could be detected simultaneously. The signal intensities were compared and quantified with the Quantity One Version 4.5.2 program. Gal-1-treated and non-treated cells were incubated with antibodies against REarranged during Transfection (RET) and phosphorylated RET(Y905). Staining reaction was performed with the avidin-biotinylated peroxidase complex (ABC) reagent.
RESULTS: We demonstrated that gal-1 inhibited RET and Janus Kinase 2 (JAK2) signals and upregulated Vascular endothelial growth factor receptor 3 (VEGFR3) signal in BeWo cells. We also showed the downregulation of phosphorylation on RET phosphotyrosine residue 905 in BeWo cells with phosphorylation specific antibodies and immunocytochemistry.
CONCLUSION: Out of a number of 71 different RTKs, the stimulation of BeWo cells with gal-1 showed a significant alteration of signal intensity in only 3 RTKs: JAK2, RET and VEGFR3. Our data suggest that phosphorylation of these RTKs could be involved in cell differentiation processes that could be responsible for the already known effect of gal-1 on BeWo cells, the inhibition of proliferation.
Sensors and Actuators B: Chemical 139: 2-8 (2009)
Protein microarray for the analysis of human melanoma biomarkers
Austrian Research Centers GmbH–ARC, Department of Bioresources, 2444 Seibersdorf, Austria
Abstract
The clinical course of malignant melanoma is notoriously variable. Current staging criteria allow assignment to risk categories but do not permit accurate assessment of prognosis. The simultaneous detection of biomarkers reflecting disease stage and likelihood of progression is therefore urgently needed for treatment decisions.
Herein we describe the development of a protein microarray platform for the parallel analysis of five melanoma serum biomarker proteins: S100B, VEGF-A, CRP, IL-6 and IL-10. The platform utilizes ARChip Epoxy as a solid support for the covalent immobilization of antibodies using contact printing. Tests are based on sandwich immunoassays with streptavidin–biotin chemistry and fluorescence detection. Using CRP as a model marker optimization of the system in respect to printing buffer, antigen probe concentration (1 mg/mL), assay binding buffer and incubation time (120 min) is outlined. High reproducibility (CV < 20%), weak cross-reactivity (<0.5% for IL-10) and low limit of detection (1.88 × 10−4 mg/L) were achieved. Fit of a four-parameter logistic quantification model to standard curves of the five on-chip assays shows excellent coefficients of determination (R2 ≥ 0.966). Achieved sensitivities are perfectly comparable to classical ELISA kits and obtained working ranges allow discrimination between normal and literature-reported elevated serum levels of melanoma markers.
Sensors, 2008 IEEE Oct. (2008)
An optical platform based on fluorescence anisotropy for C — reactive protein assay
Baldini, F. Carloni, A. Giannetti, A. Trono, C. Porro, G. Tedeschi, L.
Inst. of Appl. Phys., CNR, Firenze
Abstract
A novel optical platform for point of care testing application was developed. The core of the platform is a miniaturised polymethylmetacrylate (PMMA) chip through which the analysed sample flows. Thanks to the fluorescence anisotropy exhibited by any dipole emitting at a distance from a medium interface of the order of the emitted wavelength, a large fraction of the fluorescence emitted by the sensing layer immobilized onto the internal flow-channel travels along the thickness of the PMMA up to its end-face where it is collected by a plastic optical fibre connected to a spectrum analyser. The potentiality of the optical chip was investigated with a sandwich assay for the C-reactive protein, one of the markers for inflammatory diseases.
Anal Bioanal Chem. 392:727-36 (2008)
Development and characterization of new rat monoclonal antibodies for procalcitonin.
Krämer PM, Gouzy MF, Kess M, Kleinschmidt U, Kremmer E.
Institute of Ecological Chemistry, Helmholtz Center Munich, German Research Center for Environmental Health (GmbH), Ingolstädter Landstrasse 1, 85764, Neuherberg, Germany. kramer@helmholtz-muenchen.de
Abstract
The development of selective and sensitive biological recognition elements, e.g., antibodies, for the detection of relevant blood markers is a great challenge in the field of biosensors. In this context, five new rat monoclonal antibodies (mAbs) for procalcitonin (PCT), a marker for bacterial infection and sepsis, were developed and characterized. One mAb, PROC1 3G3, was used as capture antibody. Four mAbs, PROC4 6C6, PROC4 6B2, PROC4 1G3, and PROC4 1D6, were used as detection mAbs, either as Protein G-purified or as biotinylated mAbs. A surface plasmon resonance (SPR) biosensor was used to characterize the antigen-antibody biomolecular interactions. The capture mAb (PROC1 3G3) has an equilibrium dissociation constant (K (D)) of 3.42 x 10(-8) M. All four detection mAbs (PROC4 6C6, PROC4 6B2, PROC4 1G3, and PROC4 1D6) are of high affinity (K (A) = 2.81-6.11 x 10(8) M(-1); K (D) = 1.64-3.56 x 10(-9) M) and have moderate dissociation rate constants (k (d) = 1.70-2.40 x 10(-3) s(-1)). Four different sandwich enzyme-linked immunosorbent assays (ELISAs) with standards of human recombinant (hr) PCT, using PROC1 3G3 as capture mAb and PROC4 mAbs as detection mAbs, respectively, led to highly specific determinations of PCT without cross-reactivities to calcitonin and katacalcin. The lower limits of quantification (LLOQ) for hrPCT (in 40 mM phosphate-buffered saline (PBS), pH 7.6) with these assays ranged from 2.3 to 12.8 microg L(-1). In addition, sandwich ELISAs were set up with biotinylated PROC4 mAbs, and with hrPCT in 4% human serum albumin (diluted 1:10 in 40 mM PBS, including 1:5 (v/v) LowCross Buffer(R)). The LLOQs of these sandwich assays ranged from 4.1 to 6.0 microg L(-1) and were thus much closer together for the different assays. With the latter assay setup (PROC1 3G3 as capture mAb, PROC4 6C6-biotin as detection mAb) a first collection of five serum samples was determined (healthy volunteers, unspiked, and spiked). Recovery rates for the spiked samples ranged from 98.3 to 115.7%. The newly developed anti-PCT mAbs should find broad applications in immunosensors for point-of-care diagnostics of sepsis and systemic inflammation processes.
FASEB J. 22:1021-31 (2007)
Up-regulation of nestin in the infarcted myocardium potentially indicates differentiation of resident cardiac stem cells into various lineages including cardiomyocytes.
Scobioala S, Klocke R, Kuhlmann M, Tian W, Hasib L, Milting H, Koenig S, Stelljes M, El-Banayosy A, Tenderich G, Michel G, Breithardt G, Nikol S.
Department of Cardiology and Angiology, University Hospital of Muenster, Muenster, Germany.
Abstract
To identify proteins involved in cardiac regeneration, a proteomics approach was applied. A total of 26 proteins, which displayed aberrant expression in mouse hearts infarcted through ligation of the left anterior descending coronary artery, were identified. These included the intermediate filament protein nestin, which was up-regulated in the infarct border zone. Corresponding changes were observed for its mRNA. Nestin mRNA was also up-regulated in hearts from 17 of 19 patients with end-stage heart failure, including 4 with acute myocardial infarction in comparison with 8 donor hearts. Immunofluorescence confocal laser scanning microscopy revealed that nestin is expressed, on the one hand, in small proportions of cardiomyocytes, endothelial cells, smooth muscle cells, neuronal cells, and fibroblasts. On the other hand, it was found to be coexpressed with the stem cell markers c-kit, Sca-1, Mdr-1, and Abcg2 in small interstitial cells. In infarcted hearts from chimeric mice transplanted with bone marrow from enhanced green fluorescent protein (EGFP) transgenic mice, less than 1% of nestin-positive cells coexpressed EGFP, although EGFP-positive cells were abundant in these. Consequently, enhanced expression of nestin in the injured myocardium might reflect spontaneous regenerative processes supposedly based on the differentiation of resident cardiac stem cells into diverse cardiac cell types.
Neoplasia. 9:745-54 (2007)
Increases in c-Yes expression level and activity promote motility but not proliferation of human colorectal carcinoma cells.
Barraclough J, Hodgkinson C, Hogg A, Dive C, Welman A.
Cancer Research UK, Paterson Institute for Cancer Research, University of Manchester, Wilmslow Road, Manchester M20 4BX, UK.
Abstract
Increases in the levels and/or activity of nonreceptor tyrosine kinases c-Src and c-Yes are often associated with colorectal carcinogenesis. The physiological consequences of increased c-Yes activity during the early and late stages of tumorigenesis, in addition to the degree of redundancy between c-Yes and c-Src in colorectal cancer cells, remain elusive. To study the consequences of increases in c-Yes levels and activity in later stages of colorectal carcinogenesis, we developed human colorectal cancer cell lines in which c-Yes levels and activity can be inducibly increased by a tightly controlled expression of wild-type c-Yes or by constitutively active mutants of c-Yes, c-YesY537F, and c-Yes Delta t6aa. c-Yes induction resulted in increased cell motility but did not promote proliferation either in vitro or in vivo. These results suggest that in later stages of colorectal carcinogenesis, elevations in c-Yes levels/activity may promote cancer spread and metastasis rather than tumor growth.
