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Antibody Complexes (antibody + complex)
Selected AbstractsCapillary electrophoresis of affinity complexes between subviral 80S particles of human rhinovirus and monoclonal antibody 2G2ELECTROPHORESIS, Issue 13 2006Leopold Kremser Dr. Abstract Human rhinoviruses (HRVs), the main etiologic agents of the common cold, transform into subviral B- or 80S particles (they sediment at 80S upon sucrose density gradient centrifugation) during infection and, in,vitro, upon exposure to a temperature between 50 and 56°C. With respect to the native virion they lack the genomic RNA and the viral capsid protein VP4. 80S particles are unstable and easily disintegrate into their components, VP1, VP2, and VP3 in buffers containing SDS. However, this detergent was found to be a necessary constituent of the BGE for the analysis of these viruses and their complexes with receptors and antibodies by CE. We here demonstrate that dodecylpoly(ethyleneglycol ether) (D-PEG) a nonionic detergent, is suitable for analysis of subviral particles as it preserves their integrity, in contrast to SDS. Electrophoresis of the 80S particles in borate buffer (pH,8.3, 100,mM) containing 10,mM D-PEG resulted in a well-defined electrophoretic peak. The identity of the peak was confirmed, among other means, by complexation with mAb,2G2, which recognizes a structural epitope exclusively present on subviral particles but not on native virus. Upon incubation of the 80S particles with mAb,2G2 the peak disappeared, but a new peak, attributed to the antibody complex emerged. The separation system allowed following the time course of the transformation of intact HRV serotype,2 into 80S particles upon incubation at temperatures between 40 and 65°C. We also demonstrate that subviral particles derived from HRV2 labeled with the fluorescence dyes FITC or Cy3.5 were stable in the separation system containing D-PEG. Dye-modified particles were still recognized by mAb,2G2, suggesting that the exposed lysines that are derivatized by the reagent do not form part of the epitope of the antibody. [source] Aggresome formation by anti-Ras intracellular scFv fragmentsFEBS JOURNAL, Issue 2 2001The fate of the antigen, antibody complex Diverting the antigen from its normal intracellular location to other compartments in an antibody-mediated way represents a mode of action for intracellular antibodies [Cardinale, A., Lener, M., Messina, S., Cattaneo, A. & Biocca, S. (1998) FEBS Lett.,439, 197,202; Lener, M., Horn, I.R., Cardinale, A., Messina, S., Nielsen, U.B., Rybak, S.M., Hoogenboom, H.R., Cattaneo, A. & Biocca, S. (2000) Eur J Biochem.267, 1196,205]. In the case of p21Ras, the sequestration of the antigen in aggregated structures in the cytoplasm of transfected cells leads to the inhibition of its biological function. We have further investigated the intracellular fate of the antigen,antibody complex by analyzing the effect of proteasome inhibitors on the formation and the intracellular localization of the aggregates. Overexpression of anti-Ras scFv fragments or inhibition of proteasomes activity leads to the formation of large perinuclear aggresomes formed of ubiquitinated-scFv fragments in which p21Ras is sequestered and degraded in an antibody-mediated way. Disruption of microtubules by nocodazole completely abrogates the accumulation of scFv fragments in a single aggresome and induces the dispersion of these structures in the periphery of the cell. Cotransfection of the GFP-scFv with a myc-tagged ubiquitin and colocalization with specific anti-proteasome antibodies indicate the recruitment of exogenous ubiquitin and proteasomes to the newly formed aggresomes. Taken together these results suggest that the intracellular antigen,antibody complex is naturally addressed to the ubiquitin,proteasome pathway and that the mechanism of ubiquitination does not inhibit the antibody binding properties and the capacity to block the antigen function. [source] Role of anti-,-glucan antibody in host defense against fungiFEMS IMMUNOLOGY & MEDICAL MICROBIOLOGY, Issue 1 2005Ken-ichi Ishibashi Abstract We have recently detected an anti-,-glucan antibody in normal human and normal mouse sera. The anti-,-glucan antibody showed reactivity to pathogenic fungal Aspergillus and Candida cell wall glucan. Anti-,-glucan antibody could bind whole Candida cells. It also enhanced the candidacidal activity of macrophages in vitro. The anti-,-glucan antibody titer of DBA/2 mice intravenously administered either Candida or Aspergillus solubilized cell wall ,-glucan decreased remarkably dependent on dose. Moreover, in deep mycosis patients, the anti-,-glucan antibody titer decreased, and this change correlated with clinical symptoms and other parameters such as C-reactive protein. It was suggested that the anti-,-glucan antibody formed an antigen,antibody complex and participated in the immune response as a molecule recognizing pathogenic fungi. [source] Analysis of several fluorescent detector molecules for protein microarray useLUMINESCENCE: THE JOURNAL OF BIOLOGICAL AND CHEMICAL LUMINESCENCE, Issue 1 2003Rick Wiese Abstract The utility of several streptavidin-linked fluorescent detector molecules was evaluated on two protein microarray platforms. Tested detector molecules included: Alexa Fluor 546; R-phycoerythrin (RPE), orange fluospheres; Cy3-containing liposomes (Large Unilamellar Vesicles, LUV) labelled with Cy3; and an RPE,antibody complex. The two array architectures tested consisted of an array of murine Fc,biotin and an array of murine IgG (the murine IgG array was probed with a biotinylated rabbit anti-murine IgG). These platforms allowed for the direct comparison of detector utility by detector recognition of array-bound biotin. All of the fluorescent detectors examined demonstrated utility on each of the array platforms. For the Fc,biotin array, detector signal intensity (background adjusted) was as follows: RPE,antibody complex,>,fluospheres,>,RPE,>,liposomes,>,Alexa 546: for the IgG array: RPE/antibody complex,>,RPE,>,fluospheres,>,Alexa546,>,liposomes. The RPE,antibody complex fluoresced 67% and 150% more intensely than the next closest detector molecule for the Fc,biotin and the murine IgG arrays, respectively. A marked increase in background fluorescence (as compared to RPE alone) did not accompany the increase in signal intensity gained through RPE,antibody complex use (a true increase in signal:noise ratio). These results suggest that the RPE,antibody complex is superior to other molecules for fluorescent detection of analytes on protein microarrays. Copyright © 2002 John Wiley & Sons, Ltd. [source] Coordination chemistry of iron(III),porphyrin,antibody complexesFEBS JOURNAL, Issue 2 2002Influence on the peroxidase activity of the axial coordination of an imidazole on the iron atom An artificial peroxidase-like hemoprotein has been obtained by associating a monoclonal antibody, 13G10, and its iron(III),,,,,,,,- meso -tetrakis(ortho -carboxyphenyl)porphyrin [Fe(ToCPP)] hapten. In this antibody, about two-thirds of the porphyrin moiety is inserted in the binding site, its ortho -COOH substituents being recognized by amino-acids of the protein, and a carboxylic acid side chain of the protein acts as a general acid base catalyst in the heterolytic cleavage of the O,O bond of H2O2, but no amino-acid residue is acting as an axial ligand of the iron. We here show that the iron of 13G10,Fe(ToCPP) is able to bind, like that of free Fe(ToCPP), two small ligands such as CN,, but only one imidazole ligand, in contrast to to the iron(III) of,Fe(ToCPP) that binds two. This phenomenon is general for a series of monosubstituted imidazoles, the 2- and 4-alkyl-substituted imidazoles being the best ligands, in agreement with the hydrophobic character of the antibody binding site. Complexes of antibody 13G10 with less hindered iron(III),tetraarylporphyrins bearing only one [Fe(MoCPP)] or two meso-[ortho -carboxyphenyl] substituents [Fe(DoCPP)] also bind only one imidazole. Finally, peroxidase activity studies show that imidazole inhibits the peroxidase activity of 13G10,Fe(ToCPP) whereas it increases that of 13G10,Fe(DoCPP). This could be interpreted by the binding of the imidazole ligand on the iron atom which probably occurs in the case of 13G10,Fe(ToCPP) on the less hindered face of the porphyrin, close to the catalytic COOH residue, whereas in the case of 13G10,Fe(DoCPP) it can occur on the other face of the porphyrin. The 13G10,Fe(DoCPP),imidazole complex thus constitutes a nice artificial peroxidase-like hemoprotein, with the axial imidazole ligand of the iron mimicking the proximal histidine of peroxidases and a COOH side chain of the antibody acting as a general acid-base catalyst like the distal histidine of peroxidases does. [source] An in silico method using an epitope motif database for predicting the location of antigenic determinants on proteins in a structural contextJOURNAL OF MOLECULAR RECOGNITION, Issue 1 2006Vincent Batori Abstract Presently X-ray crystallography of protein,antibody complexes is still the most direct way of identifying B-cell epitopes. The objective of this study was to assess the potential of a computer-based epitope mapping tool (EMT) using antigenic amino acid motifs as a fast alternative in a number of applications not requiring detailed information, e.g. development of pharmaceutical proteins, vaccines and industrial enzymes. Using Gal d 4 as a model protein, the EMT was capable of identifying, in the context of the folded protein, amino acid positions known to be involved in antibody binding. The high sensitivity and positive predictive value of the EMT as well as the relevance of the structural associations suggested by the EMT indicated the existence of amino acid motifs that are likely to be involved in antigenic determinants. In addition, differential mapping revealed that sensitivity and positive predictive value were dependent on the minimum relative surface accessibility (RSA) of the amino acids included in the mapping, demonstrating that the EMTs accommodated for the fact that epitopes are three-dimensional entities with various degrees of accessibility. The comparison with existing prediction scales demonstrated the superiority of the EMT with respect to physico-chemical scales. The mapping tool also performed better than the available structural scales, but the significance of the differences remains to be established. Thus, the EMT has the potential of becoming a fast and simple alternative to X-ray crystallography for predicting structural antigenic determinants, if detailed epitope information is not required. Copyright © 2005 John Wiley & Sons, Ltd. [source] A synergistic approach to protein crystallization: Combination of a fixed-arm carrier with surface entropy reductionPROTEIN SCIENCE, Issue 5 2010Andrea F. Moon Abstract Protein crystallographers are often confronted with recalcitrant proteins not readily crystallizable, or which crystallize in problematic forms. A variety of techniques have been used to surmount such obstacles: crystallization using carrier proteins or antibody complexes, chemical modification, surface entropy reduction, proteolytic digestion, and additive screening. Here we present a synergistic approach for successful crystallization of proteins that do not form diffraction quality crystals using conventional methods. This approach combines favorable aspects of carrier-driven crystallization with surface entropy reduction. We have generated a series of maltose binding protein (MBP) fusion constructs containing different surface mutations designed to reduce surface entropy and encourage crystal lattice formation. The MBP advantageously increases protein expression and solubility, and provides a streamlined purification protocol. Using this technique, we have successfully solved the structures of three unrelated proteins that were previously unattainable. This crystallization technique represents a valuable rescue strategy for protein structure solution when conventional methods fail. [source] Macromolecular recognition in the Protein Data BankACTA CRYSTALLOGRAPHICA SECTION D, Issue 1 2007Joël Janin Crystal structures deposited in the Protein Data Bank illustrate the diversity of biological macromolecular recognition: transient interactions in protein,protein and protein,DNA complexes and permanent assemblies in homodimeric proteins. The geometric and physical chemical properties of the macromolecular interfaces that may govern the stability and specificity of recognition are explored in complexes and homodimers compared with crystal-packing interactions. It is found that crystal-packing interfaces are usually much smaller; they bury fewer atoms and are less tightly packed than in specific assemblies. Standard-size interfaces burying 1200,2000,Å2 of protein surface occur in protease,inhibitor and antigen,antibody complexes that assemble with little or no conformation changes. Short-lived electron-transfer complexes have small interfaces; the larger size of the interfaces observed in complexes involved in signal transduction and homodimers correlates with the presence of conformation changes, often implicated in biological function. Results of the CAPRI (critical assessment of predicted interactions) blind prediction experiment show that docking algorithms efficiently and accurately predict the mode of assembly of proteins that do not change conformation when they associate. They perform less well in the presence of large conformation changes and the experiment stimulates the development of novel procedures that can handle such changes. [source] Effects of 100 GHz radiation on alkaline phosphatase activity and antigen,antibody interaction,BIOELECTROMAGNETICS, Issue 3 2009A. Homenko Abstract Equipment that generates microwave radiation (MWR) spanning the frequency range of 300 MHz,100 GHz is becoming more common. While MWR lacks sufficient energy to break chemical bonds, the disagreement as to whether MWR exposure is detrimental to cellular dysfunction may be difficult to clarify using complex systems such as whole animals, cells, or cell extracts. Recently, the high frequency range of terahertz (THz) radiation has been explored and sources of radiation and its detectors have been developed. THz radiation is associated with the frequency interval from 100 GHz to 20 THz and constitutes the next frontier in imaging science and technology. In the present study, we investigated the effect of radiation in the low frequency THz range (100 GHz) on two defined molecular interactions. First, the interaction of soluble or immobilized calf alkaline phosphatase with the substrate p -nitrophenylphosphate and second, the interaction between an antibody (mouse monoclonal anti-DNP) and its antigen (DNP). Irradiation of enzyme either prior to addition of substrate or during the enzymatic reaction resulted in small but significant reductions in enzyme activity. These differences were not observed if the enzyme had previously been immobilized onto plastic microwells. Exposure of immobilized antigen to radiation did not influence the ability of the antigen to interact with antibody. However, irradiation appeared to decrease the stability of previously formed antigen,antibody complexes. Our data suggest that 100 GHz radiation can induce small but statistically significant alterations in the characteristics of these two types of biomolecular interactions. Bioelectromagnetics 30:167,175, 2009. © 2008 Wiley-Liss, Inc. [source] | |||||||||||||