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Antibody Molecules (antibody + molecule)
Selected AbstractsGold Nanoparticle-Hybridized "Nano-Sponge" Polymer Coatings to Enhance the Reliability and Sensitivity of BiosensorsMACROMOLECULAR RAPID COMMUNICATIONS, Issue 13 2009Hyung-Jun Jeong Abstract We have created a new functional biosensor coating composed of polyelectrolyte multilayers containing gold nanoparticles. This gold-hybridized polyelectrolyte multilayer film possesses a stable nanoporous structure under physiological conditions. Antibody molecules were successfully conjugated onto the gold nanoparticles within the film. This functional coating successfully extinguished false signals from non-specific binding of proteins and cells and also provided highly enhanced detection sensitivity. Furthermore, the drastic differences in protein and cellular adhesion properties between a chip coated with the nanoporous PEM film and a bare chip demonstrate that morphological control of biological interactions on chip surfaces is possible. [source] Analysis of the composition of immunoconjugates using size-exclusion chromatography coupled to mass spectrometryRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 13 2005Alexandru C. Lazar Recombinant monoclonal antibody drug products play an increasingly important role in the treatment of various diseases. Antibodies are large, multi-chain proteins and antibody preparations often contain several molecular variants, which renders them heterogeneous. The heterogeneity is further increased in immunoconjugates prepared by covalently linking several drug molecules per antibody molecule. As part of the product characterization, the molecular weights of the antibodies or their drug conjugates need to be measured. Electrospray ionization mass spectrometry (ESI-MS) is well suited for the analysis of recombinant antibodies and immunoconjugates. Sample preparation is an important element of ESI-MS analysis, in particular samples need to be freed of interfering charged species, such as salts and buffer components. In this paper, Amicon centrifugal filters, reversed-phase high-performance liquid chromatography (HPLC), and size-exclusion HPLC were evaluated for sample desalting. Size-exclusion HPLC, using aqueous acetonitrile as the mobile phase, directly coupled to ESI-MS provided the best performance and was optimized for the study of immunoconjugates. The results showed that antibodies carrying covalently linked maytansinoid molecules generated charge envelope profiles that differ from those of the non-conjugated antibody. For the determination of the distribution of the various conjugate species in an immunoconjugate sample prepared by randomly linking in the average 3.6 drug molecules per antibody molecule, the experimental conditions needed to be carefully selected to allow acquisition of the whole spectrum containing the charge envelopes of all species. Copyright © 2005 John Wiley & Sons, Ltd. [source] Expression of antibodies using single-open reading frame vector design and polyprotein processing from mammalian cellsBIOTECHNOLOGY PROGRESS, Issue 3 2009Yune Z. Kunes Abstract We describe a novel polyprotein precursor-based approach to express antibodies from mammalian cells. Rather than expressing heavy and light chain proteins from separate expression units, the antibody heavy and light chains are contained in one single-open reading frame (sORF) separated by an intein gene fused in frame. Inside mammalian cells this ORF is transcribed into a single mRNA, and translated into one polypeptide. The antibody heavy and light chains are separated posttranslationally, assembled into the functional antibody molecule, and secreted into culture medium. It is demonstrated that Pol I intein from P. horikoshii mediates protein splicing and cleavage reactions in mammalian cells, in the context of antibody heavy and light chain amino acid sequences. To allow the separation of antibody heavy chain, light chain, and the intein, we investigated a number of intein mutations designed to inhibit intein-mediated splicing but preserve cleavage reactions. We have also designed constructs in which the signal peptide downstream from intein has altered hydrophobicity. The use of some of these mutant constructs resulted in more efficient antibody secretion, highlighting areas that can be further explored in improving such an expression system. An antibody secreted using one of the sORF constructs was characterized. This antibody has correct N-terminal sequences for both of its heavy and light chains, correct heavy and light chain MW as well as intact MW as measured by mass spectrometry. Its affinity to antigen, as measured by surface plasmon resonance (SPR), is indistinguishable from that of the same antibody produced using conventional method. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009 [source] Immunosensors: (Ionic-Liquid-Doped Polyaniline Inverse Opals: Preparation, Characterization, and Application for the Electrochemical Impedance Immunoassay of Hepatitis B Surface Antigen) Adv.ADVANCED FUNCTIONAL MATERIALS, Issue 19 2009Funct. Xing-Hua Li et al. describe the preparation of ionic liquid-doped polyaniline (IL-PANI) inverse opaline film with surface assemblies of gold nanoparticles. The resulting AuNP/IL-PANI film is conjugated with Hepatitis B surface antibody molecules to fabricate a immunosensor with a low detection limit for Hepatitis B surface antigen. [source] Rhesus macaque antibody molecules: sequences and heterogeneity of alpha and gamma constant regionsIMMUNOLOGY, Issue 1 2004Franco Scinicariello Summary Rhesus macaques (Macaca mulatta) are extensively used in vaccine development. Macaques infected with simian immunodeficiency viruses (SIV) or simian-human immunodeficiency viruses (SHIV) are the best animal model currently available for acquired-immune-deficiency-syndrome-related studies. Recent results emphasize the importance of antibody responses in controlling HIV and SIV infection. Despite the increasing attention placed on humoral immunity in these models, very limited information is available on rhesus macaque antibody molecules. Therefore, we sequenced, cloned and characterized immunoglobulin gamma (IGHG) and alpha (IGHA) chain constant region genes from rhesus macaques of Indian and Chinese origin. Although it is currently thought that rhesus macaques express three IgG subclasses, we identified four IGHG genes, which were designated IGHG1, IGHG2, IGHG3 and IGHG4 on the basis of sequence similarities with the four human genes encoding the IgG1, IgG2, IgG3 and IgG4 subclasses. The four genes were expressed at least at the messenger RNA level, as demonstrated by real-time reverse transcription polymerase chain reaction (RT-PCR). The level of intraspecies heterogeneity was very high for IGHA genes, whereas IGHG genes were remarkably similar in all animals examined. However, single amino acid substitutions were present in IGHG2 and IGHG4 genes, indicating the presence of IgG polymorphism possibly resulting in the expression of different allotypes. Two IgA alleles were identified in several animals and RT-PCR showed that both alleles may be expressed. Presence of immunoglobulin gene polymorphism appears to reflect the unusually high levels of intraspecies heterogeneity already demonstrated for major histocompatibility complex genes in this non-human primate species. [source] Solid-phase biotinylation of antibodies,JOURNAL OF MOLECULAR RECOGNITION, Issue 3 2004Elizabeth Strachan Abstract Biotinylation is an established method of labeling antibody molecules for several applications in life science research. Antibody functional groups such as amines, cis hydroxyls in carbohydrates or sulfhydryls may be modified with a variety of biotinylation reagents. Solution-based biotinylation is accomplished by incubating antibody in an appropriate buffered solution with biotinylation reagent. Unreacted biotinylation reagent must be removed via dialysis, diafiltration or desalting. Disadvantages of the solution-based approach include dilution and loss of antibody during post-reaction purification steps, and difficulty in biotinylation and recovery of small amounts of antibody. Solid-phase antibody biotinylation exploits the affinity of mammalian IgG-class antibodies for nickel IMAC (immobilized metal affinity chromatography) supports. In this method, antibody is immobilized on a nickel-chelated chromatography support and derivitized on-column. Excess reagents are easily washed away following reaction, and biotinylated IgG molecule is recovered under mild elution conditions. Successful solid phase labeling of antibodies through both amine and sulfhydryl groups is reported, in both column and mini-spin column formats. Human or goat IgG was bound to a Ni-IDA support. For sulfhydryl labeling, native disulfide bonds were reduced with TCEP, and reduced IgG was biotinylated with maleimide,PEO2 biotin. For amine labeling, immobilized human IgG was incubated with a solution of NHS,PEO4 biotin. Biotinylated IgG was eluted from the columns using a buffered 0.2,M imidazole solution and characterized by ELISA, HABA/avidin assay, probing with a streptavidin,alkaline phosphatase conjugate, and binding to a monomeric avidin column. The solid phase protocol for sulfhydryl labeling is significantly shorter than the corresponding solution phase method. Biotinylation in solid phase is convenient, efficient and easily applicable to small amounts of antibody (e.g. 100,,g). Antibody biotinylated on-column was found to be equivalent in stability and antigen-recognition ability to antibody biotinylated in solution. Solid-phase methods utilizing Ni-IDA resin have potential for labeling nucleic acids, histidine-rich proteins and recombinant proteins containing polyhistidine purification tags, and may also be applicable for other affinity systems and labels. Copyright © 2004 John Wiley & Sons, Ltd. [source] Ubiquitous cancer genes: Multipurpose molecules for protein micro-arraysPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 1 2006Brigitte Altenberg Abstract Multipurpose genes in the human genome which are over-expressed in a large variety of different cancers have been identified. Forty-two of the 19,016,human genes annotated to date (0.2%) are ubiquitously over-expressed in half or more of the 36,investigated human cancers. Of these genes, 15,are involved in protein biosynthesis and folding, six of them in glycolysis. A group of 13,solid tumours over-express almost all (39,42 of 42) ubiquitous cancer genes, suggesting a common mechanism underlying these cancers. Others, such as endocrine cancers, have only a few over-expressed ubiquitous cancer genes. The proteins for which these genes code or the corresponding antibodies are candidates for small protein microarrays aiming at maximum information with only a limited number of proteins. Since the over-expression pattern varies from cancer to cancer, distinction between different cancer classes is possible using one single set of protein or antibody molecules. [source] Rapid whole monoclonal antibody analysis by mass spectrometry: An Ultra scale-down study of the effect of harvesting by centrifugation on the post-translational modification profile,BIOTECHNOLOGY & BIOENGINEERING, Issue 1 2010C.Q. Reid Abstract With the trend towards the generation and production of increasing numbers of complex biopharmaceutical (protein based) products, there is an increased need and requirement to characterize both the product and production process in terms of robustness and reproducibility. This is of particular importance for products from mammalian cell culture which have large molecular structures and more often than not complex post-translational modifications (PTMs) that can impact the efficacy, stability and ultimately the safety of the final product. It is therefore vital to understand how the operating conditions of a bioprocess affect the distribution and make up of these PTMs to ensure a consistent quality and activity in the final product. Here we have characterized a typical bioprocess and determined (a) how the time of harvest from a mammalian cell culture and, (b) through the use of an ultra scale-down mimic how the nature of the primary recovery stages, affect the distribution and make up of the PTMs observed on a recombinant IgG4 monoclonal antibody. In particular we describe the use of rapid whole antibody analysis by mass spectrometry to analyze simultaneously the changes that occur to the cleavage of heavy chain C-terminal lysine residues and the glycosylation pattern, as well as the presence of HL dimers. The time of harvest was found to have a large impact upon the range of glycosylation patterns observed, but not upon C-terminal lysine cleavage. The culture age had a profound impact on the ratio of different glycan moieties found on antibody molecules. The proportion of short glycans increased (e.g., (G0F)2 20,35%), with an associated decrease in the proportion of long glycans with culture age (e.g., (G2F)2 7,4%, and G1F/G2F from 15.2% to 7.8%). Ultra scale-down mimics showed that subsequent processing of these cultures did not change the post-translational modifications investigated, but did increase the proportion of half antibodies present in the process stream. The combination of ultra scale-down methodology and whole antibody analysis by mass spectrometry has demonstrated that the effects of processing on the detailed molecular structure of a monoclonal antibody can be rapidly determined early in the development process. In this study we have demonstrated this analysis to be applicable to critical process design decisions (e.g., time of harvest) in terms of achieving a desired molecular structure, but this approach could also be applied as a selection criterion as to the suitability of a platform process for the preparation of a new drug candidate. Also the methodology provides means for bioprocess engineers to predict at the discovery phase how a bioprocess will impact upon the quality of the final product. Biotechnol. Bioeng. 2010;107: 85,95. © 2010 Wiley Periodicals, Inc. [source] Engineered therapeutic antibodies with improved effector functionsCANCER SCIENCE, Issue 9 2009Tsuguo Kubota In the past decade, more than 20 therapeutic antibodies have been approved for clinical use and many others are now at the clinical and preclinical stage of development. Fragment crystallizable (Fc)-dependent antibody functions, such as antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and a long half-life, have been suggested as important clinical mechanisms of therapeutic antibodies. These functions are primarily triggered through direct interaction of the Fc domain with its corresponding receptors: Fc,RIIIa for ADCC, C1q for CDC, and neonatal Fc receptor for prolongation of the clearance rate. However, current antibody therapy still faces the critical issues of insufficient efficacy and the high cost of the therapeutic agents. A possible solution to these issues could be to engineer antibody molecules to enhance their antitumor activity, leading to improved therapeutic outcomes and reduced doses. Here, we review advanced Fc engineering approaches for the enhancement of effector functions, some of which are now ready for evaluation of their effectiveness in clinical trials. (Cancer Sci 2009; 100: 1566,1572) [source] | |||||||||||||||||||