The Experts below are selected from a list of 9495 Experts worldwide ranked by ideXlab platform
Vittoria Pinto - One of the best experts on this subject based on the ideXlab platform.
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a scalable method for o Antigen Purification applied to various salmonella serovars
Analytical Biochemistry, 2013Co-Authors: Francesca Micoli, Carlo Giannelli, Simona Rondini, Massimiliano Gavini, Ivan Pisoni, Luisa Lanzilao, Anna Maria Colucci, F Pippi, Luigi Sollai, Vittoria PintoAbstract:The surface lipopolysaccharide of gram-negative bacteria is both a virulence factor and a B cell Antigen. Antibodies against O-Antigen of lipopolysaccharide may confer protection against infection, and O-Antigen conjugates have been designed against multiple pathogens. Here, we describe a simplified methodology for extraction and Purification of the O-Antigen core portion of Salmonella lipopolysaccharide, suitable for large-scale production. Lipopolysaccharide extraction and delipidation are performed by acetic acid hydrolysis of whole bacterial culture and can take place directly in a bioreactor, without previous isolation and inactivation of bacteria. Further O-Antigen core Purification consists of rapid filtration and precipitation steps, without using enzymes or hazardous chemicals. The process was successfully applied to various Salmonella enterica serovars (Paratyphi A, Typhimurium, and Enteritidis), obtaining good yields of high-quality material, suitable for conjugate vaccine preparations.
Andreas Plückthun - One of the best experts on this subject based on the ideXlab platform.
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fast selection of antibodies without Antigen Purification adaptation of the protein fragment complementation assay to select Antigen antibody pairs
Journal of Molecular Biology, 2001Co-Authors: Ekkehard Mossner, Holger Koch, Andreas PlückthunAbstract:Abstract We have adapted the protein fragment complementation assay (PCA) to the screening and selection of antibodies in the single-chain Fv (scFv) format. In this assay, two interacting proteins (target and antibody) are genetically fused to the two halves of the dissected enzyme dihydrofolate reductase. Binding of the two partners reassembles this enzyme and reconstitutes its activity, thus allowing growth on minimal medium. We have optimized this system with regard to linker length and orientation, and can reach an efficiency for Antigen/antibody interactions similar to that with fused leucine zippers. Using several model antibodies specific for peptides and proteins, we show that cognate interactions give rise to about seven orders of magnitude more colonies than non-specific interactions. When transforming mixtures of plasmids encoding different Antigens and/or antibodies, all colonies tested contained plasmids encoding cognate pairs. We believe that this system will be very powerful as a routine system for generating antibodies, especially in functional genomics, since it does not require Purification and immobilization of the Antigen. The identification of an antibody specific for a cDNA or EST-encoded protein will require only cloning, transformation and plating of bacteria.
Francesca Micoli - One of the best experts on this subject based on the ideXlab platform.
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a scalable method for o Antigen Purification applied to various salmonella serovars
Analytical Biochemistry, 2013Co-Authors: Francesca Micoli, Carlo Giannelli, Simona Rondini, Massimiliano Gavini, Ivan Pisoni, Luisa Lanzilao, Anna Maria Colucci, F Pippi, Luigi Sollai, Vittoria PintoAbstract:The surface lipopolysaccharide of gram-negative bacteria is both a virulence factor and a B cell Antigen. Antibodies against O-Antigen of lipopolysaccharide may confer protection against infection, and O-Antigen conjugates have been designed against multiple pathogens. Here, we describe a simplified methodology for extraction and Purification of the O-Antigen core portion of Salmonella lipopolysaccharide, suitable for large-scale production. Lipopolysaccharide extraction and delipidation are performed by acetic acid hydrolysis of whole bacterial culture and can take place directly in a bioreactor, without previous isolation and inactivation of bacteria. Further O-Antigen core Purification consists of rapid filtration and precipitation steps, without using enzymes or hazardous chemicals. The process was successfully applied to various Salmonella enterica serovars (Paratyphi A, Typhimurium, and Enteritidis), obtaining good yields of high-quality material, suitable for conjugate vaccine preparations.
Tao Hong - One of the best experts on this subject based on the ideXlab platform.
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Purification of human respiratory syncytial virus fusion glycoprotein.
Protein Expression and Purification, 2011Co-Authors: Yan-peng Zheng, Yaning Tang, Xiaobo Wang, Tao HongAbstract:Human respiratory syncytial virus (RSV) fusion glycoprotein (F) elicits neutralizing antibodies to RSV and has therefore attracted much attention as a suitable candidate Antigen in the development of gene-based vaccines against RSV infections. However, a major obstacle in vaccine development has been the problem of Antigen Purification. To address this problem, we have developed a new method that combines sucrose gradient ultracentrifugation and a two-step chromatographic process, to purify RSV F from RSV particles propagated in HEp-2 cells. Analysis of the fractions produced using this method showed recovery of a functional homodimer with a molecular weight of 140 kDa, and 54% preservation of the original F.
Pearay L. Ogra - One of the best experts on this subject based on the ideXlab platform.
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Preparation of respiratory syncytial virus subgroup A and B Antigens for enzyme immunoassay antibody detection.
Acta medica Croatica : c̆asopis Hravatske akademije medicinskih znanosti, 1994Co-Authors: Gordana Mlinarić-galinović, T. Chonmaintree, Janak Patel, R. Abraham, Roberto P Garofalo, Pearay L. OgraAbstract:Abstract A simplified method was described for Purification of respiratory syncytial virus (RSV) subgroup A and B aimed to be used as Antigens in enzyme immunoassay (EIA). The titer of each RSV subgroup and the amount of protein was determined from the visible band in 45% sucrose gradient. The quality of prepared RSV subgroup Antigens for EIA was described in terms of the achievable final titer, the amount of protein, and EIA criss-cross titration. The RSV subgroup A and B Antigens, diluted as 1:100 (low opalescent band in 45% sucrose layer) or 1:800 (high opalescent band in 45% sucrose layer) produced a positive reaction in EIA criss-cross titration with IgG antibodies from the patient's serum (convalescent phase) diluted as 1:25,600 (for RSV A) and 1:6,400 (for RSV B). This method offers shorter and more simplified steps of viral Antigen Purification, and provides acceptable quantity and quality of viral Antigens appropriate for use in EIA.
