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Antigen Purification

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Vittoria Pinto – One of the best experts on this subject based on the ideXlab platform.

  • a scalable method for o Antigen Purification applied to various salmonella serovars
    Analytical Biochemistry, 2013
    Co-Authors: Francesca Micoli, Simona Rondini, Massimiliano Gavini, Ivan Pisoni, Luisa Lanzilao, Anna Maria Colucci, Carlo Giannelli, F Pippi, Luigi Sollai, Vittoria Pinto

    Abstract:

    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.

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Andreas Plückthun – One of the best experts on this subject based on the ideXlab platform.

  • fast selection of antibodies without Antigen Purification adaptation of the protein fragment complementation assay to select Antigen antibody pairs
    Journal of Molecular Biology, 2001
    Co-Authors: Ekkehard Mossner, Holger Koch, Andreas Plückthun

    Abstract:

    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.

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Francesca Micoli – One of the best experts on this subject based on the ideXlab platform.

  • a scalable method for o Antigen Purification applied to various salmonella serovars
    Analytical Biochemistry, 2013
    Co-Authors: Francesca Micoli, Simona Rondini, Massimiliano Gavini, Ivan Pisoni, Luisa Lanzilao, Anna Maria Colucci, Carlo Giannelli, F Pippi, Luigi Sollai, Vittoria Pinto

    Abstract:

    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.

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