Greenwell, P.

Rughooputh, S.

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     University of Westminster Eprints http://eprints.wmin.ac.uk   Isolation of Neisseria gonorrhoeae-specific antibodies from a phage display library.  Sanjiv Rughooputh Pamela Greenwell School of Biosciences    This is an electronic version of an article published in Molecular and Cellular Proteomics, 2 (9). 758, September 2003.  Molecular and Cellular Proteomics is available online at:  http://www.mcponline.org/cgi/reprint/2/9/702    The Eprints service at the University of Westminster aims to make the research output of the University available to a wider audience.  Copyright and Moral Rights remain with the authors and/or copyright owners. Users are permitted to download and/or print one copy for non-commercial private study or research.  Further distribution and any use of material from within this archive for profit-making enterprises or for commercial gain is strictly forbidden.        Whilst further distribution of specific materials from within this archive is forbidden, you may freely distribute the URL of the University of Westminster Eprints (http://eprints.wmin.ac.uk).   In case of abuse or copyright appearing without permission e-mail wattsn@wmin.ac.uk.  38.27Nuclear Localization Signals of Kaiso andthe Catenin p120ctnK. F. Kelly, A. A. Otchere, M. Graham, and J. M. DanielMcMaster University, Hamilton, ON, CanadaKaiso is a new member of the BTB/POZ (Broad complex, Tramtrak, Bric abrac/Pox virus and zinc finger) family of zinc finger transcription factorsthat are implicated in development and cancer. Kaiso was identifiedthrough a Yeast-2-Hybrid screen to identify binding partners for p120ctn, amember of the E-cadherin-catenin complex, the predominant cell adhe-sion system in epithelial cells. Like most POZ proteins, Kaiso is a tran-scriptional repressor, however it displays unique bimodal DNA bindingproperties. Interestingly, the DNA-binding and transcriptional repressionability of Kaiso are abolished in vitro in the presence of exogenous p120ctn.Despite the nuclear role for Kaiso and p120ctn in gene regulation, theirmechanism of nuclear import is currently unknown. Via deletion mutagen-esis, we have now identified one NLS (nuclear localization signal) in Kaisoand two in p120ctn. Excitingly, the Kaiso NLS is one of the first identifiedfor a BTB/POZ protein. p120ctn possesses two NLSs, one of which directsnuclear import more potently and consistently than the other. Point mu-tation of key positively charged residues to neutral alanines in each NLSabolishes their functionality. We are currently investigating the effect ofthis minimal-mutation on the nuclear localization of full length Kaiso andp120ctn. In addition, nuclear import assays using digitonin-permeablizedcells and exogenous GST-p120ctn and GST-Kaiso substrates should clar-ify the nuclear import mechanism for Kaiso and p120ctn. Our findings willcontribute to our understanding of the physiological relevance of theKaiso/p120ctn interaction as well as the mechanism of nuclear import forthe BTB/POZ family transcription factors.39.1Microfluidic Technology Applied to QualityControl of AntibodiesT. Neumann, M. Kuschel, and P. BarthmaierAgilent Technologies Deutschland GmbH, Waldbronn, GermanyAntibodies are commonly produced for diagnostics, as research tools oras therapeutics and require precise testing and quality control (QC). Thekey quality criteria include protein identification, quantitation, purity andstability. The standard methods for antibody QC include besides others,gel electrophoresis (GE) and capillary gel electrophoresis (CGE). GE hasnot changed significantly over the past 30 years and does not allow for ahigh degree of automation. CGE is a well-known alternative to GE andoffers important advantages for protein analysis: automation and fastersample throughput. Lab-on-a-chip (LOAC) technology has extended theseadvantages even further by transferring CGE principles into a glass chipformat. It integrates multiple manual experimental procedures such assample handling, separation, staining, detection and analysis in a singleautomated process. The first commercial LOAC instrument was used toverify the suitability of this technology for antibody QC. It allows sizing andanalysis of proteins from 5 to 200 kDa, depending on the application, andto determine relative concentration based on internal standards, or abso-lute quantitation based on user-defined standards. Antibodies were ana-lyzed for size, concentration, purity, and stability. The sizing and quanti-tation accuracy as well as reproducibility was determined and comparedto standard methods. The LOAC protein analysis is comparable in sensi-tivity, sizing accuracy and reproducibility to gels stained with Coomassie.Resolution and linear dynamic range are improved. Absolute quantitationaccuracy and reproducibility is superior to gels and is comparable toLowry or Bradford. The LOAC analysis allows performing antibody anal-ysis under both reducing and non-reducing conditions in one run. Addi-tional advantages include fast analysis times, ease of use, automated dataanalysis, and digital data.39.2Isolation of Neisseria gonorrhoeae-specifcAntibodies from a Phage Display LibraryS. Rughooputh and P. GreenwellSchool of Biosciences, University of Westminster, London, UnitedKingdomScFv antibodies against Neisseria gonorrhoeae were successfully isolatedfrom the Griffin phage display library (kind gift from MRC, UK). The librarywas panned against Neisseria gonorrhoeae NCTC (12700) coated onimmunotubes. After 3 rounds of selection, the phagemids were checkedagainst a wide range of microorganisms for cross-reactivity. Phagemidsexpressing non-specific antibodies were removed by panning against amixture of microorganisms. The specific antibody-producing phagemidswere subjected to two further rounds of selection against N. gonorrhoeae.The phagemids were then harvested and used to infect Escherichia coliHB2151 to produce soluble antibody. The transfected cells were plated atlow density and 22 individual clones selected for monoclonal antibodyproduction and analysis. Three clones were found to produce high-titreneisseria-specific antibody and these were selected and used in furtherexperiments. The specificity and titre of these antibodies were determinedusing Dot blot, Western blot and ELISA. The most avid antibody, prior topurification and concentration, had a titre of 1 in 100. The sizes of theantibody fragments were determined by SDS PAGE and Western blotting.The antibody-encoding insert,300bp, was amplified using PCR and se-quenced. The size of the detected antigen was analysed using SDS PAGEWestern blotting. To the best of our knowledge, this is the first report ofNeisseria-specific antibodies isolated by phage display technology. Themethodology described would be useful for the isolation of antibodiesagainst a wide range of microbes.39.3Selection of Anti-ssDNA Chicken scFvsfrom Non-immunized Animal PhageDisplay Combinatorial LibraryA. Q. Maranhao and M. M. BrigidoUniversidade De Brası´lia, Brası´lia, BrazilThe common route to obtain monoclonal antibodies is making hybridomafrom immunized animals. Recently, construction and screening of combi-natorial library displayed on filamentous phage surface has emerged as analternative to hybridoma methodology. Among vertebrates, chicken is agood source of those libraries as cloning is facilitated due to the V genesegment gene conversion rearrangements that create the variability oftheir antibodies. This makes possible to clone the whole V gene repertoireperforming a single step PCR amplification for each variable chain. Weused a chicken scFv library displayed on filamentous phage surface andpanning against ssDNA. A five round selection was doneusing oligo-dTcellulose and the soluble antigen eluted and the remaining resin boundphages were transduced. The ratio between output and input tittering rosefrom 1/105 (round 2) to 1/102 (round 5). Clones from round 4 and 5 weregrown in a 96 deep well plateand they were analyzed by their ability toproduce antibody and also by their bindingactivity. The protein productionwas probed in a dot blot assay using an anti-HA antibodyand the bindingwas probed with biotinilated oligo dT. Individual clones selectedfromthese experiments had their nucleotide sequences determined. Theanalysis of the V chainCDRs revealed that some of them share commonones and also entire V chains. These resultsshow that the enrichment ofbinders was not random and possible driven by the antigen,even thislibrary coming from non-immunized animals.HUPO 2nd Annual & IUBMB XIX World Congress, October 8–11, Montreal758 Molecular & Cellular Proteomics 2.9

Isolation of Neisseria gonorrhoeae-specific antibodies from a phage display library

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Isolation of Neisseria gonorrhoeae-specific antibodies from a phage display library

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