Gourdine, J.P.

Greenwell, P.

Smith-Ravin, E.J.

WestminsterResearch

     University of Westminster Eprints http://eprints.wmin.ac.uk   Phage display antibody against gill proteins of the tropical clam.  J-P. Gourdine1 E.J. Smith-Ravin1 Pamela Greenwell2  1 Biology, Universite des Antilles et de la Guyane, Pointe-a-Pitre, Guadaloupe, France 2School of Biosciences, University of Westminster    This is an electronic version of an article published in Molecular and Cellular Proteomics, 2 (9). 759, 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. 39.4Phage Display Antibody Against GillProteins of the Tropical ClamJ.-P. Gourdine1, E. J. Smith-Ravin1, and P. Greenwell21Biology, Universite´ des Antilles et de la Guyane, Pointe-a`-Pitre,Guadeloupe, France; and 2University of Westminster, London,United KingdomIntroduction: Codakia orbicularis (Bivalvia, Lucinidae) is a tropical clam,which lives in areas of high sulfur concentrations and which containsendosymbiotic chemoautotrophic bacteria in its gills. Gill proteins arethought to be involved in sulfur metabolism and in symbiont/host interac-tions. We used the Recombinant Phage display Antibody System (RPAS)to raise Single chain Fragment variable (ScFv) antibodies against theseproteins. Material and Methods: Griffin.1 library of phage display antibod-ies was used. Three rounds of selection of phage panning were performedon immunotubes coated with total extract of native gill proteins. Thebound M13 phages were isolated and tested by ELISA and the positiveclones were selected for ScFv production. The presence of ScFv DNAinserts was assessed by PCR. These chimeric antibodies were purified byammonium sulfate precipitation or IMAC, and used in Dot-blot and West-ern-blot assays as well as in plate antigen capture combined with SDS-PAGE. Results and Discussion: Five out of 60 isolated clones were usedfor ScFv production. Purification on IMAC gave ScFv antibodies of about30 000 Daltons at a higher yield compared to ammonium sulfate precip-itation. Antigen capture showed a strong positive reaction to a 15 000Dalton protein, a major band in the electrophoretic profile of gill proteins.These recombinant antibodies are being tested by immunohistochemistryon gill sections. Total gill extract has been used for the first time to raisephage antibodies against clam proteins, which antibodies might be usefulin the isolation and study of the properties of the said proteins.39.5A General Method to Design Primers forthe AmplificationE. Sosthe`ne Severin, Y Frobert, D. Boquet, C. Cre´minon,and J. GrassiService de Pharmacologie et d’Immunologie, DRM/DSV,CEA-Saclay, Gif sur Yvette, FranceThe great diversity of variable genes (VH and VL) of immunoglobulinsrequires, for PCR amplification of these regions, the use of ”universal“oligonucleotides primers. Nevertheless, even the most recent set of prim-ers failed to amplify without amino acids modification the VH and VLcDNA. We have designed a new set of mouse immunoglobulin primersusing well established criteria; i) by using the IMGT which permits toeliminate all abnormal sequences due to a high annotation level, ii) bydesigning a degenerated primer for each sub-group, iii) by designing thisdegenerated primer from the alignment of DNA and cDNA. This approachleads to a mix of primers for all sub-groups conserving the entire diversityof amino-acids. We have successfully tested this set of primers for theamplification of variable regions of several antibodies directed againstHistamine (mAb Hist31), Acetylcholinesterase (mAb Elec408) and prionprotein (PrP) (mAb Saf 34). We have built the recombining antibody singlechain fragment variable (ScFvHis-31) further expressed in E.coli. Theoriginality of our approach relies also on the use of a newly developedprokaryotic periplasmic expression vector, including two sites of restric-tion Sfi 1, two Tags sequences at final ends of produced ScFv. This vectorallows the fast detection of full length expressed ScFv and the corre-sponding functional clones, using 96 wells culture plate format and thefurther large-scale expression of the relevant clones in order to charac-terize their specificity and apparent affinity.39.6From Genes to Intracellular Antibodies:Dissecting the Proteome with Splint, aSingle Pot Library of Stable AntibodiesM. Visintin, I. Cannistraci, and A. CattaneoLay Line Genomics, Rome, ItalyThe Intracellular Antibody Capture Technology (IACT) is an in vivo two-hybrid based method for the selection of antibodies folded for ectopicexpression (1). The IAC technology has been successfully used for therapid identification of antigen-antibody pairs in intracellular compartmentsand for in vivo epitope mapping (2).In the original format the isolation ofintracellular antibodies or intrabodies can be done starting from existingscFv phage display, from spleen of immunized mice or from human biopsyof patients. To overcome the limitation of needing a purified proteinantigen for the in vitro panning step, if the phage display approach ischosen for the construction of the IAC library, or for the immunization ofmice if the spleen RNA approach is preferred, we have constructed aSingle Pot Library of Intrabodies (SPLINT), a nave scFv yeast library fromwhich functional intracellular antibodies can be derived directly from genesequences, with no handling of proteins. In this contribution we describethe development of SPLINT technology and the selection and character-ization of intracellular antibodies against a panel of different antigens. Theapplication of SPLINT to isolate validated intrabodies on a genome-widescale should help the systematic study of the functional organization of thecell proteome and should provides a valuable tool for the validation oftherapeutic targets in cell-wide analysis.HUPO 2nd Annual & IUBMB XIX World Congress, October 8–11, MontrealMolecular & Cellular Proteomics 2.9 759

Phage display antibody against gill proteins of the tropical clam

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Phage display antibody against gill proteins of the tropical clam

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