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This Article Full Text Full Text (PDF) Supplemental Data All Versions of this Article: M900009-MCP200v1 8/11/2544    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Glossary Google Scholar Articles by Hamsten, C. Articles by Persson, A. PubMed PubMed Citation Articles by Hamsten, C. Articles by Persson, A. Social Bookmarking                      What's this?

Molecular & Cellular Proteomics 8:2544-2554, 2009.
© 2009 by The American Society for Biochemistry and Molecular Biology, Inc.

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Research

Recombinant Surface Proteomics as a Tool to Analyze Humoral Immune Responses in Bovines Infected by Mycoplasma mycoides Subsp. mycoides Small Colony Type^*,

Carl Hamsten, Maja Neiman, Jochen M. Schwenk, Marica Hamsten, John B. March and Anja Persson^,¶

From the Department of Proteomics, School of Biotechnology, Royal Institute of Technology (KTH), AlbaNova University Center , SE-106 91 Stockholm, Sweden and
BigDNA Ltd. , Roslin Biocentre, Roslin, Midlothian EH25 9PP, United Kingdom

A systematic approach to characterize the surface proteome of Mycoplasma mycoides subspecies mycoides small colony type (M. mycoides SC), the causative agent of contagious bovine pleuropneumonia (CBPP) in cattle, is presented. Humoral immune responses in 242 CBPP-affected cattle and controls were monitored against one-third of the surface proteins of M. mycoides SC in a high throughput magnetic bead-based assay. Initially, 64 surface proteins were selected from the genome sequence of M. mycoides SC and expressed as recombinant proteins in Escherichia coli. Binding of antibodies to each individual protein could then be analyzed simultaneously in minute sample volumes with the Luminex suspension array technology. The assay was optimized on Namibian CBPP-positive sera and Swedish negative controls to allow detection and 20-fold mean signal separation between CBPP-positive and -negative sera. Signals were proven to be protein-specific by inhibition experiments, and results agreed with Western blot experiments. The potential of the assay to monitor IgG, IgM, and IgA responses over time was shown in a proof-of-concept study with 116 sera from eight animals in a CBPP vaccine study. In conclusion, a toolbox with recombinant proteins and a flexible suspension array assay that allows multiplex analysis of humoral immune responses to M. mycoides SC has been created.

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