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Originally published In Press as doi:10.1074/mcp.T300001-MCP200 on September 29, 2003.
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Molecular & Cellular Proteomics 2:1342-1349, 2003.
© 2003 by The American Society for Biochemistry and Molecular Biology, Inc.

Technology

A Nonredundant Human Protein Chip for Antibody Screening and Serum Profiling*

Angelika Lueking{ddagger}, Alexandra Possling{ddagger}, Otmar Huber§, Allan Beveridge{ddagger}, Martin Horn, Holger Eickhoff, Johannes Schuchardt||, Hans Lehrach{ddagger} and Dolores J. Cahill{ddagger},**,{ddagger}{ddagger}

From the {ddagger} Max-Planck-Institute for Molecular Genetics, Ihnestrasse 73, 14195 Berlin, Germany; § Institute of Clinical Chemistry and Pathobiochemistry, Universitätsklinikum Benjamin Franklin, Hindenburgdamm 30, 12200 Berlin, Germany; Scienion AG, Volmerstrasse 7a, D-12489 Berlin, Germany; || MicroDiscovery GmbH, Immanuelkirchstraße 12, D-10405 Berlin, Germany; and {ddagger}{ddagger} Centre for Human Proteomics, Royal College of Surgeons in Ireland, Dublin 2, Ireland and Protagen AG, Emil-Figge-Straße 76a, D-44227 Dortmund, Germany

There is burgeoning interest in protein microarrays, but a source of thousands of nonredundant, purified proteins was not previously available. Here we show a glass chip containing 2413 nonredundant purified human fusion proteins on a polymer surface, where densities up to 1600 proteins/cm2 on a microscope slide can be realized. In addition, the polymer coating of the glass slide enables screening of protein interactions under nondenaturing conditions. Such screenings require only 200-µl sample volumes, illustrating their potential for high-throughput applications. Here we demonstrate two applications: the characterization of antibody binding, specificity, and cross-reactivity; and profiling the antibody repertoire in body fluids, such as serum from patients with autoimmune diseases. For the first application, we have incubated these protein chips with anti-RGSHis6, anti-GAPDH, and anti-HSP90ß antibodies. In an initial proof of principle study for the second application, we have screened serum from alopecia and arthritis patients. With analysis of large sample numbers, identification of disease-associated proteins to generate novel diagnostic markers may be possible.

** To whom correspondence should be addressed: Centre for Human Proteomics, Royal College of Surgeons in Ireland, 123 St. Stephen’s Green, Dublin 2, Ireland. Tel.: 353-(1)-402-8550; Fax: 353-(1)-402-2453; E-mail: chp{at}rcsi.ie


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