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Design of recombinant antibody microarrays for complex proteome analysis: Choice of sample labeling-tag and solid support

Author:
  • Christer Wingren
  • Johan Ingvarsson
  • Linda Dexlin Mellby
  • Dominika Szul
  • Carl Borrebaeck
Publishing year: 2007
Language: English
Pages: 3055-3065
Publication/Series: Proteomics
Volume: 7
Issue: 17
Document type: Journal article
Publisher: John Wiley & Sons

Abstract english

Antibody-based microarray is a novel technology with great potential within high-throughput proteomics. The process of designing high-performing antibody (protein) microarrays has, however, turned out to be a challenging process. In this study, we have developed further our human recombinant single-chain variable-fragment (scFv) antibody microarray methodology by addressing two crucial technological issues, choice of sample labeling-tag and solid support. We examined the performance of a range of dyes in a one- or two-color approach on a selection of solid supports providing different surface and coupling chemistries, and surface structures. The set-ups were evaluated in terms of sensitivity specificity, and selectivity. The results showed that a one-color approach, based on NHS-biotin (or ULS-biotin) labeling, on black polymer Maxisorb slides (or Nexterion slide H) was the superior approach for targeting low-abundant (pg/mL) analytes in nonfractionated, complex proteomes, such as human serum or crude cell supernatants. Notably, microarrays displaying adequate spot morphologies, high S/Ns, minimized nonspecific binding, and most importantly a high selectivity, specificity, and sensitivity (>= fM range) were obtained. Taken together, we have designed the first generation of a high-performing recombinant scFv antibody microarray technology platform on black polymer Maxisorb slides for sensitive profiling of low-abundant analytes in nonfractionated biotinylated complex proteomes.

Keywords

  • Immunology in the medical area
  • recombinant
  • proteome analysis
  • antibody microarray
  • protein labeling
  • solid support
  • scFv

Other

Published
  • CREATE Health
  • ISSN: 1615-9861
Carl B
Carl Borrebaeck
E-mail: carl.borrebaeck [at] immun.lth.se

Professor

Department of Immunotechnology

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Director

Create Health

+46 46 222 96 13

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