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

Författare:
Publiceringsår: 2007
Språk: Engelska
Sidor: 3055-3065
Publikation/Tidskrift/Serie: PROTEOMICS
Volym: 7
Nummer: 17
Dokumenttyp: Artikel
Förlag: Wiley

Sammanfattning

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.

Disputation

Nyckelord

  • Medicine and Health Sciences
  • recombinant
  • proteome analysis
  • antibody microarray
  • protein labeling
  • solid support
  • scFv

Övriga

Published
Yes
  • ISSN: 1615-9853

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