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

Wingren, Christer LU ; Ingvarsson, Johan LU ; Dexlin Mellby, Linda LU ; Szul, Dominika LU and Borrebaeck, Carl LU (2007) In Proteomics 7(17). p.3055-3065
Abstract
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... (More)
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. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
recombinant, proteome analysis, antibody microarray, protein labeling, solid support, scFv
in
Proteomics
volume
7
issue
17
pages
3055 - 3065
publisher
John Wiley & Sons
external identifiers
  • wos:000249635200004
  • scopus:34748919097
ISSN
1615-9861
DOI
10.1002/pmic.200700025
project
CREATE Health
language
English
LU publication?
yes
id
6142ec28-12a4-4ddb-b9ed-742ccdc97018 (old id 656421)
date added to LUP
2007-12-19 09:14:30
date last changed
2017-07-09 03:33:51
@article{6142ec28-12a4-4ddb-b9ed-742ccdc97018,
  abstract     = {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.},
  author       = {Wingren, Christer and Ingvarsson, Johan and Dexlin Mellby, Linda and Szul, Dominika and Borrebaeck, Carl},
  issn         = {1615-9861},
  keyword      = {recombinant,proteome analysis,antibody microarray,protein labeling,solid support,scFv},
  language     = {eng},
  number       = {17},
  pages        = {3055--3065},
  publisher    = {John Wiley & Sons},
  series       = {Proteomics},
  title        = {Design of recombinant antibody microarrays for complex proteome analysis: Choice of sample labeling-tag and solid support},
  url          = {http://dx.doi.org/10.1002/pmic.200700025},
  volume       = {7},
  year         = {2007},
}