Design of recombinant antibody microarrays for complex proteome analysis: Choice of sample labeling-tag and solid support
(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)
Please use this url to cite or link to this publication:
https://lup.lub.lu.se/record/656421
- author
- Wingren, Christer LU ; Ingvarsson, Johan LU ; Dexlin Mellby, Linda LU ; Szul, Dominika LU and Borrebaeck, Carl LU
- organization
- publishing date
- 2007
- 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 Inc.
- external identifiers
-
- wos:000249635200004
- scopus:34748919097
- ISSN
- 1615-9861
- DOI
- 10.1002/pmic.200700025
- language
- English
- LU publication?
- yes
- id
- 6142ec28-12a4-4ddb-b9ed-742ccdc97018 (old id 656421)
- date added to LUP
- 2016-04-01 11:48:55
- date last changed
- 2022-01-26 18:41:14
@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}},
keywords = {{recombinant; proteome analysis; antibody microarray; protein labeling; solid support; scFv}},
language = {{eng}},
number = {{17}},
pages = {{3055--3065}},
publisher = {{John Wiley & Sons Inc.}},
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}},
doi = {{10.1002/pmic.200700025}},
volume = {{7}},
year = {{2007}},
}