Microarrays based on affinity-tagged single-chain Fv antibodies: sensitive detection of analyte in complex proteomes.

Christer Wingren; Cornelia Steinhauer; Johan Ingvarsson; Erik Persson; Katrin Larsson; Carl A K Borrebaeck

doi:10.1002/pmic.200401009

Microarrays based on affinity-tagged single-chain Fv antibodies: sensitive detection of analyte in complex proteomes.

Christer Wingren, Cornelia Steinhauer, Johan Ingvarsson, Erik Persson, Katrin Larsson, Carl A K Borrebaeck

Publikation: Bidrag til tidsskrift › Tidsskriftartikel › Forskning › peer review

75 Citationer (Scopus)

Abstract

Udgivelsesdato: 2005-Apr

Originalsprog	Engelsk
Tidsskrift	Proteomics
Vol/bind	5
Udgave nummer	5
Sider (fra-til)	1281-91
Antal sider	10
ISSN	1615-9853
DOI	https://doi.org/10.1002/pmic.200401009
Status	Udgivet - 2005
Udgivet eksternt	Ja

Bibliografisk note

Keywords: Humans; Immunoglobulin Fragments; Protein Array Analysis; Proteome; Recombinant Proteins; Sensitivity and Specificity

Adgang til dokumentet

10.1002/pmic.200401009

Citationsformater

@article{83ab48b057c411dd8d9f000ea68e967b,

title = "Microarrays based on affinity-tagged single-chain Fv antibodies: sensitive detection of analyte in complex proteomes.",

abstract = "Protein-based microarrays are among the novel class of rapidly emerging proteomic technologies that will allow us to efficiently perform global proteome analysis. However, the process of designing adequate protein microarrays is a major inherent problem. In this study, we have evaluated a protein microarray platform based on nonpurified affinity-tagged single-chain (sc) Fv antibody fragments to generate proof-of-principle and to demonstrate the specificity and sensitivity of the array design. To this end, we used our human recombinant scFv antibody library genetically constructed around one framework, the n-CoDeR library containing 2 x 10(10) clones, as a source for our probes. The probes were immobilized via engineered C-terminal affinity tags, his- or myc-tags, to either Ni(2+)-coated slides or anti-tag antibody coated substrates. The results showed that highly functional microarrays were generated and that nonpurified scFvs readily could be applied as probes. Specific and sensitive microarrays were obtained, providing a limit of detection in the pM to fM range, using fluorescence as the mode of detection. Further, the results showed that spotting the analyte on top of the arrayed probes, instead of incubating the array with large sample volumes (333 pL vs. 40 microL), could reduce the amount of analyte required 4000 times, from 1200 attomole to 300 zeptomole. Finally, we showed that a highly complex proteome, such as human sera containing several thousand different proteins, could be directly fluorescently labeled and successfully analyzed without compromising the specificity and sensitivity of the antibody microarrays. This is a prerequisite for the design of high-density antibody arrays applied in high-throughput proteomics.",

author = "Christer Wingren and Cornelia Steinhauer and Johan Ingvarsson and Erik Persson and Katrin Larsson and Borrebaeck, {Carl A K}",

note = "Keywords: Humans; Immunoglobulin Fragments; Protein Array Analysis; Proteome; Recombinant Proteins; Sensitivity and Specificity",

year = "2005",

doi = "10.1002/pmic.200401009",

language = "English",

volume = "5",

pages = "1281--91",

journal = "Proteomics",

issn = "1615-9853",

publisher = "Wiley - V C H Verlag GmbH & Co. KGaA",

number = "5",

}

TY - JOUR

T1 - Microarrays based on affinity-tagged single-chain Fv antibodies: sensitive detection of analyte in complex proteomes.

AU - Wingren, Christer

AU - Steinhauer, Cornelia

AU - Ingvarsson, Johan

AU - Persson, Erik

AU - Larsson, Katrin

AU - Borrebaeck, Carl A K

N1 - Keywords: Humans; Immunoglobulin Fragments; Protein Array Analysis; Proteome; Recombinant Proteins; Sensitivity and Specificity

PY - 2005

Y1 - 2005

N2 - Protein-based microarrays are among the novel class of rapidly emerging proteomic technologies that will allow us to efficiently perform global proteome analysis. However, the process of designing adequate protein microarrays is a major inherent problem. In this study, we have evaluated a protein microarray platform based on nonpurified affinity-tagged single-chain (sc) Fv antibody fragments to generate proof-of-principle and to demonstrate the specificity and sensitivity of the array design. To this end, we used our human recombinant scFv antibody library genetically constructed around one framework, the n-CoDeR library containing 2 x 10(10) clones, as a source for our probes. The probes were immobilized via engineered C-terminal affinity tags, his- or myc-tags, to either Ni(2+)-coated slides or anti-tag antibody coated substrates. The results showed that highly functional microarrays were generated and that nonpurified scFvs readily could be applied as probes. Specific and sensitive microarrays were obtained, providing a limit of detection in the pM to fM range, using fluorescence as the mode of detection. Further, the results showed that spotting the analyte on top of the arrayed probes, instead of incubating the array with large sample volumes (333 pL vs. 40 microL), could reduce the amount of analyte required 4000 times, from 1200 attomole to 300 zeptomole. Finally, we showed that a highly complex proteome, such as human sera containing several thousand different proteins, could be directly fluorescently labeled and successfully analyzed without compromising the specificity and sensitivity of the antibody microarrays. This is a prerequisite for the design of high-density antibody arrays applied in high-throughput proteomics.

AB - Protein-based microarrays are among the novel class of rapidly emerging proteomic technologies that will allow us to efficiently perform global proteome analysis. However, the process of designing adequate protein microarrays is a major inherent problem. In this study, we have evaluated a protein microarray platform based on nonpurified affinity-tagged single-chain (sc) Fv antibody fragments to generate proof-of-principle and to demonstrate the specificity and sensitivity of the array design. To this end, we used our human recombinant scFv antibody library genetically constructed around one framework, the n-CoDeR library containing 2 x 10(10) clones, as a source for our probes. The probes were immobilized via engineered C-terminal affinity tags, his- or myc-tags, to either Ni(2+)-coated slides or anti-tag antibody coated substrates. The results showed that highly functional microarrays were generated and that nonpurified scFvs readily could be applied as probes. Specific and sensitive microarrays were obtained, providing a limit of detection in the pM to fM range, using fluorescence as the mode of detection. Further, the results showed that spotting the analyte on top of the arrayed probes, instead of incubating the array with large sample volumes (333 pL vs. 40 microL), could reduce the amount of analyte required 4000 times, from 1200 attomole to 300 zeptomole. Finally, we showed that a highly complex proteome, such as human sera containing several thousand different proteins, could be directly fluorescently labeled and successfully analyzed without compromising the specificity and sensitivity of the antibody microarrays. This is a prerequisite for the design of high-density antibody arrays applied in high-throughput proteomics.

U2 - 10.1002/pmic.200401009

DO - 10.1002/pmic.200401009

M3 - Journal article

C2 - 15732136

SN - 1615-9853

VL - 5

SP - 1281

EP - 1291

JO - Proteomics

JF - Proteomics

IS - 5

ER -