Use of protein G microcolumns in chromatographic immunoassays: A comparison of competitive binding formats

Erika L. Pfaunmiller, Jeanethe A. Anguizola, Mitchell L. Milanuk, Na Tasha Carter, David S. Hage

Research output: Contribution to journalArticle

4 Citations (Scopus)

Abstract

Affinity microcolumns containing protein G were used as general platforms for creating chromatographic-based competitive binding immunoassays. Human serum albumin (HSA) was used as a model target for this work and HSA tagged with a near infrared fluorescent dye was utilized as the label. The protein G microcolumns were evaluated for use in several assay formats, including both solution-based and column-based competitive binding immunoassays and simultaneous or sequential injection formats. All of these methods were characterized by using the same amounts of labeled HSA and anti-HSA antibodies per sample, as chosen for the analysis of a protein target in the low-to-mid ng/mL range. The results were used to compare these formats in terms of their response, precision, limit of detection, and analysis time. All these methods gave detection limits in the range of 8-19 ng/mL and precisions ranging from ±5% to ±10% when using an injection flow rate of 0.10 mL/min. The column-based sequential injection immunoassay provided the best limit of detection and the greatest change in response at low target concentrations, while the solution-based simultaneous injection method had the broadest linear and dynamic ranges. These results provided valuable guidelines that can be employed to develop and extend the use of protein G microcolumns and these competitive binding formats to other protein biomarkers or biological agents of clinical or pharmaceutical interest.

Original languageEnglish (US)
Pages (from-to)91-100
Number of pages10
JournalJournal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences
Volume1021
DOIs
StatePublished - May 15 2016

Fingerprint

Competitive Binding
Immunoassay
Serum Albumin
Limit of Detection
Injections
Proteins
Biological Factors
Biomarkers
Fluorescent Dyes
Labels
Assays
Flow rate
Guidelines
Infrared radiation
Antibodies
Pharmaceutical Preparations

Keywords

  • Affinity microcolumn
  • Chromatographic immunoassay
  • Competitive binding immunoassay
  • Human serum albumin
  • Protein G

ASJC Scopus subject areas

  • Analytical Chemistry
  • Biochemistry
  • Clinical Biochemistry
  • Cell Biology

Cite this

Use of protein G microcolumns in chromatographic immunoassays : A comparison of competitive binding formats. / Pfaunmiller, Erika L.; Anguizola, Jeanethe A.; Milanuk, Mitchell L.; Carter, Na Tasha; Hage, David S.

In: Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences, Vol. 1021, 15.05.2016, p. 91-100.

Research output: Contribution to journalArticle

@article{20a9f6b53d114007bc6db2eec0134a02,
title = "Use of protein G microcolumns in chromatographic immunoassays: A comparison of competitive binding formats",
abstract = "Affinity microcolumns containing protein G were used as general platforms for creating chromatographic-based competitive binding immunoassays. Human serum albumin (HSA) was used as a model target for this work and HSA tagged with a near infrared fluorescent dye was utilized as the label. The protein G microcolumns were evaluated for use in several assay formats, including both solution-based and column-based competitive binding immunoassays and simultaneous or sequential injection formats. All of these methods were characterized by using the same amounts of labeled HSA and anti-HSA antibodies per sample, as chosen for the analysis of a protein target in the low-to-mid ng/mL range. The results were used to compare these formats in terms of their response, precision, limit of detection, and analysis time. All these methods gave detection limits in the range of 8-19 ng/mL and precisions ranging from ±5{\%} to ±10{\%} when using an injection flow rate of 0.10 mL/min. The column-based sequential injection immunoassay provided the best limit of detection and the greatest change in response at low target concentrations, while the solution-based simultaneous injection method had the broadest linear and dynamic ranges. These results provided valuable guidelines that can be employed to develop and extend the use of protein G microcolumns and these competitive binding formats to other protein biomarkers or biological agents of clinical or pharmaceutical interest.",
keywords = "Affinity microcolumn, Chromatographic immunoassay, Competitive binding immunoassay, Human serum albumin, Protein G",
author = "Pfaunmiller, {Erika L.} and Anguizola, {Jeanethe A.} and Milanuk, {Mitchell L.} and Carter, {Na Tasha} and Hage, {David S.}",
year = "2016",
month = "5",
day = "15",
doi = "10.1016/j.jchromb.2015.12.055",
language = "English (US)",
volume = "1021",
pages = "91--100",
journal = "Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences",
issn = "1570-0232",
publisher = "Elsevier",

}

TY - JOUR

T1 - Use of protein G microcolumns in chromatographic immunoassays

T2 - A comparison of competitive binding formats

AU - Pfaunmiller, Erika L.

AU - Anguizola, Jeanethe A.

AU - Milanuk, Mitchell L.

AU - Carter, Na Tasha

AU - Hage, David S.

PY - 2016/5/15

Y1 - 2016/5/15

N2 - Affinity microcolumns containing protein G were used as general platforms for creating chromatographic-based competitive binding immunoassays. Human serum albumin (HSA) was used as a model target for this work and HSA tagged with a near infrared fluorescent dye was utilized as the label. The protein G microcolumns were evaluated for use in several assay formats, including both solution-based and column-based competitive binding immunoassays and simultaneous or sequential injection formats. All of these methods were characterized by using the same amounts of labeled HSA and anti-HSA antibodies per sample, as chosen for the analysis of a protein target in the low-to-mid ng/mL range. The results were used to compare these formats in terms of their response, precision, limit of detection, and analysis time. All these methods gave detection limits in the range of 8-19 ng/mL and precisions ranging from ±5% to ±10% when using an injection flow rate of 0.10 mL/min. The column-based sequential injection immunoassay provided the best limit of detection and the greatest change in response at low target concentrations, while the solution-based simultaneous injection method had the broadest linear and dynamic ranges. These results provided valuable guidelines that can be employed to develop and extend the use of protein G microcolumns and these competitive binding formats to other protein biomarkers or biological agents of clinical or pharmaceutical interest.

AB - Affinity microcolumns containing protein G were used as general platforms for creating chromatographic-based competitive binding immunoassays. Human serum albumin (HSA) was used as a model target for this work and HSA tagged with a near infrared fluorescent dye was utilized as the label. The protein G microcolumns were evaluated for use in several assay formats, including both solution-based and column-based competitive binding immunoassays and simultaneous or sequential injection formats. All of these methods were characterized by using the same amounts of labeled HSA and anti-HSA antibodies per sample, as chosen for the analysis of a protein target in the low-to-mid ng/mL range. The results were used to compare these formats in terms of their response, precision, limit of detection, and analysis time. All these methods gave detection limits in the range of 8-19 ng/mL and precisions ranging from ±5% to ±10% when using an injection flow rate of 0.10 mL/min. The column-based sequential injection immunoassay provided the best limit of detection and the greatest change in response at low target concentrations, while the solution-based simultaneous injection method had the broadest linear and dynamic ranges. These results provided valuable guidelines that can be employed to develop and extend the use of protein G microcolumns and these competitive binding formats to other protein biomarkers or biological agents of clinical or pharmaceutical interest.

KW - Affinity microcolumn

KW - Chromatographic immunoassay

KW - Competitive binding immunoassay

KW - Human serum albumin

KW - Protein G

UR - http://www.scopus.com/inward/record.url?scp=84954305886&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84954305886&partnerID=8YFLogxK

U2 - 10.1016/j.jchromb.2015.12.055

DO - 10.1016/j.jchromb.2015.12.055

M3 - Article

C2 - 26777776

AN - SCOPUS:84954305886

VL - 1021

SP - 91

EP - 100

JO - Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences

JF - Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences

SN - 1570-0232

ER -