Detection of total and A1c-glycosylated hemoglobin in human whole blood using sandwich immunoassays on polydimethylsiloxane-based antibody microarrays

Huang Han Chen, Chih Hsing Wu, Mei Ling Tsai, Yi Jing Huang, Shu Hui Chen

研究成果: Article

26 引文 (Scopus)

摘要

The percentage of glycosylated hemoglobin A1c (%GHbA1c) in human whole blood indicates the average plasma glucose concentration over a prolonged period of time and is used to diagnose diabetes. However, detecting GHbA1c in the whole blood using immunoassays has limited detection sensitivity due to its low percentage in total hemoglobin (tHb) and interference from various glycan moieties in the sample. We have developed a sandwich immunoassay using an antibody microarray on a polydimethylsiloxane (PDMS) substrate modified with fluorinated compounds to detect tHb and glycosylated hemoglobin A1c (GHbA1c) in human whole blood without sample pretreatment. A polyclonal antibody against hemoglobin (Hb) immobilized on PDMS is used as a common capture probe to enrich all forms of Hb followed by detection via monoclonal anti-Hb and specific monoclonal anti-GHbA1c antibodies for tHb and GHbA1c detection, respectively. This method prevents the use of glycan binding molecules and dramatically reduces the background interference, yielding a detection limit of 3.58 ng/mL for tHb and 0.20 ng/mL for GHbA1c. The fluorinated modification on PDMS is superior to the glass substrate and eliminates the need for the blocking step which is required in commercial enzyme linked immunosorbent assay (ELISA) kits. Moreover, the detection sensitivity for GHbA1c is 4-5 orders of magnitude higher, but the required sample amount is 25 times less than the commercial method. On the basis of patient sample data, a good linear correlation between %GHbA1c values determined by our method and the certified high performance liquid chromatography (HPLC) standard method is shown with R2 > 0.98, indicating the great promise of the developed method for clinical applications.

原文English
頁(從 - 到)8635-8641
頁數7
期刊Analytical chemistry
84
發行號20
DOIs
出版狀態Published - 2012 十月 16

指紋

Glycosylated Hemoglobin A
Microarrays
Blood
Antibodies
Hemoglobins
Hemoglobin
Polysaccharides
Immunosorbents
baysilon
High performance liquid chromatography
Substrates
Medical problems
Assays
Plasmas
Glucose
Glass
Molecules
Enzymes

All Science Journal Classification (ASJC) codes

  • Analytical Chemistry

引用此文

@article{e603e28339c444f5bcd53bed7fd629fc,
title = "Detection of total and A1c-glycosylated hemoglobin in human whole blood using sandwich immunoassays on polydimethylsiloxane-based antibody microarrays",
abstract = "The percentage of glycosylated hemoglobin A1c ({\%}GHbA1c) in human whole blood indicates the average plasma glucose concentration over a prolonged period of time and is used to diagnose diabetes. However, detecting GHbA1c in the whole blood using immunoassays has limited detection sensitivity due to its low percentage in total hemoglobin (tHb) and interference from various glycan moieties in the sample. We have developed a sandwich immunoassay using an antibody microarray on a polydimethylsiloxane (PDMS) substrate modified with fluorinated compounds to detect tHb and glycosylated hemoglobin A1c (GHbA1c) in human whole blood without sample pretreatment. A polyclonal antibody against hemoglobin (Hb) immobilized on PDMS is used as a common capture probe to enrich all forms of Hb followed by detection via monoclonal anti-Hb and specific monoclonal anti-GHbA1c antibodies for tHb and GHbA1c detection, respectively. This method prevents the use of glycan binding molecules and dramatically reduces the background interference, yielding a detection limit of 3.58 ng/mL for tHb and 0.20 ng/mL for GHbA1c. The fluorinated modification on PDMS is superior to the glass substrate and eliminates the need for the blocking step which is required in commercial enzyme linked immunosorbent assay (ELISA) kits. Moreover, the detection sensitivity for GHbA1c is 4-5 orders of magnitude higher, but the required sample amount is 25 times less than the commercial method. On the basis of patient sample data, a good linear correlation between {\%}GHbA1c values determined by our method and the certified high performance liquid chromatography (HPLC) standard method is shown with R2 > 0.98, indicating the great promise of the developed method for clinical applications.",
author = "Chen, {Huang Han} and Wu, {Chih Hsing} and Tsai, {Mei Ling} and Huang, {Yi Jing} and Chen, {Shu Hui}",
year = "2012",
month = "10",
day = "16",
doi = "10.1021/ac301756d",
language = "English",
volume = "84",
pages = "8635--8641",
journal = "Analytical Chemistry",
issn = "0003-2700",
publisher = "American Chemical Society",
number = "20",

}

TY - JOUR

T1 - Detection of total and A1c-glycosylated hemoglobin in human whole blood using sandwich immunoassays on polydimethylsiloxane-based antibody microarrays

AU - Chen, Huang Han

AU - Wu, Chih Hsing

AU - Tsai, Mei Ling

AU - Huang, Yi Jing

AU - Chen, Shu Hui

PY - 2012/10/16

Y1 - 2012/10/16

N2 - The percentage of glycosylated hemoglobin A1c (%GHbA1c) in human whole blood indicates the average plasma glucose concentration over a prolonged period of time and is used to diagnose diabetes. However, detecting GHbA1c in the whole blood using immunoassays has limited detection sensitivity due to its low percentage in total hemoglobin (tHb) and interference from various glycan moieties in the sample. We have developed a sandwich immunoassay using an antibody microarray on a polydimethylsiloxane (PDMS) substrate modified with fluorinated compounds to detect tHb and glycosylated hemoglobin A1c (GHbA1c) in human whole blood without sample pretreatment. A polyclonal antibody against hemoglobin (Hb) immobilized on PDMS is used as a common capture probe to enrich all forms of Hb followed by detection via monoclonal anti-Hb and specific monoclonal anti-GHbA1c antibodies for tHb and GHbA1c detection, respectively. This method prevents the use of glycan binding molecules and dramatically reduces the background interference, yielding a detection limit of 3.58 ng/mL for tHb and 0.20 ng/mL for GHbA1c. The fluorinated modification on PDMS is superior to the glass substrate and eliminates the need for the blocking step which is required in commercial enzyme linked immunosorbent assay (ELISA) kits. Moreover, the detection sensitivity for GHbA1c is 4-5 orders of magnitude higher, but the required sample amount is 25 times less than the commercial method. On the basis of patient sample data, a good linear correlation between %GHbA1c values determined by our method and the certified high performance liquid chromatography (HPLC) standard method is shown with R2 > 0.98, indicating the great promise of the developed method for clinical applications.

AB - The percentage of glycosylated hemoglobin A1c (%GHbA1c) in human whole blood indicates the average plasma glucose concentration over a prolonged period of time and is used to diagnose diabetes. However, detecting GHbA1c in the whole blood using immunoassays has limited detection sensitivity due to its low percentage in total hemoglobin (tHb) and interference from various glycan moieties in the sample. We have developed a sandwich immunoassay using an antibody microarray on a polydimethylsiloxane (PDMS) substrate modified with fluorinated compounds to detect tHb and glycosylated hemoglobin A1c (GHbA1c) in human whole blood without sample pretreatment. A polyclonal antibody against hemoglobin (Hb) immobilized on PDMS is used as a common capture probe to enrich all forms of Hb followed by detection via monoclonal anti-Hb and specific monoclonal anti-GHbA1c antibodies for tHb and GHbA1c detection, respectively. This method prevents the use of glycan binding molecules and dramatically reduces the background interference, yielding a detection limit of 3.58 ng/mL for tHb and 0.20 ng/mL for GHbA1c. The fluorinated modification on PDMS is superior to the glass substrate and eliminates the need for the blocking step which is required in commercial enzyme linked immunosorbent assay (ELISA) kits. Moreover, the detection sensitivity for GHbA1c is 4-5 orders of magnitude higher, but the required sample amount is 25 times less than the commercial method. On the basis of patient sample data, a good linear correlation between %GHbA1c values determined by our method and the certified high performance liquid chromatography (HPLC) standard method is shown with R2 > 0.98, indicating the great promise of the developed method for clinical applications.

UR - http://www.scopus.com/inward/record.url?scp=84869429681&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84869429681&partnerID=8YFLogxK

U2 - 10.1021/ac301756d

DO - 10.1021/ac301756d

M3 - Article

C2 - 22967096

AN - SCOPUS:84869429681

VL - 84

SP - 8635

EP - 8641

JO - Analytical Chemistry

JF - Analytical Chemistry

SN - 0003-2700

IS - 20

ER -