DOI 10.1515/cclm-2013-0786 Clin Chem Lab Med 2014; 52(6): 869–877
Ruijie Yua, Jiaxi Songa, Jia Wu, Dongmei Niu, Lijuan Ma, Chen Zong, Huangxian Ju* and Junjun Wang*
A sensitive chemiluminescence imaging immunoassay for simultaneous detection of serum oxidized lipoprotein(a) and low density lipoprotein Abstract
Background: Oxidized lipoprotein(a) [ox-Lp(a)] and oxidized low density lipoprotein (ox-LDL) levels have been reported to be useful predictors of cardiovascular events. The study developed a chemiluminescence (CL) imaging immunoassay method for simultaneously detecting serum concentrations of ox-Lp(a) and ox-LDL. Methods: Ox-Lp(a) and ox-LDL levels were measured by CL imaging immunoassay using a disposable immunosensor array as the carrier and a charge-coupled device as the detector, and were studied in 46 acute coronary syndromes (ACS) patients, 58 stable coronary artery disease (CAD) and 61 control subjects. Results: This method showed good linear relations (R2 > 0.99) in the concentration range of 2.00 × 10–5– 2.00 × 10–1 and 2.40 × 10–4–2.40 U/mL for ox-Lp(a) and ox-LDL, respectively. The detection limits for ox-Lp(a) and ox-LDL were 2.40 × 10–6 and 3.00 × 10–5 U/mL, respectively. The intra- and inter-assay coefficients of variation (CV) were 4.90%–6.76% and 7.11%–10.06% for ox-Lp(a), and 5.01%–6.04% and 5.47%–9.77% for ox-LDL, respectively. The mean recovery was 99.31% for ox-Lp(a) and 99.57% for ox-LDL, respectively. Significant correlations were observed between ox-Lp(a) levels detected by CL imaging immunoassay and ELISA, and between ox-LDL levels detected by the two methods, respectively. Furthermore, ox-Lp(a) and ox-LDL levels increased in stable CAD, and especially in ACS. Conclusions: The CL imaging immunoassay provided a simple, sensitive and reliable method for the simultaneous determination of serum ox-Lp(a) and ox-LDL. The clinical monitoring ox-Lp(a) and ox-LDL levels may possess distinctly clinical value for assessment of CAD risk. Keywords: chemiluminescence imaging; coronary artery disease; immunoassay; lipoprotein(a); low density lipoprotein; oxidation.
a Ruijie Yu and Jiaxi Song contributed equally to this work. *Corresponding authors: Junjun Wang, Department of Clinical Laboratory, Clinical School of Medicine, Nanjing University, Jinling Hospital, #305 East Zhongshan Road, Nanjing 210002, P.R. China, Phone: +86 25 84815775, Fax: +86 25 84815775, E-mail: [email protected]; and Huangxian Ju, State Key Laboratory of Analytical Chemistry for Life Science, Department of Chemistry, Nanjing University, Nanjing 210093, P.R. China, Phone: +86 25 83593593, Fax: +86 25 83593593, E-mail: [email protected] Ruijie Yu, Jiaxi Song, Jia Wu, Dongmei Niu and Lijuan Ma: Department of Clinical Laboratory, Jinling Hospital, Clinical School of Medicine, Nanjing University, Nanjing, P.R. China Chen Zong: State Key Laboratory of Analytical Chemistry for Life Science, Department of Chemistry, Nanjing University, Nanjing, P.R. China
Introduction Dyslipidemia is known to be closely associated with atherosclerosis. Lipoprotein(a) [Lp(a)], as an atherogenic particle, structurally resembles the low density lipoprotein (LDL) but contains a molecule of apolipoprotein(a) [apo(a)] attached to apoB-100 by a disulfide bond [1]. Elevated plasma concentrations of Lp(a) have been considered as an independent risk factor for atherosclerosis [2, 3]. Apo(a) and apoB proteins of Lp(a) can both be oxidatively modified in vivo [4]. Ox-Lp(a) has been reported to play more potent roles than native Lp(a) in atherosclerosis [5]. Several lines of evidence showed that ox-Lp(a) and ox-LDL induced adhesion molecular expression on monocytes, contributing to their recruitment and adhesion to the endothelium, and subsequently promoted intracellular accumulation of cholesteryl esters in macrophages, leading to their transformation into foam cells [6–9]. Circulating ox-LDL, as a useful marker for identifying patients with atherosclerotic disease, has been reported to be closely associated with the high risk of subsequent cardiovascular events [10–13]. Our previous studies have shown that ox-Lp(a) were present in
Brought to you by | University of Iowa Libraries Authenticated Download Date | 6/7/15 9:05 AM
870 Yu et al.: CL imaging immunoassay for ox-Lp(a) and ox-LDL newborns and children [14], and increased in rheumatoid arthritis patients with excessive cardiovascular events [15]. Elevated ox-Lp(a) levels have been found to be associated with the presence of acute coronary syndromes (ACS) and stable coronary artery disease (CAD), and with the severity of ACS [16, 17]. Currently, circulating ox-LDL and ox-Lp(a) are most often determined by enzyme-linked immunosorbent assay (ELISA) [4, 18–22]. Ox-LDL levels were also measured by chemiluminescent ELISA using a chemiluminescence (CL) detector [23]. Ox-Lp(a) levels were estimated by the degree of oxidized apo(a) and/or apoB of Lp(a) [4, 21, 22]. However, the above assays cannot simultaneously detect the two oxidized lipoproteins, and their sensitivity is relatively low. Recently, CL immunoassay combines good specificity of immunoreaction with high sensitivity of CL detection and has become a powerful analytical method [24, 25]. This technique has been extensively used for development of multiplex immunoassay [26, 27]. The CL-based imaging assay can provide simple, sensitive and high-throughput method for detection when CL is coupled with a charge-coupled device (CCD) as the detector, which has been successfully applied to microbioassay [28, 29]. Our previous work fabricated a disposable protein chip for multiplex immunoassay of four tumor markers [30]. In the present study, we developed a highly sensitive CL imaging immunoassay for simultaneous detection of serum ox-Lp(a) and ox-LDL by combining a disposable immunosensor array with a cooled low-light CCD, and studied serum ox-Lp(a) and ox-LDL levels in ACS and stable CAD patients.
Materials and methods Study subjects The present study included 46 patients with ACS, 58 patients with stable CAD, and 61 control subjects. The control subjects selected from routine health examination were found normal in physical and electrocardiography and laboratory tests, and without diseases such as hyperlipemia, hypertension, diabetes mellitus, or any clinical evident sign of atherosclerosis. The CAD patients in this study were selected from admitted patients under the Department of Cardiology of Jinling Hospital from January 2011 to December 2012, who were undergoing clinically indicated coronary angiography. Angiograms of all the CAD patients showed at least 50% stenosis of one, two, or three coronary arteries. Forty-six patients with ACS included acute myocardial infarction patients and unstable angina with Braunwald classification II or III. Fifty-eight patients with angiographically documented CAD and no cardiac events/procedures for > 1 year were considered to have stable CAD. The exclusion criteria of the CAD patients included mild disease
of angiography (a stenosis of 10%–50% of the luminal diameter in all the three coronary arteries), prior coronary revascularization and the presence of renal disease. In patients with ACS, blood samples were taken on admission. Blood samples were collected at least 12 h after fasting from control subjects and patients with stable CAD. Serum was separated immediately and stored at –70°C until analysis. All laboratory assays were conducted within 1 year of blood sampling. This study protocol was approved by the Ethics Committee of Jinling Hospital, and all the subjects provided written informed consent.
Isolation and oxidation of Lp(a) and LDL Lp(a) was purified from plasma of healthy donors by sequential density ultracentrifugation, followed by a gel-filtration chromatography as described [22]. LDL (1.030
Ruijie Yua, Jiaxi Songa, Jia Wu, Dongmei Niu, Lijuan Ma, Chen Zong, Huangxian Ju* and Junjun Wang*
A sensitive chemiluminescence imaging immunoassay for simultaneous detection of serum oxidized lipoprotein(a) and low density lipoprotein Abstract
Background: Oxidized lipoprotein(a) [ox-Lp(a)] and oxidized low density lipoprotein (ox-LDL) levels have been reported to be useful predictors of cardiovascular events. The study developed a chemiluminescence (CL) imaging immunoassay method for simultaneously detecting serum concentrations of ox-Lp(a) and ox-LDL. Methods: Ox-Lp(a) and ox-LDL levels were measured by CL imaging immunoassay using a disposable immunosensor array as the carrier and a charge-coupled device as the detector, and were studied in 46 acute coronary syndromes (ACS) patients, 58 stable coronary artery disease (CAD) and 61 control subjects. Results: This method showed good linear relations (R2 > 0.99) in the concentration range of 2.00 × 10–5– 2.00 × 10–1 and 2.40 × 10–4–2.40 U/mL for ox-Lp(a) and ox-LDL, respectively. The detection limits for ox-Lp(a) and ox-LDL were 2.40 × 10–6 and 3.00 × 10–5 U/mL, respectively. The intra- and inter-assay coefficients of variation (CV) were 4.90%–6.76% and 7.11%–10.06% for ox-Lp(a), and 5.01%–6.04% and 5.47%–9.77% for ox-LDL, respectively. The mean recovery was 99.31% for ox-Lp(a) and 99.57% for ox-LDL, respectively. Significant correlations were observed between ox-Lp(a) levels detected by CL imaging immunoassay and ELISA, and between ox-LDL levels detected by the two methods, respectively. Furthermore, ox-Lp(a) and ox-LDL levels increased in stable CAD, and especially in ACS. Conclusions: The CL imaging immunoassay provided a simple, sensitive and reliable method for the simultaneous determination of serum ox-Lp(a) and ox-LDL. The clinical monitoring ox-Lp(a) and ox-LDL levels may possess distinctly clinical value for assessment of CAD risk. Keywords: chemiluminescence imaging; coronary artery disease; immunoassay; lipoprotein(a); low density lipoprotein; oxidation.
a Ruijie Yu and Jiaxi Song contributed equally to this work. *Corresponding authors: Junjun Wang, Department of Clinical Laboratory, Clinical School of Medicine, Nanjing University, Jinling Hospital, #305 East Zhongshan Road, Nanjing 210002, P.R. China, Phone: +86 25 84815775, Fax: +86 25 84815775, E-mail: [email protected]; and Huangxian Ju, State Key Laboratory of Analytical Chemistry for Life Science, Department of Chemistry, Nanjing University, Nanjing 210093, P.R. China, Phone: +86 25 83593593, Fax: +86 25 83593593, E-mail: [email protected] Ruijie Yu, Jiaxi Song, Jia Wu, Dongmei Niu and Lijuan Ma: Department of Clinical Laboratory, Jinling Hospital, Clinical School of Medicine, Nanjing University, Nanjing, P.R. China Chen Zong: State Key Laboratory of Analytical Chemistry for Life Science, Department of Chemistry, Nanjing University, Nanjing, P.R. China
Introduction Dyslipidemia is known to be closely associated with atherosclerosis. Lipoprotein(a) [Lp(a)], as an atherogenic particle, structurally resembles the low density lipoprotein (LDL) but contains a molecule of apolipoprotein(a) [apo(a)] attached to apoB-100 by a disulfide bond [1]. Elevated plasma concentrations of Lp(a) have been considered as an independent risk factor for atherosclerosis [2, 3]. Apo(a) and apoB proteins of Lp(a) can both be oxidatively modified in vivo [4]. Ox-Lp(a) has been reported to play more potent roles than native Lp(a) in atherosclerosis [5]. Several lines of evidence showed that ox-Lp(a) and ox-LDL induced adhesion molecular expression on monocytes, contributing to their recruitment and adhesion to the endothelium, and subsequently promoted intracellular accumulation of cholesteryl esters in macrophages, leading to their transformation into foam cells [6–9]. Circulating ox-LDL, as a useful marker for identifying patients with atherosclerotic disease, has been reported to be closely associated with the high risk of subsequent cardiovascular events [10–13]. Our previous studies have shown that ox-Lp(a) were present in
Brought to you by | University of Iowa Libraries Authenticated Download Date | 6/7/15 9:05 AM
870 Yu et al.: CL imaging immunoassay for ox-Lp(a) and ox-LDL newborns and children [14], and increased in rheumatoid arthritis patients with excessive cardiovascular events [15]. Elevated ox-Lp(a) levels have been found to be associated with the presence of acute coronary syndromes (ACS) and stable coronary artery disease (CAD), and with the severity of ACS [16, 17]. Currently, circulating ox-LDL and ox-Lp(a) are most often determined by enzyme-linked immunosorbent assay (ELISA) [4, 18–22]. Ox-LDL levels were also measured by chemiluminescent ELISA using a chemiluminescence (CL) detector [23]. Ox-Lp(a) levels were estimated by the degree of oxidized apo(a) and/or apoB of Lp(a) [4, 21, 22]. However, the above assays cannot simultaneously detect the two oxidized lipoproteins, and their sensitivity is relatively low. Recently, CL immunoassay combines good specificity of immunoreaction with high sensitivity of CL detection and has become a powerful analytical method [24, 25]. This technique has been extensively used for development of multiplex immunoassay [26, 27]. The CL-based imaging assay can provide simple, sensitive and high-throughput method for detection when CL is coupled with a charge-coupled device (CCD) as the detector, which has been successfully applied to microbioassay [28, 29]. Our previous work fabricated a disposable protein chip for multiplex immunoassay of four tumor markers [30]. In the present study, we developed a highly sensitive CL imaging immunoassay for simultaneous detection of serum ox-Lp(a) and ox-LDL by combining a disposable immunosensor array with a cooled low-light CCD, and studied serum ox-Lp(a) and ox-LDL levels in ACS and stable CAD patients.
Materials and methods Study subjects The present study included 46 patients with ACS, 58 patients with stable CAD, and 61 control subjects. The control subjects selected from routine health examination were found normal in physical and electrocardiography and laboratory tests, and without diseases such as hyperlipemia, hypertension, diabetes mellitus, or any clinical evident sign of atherosclerosis. The CAD patients in this study were selected from admitted patients under the Department of Cardiology of Jinling Hospital from January 2011 to December 2012, who were undergoing clinically indicated coronary angiography. Angiograms of all the CAD patients showed at least 50% stenosis of one, two, or three coronary arteries. Forty-six patients with ACS included acute myocardial infarction patients and unstable angina with Braunwald classification II or III. Fifty-eight patients with angiographically documented CAD and no cardiac events/procedures for > 1 year were considered to have stable CAD. The exclusion criteria of the CAD patients included mild disease
of angiography (a stenosis of 10%–50% of the luminal diameter in all the three coronary arteries), prior coronary revascularization and the presence of renal disease. In patients with ACS, blood samples were taken on admission. Blood samples were collected at least 12 h after fasting from control subjects and patients with stable CAD. Serum was separated immediately and stored at –70°C until analysis. All laboratory assays were conducted within 1 year of blood sampling. This study protocol was approved by the Ethics Committee of Jinling Hospital, and all the subjects provided written informed consent.
Isolation and oxidation of Lp(a) and LDL Lp(a) was purified from plasma of healthy donors by sequential density ultracentrifugation, followed by a gel-filtration chromatography as described [22]. LDL (1.030
