| Oxidized lipoproteins are thought to contribute to the development of atherosclerosis.Elevated oxidized lipoprotein?a?[ox-Lp?a?]and oxidized low density lipoprotein?ox-LDL?levels could evaluate the presence and severity of coronary artery disease?CAD?and atherosclerotic plaque stability.Currently,circulating ox-LDL and ox-Lp?a?are most often determined by enzyme-linked immunosorbent assay?ELISA?.Ox-Lp?a?levels were estimated by the degree of oxidized apo?a?or/and apoB of Lp?a?.However,the above assays cannot simultaneously detect the two oxidized lipoproteins,and their sensitivity is relatively low.Objectives:?1?To develop a chemiluminescence?CL?imaging immunoassay method for simultaneously detecting serum concentrations of ox-Lp?a?and ox-LDL.?2?To compare the ox-Lp?a?or ox-LDL levels detected by CL assay and ELISA.?3?To measure serum ox-LDL and ox-Lp?a?concentrations by CL assay,and to investigate ox-LDL and ox-Lp?a?levels in atherosclerotic cardiovascular diseases and evaluate their association with the presence and severity of atherosclerosis lesions and the clinical value in the prediction of atherosclerotic cardiovascular diseases.Methods:?1?The immunosensor array in a 4×12 format was prepared by covalently immobilizing capture antibodies on corresponding sensing sites on a silanized glass chip using screen-printing technology.Under a sandwich immunoassay,the CL signals were collected by a charge-coupled device?CCD?for simultaneous measurement of ox-Lp?a?and ox-LDL levels,using horseradish peroxidase?HRP?as a marker and luminol-hydrogen peroxide?H2O2?as the luminescent substrate.The detection time and immunoassay conditions such as the concentration of capture antibody,dilution of HRP-labeled antibodies and the incubation times were optimized and the linear ranges,precision,accuracy and detection limits were evaluated.?2?Serum ox-Lp?a?and ox-LDL levels were determined by CL assay and ELISAs in 46 acute coronary syndromes?ACS?patients,58 stable coronary artery disease?SCAD?and 61 control subjects,and the correlation analysis was performed.Results:?1?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 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.The detection limits for ox-Lp?a?and ox-LDL were 2.40×10-6 and 3.00×10-5 U/mL at a signal-to-noise ratio of 3,respectively.?2?Compared to the controls,ox-Lp?a?and ox-LDL levels were found increased in both ACS and SCAD patients[ox-Lp?a?,ACS?0.49±0.50 U/mL?;SCAD?0.26±0.30 U/mL?;controls?0.177±0.18 U/mL?;ox-LDL,ACS?4.67±2.73 U/mL?;SCAD?3.100±1.96 U/mL?;controls?1.55±1.00U/mL?;P<0.05],and ox-Lp?a?and ox-LDL concentrations in the ACS patients were significantly higher than those in the SCAD patients?P<0.05?.The univariate analysis revealed that the presence of ox-Lp?a?or ox-LDL was a risk factor for SCAD[ox-Lp?a?,OR 6-00,95%CI 1.04,34.68;ox-LDL,OR 2.29,95%CI 1.62,3.22;respectively],and especially for ACS[ox-Lp?a?,OR 26.51,95%CI 5.00,152.89;ox-LDL,OR 3.08,95%CI 2.14,4.45;respectively].In multivariate analysis,adjusting for age,sex and serum lipid levels,the presence of ox-Lp?a?or ox-LDL was still revealed to be a risk factor for both ACS and SCAD.?3?Compared to the controls,ox-Lp?a?and ox-LDL levels detected by CL assay and ELISAs were found increased in SCAD patients,and especially in ACS.Ox-Lp?a?and ox-LDL concentrations detected by CL assay in the ACS patients were higher than those detected by ELISAs.The correlation analysis revealed that significant correlations were observed between ox-Lp?a?levels detected by CL assay and ELISA?R = 0.739,P<0.001?,and between ox-LDL levels detected by the two methods?R =0.824,P<0.001?,respectively.Furthermore,the high correlation was found between ox-Lp?a?/ox-LDL levels detected by the 2 assays at low concentrations[the low-level group,ox-Lp?a?,R=0.925,P<0.001;ox-LDL,R=0.905,P<0.001;the high-level group,ox-Lp?a?,R=0.654,P<0.001;ox-LDL,R=0.728,P<0.001,respectively].The increase value of CL intensity to oxidized lipoproteins levels ratio of CL assay was greater than the increase value of absorbance to oxidized lipoproteins levels ratio of ELISA at high concentrations[ox-Lp?a?,0.17 a.u./U·mL-1 vs.0.09 AU/U·mL-1;ox-LDL,0.18 a.u./U·mL-1 vs.0.12 AU/U·mL-1,respectively],while they were similar at low concentrations.Receiver operating characteristic curve?ROC?analysis confirmed that the performance of the association of ox-LDL detected by CL assay with ACS and SCAD was superior to that of ox-LDL detected by ELISA?P<0.05?,while the performance of ox-Lp?a?detected by the 2 assays with ACS and SCAD were similar.Conclusions:?1?The CL imaging immunoassay provided high sensitivity,wide linear range and reliable method for the simultaneous determination of serum ox-Lp?a?and ox-LDL.?2?Serum ox-Lp?a?and ox-LDL levels increased in SCAD,and especially in ACS.Significant correlations were observed between ox-Lp?a?levels detected by CL assay and ELISA,and between ox-LDL levels detected by the two methods,respectively.Furthermore,the performances of ox-Lp?a?and ox-LDL detected by CL assay in predicting ACS and SCAD were superior to those detected by ELISAs at high concentrations,which accurately reflected the levels of ox-Lp?a?and ox-LDL and was appropriate for clinical application. |
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