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HomeMILLIPLEX® Multiplex for Luminex® ImmunoassaysEfficient Measurement of Cardiovascular Disease Biomarkers Using Magnetic Bead-Based Multiplex Panels

Efficient Measurement of Cardiovascular Disease Biomarkers Using Magnetic Bead-Based Multiplex Panels

Cardiovascular disease (CVD), particularly atherosclerotic vascular disease, is a leading cause of global mortality. Inflammation pathways play a vital role in the initiation, maintenance, and progression of vascular disease, and there is a strong correlation between inflammation biomarkers (such as acute phase proteins) and prognosis in coronary artery disease. See how multiplex panels are advancing CVD research through the measurement of these inflammation and CVD biomarkers.

What Are Biomarkers of Cardiovascular Disease?

Cardiovascular diseases (CVD) are one of the leading causes of morbidity and mortality. CVD includes any disease that affects the heart, arteries, or veins, but more commonly refers to atherosclerotic conditions. CVD may also coincide with metabolic syndrome.

Biomarkers of cardiovascular disease include fibrinogen, CRP, myeloperoxidase, and more. As researchers hope to facilitate early diagnosis and intervention, interest in the measurement of circulating CVD biomarkers has increased dramatically in the past decade. Most CVD biomarker discovery research has focused on arterial plaque-related conditions. This approach is reasonable because arterial plaque development is chronic and progressive, can be measured by soluble markers, and encompasses the bulk of CVD cases. Atherosclerotic CVD is a chronic inflammation in the arterial walls that leads to plaque formation and continues along the ischemic cascade.

Table 1 describes the CVD biomarkers that are associated with the various stages of CVD. Many analytes apply to multiple stages.

Table 1.Characteristic biomarkers for various stages of cardiovascular disease.

CVD Multiplex Assays

CVD multiplex assays, such as MILLIPLEX® multiplex assays, enable scientists to simultaneously analyze multiple biomarkers in a single small sample volume of serum/plasma or cell/tissue culture derived samples. MILLIPLEX® CVD multiplex assays are available for human samples as well as samples from mouse and rat models of CVD.

MILLIPLEX® immunoassays use the magnetic bead-based Luminex® xMAP® technology. Assays can be read on any Luminex® instrument.

Example of CVD Biomarker Analysis of Serum and Plasma Samples

After verifying these MILLIPLEX® multiplexed assays for sensitivity, dynamic range, and variability, serum and plasma samples from patients with and without diagnosed CVD were tested, and found that many of the CVD biomarkers were significantly elevated in CVD patients, indicating the utility of the assay panels in CVD research.

Materials and Methods

Samples

Serum and plasma samples were obtained from emergency room patients with and without a cardiovascular disease-related diagnosis. Twenty unmatched samples of each matrix were acquired from CVD-negative and CVD-positive patients at the same hospital through a commercial vendor. Basic demographic data (age, gender, and ethnicity) were provided for each sample (Table 2). Specific conditions such as chronic heart failure, coronary artery disease, chronic obstructive pulmonary disease, and hypertension were also supplied if available.

1 = Interquartile range (Q1 value, Q3 value); 2 = Chronic heart failure; 3 = Coronary artery disease; 4 = Chronic obstructive pulmonary disease; 5 = Hypertension; *p<0.01 between CVD and no CVD diagnosis by Mann-Whitney U test

Table 2. Population demographics for serum and plasma samples.

CVD Biomarker Analysis

All 80 samples were analyzed using four MILLIPLEX® Human Cardiovascular Disease Magnetic Bead panels. The multiplex assays were generated using a typical sandwich assay format using analyte-specific, capture antibody-conjugated beads and biotinylated detection antibody. Assays were verified using purified protein standards of known concentrations.

Each panel simultaneously measured multiple CVD biomarkers as shown below:

  • Panel 1 (for neat serum and plasma samples): BNP, NT proBNP, CK-MB, CXCL6, CXCL16, ESM-1, FABP3, FABP4, PlGF, LIGHT, Oncostatin M, and Troponin I
  • Panel 2 (for 1:100 diluted serum and plasma samples): ADAMTS13, D-dimer, GDF15, Myoglobin, sICAM-1, MPO, sP-Selectin, Lipocalin-2/NGAL, sVCAM-1, and SAA
  • Panel 3 (for 1:40,000 diluted serum and plasma samples): A2M, CRP, Fetuin A, AGP, Fibrinogen, sL-Selectin, SAP, Haptoglobin, PF4/CXCL4, and Adipsin
  • Panel 4 (neat serum and plasma samples): sE-Selectin, Follistatin, sPECAM-1, Pentraxin-3, Tissue Factor, Thrombomodulin, and Troponin T

Assays were performed according to the respective protocols. In general, the 96-well assay plate was washed with 200 μL assay buffer per well. To each well was added 25 μL standard/control or buffer, 25 μL matrix (if required) or sample, and 25 μL beads. Plates were incubated overnight with shaking at 4 °C. The assay plate was washed three times with wash buffer. 50 μL detection antibodies were added to each well and incubated for 1 hour at room temperature (RT). After adding 50 μL streptavidin-phycoerythrin (SAPE) to each well, the plate was incubated at RT for 30 minutes. The assay plate was then washed three times with wash buffer and beads resuspended in sheath fluid. All plates were analyzed using the Luminex® 200™ instrument.

Statistical Analysis

Statistical tests were conducted using MiniTab® 16.1.0 software. Due to the small sample sizes and non-normal distribution of the data, the Mann-Whitney U test was used to analyze continuous variables and Fisher’s Exact Test was used for categorical variables. P-values less than 0.05 were noted as statistically significant.

Results

Standard curves for each multiplexed assay panel showed approximately three orders of magnitude in dynamic range. Assay sensitivity for most analytes was in the pg/mL range, and intra-assay and inter-assay coefficients of variation were <10% and <20%, respectively.

Assay Comparison to Other Commercially Available Assays

The concentrations of selected analytes determined using the MILLIPLEX® CVD panels were compared to concentrations determined using other commercially available assays, such as ELISAs and Luminex® bead-based assays, and plotted in Figure 1. A linear regression line was fit to each data set to assess correlation. In all cases, R values exceeded 0.8, indicating a good correlation between these multiplexed assay panels and other commercially available biomarker quantitation assays.

Graphs showing concentrations of selected analytes, such as fibrinogen, determined using the MILLIPLEX® CVD panels compared to concentrations determined using other commercially available assays.

Figure 1.Concentrations of selected analytes determined using the MILLIPLEX® CVD panels compared to concentrations determined using other commercially available assays.

CVD Analyte Multiplex Analysis

Analytes were selected from each panel that showed significant elevation in CVD patients and individual value dot plots were used to visualize the range of analyte concentrations in the individual samples for both CVD and no-CVD groups. In Panel 1, biomarkers ESM-1, FABP3, PlGF, and Troponin I were elevated in CVD patients compared to no-CVD patients (Figure 2).

Graphs showing the individual value plots for selected plasma analytes (ESM-1, FABP3, PlGF, Troponin I) using MILLIPLEX® Human CVD Magnetic Bead Panel 1 (Cat. No. HCVD1MAG-67K); p<0.05 for all analytes.

Figure 2.Individual value plots for selected plasma analytes using MILLIPLEX® Human CVD Magnetic Bead Panel 1; p<0.05 for all analytes.

Among the biomarkers in Panel 2, ADAMTS13 and GDF-15 showed the most noticeable elevation in the majority of CVD patients (Figure 3).

Graphs showing the individual value plots for selected plasma analytes (ADAMTS13, D-DIMER, GDF-15, Myoglobin) using MILLIPLEX® Human CVD Magnetic Bead Panel 2 (Cat. No. HCVD2MAG-67K); p<0.05 for all analytes except for D-dimer.

Figure 3.Individual value plots for selected plasma analytes using MILLIPLEX® Human CVD Magnetic Bead Panel 2; p<0.05 for all analytes except for D-dimer.

C-Related Protein and Adipsin were significantly elevated in CVD plasma samples analyzed using Panel 3 (Figure 4), and Follistatin, PECAM-1, and PTX3 were all elevated in CVD plasma samples analyzed using Panel 4 (Figure 5).

Graphs showing the individual value plots for selected plasma analytes (CRP, AGP, Adipsin) using MILLIPLEX® Human CVD Magnetic Bead Panel 3 (Cat. No. HCVD3MAG-67K); p<0.05 for all analytes except for AGP.

Figure 4.Individual value plots for selected plasma analytes using MILLIPLEX® Human CVD Magnetic Bead Panel 3; p<0.05 for all analytes except AGP.

Graphs showing the individual value plots for selected plasma analytes using MILLIPLEX® Human CVD Magnetic Bead Panel 4 (Cat. No. HCVD4MAG-67K); p<0.05 for all analytes.

Figure 5.Individual value plots for selected plasma analytes using MILLIPLEX® Human CVD Magnetic Bead Panel 4; p<0.05 for all analytes.

Summary

Since CVD has such complex diseases involving multiple organs and systems, multiplexed biomarker analysis is crucial for research into CVD diagnosis and treatment. These magnetic bead-based multiplex assay panels enable accurate, sensitive, reproducible, simultaneous measurement of 39 CVD biomarkers in serum, plasma, and tissue culture samples. In comparison with other commercial assay kits, these new multiplex panels show good correlations. Using these human CVD biomarker assay panels, it was shown that, compared to samples from no-CVD subjects, CVD patient samples showed significantly elevated levels of many CVD biomarkers, such as ESM-1, FABP3, PlGF, Troponin I, ADAMTS13, GDF-15, Myoglobin, CRP, PECAM-1, PTX3, and others.

Due to the increased ease of use, throughput, and hands-free operation of magnetic bead-based assays compared to traditional methods of biomarker quantitation, these MILLIPLEX® multiplex panels have the potential to increase the efficiency of biomarker discovery for CVD research.

Materials

Multiplex CVD Panels

Explore our MILLIPLEX® multiplex CVD panels that were used in this research, plus more. The MILLIPLEX® CVD Panel 3 can measure acute phase proteins. Acute phase proteins are produced in the liver in response to proinflammatory cytokines. The serum concentrations of these proteins change significantly in response to inflammation, and low-grade inflammation has been correlated with cardiovascular disease.

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For Research Use Only. Not For Use In Diagnostic Procedures.

Apolipoprotein Analysis

Apolipoproteins, such as Apo AI, Apo AII, and Apo B, bind to and transport lipids through the bloodstream. Their levels may correlate with the risk of coronary heart disease.

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For Research Use Only. Not For Use In Diagnostic Procedures.

Related Instruments and Software

Find instruments and software that work with MILLIPLEX® assays below.

For Research Use Only. Not For Use In Diagnostic Procedures.