Chemiluminescence immunoassay for sensing lipoprotein-associated phospholipase A2 in cardiovascular risk evaluation
Introduction
Lipoprotein-associated Phospholipase A2 (Lp-PLA2) is a vascular-specific inflammatory enzyme belonging to the A2 Phospholipase superfamily [1]. It is predominantly produced by macrophages, lymphocytes and foam cells in atherosclerotic plaques and expressed in higher concentrations in advanced atherosclerotic lesions than early-stage lesions [[2], [3], [4]]. Lp-PLA2 can hydrolyze oxidized phosphatidylcholine on low-density lipoprotein (LDL) particles within the arterial intima. This biochemical reaction generates lysophosphatidylcholine and oxidized free fatty acids, and both of them are potent pro-inflammatory products contributing to the formation of atherosclerotic plaques [[5], [6], [7]]. Furthermore, many studies have demonstrated that Lp-PLA2 has demonstrated modest intra- and inter-individual variation, independent from traditional cardiovascular risk factors and multifarious inflammatory biomarkers, and substantially less than C-reactive protein (CRP). Elevated levels of Lp-PLA2 in human plasma and serum is a strong risk factor for coronary heart events, a finding that to be used in conjunction with clinical evaluation and patient risk assessment as an aid in predicting risk for coronary heart disease associated with atherosclerosis [[8], [9], [10], [11], [12]].
With the process to ensure Lp-PLA2 as a biomarker to evaluate cardiovascular risk, the development of detection methods for it is ongoing synchronously. At present, methods for determination of Lp-PLA2 mainly involve spectrophotometry, latex-enhanced turbidimetry, radioimmunoassay (RIA), and enzyme-linked immunosorbent assay (ELISA) [[13], [14], [15], [16], [17], [18]]. Among them, PLAC ELISA is the most common used method and there is one FDA approved product on the market. However, one important limitation for ELISA is its relatively low sensitivity due to the insufficient capture of antigen to the surface-anchored antibody in a heterogeneous system [19]. In clinic, high sensitivity is important, which can satisfy the demand of detecting the low abundance biomarkers in the early stages of diseases or after therapeutic interventions [20]. In addition, multiple steps of incubation and washing which are requiring significant manual processing are unavoidable in a typical ELISA protocol. As a consequence, the entire process usually takes several hours to days to obtain the results [21,22]. Considering the importance of Lp-PLA2 in predicting cardiovascular events in clinic, a highly-sensitive, straightforward and automated strategy for Lp-PLA2 detection is thus in urgent needs.
To address this issue, we design a sandwich chemiluminescent immunoassay to measure Lp-PLA2 through combining magnetic separation and chemiluminescence, which is the first chemiluminescent application for Lp-PLA2 (Scheme 1). We prepare magnetic beads functionalized with anti-Lp-PLA2 monoclonal antibody (MBs-Ab2) and anti-Lp-PLA2 monoclonal antibody labelled with N-(4-Aminobutyl)-N-ethylisoluminol (ABEI) (Ab1-ABEI). The functionalized antibodies are stable for minimum four weeks at 2–8 °C. Through one step incubation of Ab1-ABEI, Lp-PLA2 in the sample, and MBs-Ab2, the MBs complex is formed. After magnetic separation and luminous substrate addition, chemiluminescent signals are generated and the intensities of which correlate with the concentration of Lp-PLA2. The total time to perform this rapid assay is 17 min. We validate this platform and apply it for Lp-PLA2 quantification in clinical serum samples.
Section snippets
Materials
Carboxylated magnetic beads were from micromod GmbH ihrem Kundenkreis. ABEI, Proclin 300, MES (2-[N-morpholino] ethane sulfonic acid), 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC), N-Hydroxysuccinimide (NHS) were purchased from Sigma Aldrich. BSA, Tris and Phosphate-buffered saline (PBS, pH = 7.2) buffer were from Shanghai Sangon Company. NaN3, Tween-20 and chemiluminescent substrates (sodium hydroxide and H2O2) were bought from Sinopharm Chemical Reagent Co., Ltd. The
The sample type of Lp-PLA2 assay
The concentrations of the samples in tubes without additives were applied as controls to calculate the deviation of data for gel separation serum, heparin plasma and EDTA-2K plasma. The quantitative values of samples collected using heparin or EDTA-2K plasma collection tubes were lower than that using the additive-free blood collection tubes (Fig. 1B, C). Meanwhile, the deviation of data for heparin plasma and EDTA-2K plasma was relatively larger than that for gel separation serum (Fig. 1). To
Discussion
A lot of guidelines for Cardiovascular disease risk, such as 2010 ACCF/AHA guidelines, 2016 ESC guidelines and 2013 ACCF/AHA guidelines [[29], [30], [31]], support the statement that Lp-PLA2 has recently emerged as an independent risk factor with high consistency and precision for plaque rupture and atherothrombotic events and it can indicate vascular inflammation specifically. Accordingly, we develop a chemiluminescent immunoassay to measure Lp-PLA2 through combining magnetic separation and
Conclusions
We have developed a straightforward, automated, rapid, accurate and precise method to detect Lp-PLA2 by magnetic separation integrated with chemiluminescence. The method adopts a one-step process by mixing MBs, antigen and antibody at one time to enable the sample to answer time within 17 min. It is also possible to utilize an automated instrument to handle and measure the samples which effectively reduce human errors and ensure repeatability. We tested 122 clinical samples, showing good
Conflicts of interest
The authors declare no conflicts of interest.
Funding
Our research is supported by National Science Foundation of China (81701789 and 81702102), Natural Science Foundation of Guangdong Province (2017A030310361) and Shenzhen Basic Research Project (JCYJ20170307153359306, JCYJ20170818144006925).
Acknowledgments
We are grateful to Dr. Zhen Zhao at Cornell University for helpful discussion. We thank Jinyun Yuan and Xiaopeng Wang from New Industries Biomedical Engineering Co. Ltd. for help in the experiments.
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