Capillary-based enzyme-linked immunosorbent assay for highly sensitive detection of thrombin-cleaved osteopontin in plasma Shun-ichi Funano a,1 , Terence G. Henares a,1 , Mie Kurata b,c , Kenji Sueyoshi a , Tatsuro Endo a , Hideaki Hisamoto a,⇑ a Department of Applied Chemistry, Graduate School of Engineering, Osaka Prefecture University, Osaka 599-8531, Japan b Department of Pathogenomics, Graduate School of Medicine, Ehime University, Ehime 791-0295, Japan c Department of Cardiology, Thorax Center of Erasmus Medical Center, 3000 CA Rotterdam, The Netherlands article info Article history: Received 10 March 2013 Received in revised form 1 May 2013 Accepted 16 May 2013 Available online 2 June 2013 Keywords: Square glass capillary Enzyme-linked immunosorbent assay (ELISA) Thrombin-cleaved osteopontin (trOPN) abstract In this study, a highly sensitive capillary-based enzyme-linked immunosorbent assay (ELISA) has been developed for the analysis of picomolar levels of thrombin-cleaved osteopontin (trOPN), a potential bio- marker for ischemic stroke, in human plasma. Using a square capillary coated with 8.5 lg/ml anti-human trOPN capture antibody for ELISA, the linear range obtained was 2 to 16 pM trOPN antigen. This concen- tration range was in the detection window of trOPN antigen in plasma samples. Compared with the con- ventional microplate-based ELISA, the current capillary technique significantly reduced the amounts of reagent from milliliter to microliter, reduced the analysis time from 8 to 3 h, and had a better sensitivity and detection limit performance from approximately 50 pM down to 2 pM of trOPN antigen. These results indicate that this capillary-based immunoassay is a potential tool for biomarker detection and may be useful in clinical trials and medical diagnostic applications. Ó 2013 Elsevier Inc. All rights reserved. According to the World Health Organization, approximately 17 million people worldwide die annually of cardiovascular diseases (CVDs), 2 such as heart attack and stroke, despite awareness of the fact that many CVDs could be prevented by lifestyle changes and an- nual medical checkups [1]. This is most likely due to the late diagno- sis of the disease. Therefore, early diagnosis of such health risks by using novel biomarkers may significantly lower their incidence. Atherosclerosis or atherothrombosis is the primary pathophys- iology of CVDs and involves stenosis of the body’s arteries. Clini- cally, it has been reported that atherosclerotic lesions or plaques secrete an extracellular matrix protein, called osteopontin (OPN), in a manner that correlates with plasma OPN levels [2,3]. These findings suggest that OPN is an important component of plaque formation in the arteries. In the presence of thrombin around the atherothrombotic plaque, OPN is cleaved, released, and detected in the bloodstream as thrombin-cleaved osteopontin (trOPN). In the case of ischemic stroke, Kurata and coworkers observed that trOPN has a more distinct distribution around the intra-plaque vessels than OPN, which could also be found in macrophages, endothelial cells, lipid cores, and calcified nodules [4]. Based on their study, which involved at least 100 patients, these authors concluded that trOPN could be a biomarker that reflects the ather- othrombotic status in ischemic stroke. A common microplate- based enzyme-linked immunosorbent assay (ELISA) was used to determine the concentration of trOPN in plasma samples, which is present in picomolar levels. However, in some cases the trOPN levels were below the detection limit of this method. Furthermore, advanced clinical trials may require a very large patient popula- tion, for which a microplate-based ELISA might not be practical be- cause it would require a vast amount of expensive reagents, a long analysis time, and low sensitivity due to the large assay volume. A capillary-based ELISA could circumvent these drawbacks. This format offers nanoliter volume confinement, a short diffusion dis- tance, and a high surface-to-volume ratio. Over recent years, capil- lary-based immunoassays have been used to analyze various antigens such as C-reactive protein [5,6], digoxin [7], Escherichia coli [8], and pesticides [9]. However, most of these capillary immu- nosensors were done in cylindrical capillaries, which are prone to unwanted reflection or refraction of light that may affect optical measurements [10]. Previously, our group used a square glass cap- illary to eliminate the unfavorable features of cylindrical capillaries [11]. We previously demonstrated the use of various capillary immunosensors employing a long capillary that is cut into small sections for analysis [12–14]. Moreover, small sections of capillary biosensors, involving immunosensors and other biosensors, could 0003-2697/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ab.2013.05.021 ⇑ Corresponding author. Fax: +81 72 254 9284. E-mail address: hisamoto@chem.osakafu-u.ac.jp (H. Hisamoto). 1 These authors contributed equally to this work. 2 Abbreviations used: CVD, cardiovascular disease; OPN, osteopontin; trOPN, thrombin-cleaved osteopontin; ELISA, enzyme-linked immunosorbent assay; FDP, fluorescein diphosphate tetraammonium salt; BSA, bovine serum albumin; PBS, phosphate-buffered saline; TBS, Tris-buffered saline. Analytical Biochemistry 440 (2013) 137–141 Contents lists available at SciVerse ScienceDirect Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio