Indirect capillary electrophoresis immunoassay of membrane protein in extracellular vesicles

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Highlights

  • A sensitive capillary electrophoresis immunoassay for a membrane protein, CD63, in extracellular vesicles was developed.

  • A novel concept for an indirect capillary electrophoresis immunoassay is proven by the determination of CD63.

  • CD63 immobilized on extracellular vesicles was quantified for the first time.

  • This method requires low consumption of both reagents and samples.

Abstract

Extracellular vesicles (EVs) exist in biological fluids such as blood, urine, and cerebrospinal fluid, and these have shown promise for use as biomarkers of cancers. Conventional methods for determination of EVs include direct detection via enzyme-linked immunosorbent assay and detection of their membrane proteins via western blotting. These techniques, however, have individual shortcomings in terms of the need for large sample consumption, processes that are time-consuming, and a lack of the capacity for quantification. In this study, we developed a method to determine the EV membrane protein, CD63, by coupling capillary electrophoresis immunoassay with laser-induced fluorescence (CEIA-LIF). In this process, the EVs were isolated from a culture medium and were subsequently reacted with a fluorescently labeled anti-CD63 antibody to form a CD63 complex localized on the surface of EVs. After removing the EVs containing the CD63 immune complex by centrifugation, the supernatant containing the free fluorescent antibody was injected into a capillary to serve as a sample. A decrease in the peak area of the free fluorescent antibody became apparent when the amount of EVs was increased while that of the fluorescent antibody remained constant. The peak areas were decreased proportionally against the increased amounts of EVs. The concentration of the CD63 could then be estimated based on the slope of the linear relationship. This study is the first to quantify CD63 immobilized on EVs via CEIA-LIF, which is a novel method with the potential to determine membrane proteins localized on the surface of EVs.

Introduction

Numerous recent studies have focused on characterizing and analyzing extracellular vesicles (EVs) in order to clarify their functions, roles, and utilities in the fields of biochemistry and medical science. Most cells produce EVs with a diameter of 50 to 150 nm [1]. The beginning of research on EVs can be traced back to the 1980s when it was discovered that red blood cells release EVs containing transferrin during the maturation process [2]. That discovery attracted much attention from researchers, and important roles of EVs have been elucidated—their biogenesis [3], their cellular recognition [4], and their uptake [5]. EVs could also play the role of biomarkers for early-stage cancer diagnosis [6], [7] because their content reflects specific information about cells. For example, the EVs in urine contain many kinds of proteins related to kidney disease so that the urine-derived EVs could be a potential biomarker for that disease [8], [9]. Furthermore, EVs in the body encapsulate various types of biomolecules such as proteins, carbohydrates, and nucleic acids, and they incorporate membrane proteins on their surface [10], [11], [12], which could play a role in cancer metastasis.

Several analytical methods have permitted the determination of EVs, however, the determination of membrane proteins on EVs remains difficult. EVs are analyzed by methods that include enzyme-linked immunosorbent assay (ELISA) [13], surface plasmon resonance (SPR) [14], and western blotting [15] that are based on the immunological reactions of exosomal proteins. In addition to these methods, nanoparticle tracking analysis is available for the quantification of EVs and to identify the physical manifestations without labeling the proteins of EVs [16], [17]. More recently, capillary electrophoresis (CE) was applied to the determination and characterization of EVs [18], [19], [20], [21]. Among these methods, only western blotting permits the analysis of the proteins contained in EVs, but qualitative results require a large sample amount.

In 1991, Nielsen introduced the concept of capillary electrophoresis immunoassay (CEIA), which uses the CE technique to visualize the immunocomplex products that form between an antigen and its corresponding antibody [22]. In this method, a mixture of the antigen and antibody is injected into the end of a capillary to quickly separate the immune complex from the free antigen (or free antibody). If this method could be used to quantify the membrane proteins in EVs, it would lessen the time for analysis and require a smaller volume for samples compared with the use of western blotting. Furthermore, the coupling of CEIA with laser-induced fluorescence (LIF) enables the highly sensitive detection of fluorescent molecules because lasers are suitable for probing a volume as small as several nano-liters, which is why LIF is compatible with CEIA [23].

Transmembrane proteins are common and are often rich in EVs, which makes them suitable as markers for EVs [24], [25]. Among them, CD9, CD63, and CD81 are well-known members of the family of tetraspanin proteins. These proteins have a structure made up of four transmembrane domains [26]. Despite the fact that these proteins are well known as major EV's markers, developing a method for their determination has remained a challenging task.

Conversely, we previously demonstrated that CEIA-LIF permits the determination of the multidrug resistance-associated protein MPR1, which is a protein localized on cell membranes [27]. During CEIA-LIF, the MRP1 protein was dissolved in a surfactant solution and reacted with a fluorescently labeled antibody, which was followed by a subsequent separation of the free antibody from the immune complex. The concentration of MRP1 was estimated from the peak area of the immune complex at sub nM levels. The results indicated that CEIA-LIF shows promise as a technique that could be used to determine membrane proteins using only a small sample.

In this study, we proposed a novel CEIA-LIF method for the determination of the membrane protein CD63 in EVs. In the proposed method, EVs were isolated from a culture medium and suspended in a phosphate buffer solution. The suspension was reacted with a fluorescent antibody, a conjugate of anti-CD63 and R-phycoerythrin, to form an immune complex with CD63 on the EVs. After centrifugation of the reaction mixture, the free fluorescent antibody in the supernatant was separated via CE-LIF. The amount of CD63 on the EVs was determined successfully from the relationship between the amount of the EVs and the peak areas of the free fluorescent antibody.

Section snippets

Chemicals

All reagents used in this study were of analytical grade. Deionized water was prepared by means of an Elix water purification system (Millipore Co. Ltd., Molsheim, France). Dulbecco's phosphate-buffered saline (DPBS) was purchased from Thermo Fisher Scientific (Yokohama, Japan). An exosome precipitation solution, ExoQuick-TC, was obtained from Funakoshi Co., Ltd. (Tokyo, Japan). Other reagents were obtained from Wako Pure Chemicals (Osaka, Japan).

Cell culture

HeLa was obtained from the JCRB Cell Bank of

Selection of the internal standard

Because the sample injection was performed manually using a siphon method by lifting of the inlet vial, it was difficult to precisely inject a constant amount of the sample. Therefore, an optimal internal standard was needed in order to normalize the injected amount of the sample for quantitative analysis. Fig. 2 shows the electropherograms of the baseline, fluorescent antibody, fluorescein, rhodamine B, and fluorescent antibody using either fluorescein or rhodamine B as a fluorescent internal

Conclusions

Herein, we described the first use of indirect CEIA-LIF to quantify the EV membrane protein, CD63. The present method seems semi-quantitative due to uncertainties such as the concentration of the fluorescent antibody, the purity of the EVs, and the possible lysis of EVs. As far as we could ascertain, however, this report of the present indirect immunoassay is the first to quantify CD63 molecules immobilized on the membranes of EVs. The proposed method was used to estimate the amount of

CRediT authorship contribution statement

Yumeki Tani: Methodology, Validation, Formal analysis, Investigation, Resources, Data curation, Writing - original draft, Visualization. Takashi Kaneta: Conceptualization, Methodology, Formal analysis, Investigation, Resources, Writing - review & editing, Visualization, Supervision, Project administration, Funding acquisition.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

This research was supported by JSPS KAKENHI Grants Number JP17H05465, JP19H04675, and JP20H02766. YT is thankful for the financial support of this research from The Yakumo Foundation for Environmental Science.

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