Capturing and concentrating adenovirus using magnetic anionic nanobeads

Abstract

We recently demonstrated how various enveloped viruses can be efficiently concentrated using magnetic beads coated with an anionic polymer, poly(methyl vinyl ether-maleic anhydrate). However, the exact mechanism of interaction between the virus particles and anionic beads remains unclear. To further investigate whether these magnetic anionic beads specifically bind to the viral envelope, we examined their potential interaction with a nonenveloped virus (adenovirus). The beads were incubated with either adenovirus-infected cell culture medium or nasal aspirates from adenovirus-infected individuals and then separated from the supernatant by applying a magnetic field. After thoroughly washing the beads, adsorption of adenovirus was confirmed by a variety of techniques, including immunochromatography, polymerase chain reaction, Western blotting, and cell culture infection assays. These detection methods positively identified the hexon and penton capsid proteins of adenovirus along with the viral genome on the magnetic beads. Furthermore, various types of adenovirus including Types 5, 6, 11, 19, and 41 were captured using the magnetic bead procedure. Our bead capture method was also found to increase the sensitivity of viral detection. Adenovirus below the detectable limit for immunochromatography was efficiently concentrated using the magnetic bead procedure, allowing the virus to be successfully detected using this methodology. Moreover, these findings clearly demonstrate that a viral envelope is not required for binding to the anionic magnetic beads. Taken together, our results show that this capture procedure increases the sensitivity of detection of adenovirus and would, therefore, be a valuable tool for analyzing both clinical and experimental samples.

Keywords:

• anionic polymer
• virus concentration
• adenovirus
• capture
• magnetic beads
• poly(methyl vinyl ether-maleic anhydrate)

Supplementary materials

Figure S1 The concentrating procedure using anionic polymer-coated magnetic beads increases the sensitivity of immunochromatography for the detection of adenovirus.

Notes: Samples containing adenovirus below the detection limit for immunochromatography (Quick Navi-Adeno) (5.0×10² PFU/μL) were subjected to magnetic separation using poly(MVE-MA)-coated magnetic beads. The BD was then solubilized with lysis buffer and subjected to a conventional immunochromatography procedure. The presence of adenovirus hexon protein was interpreted on the basis of the presence or absence of a test line (T). A positive control was also included (C). Our results show the incorporation of the magnetic bead concentrating step enables the identification of adenovirus by immunochromatography in samples that would otherwise be below the detection limit for this method.

Abbreviations: BD, bead fraction; poly(MVE-MA), poly(methyl vinyl ether-maleic anhydrate); BF, supernatant after incubation with the beads; PFU, plaque forming units.

Figure S2 Adsorption of adenovirus onto anionic polymer-coated magnetic beads under different pH conditions.

Notes: Recombinant adenovirus Axcw2 (20 μL) was diluted with 500 μL of HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) buffer (pH 6.0–8.0), which was prepared by adding different volumes of 0.1 M NaOH to 0.1 M HEPES solution, and then incubated with poly(MVE-MA)-coated magnetic beads. After magnetic separation of the beads, Quick Navi-Adeno was used for the detection of adenovirus hexon protein by immunochromatography. The presence of adenovirus hexon protein was interpreted on the basis of the presence or absence of a test line (T). A positive control was also included (C). Samples were divided into the following categories: BD, sample before incubation with the beads (BF), supernatant after incubation with the beads (SP), and samples containing the same quantity of adenovirus as in the BD (total fraction, TL). All samples were solubilized with lysis buffer and subjected to immunochromatography.

Abbreviations: BD, bead fraction; poly(MVE-MA), poly(methyl vinyl ether-maleic anhydrate); HEPES, (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid).

Acknowledgments

The authors thank Mr Satoru Shigeno and Mr Shinya Iriguchi for technical assistance. This work was supported by a Grant-in-Aid for the Promotion of Basic Research Activities for Innovative Biosciences from Bio-oriented Technology Research Advancement Institution (BRAIN) and the Japan Science and Technology Agency (JST) as well as a Grant-in-Aid for Scientific Research on Innovative Areas from the Japan Society for the Promotion of Science and a Grant-in-Aid from Science and Technology Research Promotion Program for Agriculture, Forestry, Fisheries, and Food Industry.

Disclosure

The authors report no conflicts of interest in this work.