Use of anionic polymer, poly(methyl vinyl ether-maleic anhydride)-coated beads for capture of respiratory syncytial virus

Bioorganic and Medicinal Chemistry Letters

Volumn 19, Issue 15, 2009, Pages 4488-4491

  Sakudo, Akikazu ^a   Baba, Koichi ^b   Tsukamoto, Megumi ^a   Ikuta, Kazuyoshi ^a  

^a OSAKA UNIVERSITY   (Japan)

^b Baba Pediatric Clinic   (Japan)

Author keywords

Anionic polymer; Capture; Magnetic beads; Poly(methyl vinyl ether maleic anhydrate); RSV; Virus concentration

Indexed keywords

MALEIC ANHYDRIDE; NUCLEOCAPSID PROTEIN; POLYMETHYLVINYLETHER MALEIC ANHYDRIDE; UNCLASSIFIED DRUG; VIRUS FUSION PROTEIN;

ARTICLE; CHILD; CHROMATOGRAPHY; CONTROLLED STUDY; ENZYME LINKED IMMUNOSORBENT ASSAY; FEMALE; HUMAN; HUMAN CELL; MAGNETIC FIELD; MALE; NOSE MUCUS; NUCLEOTIDE SEQUENCE; POLYMERIZATION; PRESCHOOL CHILD; RESPIRATORY SYNCYTIAL PNEUMOVIRUS; REVERSE TRANSCRIPTION POLYMERASE CHAIN REACTION; VIRUS DETECTION; VIRUS GENOME; WESTERN BLOTTING;

ANIONS; ANTIVIRAL AGENTS; BLOTTING, WESTERN; CHEMISTRY, PHARMACEUTICAL; CHROMATOGRAPHY; DRUG DESIGN; ENZYME-LINKED IMMUNOSORBENT ASSAY; HUMANS; MALEATES; NUCLEOCAPSID PROTEINS; POLYETHYLENES; POLYMERS; RESPIRATORY SYNCYTIAL VIRUS INFECTIONS; RESPIRATORY SYNCYTIAL VIRUSES; REVERSE TRANSCRIPTASE POLYMERASE CHAIN REACTION;

RESPIRATORY SYNCYTIAL VIRUS; RICE STRIPE VIRUS;

EID: 67650002789     PISSN: 0960894X     EISSN: None     Source Type: Journal    
DOI: 10.1016/j.bmcl.2009.05.127     Document Type: Article

References (23)

1. World Health Organization. Available via internet. http://www.who.int/vaccine_research/diseases/ari/en/index2.html. Accessed 21 April 2009.
2. Kuroiwa Y., Nagai K., Okita L., Ukae S., Mori T., Hotsubo T., and Tsutsumi H. J. Clin. Microbiol. 42 (2004) 4812
3. Kittigul L., Khamoun P., Sujirarat D., Utrarachkij F., Chitpirom K., Chaichantanakit N., and Vathanophas K. Mem. Inst. Oswaldo Cruz 96 (2001) 815
4. Sanyal D., Kudesia G., and Corbitt G. J. Med. Microbiol. 35 (1991) 291
5. Trepanier P., Payment P., and Trudel M. J. Virol. Methods 3 (1981) 201
6. Kobayashi S., Natori K., Takeda N., and Sakae K. Microbiol. Immunol. 48 (2004) 201
7. Roth W.K., Weber M., and Seifried E. Lancet 353 (1999) 359
8. Monozide 300 nm-diameter magnetic particles (reducing sedimentation and offering a broad binding surface) with a high ferrite content (allowing face separation under a magnetic field) were prepared by grafting of poly(MVE-MA) (CAS No. 9011-16-9) in dimethyl sulfoxide/phosphate buffer 5/95 solution for 3 h at 37 °C (Flavigny, E.; Gaboyard, M.; Merel, P.; Fleury, H. Abstract of Papers, 104th General Meeting, American Society for Microbiology: New Orleans, LA, 2004; Abstract 166). The anionic magnetic beads, Viro-adembeads, available commercially (Ademtech, Pessac, France) were used in the present study.
9. Clinical nasal aspirates were collected from pediatric patients at the Baba pediatric clinic. The method of collection was described previously (Baba, K. Japan Patent 2008-119552A, 2008). Briefly, saline was introduced into the nasal cavity, and fluid was collected using a nasal aspirator, Belvital (Melisana, Nogent-sur-Marne, France). In order to remove cell debris, the nasal fluid was filtered using a stainless steel mesh [200 grids per inch (25.4 mm)]. Nasal aspirates similarly collected from healthy donors were also used. The research project for the development of diagnostic methods for respiratory infectious diseases was approved by the Ethics Committee of the Research Institute for Microbial Diseases in Osaka University and written informed consent was obtained from the patients and healthy donors.
10. Viral capture of RSV from nasal aspirates using poly(MVE-MA) was performed as follows. Briefly, after two washes with binding buffer, anionic magnetic beads (50 μl) were further washed twice with phosphate-buffered saline (PBS). Then, 20 μl of nasal aspirate diluted with 500 μl of PBS was added to the washed beads and incubated for 20 min at room temperature. Tubes containing the magnetic beads were set under a magnet field. Then, the beads were subjected to magnet separation by discarding the supernatant and washed three times with PBS. The washed beads were resuspended with PBS (20 μl) and subjected to further analyses. After the separation, four fractions were obtained as follows: a beads fraction (20 μl of anionic magnetic beads after incubation with nasal aspirate) (BD), the sample before incubation with the beads [20 μl of nasal aspirate (20 μl) diluted with PBS (500 μl)] (BF), supernatant after the incubation and wash (20 μl of supernatant) (SP), and total sample containing the same quantity (20 μl) of nasal aspirate as BD (20 μl of nasal aspirate without any treatment) (TL) (Sakudo, A.; Baba, K.; Tsukamoto, M.; Sugimoto, A.; Okada, T.; Kobayashi, T.; Kawashita, N.; Takagi, T.; Ikuta, K. Bioorg. Med. Chem. 2009, 17, 752).
11. RT-PCR. Firstly, viral RNA from beads or aliquots of samples was extracted with the QIAamp Viral RNA mini kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. To extract RNA from the magnetic beads, lysis buffer was added. Then the beads were removed after the lysis step. RNA was eluted in 60 μl of nuclease-free water. For the RT-reaction, random primers were used. After incubation at 25 °C for 10 min, RNA was reverse-transcribed at 65 °C for 50 min, followed by denaturation of the enzyme at 85 °C for 5 min. The diluted cDNA was amplified in a reaction mixture containing primers, Ex Taq (Takara bio Inc., Otsu, Japan), and Ex Taq buffer under conditions of 40 cycles of 94 °C for 1 min, 60 °C for 1 min, and 72 °C for 1 min. PCR was carried out using the following primers for the RSV fusion protein gene: RSVfusion-F: 5′-TTA ACC AGC AAA GTG TAA GA-3′; RSVfusion-R: 5′-TTT GTT ATA GGC ATA TCA TTG-3′.
12. Sakudo A., Baba K., Tsukamoto M., Sugimoto A., Okada T., Kobayashi T., Kawashita N., Takagi T., and Ikuta K. Bioorg. Med. Chem. 17 (2009) 752
13. ELISA for the RSV antigen was performed using a SERION ELISA antigen Quadrogen kit for RSV (Serion Immundiagnostica & Institut Virion/Serion GmbH, Würzburg, Germany). RSV concentration was estimated by comparison with purified RSV (Hytest Ltd, Turku, Finland).
14. Clavet C.R., Margolin A.B., and Regan P.M. J. Virol. Methods 119 (2004) 121
15. Jothikumar N., Cliver D.O., and Mariam T.W. Appl. Environ. Microbiol. 64 (1998) 504
16. Satoh K., Iwata A., Murata M., Hikata M., Hayakawa T., and Yamaguchi T. J. Virol. Methods 114 (2003) 11
17. Uchida E., Sato K., Iwata A., Ishii-Watabe A., Mizuguchi H., Hikata M., Murata M., Yamaguchi T., and Hayakawa T. Biologicals 32 (2004) 139
18. Uchida E., Kogi M., Oshizawa T., Furuta B., Satoh K., Iwata A., Murata M., Hikata M., and Yamaguchi T. J. Virol. Methods 143 (2007) 95
19. Iwata A., Satoh K., Murata M., Hikata M., Hayakawa T., and Yamaguchi T. Biol. Pharm. Bull. 26 (2003) 1065
20. Sakudo A., and Ikuta K. Biochem. Biophys. Res. Commun. 377 (2008) 85
21. Flavigny, E.; Gaboyard, M.; Merel, P.; Fleury, H. Abstract of Papers, 104th General Meeting, American Society for Microbiology: New Orleans, LA, 2004; Abstract 166.
22. Nicholls J.M., Chan R.W., Russell R.J., Air G.M., and Peiris J.S. Trends Microbiol. 16 (2008) 149
23. Hallak L.K., Kwilas S.A., and Peeples M.E. Methods Mol. Biol. 379 (2007) 15