The resulting magnetic beads were termed antibody-integrated magnetic beads (Fig. to introduce amino groups onto the surface of the GrMNPs. The GrMNPs were then conjugated with an antibody against DENV, and the antibody-integrated magnetic beads were assessed for their ability to capture DENV. Beads incubated in a cell culture medium of DENV-infected mosquito cells were separated from the supernatant by applying a magnetic field and were then washed. The adsorption of DENV serotypes 1C4 onto the beads was confirmed using reverse transcription-polymerase chain reaction, which detected the presence of DENV genomic RNA on the GrMNPs. The methodology described in the present AVE5688 study, which employed the plasma-functionalization of GrMNPs to enable antibody-integration, represents a significant improvement in the detection of DENV. antibodies (12,16,17). On the basis of this background, the present study was performed to expand on previous results examining the influenza virus to investigate DENV via the immobilization of anti-DENV antibody onto the functionalized surface of GrMNPs. The modified GrMNPs were then assessed for their ability to capture DENVs, and the concentrated virus was then detected in combination with a PCR-based amplification procedure. Materials and methods Plasma-functionalized GrMNPs and production of antibody-integrated magnetic beads The graphite-encapsulated iron compound nanoparticles were prepared using an arc discharge method by applying a 150C200 A direct current at ~20 V between an anode and cathode, as described previously (15). A graphite electrode, molded using graphibond-551R with Fe2O3 powder, was used as the anode. On the opposite side, a graphite rod (50 mm??10 mm; 99.9%) was used as the cathode. The resulting graphite-encapsulated iron compound nanoparticles were then exposed to plasma, which was produced using an RF power supply (18,19) in an atmosphere containing ammonia at 13.56 MHz and 80 W via a matching network (18,19). Initial pretreatment was performed for 10 min using Ar plasma, followed by 2 min of ammonia plasma post-treatment for amino group introduction. During the experiments, the gas pressure was maintained at 50 Pa. The amino groups on the surface of the magnetic beads were then further labelled with 0.3 em /em M of the coupling agent, em AVE5688 N /em -succinimidyl 3-(2-pyridyldithio) propionate (SPDP; Dojindo Laboratories, Kumamoto, Japan) at pH 7C8. A human mono-clonal antibody (clone no. AVE5688 D23-1G7C2) recognizing the first domain II fusion region of the DENV envelope glycoprotein (E) (20) was reduced using dithiothreitol (DTT), resulting in breakage of the S-S bonds and generation of S-H groups. The D23-1G7C2 antibody was produced from hybridomas using peripheral blood mononuclear cells from patients in the acute phase of dengue fever 5 days following the onset of illness, and exhibits neutralizing activity against DENV1-4 (20). The S-H groups on the antibody were then reacted with the SPDP-NH2-magnetic beads, resulting in covalent crosslinking of the antibody onto the surface of the beads. The resulting magnetic beads were termed antibody-integrated magnetic beads (Fig. 1). Open in a separate window Figure 1 Schematic representation of the ammonia plasma-treated GrMNPs and their binding to anti-DENV antibody, resulting in production of antibody-integrated magnetic beads. The surfaces of the GrMNPs were reacted with ammonia plasma, produced using a radiofrequency power supply, resulting in the introduction of amino groups. SPDP was reacted with the amino group-functionalized magnetic beads (NH2-beads) at pH 7-8. Anti-DENV antibody (D23-1G7C2) was reduced using dithiothreitol, resulting in the breakage of S-S-bonds and the generation of S-H groups. The S-H group of the antibody was then further reacted with the SPDP-NH2-magnetic beads. The resultant magnetic beads were termed anti-body-integrated magnetic beads. GrMNPs, graphite-encapsulated magnetic nanoparticles; DENV, dengue virus; SPDP, em N /em -succinimidyl 3-(2-pyridyldithio) propionate. Cell culture and virus A C6/36 cell culture (American Type Culture Collection, Manassas, VA, USA), derived from em Aedes albopictus /em , was maintained in Leibovitz L15 medium (Thermo Fisher Scientific, Inc., Waltham, MA, USA) containing 0.3% tryptose phosphate broth (TPB) and 10% fetal calf Rabbit Polyclonal to MRPL54 serum (FCS; Wako Pure Chemical Industries, Ltd., Osaka, Japan). The laboratory DENV strains (21), DENV1 (Mochizuki strain), DENV2 (16681 strain), DENV3 (80-2 strain) and DENV4 (H241 strain), were used to infect the C6/36 cell cultures. The C6/36 cells were cultured to ~80% confluence, then infected with the DENVs at a multiplicity of infection of 0.1 in Leibovitz L15 medium containing 0.3% TPB and 2% FCS, and were incubated for 3 days at 28C. The medium was then collected and used for viral capture experiments. DENV capture The capture of DENV1-4 was performed as follows. Briefly, 10 em /em l of the magnetic beads were washed twice with phosphate-buffered saline (PBS). A 10 em /em l sample of medium from uninfected (Mock) or DENV-infected cell cultures were added to the washed beads with 1 ml PBS, and the tube was incubated for 15 min at room temperature..