Several strains of influenza A and B viruses were used: A/Osaka/168/09 and A/Suita/01/09 as H1N1 pdm; A/Sw/Hokkaido/2/81 as swine H1N1; A/Yamagata/32/89, A/Beijing/262/95, A/New Caledonia/20/99, and A/Brisbane/59/07 as seasonal H1N1; A/Aichi/2/68, A/Guizhou/54/89, A/Wyoming/2/03, A/Hiroshima/52/05, and A/Urguay/16/07 as seasonal H3N2; and B/Malaysia/2506/04 and B/Florida/4/06 as seasonal influenza B virus. A/Osaka/168/09 was provided by Saeko Morikawa, Osaka Prefectural Institute of Public Health, Osaka, Japan.
BALB/c mice (4 weeks old, female) were intraperitoneally immunized several times with A/Osaka/168/09 or A/Suita/01/09 viral particles which had been partially purified from the culture fluid of infected MDCK cells by ultracentrifugation. Three days after the last immunization, the spleen cells from the mice were used to prepare hybridomas.
Preparation of hybridomas.
The spleen cells from immunized mice were fused with PAI myeloma cells, as described previously (27
). Specific antibody production was screened on the basis of reactions with A/Osaka/168/09 and A/Suita/01/09 but no reaction with A/New Caledonia/20/99 by indirect immunofluorescence assay (IFA). The cells in the wells producing the specific antibody were cloned by limiting dilution and then subjected to secondary screening as described above.
IFA was performed as detailed previously (24
). Briefly, 2.5 × 104
MDCK cells per well in a 96-well plate were infected with various viruses. After 6 to 12 h, the cells were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) and permeabilized with 0.1% Triton X-100 in PBS. The bound antibody was visualized by a further reaction with an Alexa Fluor 488-conjugated secondary antibody (1:1,000; Invitrogen). For the staining of infected MDCK cells with positive-control MAbs, C179 and C43 were used as anti-influenza A virus HA and NP, respectively (19
The MAbs obtained were isotyped using an IsoQuick kit for mouse monoclonal isotyping (Sigma-Aldrich Corporation).
The MDCK cells infected with A/Suita/01/09 or A/New Caledonia/20/99 were solubilized in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer and subjected to SDS-PAGE with a 10% polyacrylamide gel. Horseradish peroxidase-conjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories) was used for the secondary antibody. The peroxidase reaction was visualized using ECL plus (GE Healthcare UK Ltd., Buckinghamshire, United Kingdom).
Influenza virus HA or NP expression.
The coding regions for the HA and NP derived from A/Suita/01/09 were amplified by reverse transcriptase PCR (RT-PCR) and cloned into a pCAGGS ΙΙ expression plasmid, as described previously (5
). 293T cells transfected with the plasmids were used as viral antigens for the reaction with the MAbs in ascitic fluid by IFA.
Preparation of ascitic fluid.
BALB/c mice (6 weeks old, female) were intraperitoneally treated with pristane (Sigma-Aldrich Corporation). After 1 week, the mice were injected with hybridoma cells (about 4 × 106 cells per mouse). Ascitic fluid samples obtained at 1 to 2 weeks postinjection were used for the characterization of individual MAbs.
Assembly for rapid diagnostic testing using the IC method.
The IgG fraction purified from murine ascitic fluid was used to develop the IC test kit with a system from Alfresa Pharma Corporation, Osaka, Japan. The anti-H1N1 pdm-specific MAbs (MAbs N-SW2-6 and N-SW4-6) prepared in this study were immobilized onto a nitrocellulose membrane (0.6 and 0.4 μg/test, respectively) for the test line to capture H1N1 pdm protein. To prepare the control line, an anti-mouse IgG antibody (Nippon Biotest Laboratories, Tokyo, Japan) was immobilized onto a nitrocellulose membrane (1.8 μg/test) to capture mouse IgG. A conjugated pad containing the same N-SW2-6 or N-SW4-6 MAb used for the test line was labeled with colloidal gold, impregnated onto glass fibers, dried, and placed between the test line and the sample-dropping region. The nitrocellulose membrane and glass fiber pad were assembled with a glass fiber sample pad on a plastic sheet within a plastic case. This assembled kit was stored in a bag with desiccant at room temperature until use.
Evaluation of test kits.
The IC test kits were evaluated with samples of nasal wash from patients with influenza-like symptoms (n = 126) collected in 2009 and 2010 at Baba Pediatric Clinic, Osaka, Japan. As control samples of seasonal influenza virus, used to confirm the specificity of the primers for PCR, specimens of nasopharyngeal fluid from influenza patients (n = 42) collected before the H1N1 pdm pandemic by Baba Pediatric Clinic and Alfresa Pharma Corporation (Osaka, Japan) were used. As a control test, an existing IC kit for rapid detection of the NPs of seasonal influenza A and B viruses (Capilia Flu A+B; Alfresa Pharma Corporation) was used.
An RT-PCR-based analysis for the highly sensitive detection of viral RNA as a “gold standard” was also performed. Primer sets that were newly designed on the basis of the sequence of the NP region using swine-derived H1N1 isolates (Table ), together with those recommended by WHO (http://www.who.int/entity/csr/resources/publications/swineflu/CDCRealtimeRTPCR_SwineH1Assay-2009_20090430.pdf
) for the specific detection of the HA region of the H1N1 pdm genome, were used. The primer sets for individual seasonal A (H1N1 and H3N2) and B viruses (Table ) were prepared as described by Yamada et al. (26
) and used for multiplex PCR. Briefly, viral RNA was extracted from the clinical samples with a QIAamp viral RNA minikit (Qiagen, Tokyo, Japan). RT-PCR was performed with a one-step RT-PCR kit (Qiagen) for H1N1 pdm and a SuperScript III one-step RT-PCR system with Platinum Taq
High Fidelity (Invitrogen) for seasonal influenza viruses. The RT-PCR conditions were as follows: 50°C for 30 min; 94°C for 3 min; then 40 cycles consisting of 94°C for 30 s, 54°C for 30 s, and 72°C for 30 s; and then 72°C for 7 min.
Primer sets used to determine the subtype of influenza virus in the patients' specimens by RT-PCR
The research protocols for human samples as well as the mouse experiments for the preparation of MAbs and ascitic fluids were approved by the Ethics Committee of the Research Institute for Microbial Diseases at Osaka University.