The nucleoprotein genes of influenza virus A/Netherlands/018/94 (H3N2) and influenza virus B/Harbin/7/94 were cloned into the bacterial expression vector pMalC to yield highly purified recombinant influenza virus A and B nucleoproteins. influenza virus B-infected patients. On comparison, hemagglutination inhibition assays detected antibody titer rises in 81.3 and 72.7% of patients infected with influenza viruses A and B, respectively. In contrast to the detection of nucleoprotein-specific IgG antibodies or hemagglutination-inhibiting antibodies, the detection of nucleoprotein-specific IgA antibodies does not require paired serum samples and therefore can be considered an attractive alternative for the rapid serological diagnosis of influenza. Influenza viruses (family polymerase (5 units) in a total volume of 100 l of 1 1 buffer. The PCR cycles consisted of 1 min at 94C, 2 min at 52C, and 4 min at 72C for a total of 40 cycles. Primer sequences were based on the consensus sequence of the NP genes of recent influenza virus A and B strains obtained from the Wisconsin Sequence Analysis Package and were designed in such a way that the ultimate PCR product contained an DH5, plasmid Rabbit Polyclonal to CLM-1. DNA isolation, and agarose gel electrophoresis were performed by standard procedures (21). Production, isolation, and purification of recombinant NP. A total of 500 ml of SOB medium (21) containing ampicillin (50 g/ml) and supplemented with glucose (2 g/liter) was inoculated with 5 ml of an overnight culture of recombinant carrying the pMalC plasmid without cloned sequences to obtain recombinant MBP (rMBP) and for recombinant carrying the pMalC plasmid in which the NP gene of influenza virus A or B was cloned to obtain recombinant fusion proteins consisting of MBP and influenza virus A NP (rNPA) or influenza virus B NP (rNPB), respectively. SDS-PAGE and Western blotting. Sodium dodecyl sulfate-polyacrylamide BMS-911543 gel electrophoresis (SDS-PAGE) and Western blotting were performed by standard procedures (21). Blots were incubated with blocking buffer (2% nonfat milk powder, 0.05% Tween 20 in phosphate-buffered saline [PBS]) for 1 h, followed by 1 h of incubation with 1:100-diluted polyclonal rabbit antisera specific for influenza virus A or B. After washing of BMS-911543 the blots with PBS, the blots were incubated BMS-911543 for 1 h with 1:500-diluted horseradish peroxidase (HRP)-labeled swine anti-rabbit IgG antibodies (Dako, Glostrup, Denmark). Then, the blots were washed with PBS followed by incubation in diaminobenzidine-H2O2 in PBS (250 g of diaminobenzidine/ml, 0.002% H2O2). The reaction was stopped with H2O when protein bands became visible. Sera. Influenza virus A- and B-specific polyclonal rabbit and ferret antisera were obtained from rabbits injected with sucrose gradient-purified influenza virus A/Hong Kong/2/68 (H3N2) or B/Harbin/7/94 and from ferrets experimentally infected with influenza virus A/Netherlands/018/94 (H3N2) BMS-911543 or B/Harbin/7/94. Human sera were obtained from adult patients with acute influenza virus B (= 24) and influenza virus A (H1N1; = 2, H3N2; = 15) infection; the patients were enrolled in clinical studies during the respiratory season in March 1995 and December 1995, respectively. Influenza virus infection was confirmed by an immunofluorescence test or by virus isolation from cell culture. Sera were collected on the day of onset of clinical symptoms (day 1) and at several time points thereafter. For the influenza virus A-infected patients, additional serum samples collected on days 6, 21, and 60 were available. For the patients with influenza virus B infection, additional serum samples collected on days 6 and 28 were available. Sera were stored at ?20C until use. HI assay. One volume of serum was mixed with 5 volumes of cholera filtrate, and the mixture was incubated at 37C for approximately 16 h, followed by 1 h of incubation at 56C. To 50 l of twofold dilution series of serum in PBS, 25 l of a solution of influenza virus A/Singapore/6/86 (H1N1), A/Johannesburg/33/94 (H3N2), or B/Harbin/7/94 containing 4 hemagglutinating units was added, BMS-911543 and the mixture was incubated at 37C for 30 min. Then, 25 l of a 1% turkey erythrocyte suspension in PBS was added, followed by 1 h of incubation at 4C. Subsequently, the hemagglutination pattern was examined and was expressed as the reciprocal value of the highest serum dilution inhibiting hemagglutination. A fourfold titer rise for paired serum samples was considered indicative of a recent influenza virus infection. ELISA. (i) ELISA for detection of IgA serum antibodies (capture IgA NP ELISA). Ninety-six-well plates coated with rabbit anti-human IgA antibodies (Meddens Diagnostics, Brummen, The Netherlands) were washed with demineralized H2O containing 0.05% Tween 80, followed by incubation with patient sera diluted 1:100 in ELISA buffer (Meddens Diagnostics). After.