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All seven strains of pH1N1 virus tested were positive by pH1N1 ELISA, with an average lower detection limit of 103.0?��?0.4?tissue culture infective dose (TCID)50/mL (or 0.009?��?0.005?HA titre). Cross-reaction of the assay with seasonal influenza virus and other common respiratory pathogens was rare. GSK126 concentration In pH1N1-infected patients, the sensitivity of the pH1N1 ELISA was 92.3% (84/91, 95%?CI?84.8�C96.9%), which is significantly higher than that of the BD Directigen EZ Flu A?+?B test (70.3%, p?Transducin specialized equipment and long turn-around time. Hence, rapid influenza diagnosis tests (RIDTs) have been used Selleckchem Staurosporine on many occasions [2]. However, RIDTs cannot efficiently differentiate pH1N1 virus infection from seasonal influenza?A virus infection, as they have poor sensitivity [2�C5], with consequences for clinical management. Therefore, a sensitive and convenient immunoassay that can differentiate the pH1N1 virus from seasonal influenza virus is desirable. In this study, with haemagglutinin (HA) of pH1N1 virus as the detection target, a double-sandwich ELISA (pH1N1 ELISA) was developed and evaluated for its ability to differentiate pH1N1 virus from other respiratory pathogens, including seasonal influenza viruses. Two monoclonal antibodies (mAbs) (10B4 and 1E12) that recognize the cluster-specific epitopes in HA of pH1N1 virus were created by immunizing with a pH1N1 isolate (A/California/04/2009(H1N1)) in mice. The specificities of the two mAbs were determined in a series of influenza viral isolates, using haemagglutination inhibition assays and cell-based microneutralization assays, performed as previously described [6,7] (Table?1). mAb 10B4 was then coated on the microplate, and mAb 1E12 was conjugated with horseradish peroxidase. A schematic diagram of the principle and manipulation of pH1N1 ELISA is shown in Fig.?1.