Multiplexed detection of influenza A virus subtype H5 and H9 via quantum dot-based immunoassay.

A quantum dot-based lateral flow immunoassay system (QD-LFIAS) was developed to simultaneously detect both influenza A virus subtypes H5 and H9. Water-soluble carboxyl-functionalized quantum dots (QDs) were used as fluorescent tags. The QDs were conjugated to specific influenza A virus subtype H5 and H9 antibodies via an amide bond. When influenza A virus subtype H5 or H9 was added to the QD-LFIAS, the QD-labeled antibodies specifically bound to the H5 or H9 subtype viruses and were then captured by the coating antibodies at test line 1 or 2 to form a sandwich complex. This complex produced a bright fluorescent band in response to 365 nm ultraviolet excitation. The intensity of fluorescence can be detected using an inexpensive, low-maintenance instrument, and the virus concentration directly correlates with the fluorescence intensity. The detection limit of the QD-LFIAS for influenza A virus subtype H5 was 0.016 HAU, and the detection limit of the QD-LFIAS for influenza A virus subtype H9 was 0.25 HAU. The specificity and reproducibility were good. The simple analysis step and objective results that can be obtained within 15 min indicate that this QD-LFIAS is a highly efficient test that can be used to monitor and prevent both Influenza A virus subtypes H5 and H9.