Development of an immunochromatographic assay specifically detecting pandemic H1N1 (2009) influenza virus

Abstract

The pandemic caused by a new type of influenza virus, pandemic H1N1 (2009) influenza virus A (AH1pdm), has had a major worldwide impact. Since hemagglutinin (HA) genes are among the most specific genes in the influenza virus genome, AH1pdm can be definitively diagnosed by viral gene analysis targeting the HA genes. This type of analysis, however, cannot be easily performed in clinical settings. While commercially available rapid diagnosis kits (RDKs) based on immunochromatography can be used to detect nucleoproteins (NPs) of influenza A and B viruses in clinical samples, there are no such kits that are specific for AH1pdm. We show here that an RDK using a combination of monoclonal antibodies against NP can be used to specifically detect AH1pdm. The RDK recognized AH1pdm virus isolates but did not recognize seasonal H1N1 and H3N2 and influenza B viruses, indicating that the specificity of the RDK is 100%. A parallel comparison of RDK with a commercial influenza A/B virus kit revealed that both types of kits had equal sensitivities in detecting their respective viruses. Preliminary evaluation of clinical samples from 5 individuals with PCR-confirmed human AH1pdm infection showed that the RDK was positive for all samples, with the same detection intensity as that of a commercial influenza A/B virus kit. This RDK, together with a new vaccine and the stockpiling of anti-influenza drugs, will make aggressive measures to contain AH1pdm infections possible.

FIG. 1.

Reactivity of Ab2 and Ab3 against NPs from AH1pdm, H5N1 HPAI, seasonal H1N1, and seasonal H3N2 viruses in a sandwich ELISA. After fixing the capture polyclonal antibody, roughly 10 ng of each NP was captured on the plates, and about 50 ng of each MAb was added to the wells. Data are presented as means ± standard errors (SE).

FIG. 2.

Epitope mapping of Ab2 and Ab3 by conventional ELISA. (a) Recombinant fragments derived from the NP of AH1pdm virus used to map Ab2 and Ab3 epitopes. (b) Reactivity of fragments derived from NP with Ab2 and Ab3 as determined by conventional ELISA. Fifty nanograms of each fragment was added to the wells, and the plates were reacted with 50 ng/well of MAb. Data were normalized against the positive control (polyclonal rat anti-NP Ab). (c) Reactivity of synthetic peptides with Ab2 and Ab3, with rat IgG as the negative control, in a conventional ELISA. Five hundreds nanograms of each peptide was captured on the wells, and 500 ng/well of MAb was added. Data in b and c are presented as means ± SE.

FIG. 3.

Reactivity of Ab1 against NPs from AH1pdm, H5N1 HPAI, seasonal H1N1, and seasonal H3N2 viruses in a sandwich ELISA (a) and epitope analysis of Ab1 using chimeric NP (b and c). (a) After fixing the capture polyclonal antibody, roughly 10 ng of each NP was captured on the plates, and about 50 ng of MAb was added to the wells. Data are presented as means ± SE. (b) Structure of chimeric NPs used in this study derived from NPs of H5N1 HPAI and seasonal H3N2 viruses. (c) Reactivity of chimeric NPs with Ab1. After fixing the capture polyclonal antibody, roughly 10 ng of each chimera was captured on the plates, and 50 ng of Ab1 was added to the wells. Data are presented as means ± SE.

FIG. 4.

Prototype RDK and specific detection of AH1pdm virus. (a) Composition of the RDK. Each RDK consists of a sample dropping area and a detection area containing a test line and a control line. The direction of sample flow is indicated. (b) Representative results of RDK dropped samples. One hundred microliters of diluted AH1pdm or seasonal influenza A virus samples (ca. 1 × 10⁸ viral copies/100 μl) was dropped onto RDKs.