Dataset Information

Broad-spectrum detection of H5 subtype influenza A viruses with a new fluorescent immunochromatography system.

ABSTRACT: Immunochromatography (IC) is an antigen-detection assay that plays an important role in the rapid diagnosis of influenza virus because the protocol is short time and easy to use. Despite the usability of IC, the sensitivity is approximately 10(3) pfu per reaction. In addition, antigen-antibody interaction-based method cannot be used for the detection of influenza viruses with major antigenic change. In this study, we established the use of fluorescent immunochromatography (FLIC) to detect a broad spectrum of H5 subtype influenza A viruses. This method has improved sensitivity 10-100 fold higher than traditional IC because of the use of fluorescent conjugated beads. Our Type-E FLIC kit detected all of the H5 subtype influenza viruses that were examined, as well as recombinant hemagglutinin (HA) proteins (rHAs) belonging to the Eurasian H5 subtype viruses and the Type-N diagnosed North American H5 subtype influenza A viruses. Thus, this kit has the improved potential to detect H5 subtype influenza viruses of different clades with both Type-E and Type-N FLIC kits. Compared with PCR-based diagnosis, FLIC has a strong advantage in usability, because the sample preparation required for FLIC is only mix-and-drop without any additional steps such as RNA extraction. Our results can provide new strategies against the spread and transmission of HPAI H5N1 viruses in birds and mammals including humans.

SUBMITTER: Sakurai A

PROVIDER: S-EPMC3819354 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Broad-spectrum detection of H5 subtype influenza A viruses with a new fluorescent immunochromatography system.

Sakurai Akira A Takayama Katsuyoshi K Nomura Namiko N Munakata Tsubasa T Yamamoto Naoki N Tamura Tsuruki T Yamada Jitsuho J Hashimoto Masako M Kuwahara Kazuhiko K Sakoda Yoshihiro Y Suda Yoshihiko Y Kobayashi Yukuharu Y Sakaguchi Nobuo N Kida Hiroshi H Kohara Michinori M Shibasaki Futoshi F

PloS one 20131106 11

Immunochromatography (IC) is an antigen-detection assay that plays an important role in the rapid diagnosis of influenza virus because the protocol is short time and easy to use. Despite the usability of IC, the sensitivity is approximately 10(3) pfu per reaction. In addition, antigen-antibody interaction-based method cannot be used for the detection of influenza viruses with major antigenic change. In this study, we established the use of fluorescent immunochromatography (FLIC) to detect a broa ...[more]

PMID: 24223117

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Project description:The highly pathogenic avian influenza viruses of the H5 subtype, such as the H5N1 viral strains or the novel H5N8 and H5N2 reassortants, are of both veterinary and public health concern worldwide. To combat these viruses, monoclonal antibodies (mAbs) against H5 hemagglutinin (HA) play a significant role. These mAbs are effective diagnostic and therapeutic agents and powerful tools in vaccine development and basic scientific research. The aim of this study was to obtain diagnostically valuable mAbs with broad strain specificity against H5-subtype AIVs.We applied the hybridoma method to produce anti-HA mAbs. The cloning and screening procedures resulted in the selection of 7 mouse hybridoma cell lines and their respective antibody clones. Preliminary immunoreactivity studies showed that these newly established mAbs, all of the IgG1 isotype, had high specificity and broad-range activities against the H5 HAs. However, these studies did not allow for a clear distinction among the selected antibodies and mAb-secreting hybridoma clones. To differentiate the analyzed mAbs and determine the exact number of hybridoma clones, peptide mapping of the Fc and Fab fragments was performed using a Matrix-Assisted Laser Desorption Ionization Time of Flight (MALDI-TOF/TOF) mass spectrometer. Detailed analyses of the acquired MS and MS/MS spectra confirmed that the Fc fragments constituted highly conserved species- and isotype-immunoglobulin components, whereas the Fab fragments exhibited considerable variation in the sequences that determine antibody specificity. This approach enabled unambiguous characterization of the selected mAbs according to their peptide composition. As a result, 6 different clones were distinguished.Our work provided a unique panel of anti-H5 HA mAbs, which meets the demand for novel, high-specificity analytical tools for use in serologic surveillance. Applications of these mAbs in areas other than diagnostics are also possible. Moreover, we demonstrated for the first time that peptide mapping of antibody fragments with mass spectrometry is an efficient method for the differentiation of antibody clones and relevant antibody-producing cell lines. The method may be successfully used to characterize mAbs at the protein level.

Project description:The unprecedented spread of highly pathogenic avian influenza virus subtype H5N1 in Asia and Europe is threatening animals and public health systems. Effective diagnosis and control management are needed to control the disease. To this end, we developed a panel of monoclonal antibodies (MAbs) against the H5N1 avian influenza virus (AIV) and implemented an antigen-capture enzyme-linked immunosorbent assay (AC-ELISA) to detect the H5 viral antigen. Mice immunized with denatured hemagglutinin (HA) from A/goose/Guangdong/97 (H5N1) expressed in bacteria or immunized with concentrated H5N2 virus yielded a panel of hybridomas secreting MAbs specific for influenza virus HA. The reactivity of each MAb with several subtypes of influenza virus revealed that hybridomas 3D4 and 8B6 specifically recognized H5 HA. Therefore, purified antibodies from hybridomas 3D4 and 8B6, which secrete immunoglobulin G (IgG) and IgM, respectively, were used as the capture antibodies and pooled hyperimmune guinea pig serum IgG served as the detector antibody. The specificity of the optimized AC-ELISA was evaluated by using AIV subtypes H5 H3, H4, H7, H9, and H10. Specimens containing AIV subtype H5 subtype yielded a specific and strong signal above the background, whereas specimens containing all other subtypes yielded background signals. The detection limits of the AC-ELISA were 62.5 ng of bacterium-expressed H5N1 HA1 protein and 124, 62, and 31 50% tissue culture infective doses of influenza virus subtypes H5N1/PR8, H5N2, and H5N3, respectively. Reconstituted clinical samples consisting of H5 AIVs mixed with pharyngeal-tracheal mucus from healthy chickens also yielded positive signals in the AC-ELISA, and the results were confirmed by reverse transcription-PCR. The tracheal swab samples from H9N2-infected chickens did not give positive signals. Taken together, the newly developed MAb-based AC-ELISA offers an attractive alternative to other diagnostic approaches for the specific detection of H5 AIV.

Dataset Information

Broad-spectrum detection of H5 subtype influenza A viruses with a new fluorescent immunochromatography system.

Publications

Broad-spectrum detection of H5 subtype influenza A viruses with a new fluorescent immunochromatography system.

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