Monoclonal Antibody Targeting Neutralizing Epitope on H5N1 Influenza Virus of Clade 1 and 0 for Specific H5 Quantification

He, Fang; Kwang, Jimmy

doi:10.1155/2013/360675

Cited by 7 publications

(9 citation statements)

References 19 publications

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“…Neutralization activity of MAb or serum against H5 strains was analyzed by microneutralization assay as previously described (27). Briefly, antibody samples were serially 2-fold diluted and incubated with 100 TCID 50 s of different clades of H5 strains for 1 h at room temperature and plated in duplicate onto MDCK cells grown in a 96-well plate.…”

Section: Methodsmentioning

confidence: 99%

A Novel Humanized Antibody Neutralizes H5N1 Influenza Virus via Two Different Mechanisms

Tan

Jia

et al. 2015

J Virol

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Highly pathogenic avian influenza virus subtype H5N1 continues to be a severe threat to public health, as well as the poultry industry, because of its high lethality and antigenic drift rate. Neutralizing monoclonal antibodies (MAbs) can serve as a useful tool for preventing, treating, and detecting H5N1. In the present study, humanized H5 antibody 8A8 was developed from a murine H5 MAb. Both the humanized and mouse MAbs presented positive activity in hemagglutination inhibition (HI), virus neutralization, and immunofluorescence assays against a wide range of H5N1 strains. Interestingly, both human and murine 8A8 antibodies were able to detect H5 in Western blot assays under reducing conditions. Further, by sequencing of escape mutants, the conformational epitope of 8A8 was found to be located within the receptor binding domain (RBD) of H5. The linear epitope of 8A8 was identified by Western blotting of overlapping fragments and substitution mutant forms of HA1. Reverse genetic H5N1 strains with individual mutations in either the conformational or the linear epitope were generated and characterized in a series of assays, including HI, postattachment, and cell-cell fusion inhibition assays. The results indicate that for 8A8, virus neutralization mediated by RBD blocking relies on the conformational epitope while binding to the linear epitope contributes to the neutralization by inhibiting membrane fusion. Taken together, the results of this study show that a novel humanized H5 MAb binds to two types of epitopes on HA, leading to virus neutralization via two mechanisms. IMPORTANCERecurrence of the highly pathogenic avian influenza virus subtype H5N1 in humans and poultry continues to be a serious public health concern. Preventive and therapeutic measures against influenza A viruses have received much interest in the context of global efforts to combat the current and future pandemics. Passive immune therapy is considered to be the most effective and economically prudent preventive strategy against influenza virus besides vaccination. It is important to develop a humanized neutralizing monoclonal antibody (MAb) against all of the clades of H5N1. For the first time, we report in this study that a novel humanized H5 MAb binds to two types of epitopes on HA, leading to virus neutralization via two mechanisms. These findings further deepen our understanding of influenza virus neutralization. R ecurrence of highly pathogenic avian influenza (HPAI) virus subtype H5N1 in humans and poultry continues to be a serious public health concern because of its unabated and widespread geographic circulation (1). Since their emergence in Asia over a decade ago, HPAI H5N1 viruses have spread to Ͼ60 countries on three continents and are endemic in poultry in Southeast Asia and Africa (2). They have caused disease in several mammals, including humans, often with lethal consequences. To date, H5N1 has resulted in 667 human cases worldwide, including 393 deaths (3). Although no sustained human-to-human transmission of the virus h...

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Section: Methodsmentioning

confidence: 99%

A Novel Humanized Antibody Neutralizes H5N1 Influenza Virus via Two Different Mechanisms

Tan

Jia

et al. 2015

J Virol

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“…Induced bacteria were pelleted by centrifugation at 10,000 x g for 10 min, and analyzed by SDS-PAGE. The inclusion bodies pellet was solubilized as in previously described procedure (He and Kwang, 2013). The unfolded ARV σC antigen was dialyzed against 2 l volume of 1x PBS for 12 hr at room temperature, using dialysis tubing (Sigma, Korea).…”

Section: Methodsmentioning

confidence: 99%

“…2 ml of DMEM was then added to the cells. Neutralization titer of the egg yolk antibody against σC antigen was analyzed by microneutralization assay as previously described (He and Kwang, 2013). Briefly, undiluted, 1:10 diluted, and 1:100 diluted IgY antibodies were applied onto BHK-21 cells grown in a 6-well plate.…”

Section: Methodsmentioning

confidence: 99%

“…These results suggest that the recombinant σC could be used as an immunogen to produce specific antibody (IgY). In order to investigate the neutralization titer of IgY antibody, a neutralization test was performed with reference to the paper of He and Kwang, 2013, using avian reovirus (Fashy-Crawley clone-1, ATCC VR-826) and BHK-21 cells. ARV reacted with the recombinant σC IgY antibody, followed by inoculation into BHK-21 cells.…”

Section: Specificity and Virus Neutralizationmentioning

confidence: 99%

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Use of IgY antibody to recombinant avian reovirus σC protein in the virus diagnostics

Jung¹,

Bae²,

Jung³

et al. 2014

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Summary. -Avian reovirus (ARV) is an important agent of several diseases causing considerable losses in poultry farming. An outer capsid protein (σC) of ARV, is known as a virus-cell attachment protein essential for virus infectivity. In this study, the σC gene of ARV was cloned and expressed in Escherichia coli. The expressed recombinant protein was used as immunogen for raising a specific IgY antibody in laying hens. At 14 weeks post immunization, the antibody titers in serum and egg yolk reached 302,000 and 355,000, respectively. The IgY antibody was capable to neutralize ARV in BHK-21 cells and it strongly reacted in ELISA with ARV but not with heterologous viruses. The IgY antibody detected ARV in field samples of infected animal tissues in dot blot assay. These results suggest that an efficient, economic and rapid diagnostics of ARV can be performed routinely using the IgY antibody against a recombinant ARV σC protein.

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“…Neutralization activity of Mab against H7 strains was analyzed by microneutralization assay as previously described [ 17 ]. Briefly, Mab was serially two-fold diluted and incubated with 100 TCID50 of different clades of H7 strains for 1 h at room temperature and plated in duplicate onto MDCK cells grown in a 96-well plate.…”

Section: Methodsmentioning

confidence: 99%

Development of dual-function ELISA for effective antigen and antibody detection against H7 avian influenza virus

Prabakaran

Tan

et al. 2013

BMC Microbiol

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BackgroundOutbreaks in poultry involving influenza virus from H7 subtype have resulted in human infections, thus causing a major concern for public health, as well as for the poultry industry. Currently, no efficient rapid test is available for large-scale detection of either antigen or antibody of H7 avian influenza viruses.ResultsIn the present study, a dual function ELISA was developed for the effective detection of antigen and antibody against H7 AIVs. The test was established based on antigen-capture-ELISA and epitope blocking ELISA. The two Mabs 62 and 98 which were exploited in the assay were identified to recognize two conformational neutralizing epitopes on H7 HA1. Both of the epitopes exist in all of the human H7 strains, including the recent H7N9 strain from China and > 96.6% of avian H7 strains. The dual ELISA was able to detect all of the five H7 antigens tested without any cross reaction to other influenza subtypes. The antigen detection limit was less than 1 HA unit of H7. For antibody detection, the sensitivity and specificity of the dual ELISA was evaluated and compared to HI and microneutralization using immunized animal sera to different H7 strains and different subtypes of AIVs. Results indicated that antibodies to H7 were readily detected in immunized animal sera by the dual ELISA whereas specimens with antibodies to other AIVs yielded negative results.ConclusionsThis is the first dual-function ELISA reported for either antigen or antibody detection against H7 AIVs. The assay was highly sensitive and 100% specific in both functions rendering it effective for H7 diagnosis.

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Monoclonal Antibody Targeting Neutralizing Epitope on H5N1 Influenza Virus of Clade 1 and 0 for Specific H5 Quantification

Cited by 7 publications

References 19 publications

A Novel Humanized Antibody Neutralizes H5N1 Influenza Virus via Two Different Mechanisms

A Novel Humanized Antibody Neutralizes H5N1 Influenza Virus via Two Different Mechanisms

Use of IgY antibody to recombinant avian reovirus σC protein in the virus diagnostics

Development of dual-function ELISA for effective antigen and antibody detection against H7 avian influenza virus

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