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H5 subtype specific binding protein capable of diagnosing and monitoring H5 avian influenza

A protein-binding, specific technology for use in antiviral immunoglobulins, microorganism-based methods, biochemical equipment and methods, etc., to solve problems such as diagnosing high H5N1AIV strains

Active Publication Date: 2012-10-10
TEMASEK LIFE SCIENCES LABORATORY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

To date, no effective method for the diagnosis of highly pathogenic H5N1 AIV strains using H5 subtype monoclonal antibodies has been reported

Method used

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  • H5 subtype specific binding protein capable of diagnosing and monitoring H5 avian influenza
  • H5 subtype specific binding protein capable of diagnosing and monitoring H5 avian influenza
  • H5 subtype specific binding protein capable of diagnosing and monitoring H5 avian influenza

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0071] Generation of hybridomas

[0072] The virus called H5N1 / PR8 was obtained from the US Centers for Disease Control. It is a non-pathogenic recombinant H5N1 influenza virus containing the HA and NA genes of the AIV H5N1 virus (A / Vietnam / 1203 / 2004) that infects humans in Vietnam. Another AIV subtype H5N3 (A / chicken / Singapore / 97) was obtained from the Agri-Food and Veterinary Authority (AVA) of Singapore. These two virus stocks were used to infect 9-11 day old embryonated eggs (Chew's Poultry Farm, Singapore) and allowed to replicate for two generations. Allantoic fluid was then drawn from embryonated eggs and virus titers were determined using a hemagglutinin assay (HA). These H5N1 and H5N3 viruses were purified by centrifuging virus-containing allantoic fluid at 10,000 rpm for 30 minutes to remove debris, followed by ultracentrifugation of the supernatant at 40,000 rpm for 3 hours. Resuspend the virus pellet in PBS.

[0073] Monoclonal antibodies (IgG and IgM) were pur...

Embodiment 2

[0078] Hybridoma Screening

[0079] Hybridoma culture supernatants were screened by hemagglutinin inhibition (HI) assay and immunofluorescence assay (IFA) as described below.

[0080] hemagglutination inhibition test H5N1 / PR8 virus obtained from CDC was used to infect 9-11 day old embryonated eggs (Chew's Poultry Farm, Singapore) and incubated at 35°C for 72-96 hours. After virus multiplication, allantoic fluid was extracted from the chicken embryo and used as H5N1 virus antigen. The HI test was performed on the culture supernatant of each hybridoma as previously described (15), using chicken hemagglutination and 4 hemagglutination units of the H5N1 / PR8 strain. Serially diluted hybridoma supernatants were initially diluted 1:50 and then mixed with 4 HA units of H5N1 / PR8 virus (inactivated with 0.1% β-propiolactone) propagated in chicken embryos and 0.5% (vol / vol) Chicken erythrocyte suspension was incubated together. Antibody titers corresponding to the reciprocal of th...

Embodiment 3

[0083] Identification of H5-subtype monoclonal antibodies

[0084] Stability of mAbs Hemagglutination inhibition assays were performed on individual hybridoma supernatants obtained at different times (days 7, 30, 45, 60, 70 and 90) to measure the stability of the cell lines. Dilutions were performed to calculate the endpoint. The HI titer of the hybridoma supernatant of mAb 6B8 was 29. The titer remained stable even down to 90 days (see figure 1 ). Therefore, hybridomas secreting mAbs of H5 antigen can maintain high-efficiency value for a long time.

[0085] mAb typing (Isotyping) Typing was performed using the Mouse mAb Typing Kit (Amersham Bioscience, England) (data not shown). Isotypes 6B8, 8F10 and 2D10 were identified as IgM and 7H10 as IgGl.

[0086] mAb specificity analysis The H5-subtype mAbs cross-reacted with the related H5-subtype AIVs H5N2 and H5N3 and also with non-H5-subtype influenza viruses H3N2, H4N1, H7N1, H9N2 and H10N5. Cross-reactivity was d...

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Abstract

The invention provides a monoclonal antibody of a specific binding avian influenza virus (AIV) H5 subtype envelope glycoprotein and a binding protein relevant to the monoclonal antibody. The monoclonal antibody and the relevant binding protein can be used for detecting the H5 subtype of the AIV, including a pathogenicity H5N1 subtype. The virus can be detected in a formalin-antiseptic and paraffin-embedded sample, and a frozen sample and biological liquid, therefore, the invention provides a method for diagnosing and monitoring the infection caused by dangerous viruses.

Description

[0001] This application is a divisional application of Chinese patent application 200780053734.4 entitled "H5 subtype-specific binding protein that can be used for diagnosis and monitoring of H5 avian influenza" submitted on May 11, 2007. field of invention [0002] The invention relates to antibodies and related binding proteins for detecting avian influenza virus (AIV). More particularly, the invention relates to monoclonal antibodies and associated binding proteins useful for the detection of the highly pathogenic H5 subtype of AIV, and to methods and articles of manufacture for diagnosing and monitoring such AIV infections in animals and humans. Background of the invention [0003] Avian influenza is a common disease of poultry. AIV subtype H5N1 has caused ongoing outbreaks of avian influenza in many parts of the world (14) 1 . Affected regions include Europe, the Middle East and especially Asia. According to the World Health Organization (WHO), about 100 people have ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/10G01N33/577G01N33/569A61K39/42A61P31/16C12N7/00C12R1/93
Inventor Y·F·胡Q·Y·杜F·和J·H-S·康
Owner TEMASEK LIFE SCIENCES LABORATORY
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