Specific monoclonal antibody against epitope of clygoprotein coding inactivated feline immunodeficiency virus

A monoclonal antibody and glycoprotein technology, applied in the direction of immunoglobulin, antiviral immunoglobulin, viral antigen components, etc., can solve the problems of inability to recognize and inactivate

Inactive Publication Date: 2004-09-15
ZOETIS W LLC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although monoclonal antibodies specific for FIV-encoded antigens or epitopes of antigenic proteins are known, for example US 5,117,014 and US 5,219,725, these antibodies do not recognize inactivated FIV
This means that for currently commercialized FIV vaccines (which all utilize inactivated FIV), there are no known monoclonal antibodies that can be used for the determination of the viral load in the vaccine composition or the potency of the inactivated FIV component

Method used

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  • Specific monoclonal antibody against epitope of clygoprotein coding inactivated feline immunodeficiency virus

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Experimental program
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Embodiment 1

[0023] Preparation of Monoclonal Antibodies Specific for Inactivated FIV-Encoded Glycoprotein Epitopes

[0024] cells and viruses

[0025] FIV-Shizuoka (FIV-Shiz, D FIV subtype) is propagated in a persistently infected lymphoid cell line obtained from FIV-Shizuoka and Fet-J (ATCC deposit number CRL 11967), which is an independent The cell line for IL-2, called Shiz. A cell line persistently infected with FIV-Shizuoka is also deposited with the ATCC under accession number CRL 11976. To generate an antigen stock solution, the virus solution was inactivated with formalin and concentrated by ultrafiltration.

[0026] Antigen stocks can also be prepared from various other FIV strains and subtypes in a similar manner, such as wild isolates, FIV strain NCSU 1 (ATCC deposit number VR-2333), FIV strain UC24818 (ATCC deposit number VR-2619), FIV- Petaluma (subtype A, TCC deposit number VR-2186), FIV-Dixon (subtype A), FIV-UK8 (subtype A), FIV-Bangston (subtype B), FIV-Amori-1 (subtyp...

Embodiment 2

[0030] Application of Monoclonal Antibody mAb 1D9 as Detection Antibody in Enzyme-Linked Immunosorbent Assay

[0031] In this evaluation, Galanthus Nivalis lectin (GNA) was used to capture glycoproteins. This GNA ELISA combines high selectivity for GNA binding with its broad reactivity with HIV-1, HIV-2, SIV and FIV glycoproteins. First, a 96 microwell plate was coated with 10 pg / mL GNA (dissolved in 50 mM carbonate) at pH 9.6 at 37° C. for 1 hour. After the wells were blocked with PBS-10% FBS at 37°C for 2 hours, samples treated with 1% Empigen BB (Calbiochem) at 37°C for 1 hour were added and incubated at 37°C for 2 hours. Unbound antigen was removed by washing 3 times with PBS containing 0.1% Tween 20. The monoclonal antibody mAb 1D9 of Example 1 diluted 1:8000 was added to each well, and the plate was incubated at 37°C for 1 hour. After washing, a 1:1000 dilution of peroxidase-labeled goat anti-mouse IgG Kirkegaard & Perry Laboratories (KPL) was added and the plate was ...

Embodiment 3

[0033] Use of monoclonal antibody mAb 1D9 as a detection antibody in immunoprecipitation assays

[0034] In this evaluation, a virus stock solution enriched with formalin-inactivated FTV-Petaluma virus (less than 5% FIV protein in 0.38 mg total protein) and 5 mg Thio-NHS-LC-Bio Pierce was incubated on ice for 1 hour. After removal of unincorporated biotin reagent by dialysis, virus-containing samples were extracted with 1% Triton X-100 in 12 mL of PBS for 1 hour and centrifuged at 100,000 g for 2 hours. The supernatant was recovered and used for immunoprecipitation. Immunoprecipitation was achieved by incubating 600 μl of extracts with 80 μl of mAb 1D9 or mAbH5332 for 1 hour at 4°C. mAB H5332 (specific for Borrelia OspA protein) was used as an irrelevant antibody control. Immune complexes were collected on immobilized protein G (Pierce), washed 4 times with cold PBS-1% NP-40, resuspended in Laemmli buffer, and subjected to SDS-PAGE and Western blotting. Blots were blocked ...

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Abstract

The present invention provides a monoclonal antibody specific for an epitope which is unique to the surface protein component of an inactivated feline immunodeficiency virus (FIV) envelope glycoprotein. Said antibody is useful for the quantification of inactivated FIV or the determination of the potency of an inactivated FIV vaccine.

Description

Background technique [0001] Feline immunodeficiency virus (FIV), originally called feline T-lymphotropic lentivirus, was first reported by Pederson et al. (Science, (1987) 235:790-793) and has been reported in domestic cats and cheetahs Be identified. Cats worldwide are infected. Like HIV, FIV has also received international attention. According to the American Association of Feline Practitioners, as many as one in 12 cats may test positive for FIV. Following infection, there is a brief phase of fever, lymphadenopathy, and neutropenia. Most cats recover at this stage and appear normal for months or years before immunodeficiency develops. Because of this latent manifestation of immunodeficiency, the use of live virus vaccines to treat or prevent FIV is quite risky. Although monoclonal antibodies specific for FIV-encoded antigens or epitopes of antigenic proteins are known, eg US 5,117,014 and US 5,219,725, these antibodies do not recognize inactivated FIV. This means that...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/53C07K16/00C07K16/10C12N5/10C12N15/02C12P21/08G01N33/569G01N33/577
CPCC07K16/1063A61P31/12C07K16/00
Inventor C·M·黄
Owner ZOETIS W LLC
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