Patent FR-2966460-A1 (FR)
Title: Differentiating a vaccinated animal against Bluetongue virus of infected animal from biological sample comprises contacting biological sample with NS1 protein of BTV virus and revealing antigen-antibody complex and anti-NS1 antibody
Inventor: POURQUIER PHILIPPE
IPC: C07K16/10
Filed: 2010-10-27
Abstract: In vitro method for differentiating a vaccinated animal or population of animals vaccinated against Bluetongue virus (BTV) of infected animal or a population from animals infected by the BTV from a biological sample from the animal, comprises (a) contacting the biological sample with the protein NS1 of BTV virus, or its fragment of the protein NS1, and (b) revealing antigen-antibody complex formed in (a) between the NS1 protein, or one of its fragments, and an anti-NS1 antibody likely to be present in the biological sample. In vitro method for differentiating a vaccinated animal or population of animals vaccinated against Bluetongue virus (BTV) of infected animal or a population from animals infected by the BTV from a biological sample from the animal, comprises: (a) contacting the biological sample with the protein NS1 of BTV virus, or its fragment of the protein NS1, and (b) revealing antigen-antibody complex formed in (a) between the NS1 protein, or one of its fragments, and an anti-NS1 antibody likely to be present in the biological sample, where the revelation is carried out by a competitive immunoassay method implementing anti-NSI monoclonal antibody or one of its functional fragments, capable of specifically recognizing the protein NS1 of BTV virus, or one of its antigenic fragments, the anti-NS1 antibody is a monoclonal antibody obtained from a culture supernatant of a hybridoma, the hybridoma is obtained by fusion between a myelomatous cell and a mammalian lymphoid cell, the mammal is previously immunized with a composition or an immunogenic compound comprising the protein NS1 of BTV, native or recombinant, or one of its fragments, the anti-NS1 antibody is selected by a process comprising: (i) contacting the test monoclonal test antibodies thus obtained from culture supernatant from the hybridomas with a native protein or NS1 recombinant of the BTV, and (ii) contacting the test monoclonal antibodies thus obtained from culture supernatant of the hybridoma with a native protein or NS1 recombinant of epizootic hemorrhagic disease virus (EHDV); and (iii) selecting the tested monoclonal antibody if specifically recognizes NS1 protein of BTV, or its fragment, and not recognizes the NS1 protein of EHDV. Independent claims are included for: (1) a kit or kit reagents to differentiate vaccinated animals or population of animals vaccinated against BTV of an infected animal or a population of animals infected with BTV by BTV virus, comprising: the protein NS1 of BTV virus, or its fragment of the NS1 protein; an antibody or one of its functional fragments, directed against the NS1 protein of BTV, or against an antigenic fragment of the NS1 protein, optionally marked, where the antibody is polyclonal, monoclonal or chimeric and optionally reagents necessary for the reaction; (2) a process of selecting a monoclonal antibody to distinguish the vaccinated animal or population of animals vaccinated against BTV of an infected animal or population of animals infected with BTV, optionally used in a competitive immunoassay method, comprising optionally selecting hybridoma obtained after immunization of an animal with the antigen comprising isolated native NS1 protein and optionally purified or recombinant of BTV virus, preferably amino acid sequence of SEQ ID NO: 1 (NS1 of BTV-1) (comprising a fully defined 552 amino acid sequence as given in the specification) or SEQ ID NO: 2 (NS1 of BTV-8) (comprising a fully defined 552 amino acid sequence as given in the specification), or sequence having at least 85% of identity with SEQ ID NO: 1 or SEQ ID NO: 2 or one of its antigenic fragments, or a BTV virus, preferably BTV-1 or BTV-8 or one of its fragments containing the NS1 protein, contacting the antibody to be tested with the NS1 protein of the BTV or its fragment of the NS1 protein, a compound or composition comprising the protein, and revealing the antigen-antibody complex formed between the NS1 protein, or one of its antigenic fragment and tested monoclonal antibody and in which the revelation step of antigen-antibody complexes formed is carried out by a method of competitive immunoassay by implementing an antibody or one of its functional fragments, where the antibody is the monoclonal antibody secreted by hybridoma referred to as 9B4Id Vet deposited at CNCM, on July 7, 2009 under number 1-4198 or one of its functional fragments, optionally marked; (3) a hybridoma called 9B4Id Vet deposited at the CNCM, on July 7, 2009 under the number 1-4198 or one of its functional fragments, optionally marked; and (4) an antibody named 9B4 Id Vet secreted by hybridoma or one of its functional fragments, optionally marked.