African swine fever DIVA immunoassay
The invention relates to a diagnostic use of an African Swine Fever Vims CD2v protein, a method, a device, and a kit for the detection of the presence of ASFV antibodies in a test sample, in particular the use thereof in a DIVA immunoassay.
1 . A method for distinguishing if a swine is infected with a wild-type ASFV or vaccinated with an ASFV live attenuated virus CD2v-marker vaccine (LAV CD2v-marker vaccine), the method comprising the steps of:
adding a test sample obtained from the swine with an antigen, wherein the antigen is an isolated antigenic fragment of a ASFV CD2v protein and wherein the antigenic fragment is a polypeptide comprising an amino acid sequence with at least 95% amino acid sequence identity to SEQ ID NO: 25,
incubating the sample with the antigen for a sufficient amount of time to allow the formation of an antibody-antigen complex, comprising an antibody from the test sample and the antigen,
detecting the presence or absence of the antibody-antigen complex, and
wherein the presence of the antibody-antigen complex identifies the swine from which the test sample is obtained as being infected with a wild-type ASFV, and the absence of an antibody-antigen complex identified the swine as vaccinated with a LAV CD2v-marker vaccine.
2 . The method according to claim 1 , wherein the antigenic fragment of the extracellular domain is a polypeptide comprising an ASFV amino acid sequence consisting of an amino acid sequence with at least 95% amino acid sequence identity to SEQ ID NO: 25.
3 . The method according to claim 1 , wherein the accompanying LAV CD2v-marker vaccine comprises an ASFV CD2v-marker vaccine strain that is capable of expressing an altered CD2v protein wherein the altered CD2v protein lacks a fragment of the extracellular domain comprising an amino acid sequence with at least 95% amino acid sequence identity to SEQ ID NO: 25.
4 . The method according to claim 3 , wherein the ASFV CD2v antigen has no epitope in common with the altered CD2v protein.
5 . The method of Use according to claim 3 , the ASFV CD2v antigen and the altered CD2v protein have no overlapping amino acid sequence.
6 . The method according to claim 1 , wherein the antigenic fragment is also used to detect the presence of ASFV antibodies in a test sample obtained from a swine infected with a wild-type ASFV and/or a swine uninfected with ASFV.
7 . The method according to claim 1 , wherein the antigenic fragment of a ASFV CD2v protein is immobilized to a solid support.
8 . The method according to claim 7 wherein the solid support is a microtiter plate, vial, bead paper strip, membrane, gel or lateral flow strip.
9 . The method according to claim 1 , wherein the method comprises detecting the presence of the antibody-antigen complex by contacting the complex with an antibody comprising a label.
10 . The method according to claim 8 , wherein the antigen immobilized to a solid support is an immunoassay or is an ELISA (enzyme linked immunosorbent assay).
11 . The method according to claim 1 , wherein the test sample is diluted with a sample diluent having a stringency of at least 5.
12 . The method according to claim 1 , wherein the sample diluent has a stringency of at least 10.
13 . The method of claim 1 , wherein diluent does not contain phosphate buffer.
14 . The method of claim 13 , wherein the diluent is a low metal-salt, high detergent (LSHD) buffer that contains: 3% v/v Tween 20, 3% v/v Aminoxide WS 35, and 0.1 M magnesium-chloride.