Fusion proteins and their use in the diagnosis and treatment of leishmaniasis
The present invention relates generally to a fusion protein made from a synthetic gene construct comprising of elements derived from the Leishmania antigens K26, K39, and K9. The fusion protein is particularly useful in the diagnosis of leishmaniasis, particularly visceral leishmaniasis in animals such as humans and dogs.
1. A method for detecting asymptomatic or sub-clinical Leishmania infection in a biological sample comprising:
(a) contacting a biological sample with a fusion polypeptide comprising Leishmania antigens K26, K39, and K9, wherein the Leishmania antigen K26 comprises a sequence having at least 95% identity to SEQ ID NO:5, the Leishmania antigen K39 comprises a sequence having at least 95% identity to SEQ ID NO:6, and the Leishmania antigen K9 comprises a sequence having at least 95% identity to SEQ ID NO:7 ; and
(b) detecting in the biological sample the presence of antibodies that bind to the fusion polypeptide, thereby detecting asymptomatic or sub-clinical Leishmania infection in the biological sample.
2. The method of claim 1 , wherein the fusion polypeptide comprises an amino acid sequence as set forth in 10-262 of SEQ ID NO: 8, or a sequence haying at least 95% identity thereto along its entire length.
3. The method of claim 2 , wherein the fusion polypeptide further comprises an N-terminal amino acid sequence of MHHHHHHTS (SEQ ID NO: 21).
4. The method of claim 1 , wherein the fusion polypeptide is bound to a solid support.
5. The method of claim 4 , wherein the solid support comprises nitrocellulose, latex or a plastic material.
6. The method of claim 1 , wherein the step of detecting comprises:
(a) removing unbound sample from the solid support;
(b) adding a detection reagent to the solid support; and
(c) determining the level of detection reagent bound to the solid support, relative to a predetermined cutoff value.
7. The method of claim 6 , wherein the detection reagent comprises a reporter group conjugated to a binding agent.
8. The method of claim 7 wherein the binding agent is selected from the group consisting of anti-immunoglobulin, Protein G, Protein A and lectins.
9. The method of claim 7 wherein the reporter group is selected from the group consisting of radioisotopes, fluorescent groups, luminescent groups, enzymes, biotin and dye particles.
10. The method of claim 1 wherein the biological sample is selected from the group consisting of sera, blood, and saliva.