Protein fragments of virB10 and sero-detection of anaplasma phagocytophium
Disclosed are cloning and expression of a plurality of protein fragments of virB10, a Type IV Secretion System (TIVSS) in Anaplasma phagocytophilum . Such recombinant protein fragments are useful in the ELISA detection of anaplasma pathogen. The use of same as kits for ELISA is also disclosed.
1. A method of detecting the presence of an antibody against Anaplasma phagocytophilum in a biological sample of a mammal comprising:
(i) immobilizing an isolated polypeptide antigen onto a surface, wherein said immobilized isolated polypeptide antigen has the amino acid sequence set forth in SEQ ID NO: 13 or SEQ ID NO: 15;
(ii) contacting said immobilized isolated polypeptide antigen with the biological sample of the mammal, under conditions that allow formation of an antibody-antigen complex, said biological sample containing an antibody against Anaplasma phagocytophilum , wherein said antibody binds to said immobilized isolated polypeptide antigen and forms a complex of the antibody and the polypeptide antigen; and
(iii) detecting the formation of said antibody-polypeptide antigen complex, wherein said detected antibody-polypeptide antigen complex is indicative of the presence of said antibody against Anaplasma phagocytophilum in said biological sample.
2. The method of claim 1 , wherein said mammal is a human.
3. The method of claim 1 , wherein said antibody is an IgG or IgM.
4. The method of claim 1 , wherein said method is an ELISA.
5. The method of claim 4 , wherein said ELISA has a sensitivity of at least >70%.
6. The method of claim 4 , wherein said ELISA has a specificity of at least >70%.
7. A method of diagnosing an infection of Anaplasma phagocytophilum in a mammal by detecting the presence of an antibody against said Anaplasma phagocytophilum comprising the steps of:
(i) obtaining a biological sample from the mammal, wherein said mammal is suspected of having the Anaplasma phagocytophilum infection;
(ii) immobilizing an isolated polypeptide antigen onto a surface, wherein said immobilized isolated polypeptide antigen has the amino acid sequence set forth in SEQ ID NO: 13 or SEQ ID NO: 15;
(iii) contacting said immobilized isolated polypeptide antigen with said biological sample, under conditions that allow formation of an antibody-antigen complex, said biological sample containing an antibody against Anaplasma phagocytophilum , and wherein said antibody binds to said immobilized isolated polypeptide antigen and forms a complex of the antibody and the polypeptide antigen; and
(iv) detecting said antibody-polypeptide antigen complex, wherein said detected antibody-polypeptide antigen complex is indicative of the presence of said antibody against Anaplasma phagocytophilum in said biological sample.
8. The method of claim 7 , wherein the isolated polypeptide antigen has the amino acid sequence set forth in SEQ ID NO: 13.
9. The method of claim 7 , wherein the isolated polypeptide antigen has the amino acid sequence set forth in SEQ ID NO: 15.
10. The method of claim 7 , wherein the mammal is a human.
11. The method of claim 7 , wherein said biological sample is whole blood.
12. The method of claim 7 , wherein the antibody is IgG or IgM.
13. The method of claim 7 , wherein said contacting step is performed at room temperature for about 1 hour.