Hemolysin and its protein fragments in sero-detection of
Disclosed is the cloning, expression and purification of a hemolysin protein and its protein fragments in Anaplasma phagocytophilum . The recombinant hemolysin and its protein fragments are useful in the ELISA detection of anaplasma pathogen. The use of same as a kit 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 onto a surface, wherein said isolated polypeptide has the amino acid sequence set forth in SEQ ID NO: 17, SEQ ID NO:19, SEQ ID NO. 21, or SEQ ID NO. 23,
(ii) contacting said isolated polypeptide with a biological sample of a mammal, under conditions that allow formation of an antibody-antigen complex, said biological sample containing an antibody against Anaplasma phagocytophilum ; and
(iii) detecting the formation of said antibody-antigen complex, wherein said detected antibody-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 1 , 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, detecting the presence of an antibody against Anaplasma phagocytophilum in a mammal suspected of having Anaplasma phagocytophilum infection, comprising the steps of:
(i) obtaining a biological sample from a mammal suspected of having a Anaplasma phagocytophilum infection;
(ii) immobilizing an isolated polypeptide on to a surface, wherein said isolated polypeptide has the amino acid sequence set forth in SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, or SEQ ID NO: 23.
(iii) contacting said isolated polypeptide with said biological sample, under conditions that allow formation of an antibody-antigen complex; and
(iv) detecting said antibody-antigen complex, wherein said detected antibody-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 has the amino acid sequence set forth in SEQ ID NO: 17.
9. The method of claim 7 , wherein the mammal is a human.
10. The method of claim 7 , wherein said biological sample is whole blood.
11. The method of claim 7 , wherein the antibody is IgG or IgM.
12. The method of claim 7 , wherein said contacting step is performed at room temperature for about 1 hour.
13. An article of manufacture comprising a packaging material, and any one or more of the isolated polypeptides set forth by SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, and SEQ ID NO: 23.
14. The article of manufacture of claim 13 , wherein said packaging material further comprises an instruction for detecting the presence of antibody against Anaplasma phagocytophilum.