Recombinant 15-kDa polypeptide and use of same in detecting human infection with
Disclosed are the cloning and expression of a novel antigen of Bartonella henselae . The recombinant polypeptide is found to be highly immunogenic and is useful as a diagnostic test antigen. The polypeptide of the present invention provides the basis of a diagnostic assay that is sensitive, rapid and accurate diagnosis of infection with Bartonella henselae using patient's sera. Disclosed also are the ELISA for both IgG and IgM and allows diagnosis of early and late infection.
1. A method of using a recombinant 15-kDa polypeptide in an immunoassay to detect an infection of Bartonella henselae in a mammal, comprising the steps of:
(a) expressing a recombinant 15-kDa polypeptide having an amino acid sequence of SEQ ID NO. 2 in an E. coli cell, said expressing step comprises:
(i) introducing a 15-kDa insert DNA having a nucleotide sequence of SEQ ID NO. 1 in said E. coli cell;
(ii) growing said E. coli cell that has said 15-kDa insert DNA;
(iii) adding 5-10 mg/mL glucose followed by IPTG; and
(iv) isolating said recombinant 15-kDa polypeptide,
(b) immobilizing said recombinant 15-kDa polypeptide onto a surface;
(c) contacting said immobilized recombinant 15-kDa polypeptide with a biological sample obtained from a mammal, said mammal is suspected of an infection of Bartonella henselae , under conditions that allow formation of an antibody-antigen complex; and
(d) detecting the formation of said antibody-antigen complex,
wherein said detected antibody-antigen complex is indicative of the presence of said antibody against Bartonella henselae in said biological sample, and is indicative of an infection of Bartonella henselae in said mammal.
2. The method according to claim 1 , wherein the mammal is a human.
3. The method according to claim 1 , wherein said antibody is an IgG or IgM.
4. The method according to claim 1 , wherein said detecting step further comprising adding an indicator reagent comprising a signal generating compound after step (c).
5. The method according to claim 4 , wherein said indicator reagent is horse-radish peroxidase.
6. A method of diagnosing an infection of Bartonella henselae in a mammal, comprising the steps of:
(a) expressing a recombinant 15-kDa polypeptide having an amino acid sequence of SEQ ID NO. 2 in an E. coli cell, said expressing step comprises:
(i) introducing a 15-kDa insert DNA having a nucleotide sequence of SEQ ID NO. 1 in said E. coli cell;
(ii) growing said E. coli cell that has said 15-kDa insert DNA;
(iii) adding 5-10 mg/mL glucose followed by IPTG; and
(iv) isolating said recombinant 15-kDa polypeptide,
(b) obtaining a biological sample from a mammal suspected of having a Bartonella henselae infection;
(c) immobilizing said recombinant 15-kDa polypeptide onto a surface;
(d) contacting said immobilized recombinant 15-kDa polypeptide with said biological sample, under conditions that allow formation of antibody-antigen complex;
(e) detecting said antibody-antigen complex,
wherein said detected antibody-antigen complex is indicative of the presence of said antibody against Bartonella henselae in said biological sample, and is indicative of a diagnosis of infection of Bartonella henselae in said mammal.
7. The method according to claim 6 , wherein said mammal is either a cat or a human.
8. The method according to claim 6 , wherein said antibody is an IgG or IgM.
9. The method according to claim 6 , wherein the contacting step is performed at room temperature for about 1 hour.
10. The method according to claim 6 , wherein said detecting step further comprising adding an indicator reagent comprising a signal generating compound after step (d).
11. The method according to claim 10 , wherein said indicator reagent is horse-radish peroxidase.