Patent
The present invention relates to an immunoassay for the detection of Borrelia specific IgG, IgM and IgG/IgM antibodies in biological samples suspected of Lyme infection. The immunoassay can be performed via a standard immunoassay format or on an automated platform. In various embodiments, the immunoassay uses one or more Borrelia specific chimeric peptides VlsE-FlaB (designated pFlaB-mV), VlsE-ErpP (designated pErp59-mV), VlsE-P35 (designated pP35-mV) alone or in combination with one or more outer surface protein C (Osp C) types B or I, p58 and DbpA. Other aspects of the invention provide antigen/substrate combinations and compositions comprising combinations of the disclosed peptides and/or proteins for use in the immunoassays described herein.
1. A substrate comprising SEQ ID NO: 2 and SEQ ID NO: 5 each immobilized on said substrate.
2. The substrate according to claim 1 , wherein said substrate is glass, plastic, polystyrene or nitrocellulose.
3. The substrate according to claim 1 , wherein said substrate is: a) a particle; b) a bead; c) a chip; d) a microchip; e) a lateral flow substrate; or f) a microtiter plate.
4. The substrate according to claim 3 , wherein said substrate is a particle or bead or a population of particles or beads.
5. The substrate according to claim 4 , wherein said substrate is a population of particles or beads comprising two or more separate subpopulations of particles or beads, each subpopulation of particles or beads being distinguishable by a specific detectable physical parameter, SEQ ID NO: 2 being immobilized on one subpopulation of particles or beads, and SEQ ID NO: 5 being immobilized on a second subpopulation of particles or beads.
6. The substrate according to claim 5 , wherein each of the two or more separate subpopulations of particles or beads has a separate specific detectable physical parameter.
7. The substrate according to claim 6 , wherein the specific detectable physical parameter is a fluorescent dye, fluorophore, luminescent agent, electron-dense reagent, radioisotope or particle size.
8. The substrate according to claim 7 , wherein the specific detectable physical parameter is a fluorophore.
9. A method of detecting antibodies specific to SEQ ID NO: 2 and/or SEQ ID NO: 5 in a mammal comprising obtaining a biological sample from the mammal, contacting said biological sample with the substrate according to claim 1 and detecting the presence or the absence of the antibodies that are bound specifically to SEQ ID NO: 2 and/or SEQ ID NO: 5.
10. The method according to claim 9 , wherein said method comprises contacting the biological sample with the substrate, wherein the substrate comprises a population of particles or beads comprising two or more separate subpopulations of particles or beads, each subpopulation of particles or beads being distinguishable by a specific detectable physical parameter, SEQ ID NO: 2 being immobilized on one subpopulation of particles or beads, and SEQ ID NO: 5 being immobilized on a second subpopulation of particles or beads.
11. The method according to claim 10 , wherein each of said two or more separate subpopulations of particles or beads has a separate specific detectable physical parameter.
12. The substrate according to claim 5 , wherein said population of beads further comprises SEQ ID NO: 8 immobilized on a third subpopulation of beads or particles having a third specific detectable physical parameter.
13. The substrate according to claim 1 , said substrate further comprising SEQ ID NO: 8 immobilized on said substrate.
14. The substrate according to claim 12 , wherein the specific detectable physical parameter is a fluorescent dye, fluorophore, luminescent agent, electron-dense reagent, radioisotope or particle size.
15. The substrate according to claim 14 , wherein the specific detectable physical parameter is a fluorophore.
16. The method according to claim 10 , wherein said population of particles or beads further comprises SEQ ID NO: 8 immobilized on a third subpopulation of particles or beads having a third specific detectable physical parameter.
17. The method according to claim 10 , wherein the separate specific detectable physical parameter is a fluorescent dye, fluorophore, luminescent agent, electron-dense reagent, radioisotope or particle size.
18. The substrate according to claim 1 , wherein the immobilization comprises covalent or non-covalent binding of SEQ ID NO: 2 and SEQ ID NO: 5 to the substrate.