Sensitive and specific assay for
View Patent ↗The invention is directed to methods of using novel Babesia antigen peptides in detecting Babesia spp. in a sample. The methods may be adapted to assays suitable for high blood volume screening, use for clinical diagnosis of patients with babesiosis, or assaying blood contaminated with Babesia spp., such as B. microti.
1. A method for identifying Babesia microti in a sample, the method comprising:
(a) contacting the sample to a solid support immobilized with a combination of at least four isolated, synthetic Babesia antigen peptides comprising (a) a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 39 or consisting of the amino acid sequence set forth in SEQ ID NO: 45; (b) a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 40; (c) a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 41 or consisting of the amino acid sequence set forth in SEQ ID NO: 46; and (d) a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 42;
wherein a Babesia microti antibody present in the sample binds to at least one Babesia antigen peptide of the combination of at least four isolated, synthetic Babesia antigen peptides;
(b) contacting the bound Babesia microti antibody of step (a) with a detectable label linked to a reagent that binds to the bound Babesia microti antibody at an epitope that is different from an epitope bound by at least one Babesia antigen peptide of the combination of at least four isolated, synthetic Babesia antigen peptides;
(c) contacting the detectable label with a substrate to form a colored-reaction product; and
(d) detecting the formation of the colored-reaction product as an indication of the presence of one or more Babesia microti in the sample.
2. The method according to claim 1 , further comprising a wash step after step (a).
3. The method according to claim 1 , wherein the substrate is a horseradish peroxidase substrate.
4. The method according to claim 1 , wherein the sample is blood or a blood product.
5. A diagnostic kit for the identification of Babesia microti in a sample, the kit comprising a combination of at least four isolated, synthetic Babesia antigen peptides selected from (a) a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 39 or consisting of the amino acid sequence set forth in SEQ ID NO: 45; (b) a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 40; (c) a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 41 or consisting of the amino acid sequence set forth in SEQ ID NO: 46; and (d) a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 42, and instructions for use.
6. The diagnostic kit according to claim 5 , wherein the combination of at least four isolated, synthetic Babesia antigen peptides are affixed to a solid support.
7. The diagnostic kit according to claim 5 , wherein the combination of at least four isolated, synthetic Babesia antigen peptides comprises a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 39 conjugated to Biotin-PEG(4).
8. The diagnostic kit according to claim 5 , wherein the combination of at least four isolated, synthetic Babesia antigen peptides comprises a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 40 conjugated to Biotin-PEG(4).
9. The diagnostic kit according to claim 5 , wherein the combination of at least four isolated, synthetic Babesia antigen peptides comprises a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 41 conjugated to Biotin-PEG(4).
10. The diagnostic kit according to claim 5 , wherein the combination of at least four isolated, synthetic Babesia antigen peptides comprises a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 42 conjugated to Biotin-PEG(4).
11. The diagnostic kit according to claim 5 , wherein the combination of at least four isolated, synthetic Babesia antigen peptides comprises a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 45 conjugated to Biotin-PEG(4).
12. The diagnostic kit according to claim 5 , wherein the combination of at least four isolated, synthetic Babesia antigen peptides comprises a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 46 conjugated to Biotin-PEG(4).
13. The diagnostic kit according to claim 5 , wherein the combination of at least four isolated, synthetic Babesia antigen peptides comprises polypeptides consisting of SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 45, and SEQ ID NO: 46.
14. A composition comprising a combination of at least four isolated, synthetic Babesia antigen peptides selected from (a) a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 39 or consisting of the amino acid sequence set forth in SEQ ID NO: 45; (b) a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 40; (c) a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 41 or consisting of the amino acid sequence set forth in SEQ ID NO: 46; and (d) a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 42.
15. The composition according to claim 14 , wherein the combination of at least four isolated, synthetic Babesia antigen peptides comprises a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 39 conjugated to Biotin-PEG(4).
16. The composition according to claim 14 , wherein the combination of at least four isolated, synthetic Babesia antigen peptides comprises a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 40 conjugated to Biotin-PEG(4).
17. The composition according to claim 14 , wherein the combination of at least four isolated, synthetic Babesia antigen peptides comprises a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 41 conjugated to Biotin-PEG(4).
18. The composition according to claim 14 , wherein the combination of at least four isolated, synthetic Babesia antigen peptides comprises a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 42 conjugated to Biotin-PEG(4).
19. The composition according to claim 14 , wherein the combination of at least four isolated, synthetic Babesia antigen peptides comprises a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 45 conjugated to Biotin-PEG(4).
20. The composition according to claim 14 , wherein the combination of at least four isolated, synthetic Babesia antigen peptides comprises a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 46 conjugated to polypeptide Biotin-PEG(4).
21. The composition according to claim 14 , wherein the combination of at least four isolated, synthetic Babesia antigen peptides comprises polypeptides consisting of SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 45, and SEQ ID NO: 46.