Peptides, devices, and methods for the detection of anaplasma antibodies
The invention provides populations of isolated peptides useful for the detection of antibodies that bind to Anaplasma antigens. The peptide populations comprise peptides derived from immunogenic fragments of the Anaplasma Outer Membrane Protein proteins. The invention also provides devices, methods, and kits comprising the populations of isolated peptides useful for the detection of antibodies that bind to Anaplasma antigens and the diagnosis of anaplasmosis. Methods of identifying the particular Anaplasma species infecting a subject using the peptide populations of the invention are also disclosed.
1. A method for detecting in a sample an antibody to an epitope of an Anaplasma antigen, the method comprising:
contacting a sample with a population of isolated peptides, wherein the population comprises three or more different peptides, wherein at least one peptide in the population comprises the sequence of SEQ ID NO: 6, and wherein the population further comprises two or more different peptides selected from:
(i) SEQ ID NO: 1 or a fragment thereof, wherein X 9 is an amino acid selected from the group consisting of I, P and H, X 17 is an amino acid selected from the group consisting of I, W, and Y, X 21 is an amino acid selected from the group consisting of R, D, and N, X 28 is an amino acid selected from the group consisting of E and N, and X 31 is an amino acid selected from the group consisting of L and V;
(ii) SEQ ID NO: 2 or a fragment thereof, wherein X 3 is an amino acid selected from the group consisting of L, V and A, X 7 is an amino acid selected from the group consisting of K, N and Q, X 11 is an amino acid selected from the group consisting of R, D, and N, and X 15 is an amino acid selected from the group consisting of E, N and Q;
(iii) SEQ ID NO: 3 or a fragment thereof, wherein X 5 is an amino acid selected from the group consisting of V and A, X 7 is an amino acid selected from the group consisting of G, I and H, X 11 is an amino acid selected from the group consisting of E, N, and Q, X 18 is an amino acid selected from the group consisting of D and N, X 21 is an amino acid selected from the group consisting of R, D, and N, X 25 is an amino acid selected from the group consisting of Q, D, and E, X 28 is an amino acid selected from the group consisting of E and N, X 31 is an amino acid selected from the group consisting of L and V, X 45 is an amino acid selected from the group consisting of K and Q, X 48 is an amino acid selected from the group consisting of F and V, X 51 is an amino acid selected from the group consisting of D and N, X 54 is an amino acid selected from the group consisting of E and Q, X 57 is an amino acid selected from the group consisting of S and Q, X 60 is an amino acid selected from the group consisting of F and W, X 63 is an amino acid selected from the group consisting of I and V, and X 66 is an amino acid selected from the group consisting of Q and D;
(iv) SEQ ID NO: 4 or a fragment thereof, wherein X 6 is an amino acid selected from the group consisting of K and Q, X 9 is an amino acid selected from the group consisting of F and V, X 12 is an amino acid selected from the group consisting of D and N, X 15 is an amino acid selected from the group consisting of E and Q, X 18 is an amino acid selected from the group consisting of S and Q, X 21 is an amino acid selected from the group consisting of F and W, X 24 is an amino acid selected from the group consisting of I and V, and X 27 is an amino acid selected from the group consisting of Q and D;
(v) SEQ ID NO: 5 or a fragment thereof, wherein X 2 is an amino acid selected from the group consisting of I and V, X 10 is an amino acid selected from the group consisting of S and Y, and X 23 is an amino acid selected from the group consisting of E and N;
(vi) SEQ ID NO: 7 or a fragment thereof, wherein X 5 is an amino acid selected from the group consisting of S and Q, X 9 is an amino acid selected from the group consisting of F and Y, X 13 is an amino acid selected from the group consisting of R and H, X 16 is an amino acid selected from the group consisting of W and Y, X 18 is an amino acid selected from the group consisting of S and Q, X 22 is an amino acid selected from the group consisting of K and H, and X 27 is an amino acid selected from the group consisting of N and D;
(vii) SEQ ID NO: 8 or a fragment thereof, wherein X 10 is an amino acid selected from the group consisting of V, L and I, and X 11 is an amino acid selected from the group consisting of A and L; and
(viii) SEQ ID NO: 9 or a fragment thereof, wherein X 5 is an amino acid selected from the group consisting of V and A, X 7 is an amino acid selected from the group consisting of G, I and H, X 11 is an amino acid selected from the group consisting of E, N, and Q, X 21 is an amino acid selected from the group consisting of R, D, and N, X 25 is an amino acid selected from the group consisting of Q, D, and E, X 28 is an amino acid selected from the group consisting of E and N, and X 31 is an amino acid selected from the group consisting of L and V; and
detecting formation of an antibody-peptide complex comprising one or more peptides in the composition, wherein formation of said complex is indicative of an antibody to an epitope of an Anaplasma antigen being present in said sample.
2. The method of claim 1 , wherein said Anaplasma antigen is from Anaplasma phagocytophilum, Anaplasma platys , or Anaplasma marginate.
3. The method of claim 1 , wherein the population of isolated peptides is immobilized to a solid support optionally through a metallic nanolayer.
4. The method of claim 1 , wherein said detecting step comprises (i) performing an ELISA assay, (ii) running a lateral flow assay, (iii) performing an agglutination assay, (iv) performing a Western blot, a slot blot, or dot blot, (v) performing a wavelength shift assay, (vi) performing an Indirect Fluorescent Antibody test , or (vii) running the sample through an analytical or centrifugal rotor.
5. The method of claim 1 , wherein said sample is from a human, canine, or feline subject.
6. The method of claim 1 , wherein said sample is a blood, serum, plasma, cerebrospinal fluid, tissue extract, urine, or saliva sample.
7. The method of claim 1 , wherein the two or more different peptides comprise SEQ ID NO: 3 or a fragment thereof and SEQ ID NO: 7 or a fragment thereof.
8. A method for diagnosing anaplasmosis in a subject, the method comprising:
contacting a sample from the subject with a population of isolated peptides, wherein the population comprises three or more different peptides, wherein at least one peptide in the population comprises the sequence of SEQ ID NO: 6, and wherein the population further comprises two or more different peptides selected from:
(i) SEQ ID NO: 1 or a fragment thereof, wherein X 9 is an amino acid selected from the group consisting of I, P and H, X 17 is an amino acid selected from the group consisting of I, W, and Y, X 21 is an amino acid selected from the group consisting of R, D, and N, X 28 is an amino acid selected from the group consisting of E and N, and X 31 is an amino acid selected from the group consisting of L and V;
(ii) SEQ ID NO: 2 or a fragment thereof, wherein X 3 is an amino acid selected from the group consisting of L, V and A, X 7 is an amino acid selected from the group consisting of K, N and Q, X 11 is an amino acid selected from the group consisting of R, D, and N, and X 15 is an amino acid selected from the group consisting of E, N and Q;
(iii) SEQ ID NO: 3 or a fragment thereof, wherein X 5 is an amino acid selected from the group consisting of V and A, X 7 is an amino acid selected from the group consisting of G, I and H, X 11 is an amino acid selected from the group consisting of E, N, and Q, X 18 is an amino acid selected from the group consisting of D and N, X 21 is an amino acid selected from the group consisting of R, D, and N, X 25 is an amino acid selected from the group consisting of Q, D, and E, X 28 is an amino acid selected from the group consisting of E and N, X 31 is an amino acid selected from the group consisting of L and V, X 45 is an amino acid selected from the group consisting of K and Q, X 48 is an amino acid selected from the group consisting of F and V, X 51 is an amino acid selected from the group consisting of D and N, X 54 is an amino acid selected from the group consisting of E and Q, X 57 is an amino acid selected from the group consisting of S and Q, X 60 is an amino acid selected from the group consisting of F and W, X 63 is an amino acid selected from the group consisting of I and V, and X 66 is an amino acid selected from the group consisting of Q and D;
(iv) SEQ ID NO: 4 or a fragment thereof, wherein X 6 is an amino acid selected from the group consisting of K and Q, X 9 is an amino acid selected from the group consisting of F and V, X 12 is an amino acid selected from the group consisting of D and N, X 15 is an amino acid selected from the group consisting of E and Q, X 18 is an amino acid selected from the group consisting of S and Q, X 21 is an amino acid selected from the group consisting of F and W, X 24 is an amino acid selected from the group consisting of I and V, and X 27 is an amino acid selected from the group consisting of Q and D;
(v) SEQ ID NO: 5 or a fragment thereof, wherein X 2 is an amino acid selected from the group consisting of I and V, X 10 is an amino acid selected from the group consisting of S and Y, and X 23 is an amino acid selected from the group consisting of E and N;
(vi) SEQ ID NO: 7 or a fragment thereof, wherein X 5 is an amino acid selected from the group consisting of S and Q, X 9 is an amino acid selected from the group consisting of F and Y, X 13 is an amino acid selected from the group consisting of R and H, X 16 is an amino acid selected from the group consisting of W and Y, X 18 is an amino acid selected from the group consisting of S and Q, X 22 is an amino acid selected from the group consisting of K and H, and X 27 is an amino acid selected from the group consisting of N and D;
(vii) SEQ ID NO: 8 or a fragment thereof, wherein X 10 is an amino acid selected from the group consisting of V, L and I, and X 11 is an amino acid selected from the group consisting of A and L; and
(viii) SEQ ID NO: 9 or a fragment thereof, wherein X 5 is an amino acid selected from the group consisting of V and A, X 7 is an amino acid selected from the group consisting of G, I and H, X 11 is an amino acid selected from the group consisting of E, N, and Q, X 21 is an amino acid selected from the group consisting of R, D, and N, X 25 is an amino acid selected from the group consisting of Q, D, and E, X 28 is an amino acid selected from the group consisting of E and N, and X 31 is an amino acid selected from the group consisting of L and V; and
detecting formation of an antibody-peptide complex comprising one or more peptides in the composition,
wherein formation of the complex is indicative of the subject having anaplasmosis.
9. The method of claim 8 , wherein said subject is a human, canine, or feline.
10. The method of claim 8 , wherein the two or more different peptides comprise SEQ ID NO: 3 or a fragment thereof and SEQ ID NO: 7 or a fragment thereof.
11. A method for detecting in a sample an antibody to an epitope of an Anaplasma antigen, the method comprising:
contacting a sample with a population of isolated peptides, wherein the population comprises three or more different peptides, wherein at least one peptide in the population comprises the sequence of SEQ ID NO: 3, and wherein the population further comprises two or more different peptides selected from:
(i) SEQ ID NO: 1 or a fragment thereof, wherein X 9 is an amino acid selected from the group consisting of I, P and H, X 17 is an amino acid selected from the group consisting of I, W, and Y, X 21 is an amino acid selected from the group consisting of R, D, and N, X 28 is an amino acid selected from the group consisting of E and N, and X 31 is an amino acid selected from the group consisting of L and V;
(ii) SEQ ID NO: 2 or a fragment thereof, wherein X 3 is an amino acid selected from the group consisting of L, V and A, X 7 is an amino acid selected from the group consisting of K, N and Q, X 11 is an amino acid selected from the group consisting of R, D, and N, and X 15 is an amino acid selected from the group consisting of E, N and Q;
(iii) SEQ ID NO: 4 or a fragment thereof, wherein X 6 is an amino acid selected from the group consisting of K and Q, X 9 is an amino acid selected from the group consisting of F and V, X 12 is an amino acid selected from the group consisting of D and N, X 15 is an amino acid selected from the group consisting of E and Q, X 18 is an amino acid selected from the group consisting of S and Q, X 21 is an amino acid selected from the group consisting of F and W, X 24 is an amino acid selected from the group consisting of I and V, and X 27 is an amino acid selected from the group consisting of Q and D;
(iv) SEQ ID NO: 5 or a fragment thereof, wherein X 2 is an amino acid selected from the group consisting of I and V, X 10 is an amino acid selected from the group consisting of S and Y, and X 23 is an amino acid selected from the group consisting of E and N;
(v) SEQ ID NO: 6 or a fragment thereof;
(vi) SEQ ID NO: 7 or a fragment thereof, wherein X 5 is an amino acid selected from the group consisting of S and Q, X 9 is an amino acid selected from the group consisting of F and Y, X 13 is an amino acid selected from the group consisting of R and H, X 16 is an amino acid selected from the group consisting of W and Y, X 18 is an amino acid selected from the group consisting of S and Q, X 22 is an amino acid selected from the group consisting of K and H, and X 27 is an amino acid selected from the group consisting of N and D;
(vii) SEQ ID NO: 8 or a fragment thereof, wherein X 10 is an amino acid selected from the group consisting of V, L and I, and X 11 is an amino acid selected from the group consisting of A and L; and
(viii) SEQ ID NO: 9 or a fragment thereof, wherein X 5 is an amino acid selected from the group consisting of V and A, X 7 is an amino acid selected from the group consisting of G, I and H, X 11 is an amino acid selected from the group consisting of E, N, and Q, X 21 is an amino acid selected from the group consisting of R, D, and N, X 25 is an amino acid selected from the group consisting of Q, D, and E, X 28 is an amino acid selected from the group consisting of E and N, and X 31 is an amino acid selected from the group consisting of L; and
detecting formation of an antibody-peptide complex comprising one or more peptides in the composition, wherein formation of said complex is indicative of an antibody to an epitope of an Anaplasma antigen being present in said sample.
12. The method of claim 11 , wherein the two or more different peptides comprise SEQ ID NO: 6 or a fragment thereof and SEQ ID NO: 7 or a fragment thereof.
13. A method for diagnosing anaplasmosis in a subject, the method comprising:
contacting a sample from the subject with a population of isolated peptides, wherein the population comprises three or more different peptides, wherein at least one peptide in the population comprises the sequence of SEQ ID NO: 3, and wherein the population further comprises two or more different peptides selected from:
(i) SEQ ID NO: 1 or a fragment thereof, wherein X 9 is an amino acid selected from the group consisting of I, P and H, X 17 is an amino acid selected from the group consisting of I, W, and Y, X 21 is an amino acid selected from the group consisting of R, D, and N, X 28 is an amino acid selected from the group consisting of E and N, and X 31 is an amino acid selected from the group consisting of L and V;
(ii) SEQ ID NO: 2 or a fragment thereof, wherein X 3 is an amino acid selected from the group consisting of L, V and A, X 7 is an amino acid selected from the group consisting of K, N and Q, X 11 is an amino acid selected from the group consisting of R, D, and N, and X 15 is an amino acid selected from the group consisting of E, N and Q;
(iii) SEQ ID NO: 4 or a fragment thereof, wherein X 6 is an amino acid selected from the group consisting of K and Q, X 9 is an amino acid selected from the group consisting of F and V, X 12 is an amino acid selected from the group consisting of D and N, X 15 is an amino acid selected from the group consisting of E and Q, X 18 is an amino acid selected from the group consisting of S and Q, X 21 is an amino acid selected from the group consisting of F and W, X 24 is an amino acid selected from the group consisting of I and V, and X 27 is an amino acid selected from the group consisting of Q and D;
(iv) SEQ ID NO: 5 or a fragment thereof, wherein X 2 is an amino acid selected from the group consisting of I and V, X 10 is an amino acid selected from the group consisting of S and Y, and X 23 is an amino acid selected from the group consisting of E and N;
(v) SEQ ID NO: 6 or a fragment thereof;
(vi) SEQ ID NO: 7 or a fragment thereof, wherein X 5 is an amino acid selected from the group consisting of S and Q, X 9 is an amino acid selected from the group consisting of F and Y, X 13 is an amino acid selected from the group consisting of R and H, X 16 is an amino acid selected from the group consisting of W and Y, X 18 is an amino acid selected from the group consisting of S and Q, X 22 is an amino acid selected from the group consisting of K and H, and X 27 is an amino acid selected from the group consisting of N and D;
(vii) SEQ ID NO: 8 or a fragment thereof, wherein X 10 is an amino acid selected from the group consisting of V, L and I, and X 11 is an amino acid selected from the group consisting of A and L; and
(viii) SEQ ID NO: 9 or a fragment thereof, wherein X 5 is an amino acid selected from the group consisting of V and A, X 7 is an amino acid selected from the group consisting of G, I and H, X 11 is an amino acid selected from the group consisting of E, N, and Q, X 21 is an amino acid selected from the group consisting of R, D, and N, X 25 is an amino acid selected from the group consisting of Q, D, and E, X 28 is an amino acid selected from the group consisting of E and N, and X 31 is an amino acid selected from the group consisting of L; and
detecting formation of an antibody-peptide complex comprising one or more peptides in the composition,
wherein formation of the complex is indicative of the subject having anaplasmosis.
14. The method of claim 13 , wherein the two or more different peptides comprise SEQ ID NO: 6 or a fragment thereof and SEQ ID NO: 7 or a fragment thereof.
15. A method for detecting in a sample an antibody to an epitope of an Anaplasma antigen, the method comprising:
contacting a sample with a population of isolated peptides, wherein the population comprises three or more different peptides, wherein at least one peptide in the population comprises the sequence of SEQ ID NO: 7, and wherein the population further comprises two or more different peptides selected from:
(i) SEQ ID NO: 1 or a fragment thereof, wherein X 9 is an amino acid selected from the group consisting of I, P and H, X 17 is an amino acid selected from the group consisting of I, W, and Y, X 21 is an amino acid selected from the group consisting of R, D, and N, X 28 is an amino acid selected from the group consisting of E and N, and X 31 is an amino acid selected from the group consisting of L and V;
(ii) SEQ ID NO: 2 or a fragment thereof, wherein X 3 is an amino acid selected from the group consisting of L, V and A, X 7 is an amino acid selected from the group consisting of K, N and Q, X 11 is an amino acid selected from the group consisting of R, D, and N, and X 15 is an amino acid selected from the group consisting of E, N and Q;
(iii) SEQ ID NO: 3 or a fragment thereof, wherein X 5 is an amino acid selected from the group consisting of V and A, X 7 is an amino acid selected from the group consisting of G, I and H, X 11 is an amino acid selected from the group consisting of E, N, and Q, X 18 is an amino acid selected from the group consisting of D and N, X 21 is an amino acid selected from the group consisting of R, D, and N, X 25 is an amino acid selected from the group consisting of Q, D, and E, X 28 is an amino acid selected from the group consisting of E and N, X 31 is an amino acid selected from the group consisting of L and V, X 45 is an amino acid selected from the group consisting of K and Q, X 48 is an amino acid selected from the group consisting of F and V, X 51 is an amino acid selected from the group consisting of D and N, X 54 is an amino acid selected from the group consisting of E and Q, X 57 is an amino acid selected from the group consisting of S and Q, X 60 is an amino acid selected from the group consisting of F and W, X 63 is an amino acid selected from the group consisting of I and V, and X 66 is an amino acid selected from the group consisting of Q and D;
(iv) SEQ ID NO: 4 or a fragment thereof, wherein X 6 is an amino acid selected from the group consisting of K and Q, X 9 is an amino acid selected from the group consisting of F and V, X 12 is an amino acid selected from the group consisting of D and N, X 15 is an amino acid selected from the group consisting of E and Q, X 18 is an amino acid selected from the group consisting of S and Q, X 21 is an amino acid selected from the group consisting of F and W, X 24 is an amino acid selected from the group consisting of I and V, and X 27 is an amino acid selected from the group consisting of Q and D;
(v) SEQ ID NO: 5 or a fragment thereof, wherein X 2 is an amino acid selected from the group consisting of I and V, X 10 is an amino acid selected from the group consisting of S and Y, and X 23 is an amino acid selected from the group consisting of E and N;
(vi) SEQ ID NO: 6 or a fragment thereof;
(vii) SEQ ID NO: 8 or a fragment thereof, wherein X 10 is an amino acid selected from the group consisting of V, L and I, and X 11 is an amino acid selected from the group consisting of A and L; and
(viii) SEQ ID NO: 9 or a fragment thereof, wherein X 5 is an amino acid selected from the group consisting of V and A, X 7 is an amino acid selected from the group consisting of G, I and H, X 11 is an amino acid selected from the group consisting of E, N, and Q, X 21 is an amino acid selected from the group consisting of R, D, and N, X 25 is an amino acid selected from the group consisting of Q, D, and E, X 28 is an amino acid selected from the group consisting of E and N, and X 31 is an amino acid selected from the group consisting of L; and
detecting formation of an antibody-peptide complex comprising one or more peptides in the composition, wherein formation of said complex is indicative of an antibody to an epitope of an Anaplasma antigen being present in said sample.
16. The method of claim 15 , wherein the two or more different peptides comprise SEQ ID NO: 3 or a fragment thereof and SEQ ID NO: 6 or a fragment thereof.
17. A method for diagnosing anaplasmosis in a subject, the method comprising:
contacting a sample from the subject with a population of isolated peptides, wherein the population comprises three or more different peptides, wherein at least one peptide in the population comprises the sequence of SEQ ID NO: 7, and wherein the population further comprises two or more different peptides selected from:
(i) SEQ ID NO: 1 or a fragment thereof, wherein X 9 is an amino acid selected from the group consisting of I, P and H, X 17 is an amino acid selected from the group consisting of I, W, and Y, X 21 is an amino acid selected from the group consisting of R, D, and N, X 28 is an amino acid selected from the group consisting of E and N, and X 31 is an amino acid selected from the group consisting of L and V;
(ii) SEQ ID NO: 2 or a fragment thereof, wherein X 3 is an amino acid selected from the group consisting of L, V and A, X 7 is an amino acid selected from the group consisting of K, N and Q, X 11 is an amino acid selected from the group consisting of R, D, and N, and X 15 is an amino acid selected from the group consisting of E, N and Q;
(iii) SEQ ID NO: 3 or a fragment thereof, wherein X 5 is an amino acid selected from the group consisting of V and A, X 7 is an amino acid selected from the group consisting of G, I and H, X 11 is an amino acid selected from the group consisting of E, N, and Q, X 18 is an amino acid selected from the group consisting of D and N, X 21 is an amino acid selected from the group consisting of R, D, and N, X 25 is an amino acid selected from the group consisting of Q, D, and E, X 28 is an amino acid selected from the group consisting of E and N, X 31 is an amino acid selected from the group consisting of L and V, X 45 is an amino acid selected from the group consisting of K and Q, X 48 is an amino acid selected from the group consisting of F and V, X 51 is an amino acid selected from the group consisting of D and N, X 54 is an amino acid selected from the group consisting of E and Q, X 57 is an amino acid selected from the group consisting of S and Q, X 60 is an amino acid selected from the group consisting of F and W, X 63 is an amino acid selected from the group consisting of I and V, and X 66 is an amino acid selected from the group consisting of Q and D;
(iv) SEQ ID NO: 4 or a fragment thereof, wherein X 6 is an amino acid selected from the group consisting of K and Q, X 9 is an amino acid selected from the group consisting of F and V, X 12 is an amino acid selected from the group consisting of D and N, X 15 is an amino acid selected from the group consisting of E and Q, X 18 is an amino acid selected from the group consisting of S and Q, X 21 is an amino acid selected from the group consisting of F and W, X 24 is an amino acid selected from the group consisting of I and V, and X 27 is an amino acid selected from the group consisting of Q and D;
(v) SEQ ID NO: 5 or a fragment thereof, wherein X 2 is an amino acid selected from the group consisting of I and V, X 10 is an amino acid selected from the group consisting of S and Y, and X 23 is an amino acid selected from the group consisting of E and N;
(vi) SEQ ID NO: 6 or a fragment thereof;
(vii) SEQ ID NO: 8 or a fragment thereof, wherein X 10 is an amino acid selected from the group consisting of V, L and I, and X 11 is an amino acid selected from the group consisting of A and L; and
(viii) SEQ ID NO: 9 or a fragment thereof, wherein X 5 is an amino acid selected from the group consisting of V and A, X 7 is an amino acid selected from the group consisting of G, I and H, X 11 is an amino acid selected from the group consisting of E, N, and Q, X 21 is an amino acid selected from the group consisting of R, D, and N, X 25 is an amino acid selected from the group consisting of Q, D, and E, X 28 is an amino acid selected from the group consisting of E and N, and X 31 is an amino acid selected from the group consisting of L; and
detecting formation of an antibody-peptide complex comprising one or more peptides in the composition,
wherein formation of the complex is indicative of the subject having anaplasmosis.
18. The method of claim 17 , wherein the two or more different peptides comprise SEQ ID NO: 3 or a fragment thereof and SEQ ID NO: 6 or a fragment thereof.