Method and kits for the diagnosis of influenza
Some embodiments provided herein relate to combined assays. In some embodiments, an assay for identifying influenza type A or influenza type B is combined with an assay for determining the sensitivity of an influenza neuraminidase to an antiviral drug.
1. A method for detecting an influenza virus type A or B and detecting the presence of a neuraminidase in a sample, the method comprising:
(a) contacting a sample with an immunoassay buffer adapted for an immunoassay for detecting an influenza virus type A or an influenza virus type B, thereby obtaining an immunoassay test sample;
(b) contacting a first portion of the immunoassay test sample to a test comprising an immunoassay for detecting an influenza type A or type B, thereby detecting the presence or absence of influenza type A or type B in the sample;
(c) contacting a second portion of the immunoassay test sample with a matrix comprising a neuraminidase assay buffer, thereby obtaining a neuraminidase test sample; and
(d) contacting the neuraminidase test sample with a neuraminidase assay, thereby detecting the presence of a neuraminidase in the sample,
wherein the immunoassay buffer inhibits neuraminidase activity.
2. The method of claim 1 , wherein the immunoassay buffer is incompatible with an assay for neuraminidase activity.
3. The method of claim 1 , wherein steps (c) and (d) are performed in the same vessel.
4. The method of claim 3 , wherein the vessel comprises a chamber comprising the matrix, and a chamber comprising reagents for the neuraminidase assay.
5. The method of claim 1 , wherein the matrix comprises a cross-linked polysaccharide.
6. The method of claim 3 , wherein the vessel comprises a multiwell cartridge adapted to be read using a photomultiplier.
7. The method of claim 1 , wherein the neuraminidase assay comprises determining the sensitivity of a neuraminidase to a test compound.
8. The method of claim 7 , wherein the neuraminidase assay comprises:
(a) obtaining an inhibition value ratio, wherein the ratio comprises the level of neuraminidase activity in the presence of a test compound compared to the level of neuraminidase activity in the absence of the test compound; and
(b) comparing the inhibition value ratio to an inhibition threshold, thereby determining the sensitivity of the neuraminidase activity to the test compound.
9. The method of claim 8 , wherein the inhibition threshold is determined by the detection of a type A or a type B virus.
10. The method of claim 9 , wherein a first inhibition threshold is used if type A virus is detected, and a second inhibition threshold is used if type B virus is detected.
11. The method of claim 1 , wherein the neuraminidase assay comprises a dual enzyme assay comprising a signaling enzyme.
12. The method of claim 11 , wherein the neuraminidase assay comprises a reagent that is converted by neuraminidase activity to produce a substrate for the signaling enzyme.
13. The method of claim 11 , wherein the neuraminidase assay comprises a reagent comprising a conjugate of a N-acetylneuraminic acid and luciferin, or a derivative of a N-acetylneuraminic acid and luciferin.
14. The method of claim 11 , wherein the neuraminidase assay comprises a reagent that is converted by neuraminidase activity to produce an inhibitor for the signaling enzyme.
15. The method of claim 11 , wherein the neuraminidase assay comprises a reagent comprising a conjugate of a N-acetylneuraminic acid and a trifluoromethylketone, or a derivative of a conjugate of a N-acetylneuraminic acid and a trifluoromethylketone.
16. The method of claim 11 , wherein the signaling enzyme comprises luciferase.
17. The method of claim 16 , wherein the immunoassay buffer inhibits luciferase activity.
18. The method of claim 8 , wherein the test compound is an antiviral drug.
19. A method for detecting an influenza virus type A or B and detecting the presence of a neuraminidase in a sample, the method comprising:
(a) contacting a sample with a neuraminidase assay buffer adapted for an neuraminidase assay, thereby obtaining a neuraminidase test sample;
(b) contacting a first portion of the neuraminidase test sample to a test comprising an immunoassay for detecting an influenza type A or type B, wherein the neuraminidase test sample contacts a matrix comprising immunoassay buffer, thereby obtaining an immunoassay test sample and detecting the presence or absence of influenza type A or type B in the sample; and
(c) contacting a second portion of the neuraminidase test sample with a neuraminidase assay, thereby detecting the presence of a neuraminidase in the sample,
wherein the neuraminidase assay buffer inhibits specific binding of an antibody to an antigen selected from the group consisting of influenza type A and influenza type B.
20. The method of claim 19 , wherein the matrix comprises lyophilized or dried down immunoassay buffer.
21. The method of claim 19 , wherein the neuraminidase assay comprises determining the sensitivity of a neuraminidase to a test compound.
22. The method of claim 21 , wherein the neuraminidase assay comprises:
(a) obtaining an inhibition value ratio, wherein the ratio comprises the level of neuraminidase activity in the presence of a test compound compared to the level of neuraminidase activity in the absence of the test compound; and
(b) comparing the inhibition value ratio to an inhibition threshold, thereby determining the sensitivity of the neuraminidase activity to the test compound.
23. The method of claim 22 , wherein the inhibition threshold is determined by the detection of a type A or a type B virus.
24. The method of claim 23 , wherein a first inhibition threshold is used if type A virus is detected, and a second inhibition threshold is used if type B virus is detected.
25. The method of claim 24 , wherein the first threshold is lower than the second threshold.
26. The method of claim 22 , wherein the inhibition threshold is determined by the test compound.
27. The method of claim 22 , further comprising selecting the test compound for treating the influenza.
28. The method of claim 22 , wherein the neuraminidase assay comprises a dual enzyme assay comprising a signaling enzyme.
29. The method of claim 28 , wherein the neuraminidase assay comprises a reagent that is converted by neuraminidase activity to produce a substrate for the signaling enzyme.
30. The method of claim 22 , wherein the neuraminidase assay comprises a conjugate of a N-acetylneuraminic acid and luciferin, or a derivative of a N-acetylneuraminic acid and luciferin.
31. The method of claim 22 , wherein the neuraminidase assay comprises a reagent that is converted by neuraminidase activity to produce an inhibitor for the signaling enzyme.
32. The method of claim 22 , wherein the neuraminidase assay comprises a conjugate of a N-acetylneuraminic acid and a trifluoromethylketone, or a derivative of a conjugate of a N-acetylneuraminic acid and a trifluoromethylketone.
33. The method of claim 28 , wherein the signaling enzyme comprises luciferase.
34. The method of claim 19 , wherein the level of neuraminidase activity is measured using a photomultiplier tube.
35. The method of claim 22 , wherein the test compound is an antiviral drug.
36. The method of claim 22 , wherein the test compound is an antiviral drug selected from the group consisting of Oseltamivir, Zanamivir, Lanamivir, and Peramivir.
37. The method of claim 19 , wherein the immunoassay comprises a sandwich assay.
38. The method of claim 15 , wherein the conjugate of a N-acetylneuraminic acid and a trifluoromethylketone or the derivative a N-acetylneuraminic acid and a trifluoromethylketone conjugate, comprises a masked inhibitor compound for use in a dual enzyme influenza neuraminidase sensitivity assay having the structure of Formula (I):
wherein:
R 1 R 2 and R 3 are each independently hydrogen or C 1-5 alkyl;
n is 0, 1, 2 or 3;
R 4 is —(CH 2 ) m C(═O)CF 3 ;
m is 0 or 1;
X is —S(O) z — or —CH(R 5 )—;
R 5 is —S(O) z CH 3 ; and
z is 0, 1 or 2.
39. The method of claim 38 selected from the group consisting of:
40. The method of claim 32 , wherein the conjugate of a N-acetylneuraminic acid and a trifluoromethylketone or the derivative a N-acetylneuraminic acid and a trifluoromethylketone conjugate, comprises a masked inhibitor compound for use in a dual enzyme influenza neuraminidase sensitivity assay having the structure of Formula (I):
wherein:
R 1 R 2 and R 3 are each independently hydrogen or C 1-5 alkyl;
n is 0, 1, 2 or 3;
R 4 is —(CH 2 ) m C(═O)CF 3 ;
m is 0 or 1;
X is —S(O) z — or —CH(R 5 )—;
R 5 is —S(O) z CH 3 ; and
z is 0, 1 or 2.
41. The method of claim 40 selected from the group consisting of: