Methods 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 comprising:
(a) contacting a sample with an immunoassay buffer, wherein the immunoassay buffer inhibits neuraminidase activity and is 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 adapted to detect neuraminidase activity, wherein the neuraminidase assay comprises a reagent selected from the group consisting of 1,1,1-Trifluro-4-(4-hydroxyphenyl)butan-2-one (para), 1,1,1-Trifluro-4-(3-hydroxyphenyl)butan-2-one (meta), and 1,1,1-Trifluro-4-(2-hydroxyphenyl)butan-2-one (ortho), thereby detecting the presence of a neuraminidase in the sample.
2. The method of claim 1 , wherein steps (c) and (d) are performed in the same vessel.
3. The method of claim 2 , wherein the vessel comprises a chamber comprising the matrix, and a chamber comprising reagents for the neuraminidase assay.
4. The method of claim 1 , wherein detecting the presence of a neuraminidase in the sample comprises detecting a signal with a photomultiplier.
5. The method of claim 1 , further comprising determining the sensitivity of a neuraminidase to a test compound, comprising:
(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.
6. The method of claim 5 , wherein the inhibition threshold is determined by the detection of a type A or a type B virus.
7. The method of claim 5 , further comprising selecting the test compound for treating the influenza, wherein the test compound is an antiviral drug.
8. The method of claim 7 , wherein the antiviral drug selected from the group consisting of Oseltamivir, Zanamivir, Lanamivir, and Peramivir.
9. The method of claim 1 , wherein the neuraminidase assay comprises a dual enzyme assay comprising a signaling enzyme and a substrate for the signaling enzyme.
10. The method of claim 9 , wherein the signaling enzyme comprises luciferase, and the substrate for the signaling enzyme comprises a conjugate of a N-acetylneuraminic acid and luciferin, or a derivative of a N-acetylneuraminic acid and luciferin.
11. A method for detecting an influenza virus comprising:
(a) contacting a sample with a neuraminidase assay buffer, 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 and is adapted for a 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 by specific binding of an antibody to the antigen, 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, wherein the neuraminidase assay comprises a reagent selected from the group consisting of 1,1,1-Trifluro-4-(4-hydroxyphenyl)butan-2-one (para), 1,1,1-Trifluro-4-(3-hydroxyphenyl)butan-2-one (meta), and 1,1,1-Trifluro-4-(2-hydroxyphenyl)butan-2-one (ortho), thereby detecting the presence of a neuraminidase in the sample.
12. The method of claim 11 , wherein steps (c) and (d) are performed in the same vessel.
13. The method of claim 12 , wherein the vessel comprises a chamber comprising the matrix, and a chamber comprising reagents for the neuraminidase assay.
14. The method of claim 11 , wherein detecting the presence of a neuraminidase in the sample comprises detecting a signal with a photomultiplier.
15. The method of claim 11 , further comprising determining the sensitivity of a neuraminidase to a test compound, comprising:
(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.
16. The method of claim 15 , wherein the inhibition threshold is determined by the detection of a type A or a type B virus.
17. The method of claim 15 , further comprising selecting the test compound for treating the influenza, wherein the test compound is an antiviral drug.
18. The method of claim 17 , wherein the antiviral drug selected from the group consisting of Oseltamivir, Zanamivir, Lanamivir, and Peramivir.
19. The method of claim 15 , wherein the neuraminidase assay comprises a dual enzyme assay comprising a signaling enzyme, and a substrate for the signaling enzyme.
20. The method of claim 19 , wherein the signaling enzyme comprises luciferase, and the substrate for the signaling enzyme comprises a conjugate of a N-acetylneuraminic acid and luciferin, or a derivative of a N-acetylneuraminic acid and luciferin.