SAMPLE PRETREATMENT METHOD FOR VIRUS DETECTION

Abstract

The present invention relates to a method for detecting a virus, and more particularly, to a method for detecting a virus including a lipid bilayer envelope, wherein the method allows a surface antigen, which is a lipid bilayer envelope protein, to be effectively exposed from lipoproteins that mask all or part of the envelope protein, thereby revealing the surface antigen that is masked by the lipoproteins, so that a probe such as an antibody can be used to detect the virus with high sensitivity within a short time.

Claims

1. A method for pretreating a biological sample for virus detection, the method comprising: (1) isolating an enveloped virus including a lipid bilayer envelop from the biological sample; and (2) treating the enveloped virus with a bile acid, a bile acid derivative, and/or a pH adjusting agent.

2. The method of claim 1, wherein the treating step is carried out such that an envelope protein is exposed from the lipid bilayer.

3. The method of claim 2, wherein the envelope protein is a surface antigen of the enveloped virus.

4. The method of claim 3, wherein the envelope protein is E1 or E2 protein.

5. The method of claim 1, wherein the enveloped virus is a hepatitis virus.

6. The method of claim 5, wherein the hepatitis virus is hepatitis C virus (HCV).

7. The method of claim 1, wherein the bile acid derivative is selected from the group consisting of chenodeoxycholic acid, deoxycholic acid, lithocholic acid, taurocholic acid, glycocholic acid, taurochenodeoxycholic acid, glycochenodeoxycholic acid, glycodeoxycholic acid, taurodeoxycholic acid, glycolithocholic acid, taurolithocholic acid, and muricholic acid.

8. The method of claim 1, wherein the bile acid or the bile acid derivative is used at a concentration of about 10 to about 100 μmol/L.

9. The method of claim 1, wherein the pH adjusting agent is used to adjust the pH of a culture environment for the enveloped virus to between about 4 and about 7.

10. A composition for pretreating a biological sample for enveloped virus detection, the composition comprising a bile acid, a bile acid derivative, and/or a pH adjusting agent as an active ingredient(s).

11. The composition of claim 10, wherein the enveloped virus includes a lipid bilayer envelope.

12. The composition of claim 10, wherein the bile acid derivative is selected from the group consisting of chenodeoxycholic acid, deoxycholic acid, lithocholic acid, taurocholic acid, glycocholic acid, taurochenodeoxycholic acid, glycochenodeoxycholic acid, glycodeoxycholic acid, taurodeoxycholic acid, glycolithocholic acid, taurolithocholic acid, and muricholic acid.

13. The composition of claim 10, wherein the pH adjusting agent is used to adjust the pH of a culture environment for the enveloped virus to between about 4 to about 7.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0037] FIG. 1 illustrates results for stability of E2 surface antigen and genome of HCV, depending on HCV host and non-host humoral milieu, according to an embodiment of the present invention.

[0038] FIG. 2 illustrates results for destruction of HCV, that is, detection of HCV core antigen exposed from lipoproteins, depending on HCV host and non-host humoral milieu, according to an embodiment of the present invention.

[0039] FIGS. 3A to 3D illustrate results for interaction with lipids in the presence or absence of HCV, depending on HCV host and non-host humoral milieu, according to an embodiment of the present invention.

[0040] FIG. 4 illustrates results obtained by identifying detection levels of HCV RNA, depending on concentrations of a bile acid derivative, according to an embodiment of the present invention.

[0041] FIG. 5 illustrates results obtained by identifying detection levels of HCV E2 surface antigen, depending on concentrations of a bile acid derivative, according to an embodiment of the present invention.

[0042] FIGS. 6A and 6B illustrate results obtained by identifying detection levels of HCV RNA and E2 surface antigen, depending on pH changes in a culture environment for HCV, according to an embodiment of the present invention.

DETAILED DESCRIPTION OF INVENTION

[0043] In an embodiment of the present invention, there is provided a method for pretreating a sample for virus detection, comprising steps of: isolating an enveloped virus including a lipid bilayer envelope, from a biological sample isolated from a target individual; and treating the enveloped virus with any one or more selected from the group consisting of a bile acid, a bile acid derivative, and a pH adjusting agent.

[0044] In another embodiment of the present invention, there is provided a composition for pretreating a sample for enveloped virus detection, comprising, as an active ingredient, any one or more selected from the group consisting of a bile acid, a bile acid derivative, and a pH adjusting agent.

[0045] Hereinafter, the present invention will be described in more detail by way of examples. These examples are only for describing the present invention in more detail, and it will be apparent to those skilled in the art that according to the gist of the present invention, the scope of the present invention is not limited by these examples.

EXAMPLES

Preparation Example

Isolation of Enveloped Virus

[0046] Normal serum (hepatitis B virus (HBV)- and hepatitis C virus (HCV)-negative) and chronic hepatitis C patient's serum (HCV-positive) were received from Wonju Severance Christian Hospital (Ethics Committee Approval No. CR316312). In addition, mouse (Balb c) serum (cat no. IMSBC-SER) was purchased from Innovative Research.

[0047] Each of the above-mentioned sera was added to a tube having 3 ml of a cushion buffer that contains 20 g/L of sucrose, 50 mmol/L of Tris-HCl (pH 7.5), and 30 mmol/L of NaCl. The mixture was centrifuged for 1 hour at 220,000 g. Then, the supernatant was removed from the tube, and 5 ml of phosphate-buffered saline (PBS) solution was added to the tube for dilution. Then, the HCV RNA contained in the dilution was measured through real-time PCR using the Roche's COBAS TaqMan system, to determine a concentration of the isolated virus.

Example 1

Identification of Stability of E2 Surface Antigen and Genome of HCV

[0048] It was identified how non-host humoral milieu affects genome stability and surface antigenicity of HCV.

[0049] The HCV isolated in the preparation example was added to a serum isolated from human blood, a serum isolated from mouse blood, and Dulbecco's Modified Eagle's Medium (DMEM), which is a cell culture medium, and culture was performed for 24 hours. Then, a concentration of the HCV RNA contained in each culture medium was measured through real-time PCR using the Roche's COBAS TaqMan system.

[0050] In addition, 100 μl/well of HCV anti-E2 antibody (Santa Cruz Biotechnology, Inc., cat no. sc-57769, USA) was added to a micro ELISA plate, and the reaction was allowed to proceed at 4° C. overnight. Then, 200 μl/well of 1X ELISA/ELISPOT Diluent (Invitrogen, USA) was additionally added thereto, and the reaction was allowed to proceed for 1 hour. Each of the HCV-contained culture media was dispensed into each well, in which the reaction had been completed, and the reaction was allowed to proceed for 2 hours. Then, the HCV anti-E2 antibody diluted in a ratio of 1:30 with 100 μl of 1X ELISA/ELISPOT Diluent was added to the plate, and the reaction was allowed to proceed for 1 hour. Then, the additional reaction with 100 μl of HRP-conjugated anti-rabbit IgG was allowed to proceed for 30 minutes. Then, 100 μl of TMB solution (Invitrogen, USA) was added to each well, and absorbance was measured through a microreader at 450 nm. The results are illustrated in FIG. 1.

[0051] As illustrated in FIG. 1, the HCV RNA was present at a high level in the human serum (HS), but was present at a very low level in the mouse (MS) serum and the DMEM. The HCV E2 antigen was present at a low level in the human serum, but was present at a very high level in the mouse serum.

[0052] From the above results, it can be seen that the HCV RNA is present at a remarkably high level (that is, there are numerous viral particles) in the human (host) serum as compared with the non-host-derived serum, whereas the HCV E2 is present at a higher level in the mouse (non-host) serum, suggesting that the HCV's envelope is masked by lipoproteins or the like in the human (host) serum. On the other hand, it can be seen that due to the fact that humans and mice have different bile acid composition ratios, that is, mice have a higher proportion of dihydroxy bile acid (which is a more water-soluble bile acid), a degree of exposure of the envelope protein is affected by the types of bile acid.

Example 2

Identification of HCV Core in Non-Host Humoral Milieu

[0053] To find out if the reason why HCV RNA remarkably decreases and E2 increases in mouse (non-host of HCV) serum is destruction of HCV, it was identified whether or not HCV core protein was detected.

[0054] In the same manner as in Example 1, the HCV isolated in the preparation example was added to a serum isolated from human blood, a serum isolated from mouse blood, and Dulbecco's Modified Eagle's Medium (DMEM), which is a cell culture medium, and culture was performed for 1 hour, 2 hours, or 6 hours. Then, it was identified whether or not HCV core was detected, using the same ELISA method as described for detection of E2 antigen in Example 1. The results are illustrated in FIG. 2. Here, the antibody used for ELISA was an anti-HCV core antigen antibody (Abcam, cat no. ab50288).

[0055] As illustrated in FIG. 2, it was identified that in a case where the HCV was cultured in the mouse serum, HCV core was detected at a higher level in the mouse (non-host) serum than in the host at 1 hour, and a degree of detection of HCV core increased, over time, in the mouse (non-host) serum and DMEM, which is a cell culture medium, as compared with the human (host) serum.

[0056] From the above results, it can be seen that in view of the fact that the core surrounded by an envelope is exposed in the non-host environment, HCV viral particles are destroyed and this can lead to increased detection of the envelope.

Example 3

Identification of Interaction of HCV with Lipids

[0057] In the same manner as in Example 1, the HCV isolated in the preparation example was added to a serum isolated from human blood, a serum isolated from mouse blood, and Dulbecco's Modified Eagle's Medium (DMEM), which is a cell culture medium, and culture was performed for 24 hours. Then, amounts of lipids present in each culture medium were measured using ADVIA 1800 (Simens), and the results are illustrated in FIGS. 3A to 3D.

[0058] As illustrated in FIGS. 3A to 3D, regardless of presence or absence of HCV, total cholesterol, HDL-cholesterol, LDL-cholesterol, and triglycerides were all present at higher levels in the human serum than in the mouse serum. In addition, the cholesterol level decreased in the human serum in the presence of HCV, whereas it was not possible to identify changes in cholesterol level in the mouse serum even in the presence of HCV.

[0059] From the above results, it can be seen that an HCV envelope protein is well masked by lipids or lipoproteins in human blood because lipids adhere better to the envelope, which is HCV's surface, in human serum than in mouse (non-host) serum.

Example 4

Identification of Stability of E2 Surface Antigen and Genome of HCV, Affected by Bile Acid

[0060] From the results of the above examples, based on the fact that human serum and mouse serum have different bile acid compositions, it was identified how HCV was affected by a bile acid.

[0061] In the same manner as in Example 1, the HCV isolated in the preparation example was added to Dulbecco's Modified Eagle's Medium (DMEM), which is a cell culture medium, treatment with taurocholate (tCA) or taurochenodeoxycholate (tCDCA), which is a bile acid, at 0 to 100 μmol/L was performed, and culture was performed for 24 hours. Then, HCV RNA and HCV E2 antigen were detected in the same manner as in Example 1, and the results are illustrated in FIGS. 4 and 5.

[0062] As illustrated in FIG. 4, the HCV RNA was detected at a remarkably high level in a case where treatment with the bile acid tCA at 100 μmol/L was performed, as compared with a case where tCA was used at the other concentrations and a case where the other bile acid tCDCA was used.

[0063] As illustrated in FIG. 5, it was identified that with respect to the bile acids treated, the HCV E2 antigen increased in a dose-dependent manner; and that higher HCV surface antigenicity was observed in a case where treatment with tCDCA was performed, than in a case where treatment with tCA was performed.

[0064] From the above results, it can be seen that in a case where HCV is treated with a bile acid or a bile acid derivative, lipoproteins or the like, which mask the HCV's envelope, are removed so that HCV E2 surface antigen is exposed and can be detected, thereby achieving diagnosis of HCV infection in a more efficient manner.

Example 5

Identification of Stability of E2 Surface Antigen and Genome of HCV, Affected by pH

[0065] To DMEM, which is a cell culture medium containing no bile acid, was added the HCV isolated in the preparation example in a volume of 400 μl; and the pH of the DMEM was adjusted to pH 5, 6, 7.4, 8, 9, and 10 using HCl or NaOH. Then, culture was performed for 24 hours. Then, HCV RNA and HCV E2 antigen were detected in the same manner as in Example 1, and the results are illustrated in FIGS. 6A and 6B.

[0066] As illustrated in FIGS. 6A and 6B, the HCV RNA was detected at a higher level in a range of pH 5 to 6 (slightly acidic) and pH 7 than in pH 9 to 10 (basic). In addition, the HCV E2 antigen was detected at a higher level in a range of pH 5 to pH 7 than the basic pHs.

[0067] From the above results, it can be seen that in a case where HCV is exposed to an environment of pH 5 to pH 7, which is in a slightly acidic to neutral pH range, HCV surface antigen and RNA are exposed at high levels, and this can further facilitate detection of HCV.

[0068] Although specific parts of the present invention have been described in detail, it will be apparent to those skilled in the art that these specific descriptions are provided only for preferred embodiments and the scope of the present invention is not limited thereby. Therefore, the substantial scope of the present invention should be defined by the appended claims and equivalents thereof.

INDUSTRIAL APPLICABILITY

[0069] The method for detecting a virus of the present invention allows a surface antigen, which is a lipid bilayer envelope protein, to be effectively exposed from lipoproteins that mask all or part of the envelope protein, thereby revealing the surface antigen that is masked by the lipoproteins, so that a probe such as an antibody can be used to detect the virus with high sensitivity within a short time.