JPH0640926A - Method for removing hepatitis c virus - Google Patents

Abstract

PURPOSE:To obtain a method for removing hepatitis C virus (HCV) from blood plasma or blood serum. CONSTITUTION:The method for readily removing HCV is to add bivalent metallic ions to blood plasma or blood serum, add a polyanionic compound thereto, stir the prepared mixture and remove the resultant precipitate.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a method for removing hepatitis C virus (HCV) from plasma or serum.

[0002]

BACKGROUND OF THE INVENTION and Prior Art Viral hepatitis is orally infected with drinking water (hepatitis A and hepatitis E).
There are types of infection (hepatitis B and hepatitis C) through blood transfusion and sexual intercourse. The latter type, which is infected by blood transfusion, is called post-transfusion hepatitis, and has been attracting attention because it tends to become chronic and is a risk factor for hepatocellular carcinoma. The causative viruses are hepatitis B virus (HBV) and hepatitis C virus (HCV). Regarding hepatitis B, HBV, which is the causative virus, has been confirmed to have virus particles from an early stage, and a vaccine has been put to practical use. Also, the inspection system at the time of blood donation is being completed soon. On the other hand, for hepatitis C, until recently, the pathogen was unknown, so that the detection method of the antigen and the antibody had to rely on exclusion diagnosis as non-A non-B hepatitis. However, the HCV cDNA was determined by Choo et al. Of Chiron, USA (Science, 244, p359, (1989)).
Since the separation of A, research on HC and HCV has progressed rapidly, and many findings are being accumulated.

An HCV antibody detection system using peptides obtained by gene recombination or chemical synthesis from various epitopes obtained by immunoscreening from HCV-RNA has been developed, and part of them has been put to practical use. . Since November 1989, the Japanese Red Cross Society has been the first in the world to screen blood for transfusion using an Ortho HC antibody detection kit using the C100-3 antigen. 1
As a result of measuring the samples of about 8 million blood donors until November 990, the ratio of HCV antibody-positive people was 1.15%, and the older the elderly, the higher the positive rate. If the results are revised according to the age structure, the number of HCV-positive persons in Japan is about 1
It is estimated that 700,000 people, 1.41% of the population.

The blood determined to be positive for HCV antibody by the screening is prohibited to be used not only for blood transfusion but also as a raw material for blood products. However, it has been revealed that the HC antibody detection kit cannot detect all the sera of HC patients in Japan due to the difference in antigenicity between strains that are prevalent in Japan and the US (Japan Society of Hepatology 1990 Year).

Therefore, in order to prevent infection with hepatitis C, it is strongly desired to completely remove HCV not only from blood for transfusion and various blood products but also from serum for quality control in terms of safety. It is rare.

As a method for removing HCV, an ultrafiltration method utilizing a polymer multilayer structure membrane (BMM, Asahi Kasei) is known. BMM has been put to practical use as a method for removing HCV from plasma fractionated preparations, especially coagulation factor preparations, and it is used at the final stage of purification of coagulation factors, and HCV can be extracted from a large amount of plasma or serum containing a high concentration of protein. Is not suitable for removing.

[0007]

The object of the present invention is to efficiently and simply remove HCV from plasma or serum containing HCV. It may also be applicable to the fractionation of HCV or its constituents, especially envelope proteins, obtained from tissue culture methods or gene recombinants. The present invention is characterized in that HCV can be removed from a large amount of plasma or serum at a very low cost without requiring any special equipment.

[0008]

The method for removing HCV of the present invention can be carried out very simply as follows. This can be achieved by first dissolving the divalent metal ion in plasma or serum, adding the polyanion compound, stirring the mixture, allowing it to stand overnight in air-conditioning, cooling and centrifuging, and removing the precipitate. here,
Representative examples of the polyanion compound used in the present invention include heparin, dextran sulfate, chondroitin sulfate, chondroitin polysulfate, heparanic acid, amylopectin sulfate, keratan sulfate, heparitin sulfate, xylan sulfate, caronine sulfate, cellulose sulfate, chitin sulfate, chitosan. Sulfuric acid, pectin sulfate, inulin sulfate, alginate sulfate, glycogen sulfate, polylactose sulfate, carrageenin sulfate, starch sulfate, polyglucose sulfate, laminarin sulfate, galactan sulfate, levan sulfate, mepesulfate sulfate polysaccharides, phosphotungstic acid, Examples thereof include polysulfated anethole, polyvinyl alcohol sulfuric acid, polyphosphoric acid and the like. As the divalent metal ion to be coexistent, ions of calcium, magnesium, manganese, cobalt and the like can be used.

Polyanion compounds such as sulfated polysaccharides are capable of binding to lipoproteins, particularly low density lipoproteins (LDL) and very low density lipoproteins (VLDL) in the presence of divalent metal ions to form precipitates. Are known. Regarding the mechanism of precipitation, the negative charge of the polyanion and the positive charge of the protein produce a soluble lipoprotein-polyanion complex, and a divalent metal ion is bound to the lipid component of the lipoprotein to form an insoluble lipoprotein- It is said to form a polyanion-metal ion complex. The method of applying this phenomenon to precipitate LDL and VLDL in plasma or serum and measuring the high density lipoprotein (HDL) remaining in the supernatant is widely used in clinical tests. The present inventors have conducted extensive studies based on the above-mentioned separation method for the purpose of separating and removing HCV, and as a result, provide a technique capable of efficiently separating HCV from plasma or serum containing HCV. It came to completion.

As a method for confirming that HCV has been removed by the above treatment, the presence or absence of HCV can be determined by applying the PCR method and the electrophoresis method to the supernatant and the precipitate dissolved in a sodium oxalate solution. it can.

Ampli-for amplification of HCV-cDNA
Double PCR is performed using taq polymerase and NCR1-4 primers. That is, 1st PCR is performed using a primer set outside NCR1 and 4, and then 2nd PCR is performed using an inside primer set of NCR2 and 3 using a part of the sample as a sample.

Each HCV gene fragment thus amplified was subjected to electrophoresis, and 1stPCR242bp, 2ndPC
HCV-RN in the sample with or without the R145bp band
The presence of A is determined. As a result, the supernatant after the HCV removal treatment of the present invention was HCV-RNA negative, but the precipitate fraction was HCV-RNA positive. This indicates that HCV was completely removed from plasma or serum by performing the removal treatment.

As described above, according to the method for removing HCV of the present invention, it has become possible to remove HCV existing in plasma or serum very easily and reliably.
When the HCV removal method of the present invention is applied to, for example, the production process of quality control serum highly utilized in clinical tests,
A safe serum for quality control that does not contain HCV infectious particles can be obtained easily and inexpensively.

[0014]

【Example】

Example 1 Dextran Sulfate-Calcium Chloride Treatment HTLV-I antibody, HIV antibody and HBs antigen negative, HCV antibody and HCV-RNA positive serum 200 m
1.11 g of calcium chloride was dissolved in 1 and 2.02 ml of a 10% dextran sulfate (Pharmacia) solution was added with stirring. After standing in a cold room overnight, a cooling centrifuge (Hitachi 6PR-5
After centrifugation at 4,000 rpm for 30 minutes at 2), a supernatant and a precipitate were obtained.

Example 2 Heparin Sodium-Manganese Chloride Treatment Serum 200 m positive for HCV antibody and HCV-RNA, negative for HTLV-I antibody, HIV antibody and HBs antigen
1.98 g of manganese chloride was dissolved in l, and 4.04 ml of 10% heparin sodium (Wako Pure Chemical Industries) solution was added with stirring. Thereafter, the same treatment as in Example 1 was carried out to obtain a supernatant and a precipitate.

Example 3: Phosphotungstic acid-magnesium chloride treatment Sera 200m positive for HCV antibody and HCV-RNA, all negative for HTLV-I antibody, HIV antibody and HBs antigen
2.03 g of magnesium chloride hexahydrate was dissolved in l, and 8.08 ml of a 10% solution of phosphotagustonic acid (Wako Pure Chemical) while stirring
Was added. Thereafter, the same treatment as in Example 1 was carried out to obtain a supernatant and a precipitate.

Example 4: HCV removal confirmation test (1) Sodium oxalate dissolution To the precipitate fractions obtained in Examples 1, 2 and 3, 10 ml of 0.1M sodium oxalate was added and shaken vigorously until the precipitate was dissolved. did. DEAE Dextran (Pharmacia) 10
% Solution was added at 25% v / v, and after stirring, the mixture was allowed to stand overnight in a cold room. After centrifugation at 4,000 rpm for 20 minutes in a cooling centrifuge, the supernatant was gently aspirated and collected. It was dialyzed against physiological saline to obtain a lipid fraction.

(2) Preparation of Specimen Solution 50 μl of the supernatant and the precipitation treatment solution obtained in Examples 1, 2, 3 and 4 (1) were each supplemented with 50% guanidine thiocyanate and 0.5 M 2-mercaptoethanol. Add 500 μl of 0.75 M citrate buffer (pH 7.0)
50 μl of M sodium acetate and 100 μl of chloroform / isoamyl alcohol (49: 1) were added and stirred.
After cooling in ice for 15 minutes, the ultracentrifuge (Tomy MC
-150) was subjected to ultracentrifugation at 15,000 rpm for 30 minutes to collect an aqueous layer. Glycogen (2 μl) was added and stirred, and then 2-propanol (600 μl) was added and stirred. After standing at 20 ° C for 1 hour, 15,000 rpm,
Ultracentrifugation was performed for 30 minutes to obtain an RNA pellet. The pellet was dried at 55 ° C. under reduced pressure, and then 10 μl of deionized water was added and dissolved to obtain a sample solution.

(3) cDNA synthesis 4 μl of 250 mM Tris-HCl buffer (pH 8.3) containing 375 mM KCl and 15 mM MgCl ₂ was added to ₄ μl of dATP, dTTP and dC.
0.4 μl of an equal volume mixture of TP and dGTP (both Takara Shuzo) (hereinafter dNTPs), random primer (Takara Shuzo)
1μl, RNase inhibitor (Takara Shuzo) 0.2μ
l, M-MLV reverse transcriptase (BR
L) 1 μl and 3.4 μl of deionized water were added to prepare a mixed solution for cDNA synthesis. After heating 10 μl of each sample solution obtained in Example 4 (2) at 65 ° C. for 5 minutes, 10 μl of the cDNA synthesis mixture was added, and heated at 37 ° C. for 90 minutes, and c
DNA was synthesized.

(4) PCR method 100 mM containing 500 mM KCl, 15 mM MgCl ₂ , 0.2% BSA
0.2μ of dNTPs in 5μl of Tris-HCl buffer (pH 8.3)
l, Taq polymerase (Takara Shuzo), primer N
CR-1 (synthetic DNA complementary to 7 to 26 bp of HCV-RNA, homemade, hereinafter the same) and NCR-4 (229 to 248 of the same)
0.2 μl of an aliquot of 1) complementary to bp) and 39.1 deionized water
μl was added to prepare a mixed solution for 1st PCR. Example 4
5 μl of each cDNA solution obtained in (3) was placed in a small Eppendorf tube, and 45 μl of the mixed solution for 1st PCR was added and mixed. After dropping 2 drops of mineral oil to prevent evaporation, program control system PC-70
Using 0 (Astec), 35 cycles of DNA amplification were performed (1st PCR).

Then, dNTPs were added to 5 μl of the above buffer solution.
0.2 μl, Taq polymerase 0.2 μl, primer NC
R-2 (complementary to 46-65 bp) and NCR-3 (complementary)
0.5 μl of an equal volume mixture of 171 to 190 bp)
This was used as a mixed solution for ndPCR. 1 from 1st PCR reaction solution
Transfer μl to a small Eppendorf tube and use 2nd PCR
The mixed solution (49 μl) was added and mixed. Below, 1st PCR
2nd PCR was performed in the same manner as in.

(5) Electrophoresis A 2% agarose gel (0.089M Tris-borate buffer (pH7.3) 20 mM EDTA) was prepared in a mini gel electrophoresis system Mupid-2 (Advance), and after solidification, it was placed in an electrophoresis tank. The same buffer was filled. 10 μl of the sample (1st PCR reaction solution and 2nd PCR reaction solution) obtained in Example 4 (4), to which 2 μl of the dye solution Marker V (Hanui) was added, was applied and electrophoresed at 120V. Stop the electrophoresis when the marker is about 1 cm from the center, and remove the taken gel in 0.025% ethidium bromide fluorescent staining solution.
Shake for 0 minutes and stain. The presence of HCV-RNA in the sample was determined by irradiating the stained gel with a UV lamp and observing a band at the same position as the positive control. As a result, as shown in FIGS. 1 and 2 and Table 1, for the precipitates obtained in Examples 1, 2, and 3, a band was observed at the same position as that of the positive control. No band was observed in any of the samples in the supernatant. This means that the precipitated fraction is HCV positive and the supernatant is HCV negative, and by performing the polyanion-metal salt treatment of the present invention, HCV in serum can be precipitated and HCV can be easily removed. It shows that.

[0023]

[Table 1]

[Brief description of drawings]

FIG. 1 is a diagram showing an electrophoresis result of each sample obtained by a 1st PCR reaction.

FIG. 2 is a diagram showing an electrophoresis result of each sample obtained by the 2nd PCR reaction.

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Claims (3)

[Claims] 1. A method for removing hepatitis C virus, which comprises adding a divalent metal ion and a polyanion compound to plasma or serum and stirring and removing the resulting precipitate. 2. The polyanion compound is at least one compound selected from sulfate polysaccharides such as heparin, dextran sulfate, chondroitin sulfate, chondroitin polysulfate, heparanic acid, and amylopectin sulfate, phosphotungstic acid, and polyphosphoric acid. Item 1. The removal method according to Item 1. 3. The removal method according to claim 1, wherein the divalent metal ion is at least one metal ion selected from ions of calcium, magnesium, manganese, and cobalt.