JP2001004621A - Virion internal protein discharging means - Google Patents

Abstract

PROBLEM TO BE SOLVED: To break an envelope of a virus so as to allow discharge of core protein by treating a specimen containing a virus having an envelope with alkali under the existence of a protein modifying agent and a non-ionic surfactant. SOLUTION: To a specimen such as serum, a solution containing a protein modifying agent and a non-ionic surfactant and an alkaline solution are added directly without performing concentration deproteinization and the like, and inactivation of an antibody and breakage of an envelope are carried out at the same time. After the lapse of a fixed time, an acid solution is added to return the specimen treating solution to neutral and a sample for a detection system is prepared. As an objective specimen, humor such as whole blood, plasma, serum, urine, cerebrospinal fluid, sperm, saliva, maternal milk, or mucus, feces, lesioned tissue, pus, sputum and a virus cultivation system sample is used. In this treating means, a protein modifying agent is added to a specimen, a non-ionic surfactant is added, and then, the specimen is treated with an alkaline solution. These three kinds of treatment are carried out on the specimen simultaneously.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to the diagnosis of viral infections,
The present invention relates to the identification and quantification of a virus for the purpose of follow-up, treatment policy determination, and therapeutic effect evaluation, and particularly to means for detecting or quantifying an internal protein of an enveloped virus, and the release of a viral internal protein in a sample containing the virus. About the means.

[0002]

2. Description of the Related Art In recent years, identification and quantification of viruses by genetic testing have been actively performed in the diagnosis, follow-up, treatment course determination, and treatment effect determination of viral infectious diseases. Problems have been pointed out, such as high difficulty, long time required for inspection, high inspection cost, and poor reproducibility of inspection results.

[0003] In addition, a method of measuring a virus surface protein as a virus antigen, which is a conventionally known method for identifying and quantifying a virus, is used for clinical tests and blood donation screening for some viruses. However, in many viral infections, the measurement of surface antigens is hampered by antibodies to the virus surface antigens produced by the host's immune response, which can interfere with the assay or mutate the viral surface antigens to escape host immunity. In many cases, it is not suitable for virus detection and quantification.

[0004] In the field of clinical testing, there is a test for measuring the activity of HBV DNA polymerase as a device for measuring the internal protein of a virus. However, a special test requires a complicated and special device, and is widely used. I haven't.

As an alternative to the above-described virus identification and quantification method, a kit for measuring the core protein of HCV by the EIA method after subjecting the specimen to pretreatment has recently been developed.
Since 998, health insurance has been applied and used for general clinical tests. However, since the sample preprocessing operation is complicated, a simpler preprocessing method is desired.

Hepatitis C virus (HCV) has a diameter of about 6
0 nm RNA virus with an envelope. HC
VRNA is a single-stranded plus strand, consisting of about 9500 base sequences. The gene structure is similar to flavivirus, with an untranslated region (UTR) at the 5 'end and 3' end, between which there is a translated region encoding about 3000 amino acids. The translation region includes, from the 5 ′ side, a core region encoding a structural protein, an envelope region 1 and an envelope region 2, followed by a non-structural region.

[0007] Hepatitis C is usually diagnosed by measuring HCV antibodies in the blood. For this measurement, for example, expressed proteins in the non-structural region and the core region are used as antigens. Many kits for this measurement system have already been marketed and widely used as a means for diagnosis and treatment of acute and chronic hepatitis.
However, this measurement is only for knowing the presence or absence of HCV antibodies in blood, and is not for measuring HCV in blood.

[0008] The means for measuring the core protein of HCV is a system developed for measuring the amount of virus in the blood, and kits have already been marketed and used for clinical tests. The kit includes a sample pretreatment step of releasing HCV core protein from HCV in a sample, and an HCV in the sample pretreatment solution.
Measuring the V core protein by enzyme immunoassay (EIA) using a monoclonal antibody thereto.

In this sample pretreatment method, first, a polyethylene glycol solution is added to a serum sample, and HCV
Is insolubilized, and then centrifuged to collect as a precipitate fraction. The supernatant is removed to concentrate HCV particles and remove / reduce other serum components (reaction-interfering components). Next, the precipitate fraction is redissolved, alkali is added, and the mixture is left for a certain time. At this time, H in the sample which acts competitively during the subsequent EIA reaction
The CV antibody (core antibody) is inactivated. Next, an acidic solution containing a nonionic surfactant is added to neutralize the sample processing solution, and at the same time, the envelope of the HCV particles is peeled to expose the core protein. The above process is the sample pretreatment step, but the use of a urea solution at the time of redissolving the precipitate fraction is effective in releasing core protein from HCV particles,
The above-described sample pretreatment method including the addition of urea is a known technique currently used in clinical tests (Japanese Patent Laid-Open No. 8-2).
9427) (Medical Science and Pharmacy, 36 , (1), 1996).

[0010] Another method for measuring the internal antigen of an enveloped virus includes proteolytic enzyme treatment,
It has been proposed to use a nonionic surfactant in order to destroy the envelope portion after inactivating the antibody present in the sample by a treatment such as heat treatment (Japanese Patent Application Laid-Open No. Hei 8-50).
133).

[0011] As a newer proposal, a specimen containing HCV is treated with a treatment solution containing a chaotropic ion, an acidifying agent and a nonionic surfactant to inactivate antibodies present in the specimen and reduce the envelope. A method that enables simultaneous destruction has been proposed (JP-A-11-5194).
0).

[0012]

The problem to be solved by the present invention is to take into account the above-mentioned prior art documents, and to provide a simpler and more reliable detection of a virus particle internal protein, especially HCV. It is to provide a pretreatment means for releasing a core protein. In more detail, while following the method established previously, the process is simplified, the economics of pretreatment are increased, and at the same time, the specificity of the previous measurement is maintained, so that the method for measuring HCV core protein can be used. It is to provide a processing means.

[0013]

According to the present invention, a virus-containing envelope is destroyed by subjecting a specimen containing a virus having an envelope to alkaline treatment in the presence of a protein denaturing agent and a nonionic surfactant. The inventors have found that the protein is released, and completed the present invention.

[0014]

BEST MODE FOR CARRYING OUT THE INVENTION According to the present invention, a solution containing a protein denaturant and a nonionic surfactant and an alkali solution are directly added without subjecting a sample such as serum to a treatment such as concentration and deproteinization. The inactivation of the antibody and the destruction of the envelope are simultaneously carried out, and after a certain period of time, an acidic solution is added to return the sample treatment solution to neutrality, thereby obtaining a sample for the detection system.

The subject of the present invention includes whole blood, plasma, serum, urine, cerebrospinal fluid, semen, saliva, breast milk, mucus and other bodily fluids and feces, diseased tissue, pus, sputum, and virus culture system. Sample.

[0016] Target viruses include herpes virus (HSV, CMV, ZVZ, EBV, HHV6, etc.), influenza virus, human immunodeficiency virus (HIV), and human adult T-cell leukemia virus (HTL).
V), hepatitis virus (HBV, HCV, HDV, HG
V) and other pathogenic viruses for various diseases such as common cold syndrome, digestive system diseases, central nervous system diseases, respiratory system diseases, and hemorrhagic fever.

The processing means of the present invention does not require a conventional complicated operation or special device, and the required time is greatly reduced. In addition, in order to obtain a sample amount necessary for the detection system, 200 μL of the sample was required in the conventional method, but in the present invention, the measurement can be performed in 40 μL.

The processing means of the present invention adds a protein denaturant to a specimen. Urea and guanidine hydrochloride are generally well known as protein denaturants, but chaotropic agents such as sodium thiocyanate and sodium perchlorate are also effective in releasing core protein. The concentration of the protein denaturant during sample processing is preferably 0.5 M or more, and more preferably 1 to 5 M.

The processing means of the present invention adds a nonionic surfactant to a specimen. As the nonionic surfactant, a polyoxyethylene derivative is generally used, and examples thereof include Triton X-100, Nonidet P40, Tween 20, Tween 80, and Bridge 35. More preferably, it is Tween 80, and the concentration during processing is 1 to 10%, more preferably 2 to 4%.

[0020] The processing means of the present invention performs the processing on the sample with an alkaline solution. As the alkali, sodium hydroxide or potassium hydroxide is generally used, and the alkalinity at the time of the treatment is 0.05 to 0.5 N, more preferably 0.1 to 0.4 N.

The processing means of the present invention simultaneously performs the above three processes on a sample. Processing temperature and time are 10
It is about 5 to 30 minutes at ３０30 ° C., and preferably 10 to 20 minutes at about 20 ° C. If the treatment temperature is too low or the reaction time is too short, the antibody is inactivated, the envelope is destroyed, and the core protein is not released sufficiently.On the other hand, if the treatment temperature is high and the treatment time is long, the denaturation of the core protein becomes excessive. , Causing a decrease in measured values.

After inactivating the antibody and destroying the envelope, the alkali is neutralized by adding an appropriate acidic solution to the sample processing solution. The pH after neutralization is about 6 to about 8, more preferably around 7. The sample pre-processing is completed by this neutralization processing, and is applied to the subsequent detection system. By this pretreatment, the sample pretreatment liquid is diluted from the original sample. Considering the interference of serum components to the subsequent detection system, etc.
It is desirable to dilute it several times as much as the original sample, but it is optimally about 2 to 4 times considering the detection limit.

Thus, the sample which has been subjected to the pretreatment means provided has a minimum detection limit of 8 pg as before due to the dilution treatment.
/ ML to 20 to 40 pg / mL, it is desirable to use a detection system having a detection sensitivity 2.5 to 5 times higher than that of the conventional detection system. It is considered that a means for increasing the sensitivity of the detection system can be achieved by employing a highly sensitive luminescent substrate or using an antibody having a higher affinity. (Clinical Chemistry,
23 , 259-267, 1994)

Hereinafter, HCV will be specifically described as an embodiment of the present invention. However, the invention is not limited to this embodiment as long as it has an envelope.

Example 1 One sample of HCV antibody and HCV-RNA negative panel serum and two cases of HCV-RNA positive panel serum were used as samples.
Sample pretreatment was performed. For each 40 μL of sample, urea 6
60 μL of M, Tween 806 6% solution was added, and then 20 μL of 2.4N sodium hydroxide solution was added and stirred. This sample pretreatment liquid was used at 10 ° C, 20 ° C, 30 ° C,
Under four temperature conditions of 40 ° C., 1 minute, 5 minutes, 10 minutes,
After processing under the conditions of 20 minutes, 30 minutes and 60 minutes,
40 μL of a 0.4 M citric acid solution was added and stirred to obtain 160 μL of a sample pretreatment liquid. Using this sample pretreatment liquid as a sample, HCV core protein was measured using a conventional detection system (Medical and Pharmacy, 36 , (1), 1996). The results are shown in Table 1 and FIG.

In order to obtain the maximum value for both positive panels, a certain processing time is required. The lower the processing temperature, the longer the required time. However, when the processing temperature is increased, the measured value after the optimum processing time becomes longer. Decrease more rapidly. It is determined that a treatment at a treatment temperature of 20 ° C. for 10 to 20 minutes is a preferable condition in which the maximum value is obtained and the measured value does not change with time. This is a sodium hydroxide concentration of 0.4
The optimum conditions for the treatment with N are as follows. When the concentration of sodium hydroxide is low, the optimum condition shifts to a higher temperature or for a longer time, and when the concentration of sodium hydroxide is high, the optimum condition is changed. Shifts to lower temperatures and shorter times.

[0027]

[Table 1]

Example 2 One HCV antibody and HCV-RNA negative serum, HC
Using two V-RNA positive panel sera as samples, sample pretreatment was performed 10 times each by the following operation. To each 40 μL of the sample, 60 μL of a 6 M urea and 6% solution of Tween 80 were added, followed by 20 μL of a 2.4 N sodium hydroxide solution, followed by stirring. After treatment at room temperature for 10 minutes, 40 μL of a 0.4 M citric acid solution was added and stirred to obtain 160 μL of a sample pretreatment liquid. Using this sample pretreatment liquid as a sample, HCV core protein measurement was performed using a conventional detection system (Medical Science and Pharmacy, 36 , (1), 1996). The results are shown in Table 2.

All negative panels have a detection limit (40 pg / m
L), and the CV of the measured value of the positive panel was about 4%. The result of the EIA measurement including the pretreatment operation was a satisfactory result.

[0030]

[Table 2]

Example 3 In each of 30 sera of healthy persons and 30 sera of HCV-RNA-positive patients, urea 6M, Tween 8
60 μL of a 06% solution was added, followed by 20 μL of a 2.4N sodium hydroxide solution, followed by stirring. 10 at room temperature
After treatment for 40 minutes, 40 μL of a 0.4 M citric acid solution was added and stirred to obtain 160 μL of a sample pretreatment liquid. Using this sample pretreatment liquid as a sample, HCV core protein was measured using a conventional detection system. Thirty HCV-RNA-positive patient sera were pretreated by the conventional sample pretreatment method (PEG method), and the same HCV core protein was measured (Medical Science and Pharmacy, 36 , (1), 1996). The results are shown in Tables 3 and 4.

The sera of 30 healthy subjects were all of the detection limit (40
pg / mL), and no specificity problem was observed. For the sera of 30 HCV-RNA-positive patients, the values measured by the pretreatment method of the present invention and the values of the previous treatment method (PE
G), the ratio of the measured values was 1.0 to 3.1 (average value 1.8). The higher value than the conventional method was presumed to be due to the fact that 100% of HCV was not recovered in the precipitated fraction by the PEG treatment in the conventional pretreatment method, and the recovery rate was different depending on the serum state for each sample. . Therefore, it is suggested that the pretreatment method of the present invention can obtain a result reflecting the amount of virus more than the conventional pretreatment method.

[0033]

[Table 3]

[0034]

[Table 4]

[0035]

As described above, the sample pretreatment means of the present invention enables a simplified process and a short processing time as compared with the conventional method. The processing that has been performed can be achieved in about 20 minutes, and greatly enhances the efficiency of core protein testing for viruses having an envelope such as HCV.

[Brief description of the drawings]

FIG. 1 is a graph showing the influence of sample processing time and sample processing temperature on measured values of HCV core protein in two cases of HCV-RNA positive panel sera.

Claims (3)

[Claims] 1. A virus internal protein release treatment means, wherein a sample containing an enveloped virus is neutralized after being subjected to an alkali treatment in the presence of a protein denaturing agent and a nonionic surfactant. . 2. The virus internal protein release processing means according to claim 1, wherein the viral internal protein to be subjected to the release treatment is a core protein of hepatitis C virus (HCV). 3. An early method for diagnosing a viral infectious disease, comprising using the means of claim 1 or 2 to directly measure a virus internal protein on a sample without performing a preliminary screening by an antibody test.