JP4890399B2 - Reagent for quality control for detecting HBV antigen and method for producing the same - Google Patents

Description

  The present invention relates to a quality control reagent that can be used in a method for detecting a hepatitis B virus (HBV) antigen and a method for producing the same.

In general, diagnosis of the presence or absence of infection with hepatitis B virus (HBV) and the type of infection (transient or persistent infection), observation of the course of treatment, investigation of the history of infection, etc. HBV markers such as hepatitis virus s antigen (HBsAg), hepatitis B virus e antigen (HBeAg), antibody against HBsAg (HBs antibody), antibody against hepatitis B virus nuclear antigen (HBc antibody), antibody against HBeAg (HBe antibody) Can be done by examining
For example, a positive HBs antibody or HBc antibody indicates that the patient has been infected with HBV in the past, but first, HBsAg is examined in order to examine the presence or absence of infection. Further, by examining the amount of HBeAg, it is possible to know the degree of infectivity of the virus in the patient's body.

These HBV markers are generally known to be detectable and quantifiable by immunoassays such as radioimmunoassay (RIA) and enzyme immunoassay (EIA) using antibodies or antigens against these antigens or antibodies.
In order to confirm that such measurement of the HBV marker is accurately performed, a quality control reagent (so-called positive control) including the HBV antigen or antibody to be measured is used.

  Conventionally, the HBV antigen contained in the quality control reagent for measuring HBsAg and HBeAg has been obtained from human blood infected with HBV. However, in this case, there are problems such as it is difficult to stably obtain human blood as a raw material and there is a risk of infection during purification of the antigen.

Therefore, research for producing these HBV antigens by gene recombination technology is underway.
For example, Japanese Patent Application Laid-Open No. 58-52228 (Patent Document 1) discloses a method for obtaining HBeAg by treating hepatitis B virus c antigen (HBcAg) expressed in Escherichia coli with a gene recombination technique with a protease. Has been. JP-A-7-203971 (Patent Document 2) discloses recombinant HBeAg to be expressed in yeast.
JP 2001-294599 A (Patent Document 3) discloses that HBsAg is obtained by a gene recombination technique.

  Currently, quality control reagents containing HBsAg as described above are manufactured as quality control reagents for HBsAg measurement, and quality control reagents containing HBeAg are used as quality control reagents for HBeAg measurement, respectively. Are sold. However, there is no known quality control reagent that can be used in common for the measurement of these two types of antigens.

If a quality control reagent that can be commonly used for the measurement of two types of antigens, HBsAg and HBeAg, can be obtained, one type of quality control reagent can be used for two types of measurement, so the number of reagents can be reduced. Thus, the complexity of the inspection can be reduced. Therefore, development of a quality control reagent that can be commonly used for the measurement of HBsAg and HBeAg is desired.
JP 58-52228 A JP-A-7-203971 JP 2001-294599 A

An object of the present invention is to provide a quality control reagent that can be used in common for the measurement of HBsAg and HBeAg.

The present invention provides a quality control reagent for detecting HBV antigens, which contains both HBsAg and HBeAg obtained by treating HBcAg with protease.
The present invention also provides a method for producing a quality control reagent for HBV antigen detection, which comprises a step of mixing HBsAg and HBeAg obtained by subjecting HBcAg to protease treatment.

Since the quality control reagent for detecting HBV antigen of the present invention contains HBsAg and HBeAg, it can be used in both the measurement of HBsAg and the measurement of HBeAg. Thereby, when measuring about both antigens, it can avoid that the number of reagents increases and a measurement becomes complicated.
Further, HBsAg and HBeAg contained in this quality control reagent have good stability. Thereby, the reagent for quality control excellent in stability can be provided.

  The present inventors considered that a reagent for quality control that can be commonly used for measurement of HBsAg and HBeAg can be obtained by including two antigens of HBsAg and HBeAg in the reagent. However, conditions for good stability of HBsAg and HBeAg are not necessarily the same. For example, the stability of HBsAg is good around pH 7.0, whereas the stability of HBeAg is poor. On the other hand, the stability of HBeAg is relatively good around pH 8.5, whereas the stability of HBsAg is deteriorated. For this reason, even if a quality control reagent containing these two types of antigens is simply prepared, only one of the antigens with poor stability can be prepared. Cannot be fully guaranteed.

  Therefore, as a result of intensive studies to obtain a quality control reagent containing both antigens of HBsAg and HBeAg under stable conditions, the present inventors use HBeAg obtained by protease treatment of HBcAg. Thus, it was found that these two types of antigens can be stably contained in the same quality control reagent, and the present invention was completed.

  In this specification, the “quality control reagent for detecting HBV antigen” means a quality control substance used for assuring the accuracy of measurement for detecting an HBV antigen, so-called positive control.

The quality control reagent of the present invention contains HBsAg and HBeAg obtained by subjecting HBcAg to protease treatment.
HBsAg is a protein that constitutes the outer shell of HBV and is called HBV surface antigen. The HBsAg used for the quality control reagent of the present invention may be obtained by purifying from the blood of a person infected with HBV, but it can be obtained of a certain quality by genetic recombination technology. The resulting recombinant HBsAg is preferred.
HBsAg may be an antigen fragment obtained by excising a portion having antigenicity as the s antigen.
A commercial item can be used as HBsAg.

HBeAg used for the above-mentioned quality control reagent can be obtained by subjecting HBcAg to a protease treatment. HBeAg is a protein that constitutes the core particle of HBV, and when HBV grows, it is released into the blood of humans infected with HBV, so the state of infection can be examined by detecting this antigen. .
Since HBcAg is usually present inside the HBV outer shell, it is almost impossible to detect HBcAg in the blood of humans infected with HBV.
Therefore, the HBcAg used for obtaining the above-mentioned HBeAg is preferably a recombinant HBcAg obtained by a gene recombination technique.
A commercially available product can be used as the recombinant HBcAg.

  As a method for obtaining HBeAg by treating protease with HBcAg, a known method was used (the method described in Patent Document 1).

  The protease used for obtaining HBeAg is not particularly limited as long as it is a protease usually used for obtaining HBeAg from HBcAg. Trypsin, pronase, subtilisin, carboxypeptidase A, carboxypeptidase B, papain, chymopine, promeline, protease Those selected from the group consisting of K and thermolysin can be suitably used. More preferred is trypsin.

  The respective concentrations of HBsAg and HBeAg in the above-mentioned quality control reagent can be appropriately set according to the type of measurement method using the quality control reagent, the type of reagent and apparatus used for the measurement, and the like.

HBsAg is generally considered to be stable at a neutral pH such as pH 6.5 to 7.5. On the other hand, HBeAg is generally considered to be stable at a pH near weak alkalinity such as pH 8.0 to 9.0. However, when the present inventors investigated the stability of HBeAg obtained by the protease treatment as described above, it was found that HBeAg obtained by the protease treatment was also stable at a pH near neutral.
For these reasons, it is preferable that the quality control reagent has a pH around neutral. As specific pH, 6.5-7.5 are preferable and 6.8-7.2 are more preferable.
The above-mentioned quality control reagent preferably further contains a buffer for maintaining the pH in the above-described range in addition to the above-described HBsAg and HBeAg. Examples of the buffer include triethanolamine (TEA), sodium phosphate, and Good buffer such as PIPES, ACES, and BES. The concentration of the buffer is not particularly limited as long as the pH can be maintained.

The above-mentioned quality control reagent preferably further contains a protein protective agent for preventing HBsAg and HBeAg in the quality control reagent from adsorbing to a storage container or the like. A conventionally well-known thing can be used for such a protein protective agent, BSA, casein, collagen, gelatin, polyvinyl alcohol (PVA) etc. are mentioned. By including a protein protective agent in the quality control reagent, it is possible to prevent the HBsAg and HBeAg in the quality control reagent from being adsorbed to a storage container or the like.
The protein protecting agent is preferably contained in the quality control reagent in an amount of 0.001 to 5% by weight, more preferably 0.05 to 1% by weight.

  In addition to the above-described components, the above-mentioned quality control reagent can contain additive components known to be contained in conventional quality control reagents. Examples of such additive components include preservatives (for example, sodium azide), excipients (for example, sucrose), chelating agents (for example, EDTA), and the like.

  The above quality control reagent may be in a liquid state, in a frozen state in which the liquid is frozen, or in a solid state obtained by lyophilization from the liquid. For example, when it is liquid, the quality control reagent can be used for measurement in that state. When in a frozen state, the reagent for quality control can be used for measurement after liquefying by thawing before use. When it is in a solid form, it can be used for measurement after it is made liquid by dissolving in an appropriate solvent such as water or a buffer before use.

  The above-described reagent for quality control stably holds HBsAg and HBeAg. The term “stable” as used herein refers to a change in antigenicity of HBsAg and HBeAg in the reagent when the accuracy control reagent is stored in a liquid refrigerated (2 to 8 ° C.) condition for approximately 6 months, preferably approximately 1 year. It means within ± 10% range, preferably within ± 5% range compared with that before storage. Such stability can be easily predicted by examining the antigenicity of HBsAg and HBeAg when the quality control reagent is stored in a liquid state at 45 ° C. for 3 days. In this simple method, if these antigenicities in the quality control reagent are maintained at 90% or more, preferably 95% or more with respect to the control, HBsAg and HBeAg in the reagent are stably retained. Predictable. The antigenicity can be measured by a method for measuring an antigen, for example, using an immunoassay method using an antibody against the antigen. As said control | contrast, what preserve | saved the quality control reagent for the same period on the conditions of refrigeration (2-8 degreeC) or freezing (less than -30 degreeC) can be used.

  When the above-mentioned quality control reagent is liquid, it is a reagent with excellent storage stability of HBsAg and HBeAg in the reagent solution. As described above, the reagent may be in a frozen or solid state in addition to liquid form. However, when the measurement is performed, the accuracy control reagent in a frozen or solid state is usually melted or dissolved in an aqueous solution to form a liquid and then used for the measurement. And the quality control reagent in a frozen or solid state is a reagent excellent in storage stability of HBsAg and HBeAg when it is made liquid.

The above-mentioned reagent for quality control can be produced by mixing HBeAg obtained from protease treatment with HBcAg and HBsAg. The HBsAg described above can be used as the HBsAg used in the production.
In the mixing step, these antigens can be mixed in a liquid state or can be mixed in a solid state. Alternatively, one of the antigens in a solid state and the other antigen in a liquid state can be mixed. The liquid mixture obtained in the liquid state can be frozen or lyophilized.

  In the above method for producing a quality control reagent, in the mixing step, any of the above components, that is, a buffer, a protein protecting agent, an additive component, and the like can be mixed together.

  Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples.

Reference Example 1 Stability of HBsAg and HBeAg by pH (accelerated test at 45 ° C.)
In order to confirm the stability of HBs antigen (HBsAg) and HBe antigen (HBeAg) with respect to pH, the following experiment was performed.
Commercially available recombinant HBsAg (adr type, CORTEX BIOCHEM) and commercially available recombinant HBeAg (Fitzgerald) were mixed with 0.1% bovine serum albumin (BSA) pH 7.0 or 7.5 triethanolamine ( TEA) was dissolved in buffer or Tris-HCl buffer at pH 8.0 or 8.5, respectively.
Each antigen in the solution after the obtained HBsAg / HBeAg-containing solution was stored at 45 ° C. for 1 to 3 days was used for recombinant HBsAg (hereinafter, also referred to as rHBsAg), for Elsia FS-HBs antigen (for HBsAg measurement). Reagents, Sysmex) and recombinant HBeAg (hereinafter also referred to as rHBeAg) were measured using Elsia FS-HBe antigen (reagent for measuring HBeAg, Sysmex). ELISA-FS1200 (Sysmex Corporation) was used as a measuring device.

Furthermore, the measured value obtained by measuring the HBsAg / HBeAg-containing solution was determined as a relative value to the value measured on the 0th day.
The results are shown in Table 1 and FIG.

Table 1 shows the relative values of rHBsAg and rHBeAg. FIG. 1 shows (A) a change in the relative value of rHBsAg and (B) a change in the relative value of rHBeAg.
From the results of Table 1 and FIG. 1, when stored at 45 ° C., rHBsAg in a solution of pH 7.0 to 8.0 showed a relative value of 80% or more even after 3 days of storage, but pH 8.5 The rHBsAg in this solution was a relative value of about 60% after 3 days of storage. From the above, it can be seen that rHBsAg is relatively stable around pH 7.0, but not stable around pH 8.5. On the other hand, when stored under the condition of 45 ° C., the relative value of rHBeAg decreased in 1 day after storage. This may be due to the special condition of 45 ° C. In addition, when the result of each pH is compared, it is estimated that rHBeAg is comparatively stable on the alkali side of pH 7.5 to 8.5 rather than pH 7.0. From the above, it is expected that even if these two types of recombinant antigens are stored in the same solution, they cannot be used as stable quality control reagents.

Reference Example 2 HBsAg and HBeAg pH stability (long-term stability)
The same commercially available recombinant HBsAg and commercially available recombinant HBeAg as used in Reference Example 1 above were dissolved in pH 8.0 triethanolamine (TEA) buffer solution (containing 0.1% BSA), respectively. A solution containing HBsAg / HBeAg was obtained. Each antigen in the solution when this solution was stored at 9 ° C. for 3, 6 and 9 months was measured in the same manner as in Reference Example 1.
As a control, an HBsAg / HBeAg-containing solution that was stored frozen (−80 ° C.) was used.
In addition, measurement is performed 3 times and the average value is shown as a result of the measured value.

The relative value was calculated from the measured value of each antigen measured with the measured value of the control as 100%.
The results are shown in Table 2 and FIG.

  Table 2 shows (A) the relative value of rHBsAg and (B) the relative value of rHBeAg. FIG. 2 shows changes in the relative values of rHBsAg and rHBeAg. From the results in Table 2 and FIG. 2, it can be seen that the relative value of rHBsAg decreased while the relative value of rHBeAg increased as the period passed. That is, it can be seen that neither the storage stability of rHBsAg nor rHBeAg is good in the HBsAg / HBeAg-containing solution of this comparative example. From the above, it has been found that it is difficult to obtain a quality control reagent in which HBsAg and HBeAg are stable even if two antigens are used in combination.

Example 1
As a method for obtaining HBeAg by protease treatment (trypsin treatment) of HBcAg, a known method was used (method described in Patent Document 1). Hereinafter, the HBeAg thus obtained is also referred to as trypsin-treated HBeAg.

Mixing trypsin-treated HBeAg and commercially available recombinant HBsAg (adr type, CORTEX BIOCHEM) in 0.1M TEA buffer (containing 0.1% BSA) at pH 7.0 to obtain a quality control reagent It was. Antigens in the reagents were measured when the precision control reagents were stored at 37, 45 and 50 ° C. for the number of days shown in Table 3, respectively.
As a control, the above-mentioned reagent for quality control that was refrigerated (2 to 8 ° C.) was used.

The amount of HBeAg was measured in the same manner as in Reference Example 1. On the other hand, the amount of HBsAg was measured according to the immunoassay for HBsAg described below.
<Immunoassay of HBsAg>
(1) Reagent preparation Anti-HBsAg mouse monoclonal antibody of international accession number FERM BP-10582 is biotinylated using a commercially available kit and biotinylated anti-HBsAg mouse monoclonal antibody solution (2 μg / mL, 0.1 M MES buffer (pH 6) .5)) was obtained. Further, alkaline phosphatase (ALP) was bound to an anti-HBsAg mouse monoclonal antibody of international accession number FERM BP-10583, and an alkaline ALP-conjugated anti-HBsAg mouse monoclonal antibody solution (200 mU / mL, 0.1 M MES buffer (pH 6.5)). ) Commercially available streptavidin magnetic particles were suspended in 20 mM MES buffer (pH 6.5, 0.1% BSA) so as to be 0.5% to prepare a streptavidin magnetic particle suspension.

(2) Immunoassay Method 50 μL of a biotinylated anti-HBsAg mouse monoclonal antibody solution and 20 μL of a quality control reagent were mixed and incubated at 42 ° C. for about 2 minutes. Next, 30 μL of 0.5% streptavidin magnetic particle suspension was added and incubated at 42 ° C. for about 1 minute. After washing the magnetic particles with a washing solution (20 mM sodium phosphate buffer (pH 7.5)), 100 μL of ALP-conjugated anti-HBsAg mouse monoclonal antibody solution was added and incubated at 42 ° C. for about 1 minute. After washing the magnetic particles with the above washing solution, 100 μL of CDP-Star (registered trademark) (Applied Biosystem), which is a substrate for ALP, was added to emit light. Luminescence intensity was measured using LUMI-COUNTER 700 (Microtech Nichion Co., Ltd.).

The relative value was calculated from the measured value of each antigen measured with the measured value of the control as 100%.
The results are shown in Table 3 and FIG.

Table 3 shows (A) the relative value of rHBsAg and (B) the relative value of trypsin-treated HBeAg. FIG. 3 shows (A) change in the relative value of rHBsAg and (B) change in the relative value of trypsin-treated HBeAg.
Furthermore, when an Arrhenius plot was created from the measured values at each temperature condition and the deterioration of each antigen in the produced quality control reagent was predicted, rHBsAg was 97.9% when stored at 9 ° C. for 395 days, It was predicted that trypsin-treated HBeAg was 99% or more and the activity of each antigen remained. From the above, it can be predicted that the quality control reagent containing rHBsAg and trypsin-treated HBeAg is excellent in storage stability.

Example 2
Each antigen in the reagent when the quality control reagent prepared in Example 1 was stored at 9 ° C. for 3, 5 or 6 months was measured in the same manner as in Example 1. As a control, a quality control reagent stored at −80 ° C. was used.
In addition, measurement is performed 3 times and the average value is shown as a result of the measured value.
The relative value was calculated from the measured value of each antigen measured with the measured value of the control as 100%.
The results are shown in Table 4 and FIG.

Table 4 shows (A) the relative value of rHBsAg and (B) the relative value of trypsin-treated HBeAg. FIG. 4 shows (A) change in relative value of HBsAg and (B) change in relative value of trypsin-treated HBeAg. From the results in Table 4 and FIG. 4, it can be seen that even when the period elapses, the measured values of rHBsAg and trypsin-treated HBeAg are hardly changed. That is, it can be seen that the storage stability of rHBsAg and trypsin-treated HBeAg is good in the quality control reagent.
From the results of Examples 1 and 2, it can be seen that both HBsAg and HBeAg can exist stably for a long period of time in a quality control reagent prepared by combining HBsAg and HBeAg obtained by protease treatment.

Reference examples 3 and 4
Commercially available recombinant HBsAg (adr type, CORTEX BIOCHEM), or HBeAg prepared by trypsinization from HBcAg as described in Example 1 was separately prepared at pH 6.5, 6.8, 6.9, respectively. An HBsAg-containing solution and an HBeAg-containing solution were prepared by dissolving in 7.0.7.1 and 7.2 0.1M TEA buffer (containing 0.1% BSA).
The antigens in the solutions when these solutions were stored at 45 ° C. for 2 and 7 days were measured in the same manner as in Reference Example 1.
As a control, an HBsAg-containing solution or an HBeAg-containing solution that was refrigerated (2 to 8 ° C.) was used.
The relative value was calculated from the measured value of each antigen measured with the measured value of the control as 100%.
The results are shown in Table 5 and FIG.

Table 5 shows (A) the relative value of rHBsAg in the HBsAg-containing solution, and (B) the relative value of trypsin-treated HBeAg in the HBeAg-containing solution. FIG. 5 shows (A) change in the relative value of HBsAg in the HBsAg-containing solution, and (B) change in the relative value of trypsin-treated HBeAg in the HBeAg-containing solution. From Table 5 (A) and FIG. 5 (A), it can be seen that the stability of rHBsAg in the HBsAg-containing solution is good under the conditions of pH 6.5 to 7.2. Moreover, it can be seen from Table 5 (B) and FIG. 5 (B) that the stability of trypsin-treated HBeAg in the HBeAg-containing solution is good under the conditions of pH 6.5 to 7.2.
From the above, it can be seen that each antigen in the quality control reagents of Examples 1 and 2 has good stability even at the above pH.

It is a graph which shows the stability in 45 degreeC in each pH of HBsAg in the HBsAg * HBeAg containing solution of Reference Example 1 and HBeAg. 5 is a graph showing long-term storage stability of HBsAg and HBeAg in the HBsAg / HBeAg-containing solution of Reference Example 2. 4 is a graph showing storage stability in an accelerated test of HBsAg and HBeAg in the quality control reagent of Example 1. FIG. 3 is a graph showing long-term storage stability of HBsAg and HBeAg in the quality control reagent of Example 2. It is a graph which shows the storage stability in the accelerated test of each antigen in a HBsAg containing solution and the HBeAg containing solution obtained by the trypsin treatment.

Claims (8)

  A type B comprising both hepatitis B virus s antigen (HBsAg) and hepatitis B virus e antigen (HBeAg) obtained by protease treatment of hepatitis B virus c antigen (HBcAg) Reagent for quality control for detection of hepatitis virus (HBV) antigen.   The quality control reagent according to claim 1, further comprising a buffer for maintaining the pH at 6.5 to 7.5.   The quality control reagent according to claim 1 or 2, further comprising a protein protecting agent.   The quality control reagent according to any one of claims 1 to 3, wherein the protease is trypsin.   The reagent for quality control according to any one of claims 1 to 4, wherein the HBsAg is a recombinant HBsAg.   The quality control reagent according to any one of claims 1 to 5, wherein the HBcAg is a recombinant HBcAg.   A method for producing a quality control reagent for detecting HBV antigen, comprising a step of mixing HBsAg and HBeAg obtained by subjecting HBcAg to protease treatment.   The production method according to claim 7, wherein a buffering agent for maintaining the pH at 6.5 to 7.5 is further mixed in the mixing step.

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