JP4568335B2 - Antibody-containing solution control reagent - Google Patents

Description

  The present invention relates to an antibody-containing solution control reagent that can prevent inactivation of an antibody in a solution containing an antibody against a virus-derived antigen and can be stably stored.

  When an antigen or antibody in a specimen (patient serum or the like) is immunoassayed, it is generally performed to measure the control reagent containing the antigen or antibody at the same time and perform accuracy control. Furthermore, when immunoassay for antibodies against virus-derived antigens, control reagents containing virus antibodies are measured and compared with the measurement results to determine the presence or absence of virus antibodies or to quantify the amount of virus antibodies, etc. It can also be used as a standard product (calibrator). Since these control reagents desirably have the same physical properties as the specimen, they are generally prepared from human-derived plasma or serum.

Since human-derived plasma or serum contains proteolytic enzymes, lipids, etc., the control reagent in the solution state has poor storage stability, generation of insoluble matter, inactivation of the antigen or antibody to be measured, etc. It was. In addition, since plasma or serum containing virus antibodies is highly likely to contain infectious viruses, virus inactivation treatment is necessary, and this treatment may further inactivate virus antibodies in the control reagent. It was. In order to solve these problems, a method for removing insoluble substances in advance (Patent Document 1), a method using a special additive (Patent Document 2), and a method for preparing a control reagent by an inactivation method of a virus that hardly causes inactivation (Nonpatent literature 1, patent literature 3) etc. are proposed.
Japanese Patent Laid-Open No. 9-80049 JP-A-7-209298 JP 2001-13137 A Instruments / Reagents, pages 349-353, 22 (5), 1999

  However, in the conventional method, the storage stability in a refrigerated state (2 to 10 ° C.) is good as a solution control reagent, but the stability at a high storage temperature (for example, 20 to 40 ° C.) is not good. Since the control reagent is usually used at room temperature, even if it is stable in refrigerated storage, it may be deactivated by frequent use at room temperature, resulting in erroneous measurement results. That is, an object of the present invention is to provide a solution control reagent containing a viral antibody that is stable even at room temperature.

The antibody-containing control reagent of the present invention is characterized by comprising human-derived plasma or serum containing an antibody against a virus-derived antigen and 0.5 to 50 g / L of polyvinyl alcohol per control reagent volume. The gist.

  The antibody-containing solution control reagent of the present invention does not inactivate viral antibodies not only in refrigerated storage but also at room temperature storage. Therefore, the antibody-containing solution control reagent of the present invention has an effect that no erroneous measurement results are obtained.

  As antibodies against the virus-derived antigens of the present invention (hereinafter referred to as virus antibodies), conventionally known antibodies can be used. For example, antibody against hepatitis B virus surface antigen (HBs antibody), antibody against hepatitis B virus core antigen (HBc antibody), antigen derived from hepatitis C virus {core antigen, NS3 antigen, NS4 antigen, NS5 antigen, etc.} Antibody (HCV antibody), antibody against antigen derived from Treponema paridum (TP antibody), antibody against antigen derived from human immunodeficiency virus (HIV antibody), antibody against antigen derived from adult T-cell leukemia virus (ATL antibody), cytomegalo Examples thereof include an antibody against a virus-derived antigen (CMV antibody) and an antibody against a parvo B19 virus-derived antigen (PB19 antibody). Of these, HBs antibody, HBc antibody, antibody against an antigen derived from hepatitis C virus (HCV antibody), TP are preferable from the viewpoint of the necessity of a control reagent with high clinical frequency and higher stability. Antibodies and HIV antibodies, more preferred are HBs antibodies and HCV antibodies, and particularly preferred are HCV antibodies.

  As plasma or serum derived from humans containing virus antibodies, plasma or serum obtained from a virus-infected person can be used. For example, Golden West Biologicals, Inc., ProMedDx, LLC. It can obtain from. Viral antibodies contained in plasma or serum from individual virus-infected persons may have different responsiveness to virus-derived antigens depending on the individual. Therefore, it is preferable to pool and use plasma or serum from a plurality of virus-infected persons in order to make the reactivity uniform.

  The amount of virus antibody in plasma or serum obtained from a virus-infected person shows various amounts, but generally greatly exceeds the amount of antibody necessary as a control reagent. Accordingly, dilution with plasma or serum obtained from a healthy person (a person who is not infected with a virus) is performed as necessary. Healthy human plasma or serum is also available.

  These plasma or serum are preferably subjected to treatment such as degreasing from the viewpoint of storage stability of the solution control reagent, in particular, in order to prevent the generation of insoluble matter. Examples of the degreasing treatment include, for example, a method in which serum or plasma is stored at low temperature for several months and the precipitated insolubilized components are removed by centrifugation, freeze-thawing is repeated until no insolubilized components are deposited, organic solvent A method of removing by extraction with shaking with a mixture {for example, the method described in Clin. Chem. 29, 120-125 (1983)} is known.

  In the present invention, human-derived plasma or serum containing a virus antibody is preferably used after being subjected to a treatment for inactivating the virus. Virus inactivation methods are known in the following literature. Liquid heat treatment (Y. Uemura et al., Vox Sang 57 (1) 1-3, 1989), dry heat treatment (M. Savage et al., Biologicals 26 (2) 116-124, 1998), surfactant treatment { (Y. Uemura et al, Vox Sang 67 (3) 246-254, 1994), (B. Horowitz et al., Transfusion 25 (6) 516-522, 1985)}, acid treatment (Prokudina EN et al., Vopr Virus 34 (3) 280-283, 1989), alkali treatment (Ests MK et al., J. Gen Virol 43 (2) 403-409, 1979), alkylating agent treatment (B. Horowitz et al., Transfus). on 25 (6) 516~522,1985) and virus removal membrane treatment (S. Manabe, Dev.Biol.Stand. 88 81~90,1996).

In the virus inactivation treatment method, the most preferable for the present invention is a combination of alkylating agent treatment, surfactant treatment and liquid heat treatment.
Specifically, human plasma or serum containing viral antibodies is treated with 0.1% to 2% (volume / volume of plasma or serum) of tri (n-butyl) phosphate as one of the alkylating agent treatments, preferably 0.3% to 0.5% (volume / volume of plasma or serum) and 0.1% to 3% (volume / plasma or volume of polyoxyethylene sorbitan monooleate) as one of the surfactant treatments. Serum volume), preferably 0.5-2% (volume / plasma or serum volume) and treated at 15-37 ° C. for 4-24 hours, then 50-70 ° C., preferably about 56-60 ° C. The liquid heat treatment can be performed for 1 to 10 hours.

  Further, if necessary, the plasma or serum subjected to virus inactivation treatment is passed through an adsorption resin column, and the added inactivating agent is substantially removed. For example, the inactivating agent added by passing through an Amberlite XAD-2 resin column can be adsorbed and removed. The purpose of removing the inactivating agent is to reduce the extent to which these substances affect the stability of the control reagent. However, according to the present invention, the stability can be maintained without removing the inactivating agent. There is a merit.

  In the present invention, conventionally known polyvinyl alcohol can be used, but from the viewpoint of availability and solubility during preparation of the control reagent, the saponification degree is 70 to 90% and the polymerization degree is 100 to 10,000. Those having a polymerization degree of 300 to 4,000 are more preferable.

  The polyvinyl alcohol is preferably contained in an amount of 0.5 to 50 g / L, more preferably 1 to 40 g / L, still more preferably 5 from the viewpoint of storage stability of the control reagent and suppression of increase in viscosity. It is contained at ˜20 g / L.

  In the present invention, the antibody-containing solution control reagent includes human-derived plasma or serum containing a viral antibody, plasma or serum derived from the above-mentioned healthy person, if necessary, surfactant, preservative, etc. Can be included. For example, surfactants include 0.1 to 3% (volume / volume of control reagent) of polyoxyethylene sorbitan monooleate, which is also used as an inactivator. For example, 0.05 to 0.08% (weight / volume of control reagent) of sodium chloride is included.

  In the present invention, the amount of virus antibody in the antibody-containing solution control reagent can be appropriately set depending on the purpose of use of the control reagent. Specifically, the amount of virus antibody is adjusted by diluting plasma or serum containing the inactivated virus antibody with an appropriate solution (usually plasma or serum derived from a healthy person). The amount of the virus antibody is a commercially available virus antibody measurement or detection kit. For example, the HCV antibody is “Sphere Light HCV Antibody” (manufactured by Sanyo Chemical Industries), and the HBs antibody is “Sphere Light HBs Antibody” [Wako Pure Chemical Industries, Ltd.] It can be measured by “made by Co., Ltd.”

The method for producing the antibody-containing solution control reagent of the present invention can be appropriately set by combining methods known to those skilled in the art. For example, it can be manufactured through the following steps (1) to (6).
(1) Step of pooling serum containing virus antibodies, inactivating and degreasing (2) Step of pooling healthy human serum (serum not containing virus antibodies) and degreasing (possibility of containing unknown virus) Because there is, you may carry out inactivation processing)
(3) Steps (1) and (2) are mixed with the serum, and the virus antibody amount is adjusted to the target amount (4) Step (3) in which polyvinyl alcohol is dissolved in the product obtained in step (3) ( Add preservatives if necessary)
(5) Filtration, subdivision, labeling, etc. (6) Final quality inspection process

The antibody-containing solution control reagent of the present invention performs immunoassays such as enzyme immunoassay, radioimmunoassay, or immunoturbidimetric assay in the field of clinical tests for diagnosing viral infection by detecting or quantifying viral antibodies. Sometimes it can be used as a control for comparison. Specifically, the following examples are given as usage.
(1) When using as a standard solution for calibration curve Use multiple antibody-containing solution control reagents with different viral antibody amounts for immunoassay, create a calibration curve from the signal and viral antibody amount obtained as a result of the measurement, Used to determine the concentration value from the measurement signal.
(2) When used as a cut-off control: Prepare an antibody-containing solution control reagent that shows a cut-off (the value that is judged to have a virus antibody if the measurement signal is greater than this), and measure it at the same time as the unknown sample. Is used to determine the presence or absence of a virus antibody by comparing the signal of the control signal with the signal of the control reagent.
(3) When used as a control for accuracy control When performing immunoassays, measure the same antibody-containing solution control reagent in order to grasp changes in analyzers and reagent lots over time. Used for accuracy control.

EXAMPLES Hereinafter, although an Example demonstrates this invention further, this invention is not limited to this. Examples 8 and 9 below are reference examples.
<Example 1>
a) Inactivation of HCV antibody-containing serum Ten HCV antibody-positive human sera obtained from Golden West Biologicals, Inc. (10 mL each) were pooled to obtain 100 mL of HCV antibody-containing serum. It was. To this, 0.3 mL of tri (n-butyl) phosphoric acid and 1 mL of polyoxyethylene sorbitan monooleate were added, mixed and dissolved uniformly, and then allowed to stand at 25 ° C. for 8 hours. Furthermore, inactivated HCV antibody-containing serum was obtained by allowing the serum temperature to reach 56 ° C. in a 56 ° C. water bath for 2 hours.
b) Preparation of HCV antibody-containing control reagent 100 mL of the inactivated HCV antibody-containing serum obtained above and 900 mL of defatted human normal pool serum (HCV antibody negative, obtained from Golden West Biologicals) were mixed, and diluted HCV antibody-containing serum Got. To 100 mL of diluted HCV antibody-containing serum, 1 g of polyvinyl alcohol (saponification degree 80%, polymerization degree about 500) [manufactured by Wako Pure Chemical Industries, Ltd.], 0.08 g of sodium azide [manufactured by Wako Pure Chemical Industries, Ltd.] In addition, it was stirred and dissolved uniformly. This solution was filtered through a membrane filter having a pore size of 0.25 μm to obtain an HCV antibody-containing control reagent (1).
c) Confirmation of HCV antibody titer (antibody amount) The HCV antibody titer of the HCV antibody-containing control reagent (1) was determined using an analytical device “SphereLight 180” using a clinical test drug kit “Sphere Light HCV Antibody” (manufactured by Sanyo Chemical Industries). Measurement was performed using [Olympus Optical Co., Ltd.]. The measurement operation was performed according to the instructions attached to the kit. As a result of the measurement, the HCV antibody titer of the HCV antibody-containing control reagent (1) was a cut-off index (hereinafter abbreviated as COI) 3.60.
d) Evaluation of storage stability 2 mL of HCV antibody-containing control reagent (1) was dispensed into a 5 mL polyethylene bottle, sealed, and stored at 8 ° C., 25 ° C. and 40 ° C. for 2 months. The antibody titer was measured. The results are shown in Table 1.

<Examples 2 to 9 and Comparative Example 1>
In Example 1, except that the concentration of polyvinyl alcohol was changed to the value shown in Table 1, in the same manner as in Example 1, the HCV antibody-containing control reagents (2) to (9) and the comparative HCV antibody-containing control reagent (1) were used. Obtained. These were evaluated in the same manner as in Example 1, and the results are shown in Table 1.

<Comparative Example 2>
In Comparative Example 1, the inactivated HCV antibody-containing serum was further passed through an Amberlite XAD-2 resin column, so that the added tri (n-butyl) phosphate and polyoxyethylene sorbitan monooleate were removed by adsorption. In the same manner as in Comparative Example 1, a comparative HCV antibody-containing control reagent (2) was obtained. This was evaluated in the same manner as in Example 1, and the results are shown in Table 1.

  The HCV antibody-containing control reagent of the present invention is less susceptible to a decrease in reactivity due to an increase in viscosity or the like than the comparative HCV antibody-containing control reagent, and is significantly improved in stability at 25 ° C. and 40 ° C. storage. I understood. Particularly, in Examples 1, 4, and 5, suppression of immunoreactivity was hardly observed, and the storage stability was 40% at 90 ° C. after storage for 2 months or more, which was good.

The antibody-containing solution control reagent of the present invention can stably store an antibody against a virus-derived antigen in a solution state even at room temperature. Therefore, there is no problem of inactivation due to frequent use, and there is an effect that there is no wrong measurement.
Therefore, the antibody-containing solution control reagent of the present invention performs immunoassays such as enzyme immunoassay, radioimmunoassay, or immunoturbidimetric assay in the field of clinical tests for diagnosing virus infection by detecting viral antibodies. Sometimes it can be used as a control for comparison.

Claims (2)

An antibody-containing solution control reagent comprising human-derived plasma or serum containing an antibody against a virus-derived antigen and 0.5 to 50 g / L of polyvinyl alcohol per control reagent volume . The antibody-containing solution control reagent according to claim 1, wherein the antibody is an antibody against an antigen derived from hepatitis C virus.