Compositions, indicators and methods for evaluating antiviral performance, and methods for searching cleansing conditions

Abstract

To provide methods for evaluating HBV removal performance with safety and in a short time.SOLUTION: The invention relates to a composition for evaluating antiviral performance comprising an envelope type bacteriophage and impurity derived from a living body. Preferably the impurity derived from a living body comprises serum proteins. The invention also encompasses an indicator for evaluating antiviral performance where the indicator comprises the composition for evaluating antiviral performance on its substrate surface and a method for evaluating antiviral performance of medical devises. The method comprises cleansing a medical devise and the indicator together where the medical devise is stained with a body fluid, and then collecting and culturing the residue on the indicator surface to determine the amount of the envelope type bacteriophage.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a composition for antiviral evaluation, an indicator for antiviral evaluation, a method for antiviral evaluation, and a method for searching cleaning conditions. [Background technology]

[0002] Medical equipment used in medical institutions may become contaminated with body fluids such as blood, or biological dirt such as tissue fragments. To prevent infection by viruses and bacteria, medical equipment must be washed after use before reuse, and the degree of cleaning must be checked before reuse.

[0003] For example, Patent Document 1 discloses an indicator for evaluating the cleaning of medical equipment, which comprises a coating film containing vegetable protein as a pseudo-contaminant formed on the surface of a sheet substrate. In Patent Document 1, the extent to which vegetable protein, a pseudo-contaminant contained in the coating film on the surface of the sheet substrate, has been removed by cleaning is evaluated, and this is used to determine whether many parameters related to cleaning are properly reflected in the cleaning device. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] JP 2009-039192 A Summary of the Invention [Problem to be solved by the invention]

[0005] Among the viruses contained in biological dirt, hepatitis B virus (HBV) is known to have an extremely high risk of infection, and there is a risk that something with biological dirt containing HBV attached to it may become infected due to medical procedures or through a wound. In situations where invasive procedures such as surgical procedures and dental procedures are common, medical tools or medical equipment are likely to be contaminated by the patient's blood or droplets, and it is desirable to be able to evaluate the risk of HBV infection not only in medical settings but also in food factories, nursing homes, public facilities (such as toilets), and the like. Several contamination models and indicators for cleaning evaluation have been proposed, but as shown in the above-mentioned Patent Document 1, cleaning is evaluated based on the removability of pseudo-contaminants such as proteins, and the removability of the virus itself has not been evaluated.

[0006] In addition, there have been several reported cases of antiviral evaluation using HBV, but this has the disadvantages of requiring special equipment to reduce the risk of infection during evaluation, and the evaluation period being long.

[0007] Therefore, an object of the present invention is to provide a method capable of evaluating the removal ability of HBV itself safely and in a short period of time. [Means for solving the problem]

[0008] The present invention which achieves the above object is as follows. [1] A composition for antiviral evaluation comprising an enveloped bacteriophage and biological contaminants. [2] The composition for antiviral evaluation according to [1], wherein the biological contaminants include serum proteins. [3] An indicator for antiviral evaluation having the composition for antiviral evaluation according to [1] or [2] on a substrate surface. [4] A method for antiviral evaluation of a medical device, comprising washing the indicator for antiviral evaluation described in [3] together with a medical device contaminated with a body fluid, recovering and culturing the residue on the surface of the indicator for antiviral evaluation after washing, and measuring the amount of the enveloped bacteriophage, thereby performing an antiviral evaluation of the medical device. [5] A method for searching for washing conditions, which comprises repeating the steps of washing the indicator for antiviral evaluation described in [3], recovering and culturing a residue on the surface of the indicator for antiviral evaluation after washing, and measuring the amount of the enveloped bacteriophage while changing the washing conditions. Effect of the Invention

[0009] According to the present invention, since bacteriophage is used, the risk of HBV infection can be eliminated and the HBV removal ability can be alternatively evaluated in a short period of time. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] As a result of the inventors' investigations, they discovered that a composition containing a bacteriophage that has the same membrane characteristics as HBV but has low pathogenicity to humans, and contaminants of biological origin, can be used as an alternative evaluation of the removal of HBV, and thus completed the present invention.

[0011] (1) Bacteriophage In the present invention, an enveloped bacteriophage is used as an alternative to evaluating the removal of HBV by washing. Bacteriophage is a virus that infects bacteria, and since it only infects limited bacteria, it is a virus with low pathogenicity to humans. Since HBV is an enveloped virus, the use of an enveloped bacteriophage having a similar structure makes it possible to evaluate the extent to which HBV can be removed by washing (hereinafter, sometimes referred to as antiviral effect) while ensuring safety to humans. It is more preferable to use φ6 phage as the bacteriophage.

[0012] (2) Biological contaminants When HBV adheres to medical devices, etc., it is usually accompanied by contaminants of biological origin. Therefore, by using enveloped bacteriophage and contaminants of biological origin, it is possible to faithfully simulate the actual presence of HBV on medical devices, etc., and to perform an alternative evaluation of HBV removal ability.

[0013] The biological contaminants (hereinafter sometimes simply referred to as contaminants) are preferably, for example, blood-derived contaminants, more preferably contaminants containing serum proteins, and even more preferably contaminants containing albumin. Specific examples of biological contaminants include fetal bovine serum (FBS), bovine serum albumin (BSA), and whole sheep blood. Biological contaminants can be obtained from animals such as cows, sheep, horses, pigs, and mice by conventional methods, or commercially available products can be used.

[0014] (3) Composition for antiviral evaluation The antiviral evaluation composition contains the above-mentioned enveloped bacteriophage and impurities. As shown in the examples below, the composition for antiviral evaluation of the present invention and a serum containing actual HBV are mixed using the same detergent at the same dilution ratio, and the antibacteriophage activity value is obtained by comparing the amount of bacteriophage before and after mixing, and the anti-HBV activity value is obtained by comparing the amount of HBV before and after mixing. When the antibacteriophage activity value and the anti-HBV activity value are 2.0 or more, it is evaluated that there is an antiviral effect, and when it is less than 2.0, it is evaluated that the antiviral effect is insufficient, and the antiviral evaluation of both is well consistent. Therefore, the composition for antiviral evaluation of the present invention can be suitably used for the antiviral evaluation of HBV.

[0015] The antiviral evaluation composition usually contains a liquid such as water. The concentration of the enveloped bacteriophage in the antiviral evaluation composition is 10 5 ~10 9 PFU / mL is preferred, and 10 7 ~10 9 It is more preferable that the concentration is PFU / mL. When the composition for antiviral evaluation contains water, the amount of water in 100% by mass of the composition is, for example, 0.01 volume / volume% or more and 99.99 volume / volume% or less.

[0016] The contaminants may be one type only or two or more types may be contained. In a preferred embodiment, the contaminants contain serum proteins, and in such a case, the amount of the proteins is preferably 10 to 130 mg / mL, more preferably 40 to 120 mg / mL, relative to the total volume of the composition for antiviral evaluation.

[0017] When the impurity is bovine serum albumin (BSA), the concentration is preferably 20 to 80 mg / mL relative to the total volume of the composition for antiviral evaluation. When the impurity is fetal bovine serum (FBS), the concentration is preferably 0.6 to 0.9 mL / mL relative to the total volume of the composition for antiviral evaluation, and the protein amount is preferably 20 to 70 mg / mL. Furthermore, when the impurity is whole sheep blood, the concentration is preferably 0.2 to 0.9 mL / mL relative to the total volume of the composition for antiviral evaluation, and the protein amount is preferably 10 to 130 mg / mL, and more preferably 40 to 120 mg / mL.

[0018] The antiviral evaluation composition is obtained by mixing the enveloped bacteriophage and the contaminants derived from the living body, and preferably also mixing water as necessary. The enveloped bacteriophage and the contaminants derived from the living body may be mixed after preparing aqueous liquids (aqueous solutions or water suspensions) containing them. The antiviral evaluation composition may also contain other components. In particular, when commercially available contaminants are used, the composition of the present invention may contain sodium chloride, sodium azide, glycine, etc., and when blood containing an anticoagulant such as sodium heparinate is used, it may contain an antagonist of the anticoagulant such as protamine sulfate or protamine hydrochloride. These other components, i.e., components other than the enveloped bacteriophage, the contaminants derived from the living body, and water, are preferably 1 mass / volume % or less in total in the composition, more preferably 0.5 mass / volume % or less.

[0019] The composition for antiviral evaluation can be used in a form in which an indicator is prepared by applying it to a substrate and solidifying it as described below, and then the indicator is put into a cleaning device together with an evaluation target to perform antiviral evaluation, and can also be used when cleaning is performed without using a cleaning device. That is, in a cleaning operation including a step of applying a cleaning agent to a target cleaning target contaminated with a body fluid by spraying, pouring, rolling, or the like, and a step of rinsing with water and / or wiping, the composition of the present invention can be applied to a location other than the target cleaning target location and solidified, and the same cleaning operation is performed on the solidified product of the composition of the present invention, and then the washed location of the solidified product of the composition of the present invention is scraped off and cultured for evaluation.

[0020] (4) Indicator for evaluating antiviral properties The indicator for antiviral evaluation has an antiviral evaluation composition containing the above-mentioned enveloped bacteriophage and biological contaminants on the surface of a substrate. Specifically, the indicator for antiviral evaluation can be produced by applying a liquid containing the enveloped bacteriophage and biological contaminants to a substrate and solidifying it. Solidification is usually carried out by drying the substrate to which the liquid has been applied, and may be allowed to stand at room temperature for about 1 to 24 hours. The amount of application of the composition containing the enveloped bacteriophage and biological contaminants (preferably a liquid composition containing water) is 1 cm 2 of the substrate. 2 For example, the volume is preferably 1 to 27 μL and the mass is preferably 1 to 27 mg per unit area.

[0021] The material of the substrate is not particularly limited, and may be a resin such as polyethylene, polypropylene, polyester resin, silicone resin, or fluororesin, a metal such as stainless steel, aluminum, or titanium, a ceramic such as alumina or zirconia, or glass, but is preferably a metal, and more preferably stainless steel. The shape of the substrate is also not limited, and may be a plate or rod, and when it is a plate, the size may be about 1 to 4 cm in short side length, 5 to 10 cm in long side length, and 0.1 to 2 mm in thickness.

[0022] (5) Antiviral evaluation method The indicator is washed together with the medical equipment contaminated with body fluids, and the residue on the indicator surface after washing is collected and cultured, and the amount of bacteriophage is measured, thereby enabling the antiviral effect of the medical equipment contaminated with body fluids to be evaluated.

[0023] The indicator may be placed directly into the cleaning device, or may be inserted into a holder and then placed, and may or may not be fixed to the cleaning device; however, it is preferable that the indicator be placed in the same position as the medical device to be cleaned.

[0024] The cleaning can be performed by a cleaning method using a known or commercially available device. The cleaning agent used can be a known or commercially available cleaning agent capable of inactivating HBV, such as Riser 4 and Thaifresh Ace NEO manufactured by Taihei Chemical Industry Co., Ltd. The cleaning method is not particularly limited, and examples thereof include immersion cleaning, ultrasonic cleaning, washer-disinfector, and manual cleaning.

[0025] The body fluids include blood, lymph, saliva, secretions, tissue fluid, and other body fluids. The medical tools are not particularly limited as long as they are used in surgery, examination, treatment, etc. Examples of the medical tools include tweezers, forceps, scissors, suction tubes, endoscopes, catheters, injection needles, etc., as well as clothing such as surgical gowns and examination gowns.

[0026] When the host bacteria and bacteriophage are mixed and cultured on an agar plate medium in which the host bacteria can grow, the bacteriophage grows and the host bacteria is lysed by the bacteriophage, forming transparent lysis spots (plaques). The amount of bacteriophage can be evaluated by counting the number of these plaques (Plaque Forming Units, PFU). A more specific procedure is as follows, for example. First, the indicator surface after washing is collected with a swab, and the collected material is eluted into SCDLP (Soybean-Casein Digest with Lecithin & Polysorbate) liquid medium. A portion of the eluate is further mixed with SCDLP liquid medium, and this process is repeated until 100% of the eluate is dissolved in SCDLP liquid medium by volume. -5 The indicator is diluted to 100% and mixed with the host bacteria in an equal volume to the diluted solution, then mixed with LB (Luria-Bertani) liquid medium and cultured on an LB agar plate medium. The culture conditions are preferably, for example, at 25 to 30°C for 20 to 30 hours. The indicator is also washed in the same manner except that water is used instead of the detergent, and the amount of bacteriophage is measured in the same manner as above, and the antibacteriophage activity value is calculated using the following formula. If the activity value is 2.0 or more, it can be evaluated as having an antiviral effect against HBV, and if it is less than 2.0, it can be evaluated as having no antiviral effect against HBV. Antibacteriophage activity value = log 10 {Number of phages remaining on plate after washing with water (PFU) / Number of phages remaining on plate after washing with detergent (PFU)}

[0027] (6) Search method for cleaning conditions The steps of washing the indicator, recovering and culturing the residue on the indicator surface after washing, and measuring the amount of the bacteriophage are repeated under different washing conditions, whereby the washing conditions can be explored.

[0028] The amount of bacteriophage in the screening method may be measured by employing the procedure specifically explained in the antiviral evaluation method described above.

[0029] According to the present invention, anti-HBV evaluation of sites contaminated with body fluids can be performed safely and quickly, and the present invention can be usefully used in medical settings, food factories, nursing homes, public facilities, etc. EXAMPLES

[0030] The present invention will be described in more detail below with reference to examples. The present invention is not limited to the following examples, and can of course be modified within the scope of the above and below-mentioned aims, and all such modifications are within the technical scope of the present invention.

[0031] (1) Measurement of anti-hepatitis B virus (HBV) activity For the evaluation, PXB-cells (registered trademark, donor: Hispanic, 1Y, female), which are fresh human hepatocytes isolated from human hepatocyte chimeric mice (PXB mice), were used. For the examination of the inactivation (antiviral) effect, Taifresh-Ace NEO and Lyser 4 manufactured by Taihei Chemical Industry Co., Ltd. were used as anti-rust cleaning solutions (detergents) for instruments. Serum containing HBV (HBV amount 5GEq / cell, protein amount about 35-75mg / mL) was used as an infection source and mixed with each detergent (mixing ratio of serum containing HBV to detergent was 1:1 by volume), reacted at room temperature for 30 minutes, and then inoculated into PXB-cells. 12 days after inoculation, HBV-related parameters (HBsAg) in the culture supernatant and cells were measured by CLEIA (chemiluminescence enzyme immunoassay) to calculate the anti-HBV activity value. The calculation method of the anti-HBV activity value is as follows. Anti-HBV activity value = log 10 {HBsAg amount in supernatant without detergent (IU / mL) / HBsAg amount in supernatant with detergent (IU / mL)}

[0032] The anti-HBV activity values ​​of each detergent are shown in Table 1.

[0033] [Table 1]

[0034] The above-mentioned anti-HBV activity values ​​indicate that the virus amount when detergent was added is sufficiently reduced compared to the virus amount when no detergent was added, and the anti-viral effect is better. As shown in Table 1, the anti-HBV activity value of 2-fold dilution of Lyser 4 was 2.8, and the anti-HBV activity value of 10-fold dilution of Thaifresh Ace NEO was 3.1, both of which were determined to have anti-HBV activity values ​​of 2.0 or more, and therefore they were determined to have anti-viral effect. On the other hand, the anti-HBV activity value of 10-fold dilution of Lyser 4 was 0, and the anti-HBV activity value of 100-fold dilution of Thaifresh Ace NEO was 0.1, and both of which were determined to have anti-HBV activity values ​​of less than 2.0, and therefore they were determined to have no anti-viral effect.

[0035] (2) Measurement of antibacteriophage activity value of antiviral evaluation composition Example 1-1, Example 1-2 First, a φ6 bacteriophage solution and a bovine serum albumin (BSA) solution were prepared according to the following procedure. The bacteriophage solution was prepared as follows: the phage (Bacteriophage φ6 (NBRC No. 105899)) was added to a solution of the host bacteria (Pseudomonas Syringae (NBRC No. 14084)) that had been previously cultured in LB liquid medium, and the culture was cultured with shaking at 27°C for 4 hours, and then the culture was left to stand at 4°C for 24 hours. This culture was centrifuged at 10,000G for 20 minutes, and the supernatant was passed through a 0.45 μm filter (Poly Vinylidene Difluoride) to obtain an aqueous suspension of the bacteriophage. A mixture of 20 μL of φ6 bacteriophage solution and 180 μL of bovine serum albumin (BSA) solution prepared by the above procedure was mixed with 200 μL of anti-rust cleaning solution for instruments (detergent) (Ryzer 4 or Taifresh Ace NEO, manufactured by Taihei Chemical Industry Co., Ltd.) in a sterilized tube and reacted for 30 minutes at 27°C. The final concentration of φ6 (concentration in the mixture of bacteriophage solution, BSA solution, and detergent) was 10 7 ~10 9The 180 μL of BSA solution was prepared by dissolving 800 mg of BSA (Nacalai Tesque, Inc., bovine serum albumin (fatty acid free), model number: 012-66) in a measuring bottle to make 10 mL, preparing an 8.0 mass / volume% BSA aqueous solution, and dissolving 100 μL of this BSA aqueous solution in 80 μL of sterile water (final concentration of BSA: 2 mass / volume%), or dissolving 150 μL of the 8.0 mass / volume% BSA aqueous solution in 30 μL of sterile water (final concentration of BSA: 3 mass / volume%). For Lyser 4, a solution prepared to give a 2-fold or 10-fold final concentration was used, and for Thaifresh Ace NEO, a solution prepared to give a 10-fold or 100-fold final concentration was used. After 30 minutes of reaction, 100 μL of the liquid was taken and added to 900 μL of SCDLP medium. -1 This procedure was repeated to prepare a dilution solution of 10 -5 The diluted solution was serially diluted up to 100 μL. 100 μL of the host bacterium (Pseudomonas Syringae) was mixed with 3.5 mL of soft agar medium (LB medium), and the entire amount was spread on a plate medium and cultured at 27°C for 24 hours. After the culture, the plaques formed were counted, and the antibacteriophage activity value was calculated from the number of phages obtained. The antibacteriophage activity value was calculated using the following formula. Antibacteriophage activity value = log 10 {Initial phage number (PFU / mL) / phage number after reaction with detergent (PFU / mL)} If the antibacteriophage activity value calculated by the above formula was 2.0 or more, it was determined that there was an antiviral effect, and if the antibacteriophage activity value was less than 2.0, it was determined that there was no antiviral effect.

[0036] Example 2-1, Example 2-2 The plaques formed after culture were measured in the same manner as in Example 1-1, except that 180 μL of bovine serum albumin (BSA) solution was replaced with 180 μL of an FBS aqueous solution prepared by mixing 160 μL of fetal bovine serum (FBS) (FETAL BOVINE SERUM, model number: 171012, Nichirei Biosciences, Inc.) and 20 μL of water, or 180 μL of the fetal bovine serum (FBS final concentration, i.e., concentration in the mixed solution of the bacteriophage solution, FBS aqueous solution, and detergent was 40 volume / volume% or 45 volume / volume%). The anti-bacteriophage activity value was calculated from the number of phages obtained.

[0037] Examples 3-1 to 3-4 First, 100 mg of protamine sulfate was diluted to 10 mL, to prepare a 1.0 mass / volume % aqueous solution of protamine sulfate. The plaques formed after culture were measured and anti-bacteriophage activity values ​​were calculated from the number of phages obtained in the same manner as in Example 1-1, except that 180 μL of a mixture of sheep whole blood (Japan Bioserum Co., Ltd., sodium heparin sheep whole blood, model number: 027-H0043) and 1.0 mass / volume% aqueous protamine sulfate solution at a volume ratio of 10:1 was used instead of 180 μL of bovine serum albumin (BSA) solution, so that the final concentration of the mixture was 20 volume / volume%, 30 volume / volume%, 40 volume / volume%, or 45 volume / volume%. The components of the 180 μL mixture of the sheep whole blood and the 1.0 mass / volume % aqueous solution of protamine sulfate are as follows: For a final concentration of 20 volume / volume%, 7.3 μL of 1.0 mass / volume% protamine sulfate aqueous solution, 72.7 μL of sheep whole blood, and 100 μL of sterile water. For a final concentration of 30 volume / volume%, 10.9 μL of 1.0 mass / volume% protamine sulfate aqueous solution, 109.1 μL of sheep whole blood, and 60 μL of sterile water. For a final concentration of 40 volume / volume%, 14.5 μL of 1.0 mass / volume% protamine sulfate aqueous solution, 145.5 μL of sheep whole blood, and 20 μL of sterile water. When the final concentration is 45 volume / volume%, 16.4 μL of 1.0 mass / volume% protamine sulfate aqueous solution, 163.6 μL of sheep whole blood

[0038] Comparative Example 20 μL of the same φ6 bacteriophage solution as in Examples 1-1 and 1-2 (final concentration of φ6 is 10 7 ~10 9 The antibacteriophage activity value was calculated in the same manner as in Example 1-1, except that a mixture of 180 μL of phage dilution solution (10 mg / L polypeptone and 0.85 mg / L sodium chloride) and 200 μL of anti-rust cleaning solution for instruments (Riser 4 or Taifresh Ace NEO, manufactured by Taihei Chemical Industry Co., Ltd.) was mixed in a sterile tube and allowed to react at 27°C for 30 minutes.

[0039] The results of Examples 1-1, 1-2, 2-1, 2-2, 3-1 to 3-4, and Comparative Examples are shown in Table 2. In Table 2, "w / v%" means mass / volume%, and "v / v%" means volume / volume%.

[0040] [Table 2]

[0041] For each of Examples 1-1, 1-2, 2-1, 2-2, and 3-1 to 3-4, the results showed that a 2-fold dilution of Lyser 4 had an antiviral effect (antibacteriophage activity value of 2.0 or more), and a 10-fold dilution of Lyser 4 had no antiviral effect (antibacteriophage activity value of less than 2.0). Also, a 10-fold dilution of Thai Fresh Ace NEO had an antiviral effect (antibacteriophage activity value of 2.0 or more), and a 100-fold dilution of Thai Fresh Ace NEO had no antiviral effect (antibacteriophage activity value of less than 2.0). These results were consistent with the evaluation of the antiviral effect against hepatitis B virus (HBV) shown in Table 1 above. On the other hand, in the comparative example that did not contain impurities, the results of 10-fold dilution of Lyser 4 and 100-fold dilution of Thaifresh Ace NEO did not match the evaluation of the antiviral effect against hepatitis B virus (HBV) shown in Table 1 above.

[0042] As shown in the comparative example, phages are well removed under any of the cleaning agent conditions in Table 2 when no contaminants are present. However, at actual HBV-contaminated sites, contaminants such as serum are present, and under such conditions, sufficient antiviral effects cannot be obtained when Lyser 4 is diluted 10-fold or Thaifresh Ace NEO is diluted 100-fold (Table 1). As shown in the examples, the method of the present invention, which uses a composition containing contaminants together with enveloped bacteriophages and evaluates the antibacteriophage activity value of the phage after mixing the composition with a cleaning agent, is consistent with the evaluation of the antiviral effect of HBV, i.e., it can be said that the actual situation of HBV contamination can be faithfully simulated, and the method is suitable as an alternative method for antiviral evaluation of HBV, and can perform antiviral evaluation of HBV safely, easily, and in a short period of time.

[0043] The protein amounts in Examples 2-1 and 2-2, and Examples 3-1 to 3-4 shown in Table 2 were determined by measuring the protein amounts in fetal bovine serum and whole sheep blood by the Bradford method. Sterile water was added to a 2 mg / mL standard BSA solution (Thermo Fisher Scientific PierceTM Bovine Serum Albumin Standard Ampules, 2 mg / mL, model number: 23209) to prepare a standard solution for creating a calibration curve. 20 μL of this standard solution for creating a calibration curve and 380 μL of Protein Assay Bradford Reagent (Fujifilm Wako Pure Chemical Industries, Ltd., model number: 168-25911) were mixed in a test tube and allowed to stand at room temperature for 10 minutes. After allowing to stand at room temperature for 10 minutes, the mixture was subjected to measurement of absorbance at a wavelength of 595 nm using a spectrophotometer (Hitachi, Ltd., model number: U-3310) to obtain a calibration curve. 20μL of fetal bovine serum or whole sheep blood was mixed with 380μL of protein assay Bradford reagent in a test tube, and the absorbance was measured in the same manner. The protein amount was calculated from the calibration curve that had been prepared. The protein amount in 1mL of fetal bovine serum was 66.4mg, and the protein amount in 1mL of whole sheep blood was 130mg.

[0044] (3) Measurement of antibacteriophage activity using an indicator for evaluating antiviral activity Example 4 First, 200 mg of protamine sulfate was diluted to 10 mL, to prepare a 2.0 mass / volume % aqueous solution of protamine sulfate. Next, a 2.0 mass / volume% protamine sulfate aqueous solution and the same φ6 bacteriophage solution as in Examples 1-1 and 1-2 were mixed at a volume ratio of 1:1 to obtain 9.1 μL of a mixed solution (a mixture of protamine sulfate aqueous solution and φ6 bacteriophage solution) with a protamine sulfate concentration of 1 mass / volume%. 9.1 μL of this mixed solution was mixed with 90.9 μL of the same sodium heparinate-treated sheep whole blood used in Examples 3-1 to 3-4 (volume ratio 1:10), and 100 μL was applied to a SUS plate and dried at room temperature for 2 hours. The final concentration of protamine sulfate in the solution applied to the SUS plate was 1 mass / volume%, and the final concentration of φ6 bacteriophage was 10. 7 ~10 8 The test piece was immersed in an anti-rust cleaning solution for instruments (a 2x dilution of Raiser 4, a 10x dilution of Thaifresh Ace NEO, or a 100x dilution of Thaifresh Ace NEO) for 30 minutes, after which the surface of the plate was swabbed and eluted in 1 mL of SCDLP medium. 100 μL of the eluate was added to 900 μL of SCDLP medium, and 10 -1 The diluted solution was prepared. This was repeated 10 times. -5 The diluted solution was serially diluted up to 100 μL. 100 μL of the host bacterium (Pseudomonas Syringae) was mixed with 3.5 mL of soft agar medium (LB liquid medium), and the entire amount was spread on a plate medium (LB agar medium) and cultured at 27° C. for 24 hours. After the culture, the plaques formed were measured, and a test was also conducted in which the solution was immersed in water instead of the detergent, and the number of phages was similarly measured, and the antibacteriophage activity value was calculated from these phage numbers. The method for calculating the antibacteriophage activity value is as follows: Antibacteriophage activity value = log 10 {Number of phages remaining on the plate after immersion in water (PFU) / Number of phages remaining on the plate after immersion in each detergent (PFU)}

[0045] If the antibacteriophage activity value calculated by the above formula was 2.0 or more, it was judged to have an antiviral effect, and if it was less than 2.0, it was judged to have no effect. The results are shown in Table 3.

[0046] [Table 3]

[0047] According to Table 3, the method of using an indicator having contaminants and enveloped bacteriophages on its surface to evaluate the amount of phage on the indicator after washing also yielded evaluations of the antiviral effect against hepatitis B virus (HBV) consistent with those shown in Table 1 above.

[0048] In this example, the amount of protein eluted onto the plate and into the immersion solution was quantified in terms of BSA by the Bradford method. The results are shown in Table 4.

[0049] [Table 4]

[0050] Table 4 shows that proteins remain on the plate even after cleaning. Conventional cleaning evaluation (indicator) is based on the idea that infectious viruses can be completely removed by completely removing proteins attached to instruments by cleaning, and it was judged that viruses were also completely removed by reducing the protein to a specified level. However, it is difficult to completely remove proteins in clinical practice, and the Cleaning Evaluation Judgment Guidelines of August 2012 (Japan Society of Medical Devices, Sterilization Technician Certification Committee) set the allowable value (200 μg / machine) and target value (100 μg / machine) for proteins remaining on washed items after washer-disinfectors and ultrasonic cleaning, and even if the amount of protein is below the allowable value and target value, infectious viruses may still exist in the remaining protein. This evaluation method can directly evaluate the inactivation effect of viruses, not the amount of remaining proteins, so it can be confirmed whether viruses remain or have been reduced on the surfaces of instruments and environments after surgical procedures (even if proteins remain).

Claims

1. A composition for antiviral evaluation comprising an enveloped bacteriophage and biological contaminants.

2. The composition for antiviral evaluation according to claim 1 , wherein the contaminants derived from a living body include serum proteins.

3. 3. An indicator for antiviral evaluation, comprising the composition for antiviral evaluation according to claim 1 or 2 on a surface of a substrate.

4. 4. A method for antiviral evaluation of a medical device, comprising washing the indicator for antiviral evaluation according to claim 3 together with a medical device contaminated with a body fluid, recovering and culturing a residue on the surface of the indicator for antiviral evaluation after washing, and measuring the amount of the enveloped bacteriophage, thereby performing an antiviral evaluation of the medical device.

5. A method for searching for washing conditions, which comprises repeatedly changing the washing conditions and carrying out the steps of washing the indicator for antiviral evaluation according to claim 3, recovering and culturing a residue on the surface of the indicator for antiviral evaluation after washing, and measuring the amount of the enveloped bacteriophage.

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