JP2023049243A - Protein release agent composition - Google Patents

Abstract

To realize a release agent capable of strongly releasing protein adhering to a medical care instrument and to evaluate at high accuracy cleanliness of a medical care instrument after cleaning by using the release agent.SOLUTION: A protein release agent composition contains: dodecyl sulfate as an anionic surface active agent; tris(2-carboxyethyl)phosphine hydrochloride as a reductant capable of irreversibly reducing a disulfide bond in protein; 2-[4-(2-hydroxyethyl)-1-piperazinyl] ethanesulfonic acid salt as a Good buffer agent; and water as a solvent for dissolving each component. A medical care instrument after being cleaned is soaked in the composition so as to obtain a collection liquid from which protein remaining in the medical care instrument is released, and protein contained in the collection liquid is quantified by a BCA method after the thiol group is alkylated so as to evaluate at high accuracy cleanliness of the medical care instrument.SELECTED DRAWING: None

Description

The present invention relates to stripping compositions, particularly compositions for stripping proteins adhering to medical devices.

In general cleanliness evaluation of medical instruments that have been cleaned for reuse, proteins remaining in the medical instruments after cleaning are sampled, and the sampled proteins are quantitatively analyzed. Specifically, a stripping agent is applied to the medical device to strip the residual protein, and the residual protein contained in the resulting stripping agent solution is quantified by, for example, the Bradford method or the BCA method.

As a stripping agent for stripping and collecting residual protein from a medical device, it is usually purified water, 0.2 M sodium hydroxide aqueous solution (e.g., Non-Patent Document 1) or 1% sodium dodecyl sulfate aqueous solution [pH 11] (e.g., , Non-Patent Document 2) is used. When reusing medical equipment, it is common to wash the medical equipment after use, but from the viewpoint of enhancing the sterilization effect, if the medical equipment is washed at a high temperature, especially a high temperature exceeding 60 ° C, it will adhere to the medical equipment. Proteins, which are a component of blood, are denatured by heat. Since the heat-denatured protein is difficult to be removed by the aforementioned stripping agent and tends to remain on the medical device, the cleanliness evaluated by quantitative analysis of the protein collected in the aforementioned stripping agent is unreliable.

A sample buffer for SDS-PAGE, which is a polyacrylamide gel electrophoresis method capable of separating proteins in order of molecular weight, is known as a stripping agent having a strong stripping force for proteins. The sample buffer for SDS-PAGE contains SDS (sodium dodecyl sulfate) as an anionic surfactant, DTT (dithiothreitol) as a reducing agent, and Tris (trishydroxymethylaminomethane) as a buffer at predetermined concentrations. It is an aqueous solution containing (for example, Non-Patent Document 3) and is used to extract proteins from cells and biological tissues and apply them to SDS-PAGE, but the component SDS has a strong dissolving power for proteins , it is expected to be a useful exfoliant for removing proteins remaining on medical equipment.

However, since the residual protein collection solution using the sample buffer for SDS-PAGE inevitably contains SDS, DTT and Tris, the quantification by the Bradford method is affected by the interference of detergent (SDS). Accuracy is compromised and quantitation accuracy is compromised by interference from reducing agents (DTT) and Tris (buffer) when using the BCA method. Regarding the Bradford method, an improved method that suppresses the influence of surfactants has been proposed, but when the residual protein in the sample buffer for SDS-PAGE is quantified by this improved method, the measurable range of the residual protein is narrow. Quantitation of residual protein in low concentration regions is practically difficult.

Cleaning Evaluation Judgment Guideline, Japan Society of Medical Devices, First Edition, August 15, 2012 Validation guidelines for manual cleaning and disinfection of medical devices, mhp-Verlag GmbH, Central Service Suppl. 2013, Appendix 8: Confirmation of cleaning, page 24 R. Novy, B. Morris (2001). Preparation of protein samples for SDS-polyacrylamide gel electrophoresis: procedures and tips.

The present invention aims to realize a stripping agent that can strongly strip proteins adhering to medical instruments, and to enable highly accurate evaluation of cleanliness of medical instruments after washing by using the stripping agent. is.

TECHNICAL FIELD The present invention relates to a composition for exfoliating proteins adhered to medical equipment. The stripper composition contains dodecyl sulfate, a reducing agent capable of irreversibly reducing disulfide bonds in proteins, Good's buffer, and water as a solvent for dissolving each component.

A reducing agent contained in the stripper composition of the present invention is, for example, tris(2-carboxyethyl)phosphine hydrochloride. Also Good's buffer is, for example, 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonate.

One form of the stripper composition of the present invention is an aqueous solution containing 0.2 to 5% by mass of dodecyl sulfate, 2 to 50 mM of reducing agent and 2 to 100 mM of Good's buffer, and having a pH of 5 to 9. be.

Another aspect of the present invention relates to a method for peeling and collecting proteins remaining on a medical device for cleanliness evaluation of the medical device after washing. This collection method includes the step of applying the protein stripper composition of the present invention to the medical device after washing to obtain a sampled liquid from which the protein remaining on the medical device has been stripped.

In still another aspect, the present invention relates to a method for evaluating cleanliness of medical equipment after washing. This cleanliness evaluation method includes a step 1 of obtaining a sampled liquid from which the protein remaining in the medical device after washing has been removed by the sampling method according to the present invention, and a step of quantifying the protein contained in the sampled liquid obtained in step 1. 2.

In one embodiment of the cleanliness evaluation method of the present invention, protein is quantified by the BCA method after adding an alkylating agent to the collected liquid in step 2. Alkylating agents used in this form are, for example, organic iodide salts. Organic iodide salts are, for example, iodoacetamide or iodoacetic acid. Also, in this form, the alkylating agent is added to the collection liquid in an amount of at least 5 times the molar equivalent of the reducing agent contained in the protein stripping agent composition.

In still another aspect, the present invention relates to a method for cleaning medical equipment after use. This cleaning method includes a step A of applying the protein remover composition of the present invention to the medical device after use, and a step B of washing off the protein remover composition adhering to the medical device after step A.

Since the protein remover composition of the present invention has a specific component composition, it can strongly remove proteins adhering to medical equipment.

Since the method for collecting residual protein according to the present invention uses the protein remover composition of the present invention, the protein remaining on the medical device after washing is strongly peeled and collected, and the residual protein obtained thereby is A protein harvest can be applied for quantification of residual protein.

The cleanliness evaluation method according to the present invention is capable of highly accurately evaluating the cleanliness of medical devices after washing because the proteins remaining on medical devices after washing are separated and collected by the protein collection method of the present invention. is.

In the cleaning method according to the present invention, the protein remover composition of the present invention is applied to the used medical equipment, so that the protein adhering to the used medical equipment can be strongly removed. can enhance the cleaning effect of

The stripping agent composition of the present invention strips proteins from medical devices in order to quantify or remove proteins adhered to the medical devices. Medical instruments to which the exfoliating composition of the present invention can be applied are not particularly limited, and include cutlery (scalpel), forceps, scissors, forceps, needle holders, hooks, retractors, suction tubes, and various drains. Examples include tubes, vascular sealing devices, various catheters, various syringes, various tubes, trays, pus basins, microsurgery instruments, and members and parts thereof. The target medical device may be one intended for reuse from the beginning, or may be a single-use medical device (SUD/Single Use Devices) for which remanufacturing is permitted exceptionally. The stripping composition of the present invention is particularly useful when used for medical instruments to which adhesion of protein, especially blood or other bodily fluids, is unavoidable.

The material of the target medical equipment is not particularly limited. For example, metals such as stainless steel, aluminum, copper, brass and titanium, glass, polyolefin resins such as polyethylene resins and polypropylene resins, silicone resins, and polychlorinated Vinyl resin, fluorine resin such as polytetrafluoroethylene, polycarbonate resin, polyester resin, polymethacrylate resin, polystyrene resin, ABS resin, polyamide resin, acetal resin, acrylic resin, methylenepentene resin, polyurethane resin, phenol resin, melamine resin and Resins, such as an epoxy resin, are mentioned.

The stripper composition of the present invention is an aqueous solution containing dodecyl sulfate, a reducing agent, a buffer and water as a solvent for dissolving them.

Various salts can be used as dodecyl sulfate as long as they function as an anionic surfactant. Alkali metal salts such as sodium dodecylsulfate and potassium dodecylsulfate are typically used, with sodium dodecylsulfate being particularly preferred. Two or more kinds of dodecyl sulfate may be used in combination.

The dodecyl sulfate concentration in the stripper composition of the present invention is preferably set to 0.2 to 5% by mass, more preferably 0.5 to 2% by mass. If the concentration of dodecyl sulfate is less than 0.2% by mass, the releasability of proteins adhering to medical equipment may be reduced. On the other hand, if the dodecyl sulfate concentration exceeds 5% by mass, protein measurement may become difficult when the stripping agent composition of the present invention is used for protein quantification.

As the reducing agent, those capable of irreversibly reducing disulfide bonds (-SS-) in proteins, specifically, those capable of irreversibly reducing disulfide bonds to thiol groups (-SH) are used. be done. Examples of such reducing agents include tris(2-carboxyethyl)phosphine hydrochloride and tris(hydroxypropyl)phosphine. Among these, tris(2-carboxyethyl)phosphine hydrochloride is particularly preferred. Two or more reducing agents may be used in combination.

The reducing agent concentration in the stripping agent composition of the present invention is preferably set to 2 to 50 mM, more preferably 5 to 20 mM. If the concentration of the reducing agent is less than 2 mM, the disulfide bonds in the protein adhered to the medical device are less likely to be reduced to thiol groups, and the strippability of the protein by the stripping agent composition of the present invention may decrease. On the other hand, if the concentration of the reducing agent exceeds 50 mM, it may become uneconomical because the effect associated with the amount used cannot be expected.

Good's buffer is used as the buffer. Examples of Good's buffers that can be used include N-(2-acetamido)-2-aminoethanesulfonic acid (ACES), N-(2-acetamido)iminodiacetic acid (ADA), N,N-bis(2- Hydroxyethyl)-2-aminoethanesulfonic acid (BES), N,N-bis(2-hydroxyethyl)glycine (Bicine), bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane (Bis-Tris), N - cyclohexyl-3-aminopropanesulfonic acid (CAPS), N-cyclohexyl-2-hydroxy-3-aminopropanesulfonic acid (CAPSO), N-cyclohexyl-2-aminoethanesulfonic acid (CHES), 3 -[N,N-bis(2-hydroxyethyl)amino]-2-hydroxypropanesulfonic acid (DIPSO), 3-[4-(2-hydroxyethyl)-1-piperazinyl]propanesulfonic acid (EPPS), 2 -[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES) or salts thereof (e.g. alkali metal salts such as sodium salts), 2-hydroxy-3-[4-(2-hydroxyethyl )-1-piperazinyl]propanesulfonic acid monohydrate (HEPPSO), 2-morpholinoethanesulfonic acid monohydrate (MES), 3-morpholinopropanesulfonic acid (MOPS), 2-hydroxy-3-morpholinopropanesulfone acid (MOPSO), piperazine-1,4-bis(2-ethanesulfonic acid) (PIPES), piperazine-1,4-bis(2-hydroxy-3-propanesulfonic acid) dihydrate (POPSO), N -tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS), 2-hydroxy-N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPSO), N-tris(hydroxymethyl)methyl- 2-Aminoethanesulfonic acid (TES) and N-[tris(hydroxymethyl)methyl]glycine (Tricine) can be mentioned. Among these Good's buffers, 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid or salts thereof are particularly preferred. Two or more Good buffers may be used in combination.

The buffer concentration in the stripping agent composition of the present invention is preferably set to 2 to 100 mM, more preferably 1 to 50 mM. If the buffer concentration is less than 2 mM, the pH of the stripper composition may become unstable. On the other hand, if the concentration of the buffering agent exceeds 100 mM, it may be uneconomical because the effect associated with the amount used cannot be expected in terms of stabilizing the pH of the stripping agent composition.

Purified water such as distilled water or pure water is used as water as the solvent.

The stripper composition of the present invention can be prepared by mixing and dissolving dodecyl sulfate, a reducing agent and Good's buffer in water as a solvent. is adjusted to near neutrality of 5 to 9 by controlling in the range of . Particularly preferred stripping compositions of the present invention are sodium dodecyl sulfate as dodecyl sulfate, tris(2-carboxyethyl)phosphine hydrochloride as reducing agent, and 2-[4-(2-hydroxyethyl) as Good's buffer. It is an aqueous solution with a pH of 5 to 9 prepared by dissolving sodium-1-piperazinyl]ethanesulfonate in purified water so that the concentration range is within the above-described range.

The stripping agent composition of the present invention can be used to strip and collect proteins remaining on medical instruments for evaluating the cleanliness of medical instruments that have been washed for reuse after use. Medical equipment, which is subject to cleanliness evaluation, is not easily restricted in terms of treatment temperature during cleaning. For example, medical instruments that have been washed at high temperatures above 60° C., which can denature proteins in order to increase sterilization effect, can also be evaluated for cleanliness by using the stripping agent composition of the present invention.

A method for collecting protein remaining on a medical device after washing using the stripping agent composition of the present invention and evaluating the cleanliness of the medical device after washing comprises the following two steps.

Step 1:
In this step, the residual protein collection method according to the present invention is applied to the medical instrument after washing. Specifically, the remover composition of the present invention is applied to the medical device after washing, and the protein remaining on the medical device is peeled off and collected. That is, a sampled liquid containing residual protein peeled off from the medical device is obtained.

In this step, the entire medical device to be evaluated is generally immersed in the release agent composition of the present invention in a container. Then, if necessary, the stripping agent composition is heated, or the container is shaken, microvibration is applied to the medical device in the container, or ultrasonic waves are applied to remove the protein remaining on the medical device. Stripping is caused mainly by the surfactant action of dodecyl sulfate in the stripping agent composition. Treatments such as heating and shaking may be combined as appropriate. As a result, the protein on the medical device migrates into the stripping agent composition to obtain a protein extract. In this process, the disulfide bonds in the structure of the protein on the medical device are reduced to thiol groups by the action of the reducing agent, increasing the solubility in the release agent composition. It is received and smoothly transferred into the release agent composition.

The container used in this step is, for example, a tray made of metal or resin, a bag made of resin, or a container made of glass.

Step 2:
In this step, the protein contained in the collected liquid obtained in step 1 is quantified. As a method for quantifying protein, a known method that is less likely to be interfered with by dodecyl sulfate, which is a surfactant contained in the collected liquid, can be used, for example, a modified Bradford method, a BCA method, an OPA method, or the like.

An improved method of the Bradford method is to quantify proteins by utilizing the fact that Coomassie Brilliant Blue G250 (CBB G250), a triphenylmethane-based dye, binds to proteins, resulting in a color change from reddish purple to blue. . According to this method, an XL-Bradford reagent containing CBB G250 is added to the collected liquid, and the collected protein can be quantified by measuring the change in absorbance of the collected liquid at a wavelength corresponding to blue (usually 595 nm). can.

The BCA method forms a chelate complex between a protein and divalent copper ions (Cu ²⁺ ) (Burett reaction). Then, when this chelate complex is reduced by protein, bicinchoninic acid (BCA), a colorimetric reagent, is coordinated with monovalent copper ions (Cu ⁺ ) that are produced in proportion to the amount of protein, resulting in blue The protein is quantified using the formation of a purple complex. According to this method, the collected protein can be quantified by measuring the absorbance change at the wavelength (usually 562 nm) corresponding to the blue-violet complex formed by adding the BCA reagent to the collected solution.

In the BCA method, the thiol group of the protein generated by the action of the reducing agent in step 1 becomes a thiol anion, which strongly binds to monovalent copper ions. The formation of a complex with is inhibited, and the change in absorbance due to the formation of the complex becomes difficult to accurately reflect the amount of protein in the sample solution. Therefore, in the case of the BCA method, an alkylating agent is added to the collected liquid obtained in step 1. The alkylating agent added here is capable of alkylating the thiol groups of the protein collected in the collection solution. Such alkylating agents are usually organic iodide salts or alkylene oxides such as ethylene oxide, preferably organic iodide salts. In particular, it is preferable to use iodoacetamide or iodoacetic acid as the organic iodide salt.

By adding an alkylating agent, the thiol group of the protein in the collected solution is alkylated to form a structure with a highly stable amide group, which suppresses the binding with monovalent copper ions. Highly accurate quantification by the BCA method becomes possible.

The amount of the alkylating agent added to the collected liquid is preferably set to at least 5 times the molar equivalent of the reducing agent contained in the stripping agent composition used in step 1 in order to smoothly promote the alkylation of the thiol groups of the protein. preferable.

Either the fluorescence method or the ultraviolet absorption method (UV method) can be used for the OPA method, but the UV method is preferred. The fluorescence method quantifies proteins by utilizing the fact that ortho-phthalaldehyde (OPA) reacts with primary amines in proteins to produce blue fluorescent substances. In this method, the collected protein is quantified by measuring the change in fluorescence intensity that occurs when the fluorescent substance generated by adding the OPA reagent to the collected solution is irradiated with light with a blue wavelength (usually 455 nm). can do. On the other hand, the UV method has the same reaction principle as the fluorescence method in that OPA generates a blue fluorescent substance, but the type of reducing agent in the OPA reagent used during measurement is different, and the ultraviolet wavelength for the fluorescent substance It quantifies proteins by measuring changes in transmitted light (usually 340 nm).

A medical device evaluated as acceptable for cleanliness can be reused by washing away the adhered remover composition according to step B of the cleaning method described below.

Since the remover composition of the present invention has excellent ability to remove proteins adhering to medical equipment, it can also be used as a cleaning agent for reusing used medical equipment. A method for cleaning medical equipment using the stripping agent composition of the present invention includes the following two steps.

Step A:
In this step, the stripper composition of the present invention is applied to the medical device. For example, the entire medical device to be cleaned is immersed in the stripper composition of the present invention in a cleaning tank. Then, if necessary, the remover composition is heated, or the washing tank is shaken, or the medical equipment in the washing tank is micro-vibrated or ultrasonic waves are applied to the medical equipment so that the Stains such as proteins are removed mainly by the surfactant action of dodecyl sulfate in the remover composition. Treatments such as heating and shaking may be combined as appropriate. The action of the reducing agent in the stripping composition in this process is as explained in step 1 of the cleanliness evaluation method.

Used medical instruments to which this step is applied may have been pre-washed, for example, washed and sterilized using a surfactant-based medical instrument washer.

Step B:
In this step, the remover composition adhering to the medical device that has undergone step A is washed away. For example, the stripping agent composition is discharged from the cleaning bath used in step A. Then, purified water for rinsing the medical equipment is supplied into the washing tank, and the remover composition adhering to the medical equipment is rinsed off with this purified water. The purified water used here is the same as the water used as the solvent in the release agent composition, and it is particularly preferred to use sterile pure water.

The medical device that has undergone step B can be reused after drying.

EXAMPLES The present invention will be specifically described below with reference to experimental examples, but the present invention is not limited by the experimental examples.

The release agents used in the experimental examples are as follows.
・Removing agent R1
Ultrapure water was prepared using Merck's ultrapure water production apparatus (product name "Mili-Q"), and this was used as release agent R1.
・Removing agent R2
Sodium dodecyl sulfate (SDS) was added to the ultrapure water used as the stripping agent R1 to a concentration of 1% by mass and dissolved to prepare an aqueous solution of pH 11, which was used as the stripping agent R2.
・Removing agent R3
Sodium dodecyl sulfate (SDS), dithiothreitol (DTT) and trishydroxymethylaminomethane (Tris) are added and dissolved in ultrapure water used as the stripping agent R1 to prepare an aqueous solution of pH 8.5, which is stripped. It was designated as agent R3. In this stripping agent, the SDS concentration was set to 2 mass %, the DTT concentration was set to 100 mM, and the Tris concentration was set to 60 mM. DTT used here is a reducing agent capable of reversibly reducing disulfide bonds in proteins.
・Removing agent R4
In stripping agent R3, the concentration of SDS was changed to 1% by mass and the concentration of DTT was changed to 100 mM, and this was used as stripping agent R4.
・Removing agent R5
Sodium dodecyl sulfate (SDS), tris(2-carboxyethyl)phosphine hydrochloride (TCEP) and sodium 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonate in ultrapure water used as stripping agent R1 (HEPES-Na) was added and dissolved to prepare an aqueous solution of pH 7, which was used as a release agent. In this stripping agent, the SDS concentration was set to 1 mass %, the TCEP concentration was set to 10 mM, and the HEPES-Na concentration was set to 10 mM.
・Removing agent R6
Sodium dodecyl sulfate (SDS) and sodium 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonate (HEPES-Na) were added and dissolved in ultrapure water used as the stripping agent R1 to adjust the pH to 7. An aqueous solution was prepared and used as a release agent. In this stripping agent, the SDS concentration was set to 1 mass %, and the HEPES-Na concentration was set to 10 mM.

[Experimental example A]
Preparation of simulated contamination specimens:
A simulated contaminant was prepared by adding 500 μL of 0.025 M calcium chloride solution to 500 μL of citrated pig blood and mixing. A pseudo-contamination test piece was prepared by applying 200 μL of the pseudo-contamination before coagulation of pig blood to one side of a plate-shaped test piece made of stainless steel (50 mm×15 mm×1 mm) and drying. Here, the following two types of pseudo-contamination test specimens were prepared using different drying methods for the pseudo-contamination.

・ Pseudo-contamination test piece T1
A test piece coated with a pseudo-contaminant is left to stand at room temperature (20°C) for 16 hours to dry. This pseudo-stained test strip maintains the native state of the proteins in the porcine blood.
・ Pseudo-contamination test piece T2
A test piece coated with a simulated contaminant is left to stand in a steam oven at 95°C for 10 minutes to dry. This pseudo-contamination test piece is obtained by wet heat denaturation of proteins in pig blood.

Experimental Examples A1 to A4:
A pseudo-contamination test piece was placed in a resin test tube containing 5 mL of a release agent and immersed at room temperature (20° C.). After 16 hours, the resin test tube was shaken, the pseudo-stained test piece was taken out, and the amount of protein in the release agent was measured using the commercially available Bradford method protein quantification kit (Antegral Inc.) for performing the improved Bradford method. (trade name “XL-Bradford (SDS-PAGE compatible)”)), and quantified against a calibration curve prepared in advance. Then, based on this quantitative result, the ratio (recovery rate) of protein transferred from the pseudo-contaminated test piece to the release agent was calculated.

Table 1 shows the results of performing each experimental example three times. Experimental examples A1 to A3 are comparative examples, and experimental example A4 is an example.

[Experimental example B]
Preparation of sham collection fluid:
Bovine serum albumin was diluted using the detachment agent shown in Table 2 to prepare simulated collection solutions S1 to S3 having a bovine serum albumin concentration of 100 μg/mL.

Experimental Examples B1-B3:
25 μL of the simulated sample was placed in a well plate, and 200 μL of commercially available BCA protein assay reagent (trade name “Pierce BCA” by Thermo Fisher Scientific Inc.) was added and incubated at 37° C. for 30 minutes. Absorbance at 562 nm was measured within 10 minutes after incubation, and protein in the simulated sample was quantified in light of a previously prepared calibration curve.

Table 3 shows the results. Experimental examples B1 to B3 are all comparative examples. In Experimental Example B2, the absorbance exceeded the upper limit of the calibration curve, and the protein could not be quantified.

Experimental Examples B4-B6:
The same amount of 4% by mass iodoacetamide aqueous solution was added to 50 μL of the simulated sample liquid, mixed, and incubated at 37° C. for 20 minutes to alkylate the thiol groups of proteins in the simulated sample liquid. 25 μL of the thus-treated pseudo-collected liquid was placed in a well plate, and 200 μL of a commercially available BCA method protein determination reagent (Thermo Fisher Scientific Inc., trade name “Pierce BCA”) was added and incubated at 37° C. for 30 minutes. Incubate for 1 minute. Absorbance at 562 nm was measured within 10 minutes after incubation, and protein in the simulated sample was quantified in light of a previously prepared calibration curve.

Table 3 shows the results. Experimental examples B4 and B5 are comparative examples, and experimental example B6 corresponds to an example. In Experimental Example B5, the absorbance exceeded the upper limit of the calibration curve, and protein could not be quantified.

[Experimental example C]
Bovine serum albumin was diluted with stripping agent R5 to prepare a simulated sample solution having a bovine serum albumin concentration of 25 μg/mL. 100 μL of the simulated sample was placed in a well plate, and 16 mass % iodoacetamide aqueous solution was added and mixed, followed by incubation at 37° C. for 20 minutes to alkylate the thiol groups of proteins in the simulated sample. After incubation, 100 μL of a commercially available BCA method protein quantitation reagent (Thermo Fisher Scientific Inc., trade name “Pierce Micro BCA”) was added to the simulated sample and incubated at 37° C. for 120 minutes. Absorbance at 562 nm was measured within 10 minutes after incubation, and protein in the simulated sample was quantified in light of a previously prepared calibration curve.

The same experiment was repeated four times and the protein quantification result was 25.86±0.83 μg/mL. According to this result, it can be seen that in this experimental example (corresponding to the example), it is possible to quantify a minute amount of protein in the pseudo-collected liquid.

Claims (11)

A composition for stripping proteins attached to a medical device,
dodecyl sulfate;
a reducing agent capable of irreversibly reducing disulfide bonds in the protein;
Good buffer and
water as a solvent for dissolving each component;
A protein stripper composition comprising: 2. The protein stripper composition of claim 1, wherein said reducing agent is tris(2-carboxyethyl)phosphine hydrochloride. 3. The protein stripper composition of claim 1 or 2, wherein said Good's buffer is 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonate. 4. Any one of claims 1 to 3, which is an aqueous solution containing 0.2 to 5% by mass of said dodecyl sulfate, 2 to 50 mM of said reducing agent and 2 to 100 mM of said Good's buffer, and having a pH of 5 to 9. 2. The protein remover composition according to claim 1. A method for exfoliating and collecting proteins remaining on a medical device for cleanliness evaluation of the medical device after washing, comprising:
A step of applying the protein stripping agent composition according to any one of claims 1 to 4 to the medical device to obtain a collected liquid from which proteins remaining on the medical device are stripped,
A method for collecting proteins remaining on medical instruments after cleaning. A step 1 of obtaining a sampled liquid from which the protein remaining in the washed medical device is stripped by the sampling method according to claim 5;
A step 2 of quantifying the protein contained in the collected liquid;
A method for evaluating the cleanliness of medical equipment after washing, including 7. The method for evaluating cleanliness of medical equipment after washing according to claim 6, wherein protein is quantified by the BCA method after adding an alkylating agent to the collected liquid in step 2. 8. The method for evaluating the cleanliness of medical equipment after washing according to claim 7, wherein the alkylating agent is an organic iodide salt. 9. The method for evaluating the cleanliness of medical equipment after washing according to claim 8, wherein the organic iodide salt is iodoacetamide or iodoacetic acid. Any one of claims 7 to 9, wherein the alkylating agent is added to the collecting solution in an amount of at least 5 times the molar equivalent of the reducing agent contained in the protein stripping agent composition used in the collecting method according to claim 5. A method for evaluating the cleanliness of medical devices after cleaning as described. A step A of applying the protein remover composition according to any one of claims 1 to 4 to the used medical device;
A step B of washing away the protein remover composition adhering to the medical device that has undergone step A;
A method for cleaning medical equipment after use, including: