JP2006145271A - Protein detection method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a protein detection method for highly accurately and simply detecting protein deposited on a medical apparatus. <P>SOLUTION: This protein detection method comprises a process for immersing the medical apparatus in an alkaline solution to extract protein therefrom, and a process for putting an extract obtained in the protein extraction process into contact with a protein dying agent. The alkaline solution is a solution of sodium hydroxide and/or of potassium hydroxide, and the concentration of the alkaline water solution is 0.05 to 2 N. The protein extraction process is executed at 25 to 90°C. The dying agent is Coomassie brilliant blue G-250. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a method for detecting proteins attached to various medical instruments used in medical institutions.

  The medical instruments used in outpatient clinics, surgical departments, examination departments, wards, etc. of medical institutions have protein stains mainly composed of proteins such as blood, body fluids, and tissue fragments. The contaminated medical device is reused through a sterilization process, a cleaning process, and a sterilization process. Therefore, in order to maintain the smooth operability of the medical device and prevent infection by the medical device, it is important and indispensable to clean the medical device to remove protein stains.

  Cleaning methods for medical instruments that are used in medical institutions and have protein stains on them include immersion cleaning, mechanical cleaning using ultrasonic cleaners, washer-disinfectors, etc., based on the cleaning equipment owned by each medical institution. ing. In addition, endoscope-specific cleaning machines and the like corresponding to the structure of a medical instrument such as an endoscope scope are also used.

  However, even if washing is performed as described above, protein stains may remain on the medical device, and it is necessary to check the cleanliness of the medical device. At present, the medical device cleanliness method for confirming the cleanliness of a medical device is to immerse the cleaned medical device in the amide black 10B staining solution, which is a protein stain, and then wash it with water. It is common to qualitatively determine the presence or absence of protein on the surface of a medical device with the naked eye by examining whether or not there is a blue staining mark stained with 10B.

Another method for confirming the cleanliness of the medical device after washing is to bring the acidic dye solution into contact with the surface of the medical device, and then wash and remove the acidic dye solution from the surface of the medical device. A method has been proposed in which a predetermined amount of an alkaline extract is brought into contact with the surface, the extract is collected, and the presence or degree of protein adhering matter on the surface of the medical device is detected by an acidic dye eluted in the extract ( Patent Document 1).
Japanese Patent No. 3165668

  However, the protein detection method proposed above stains the surface of the protein adhering to the medical device with an acid dye, and performs detection based on the amount of the acid dye adhering to the surface of the adhering protein. However, it does not detect the total amount of protein adhering to the medical device. Therefore, particularly when the protein contamination of the medical device is severe, there is a problem that a detection result far from the actual amount of fixation can be obtained.

  In addition, the protein detection method proposed above has a step of cleaning and removing excess acidic dye solution from the surface of the medical device, and the cleaning here is performed until the discharged cleaning solution is completely colorless and transparent. It is supposed to be done. However, it is difficult to visually determine whether or not the discharged cleaning liquid is completely colorless and transparent, and it is difficult to perform complete cleaning.

  The present invention has been made in view of such circumstances, and an object of the present invention is to provide a protein detection method capable of accurately and easily detecting a protein attached to a medical instrument.

In order to solve the above problems, the present invention provides:
(1) A protein comprising: a step of immersing a medical device in an alkaline solution to extract a protein; and a step of bringing the extract obtained in the protein extraction step into contact with a protein stain. Detection method.
(2) The method for detecting a protein according to (1), wherein the alkaline solution is a solution of sodium hydroxide and / or potassium hydroxide,
(3) The method for detecting a protein according to (1) or (2) above, wherein the concentration of the alkaline solution is 0.05 to 2N.
(4) The protein detection method according to any one of (1) to (3), wherein the protein extraction step is performed at 25 to 90 ° C.
(5) The protein detection method according to any one of (1) to (4) above, wherein the protein stain is Coomassie Brilliant Blue G-250,
(6) The method according to any one of (1) to (5), further comprising a step of measuring the absorbance of the contact liquid obtained by bringing the extract obtained in the extraction step into contact with the protein stain. A method for detecting the protein according to 1,
About.

  Since the detection method of the present invention includes the step of immersing the medical device in an alkaline solution and extracting the protein adhering to the medical device, the medical device is severely contaminated regardless of the shape of the medical device. Even in this case, the protein adhering to the medical instrument can be detected with high accuracy and simply, and the detection accuracy required in the medical field is sufficiently provided.

  The method for detecting a protein according to the present invention mainly detects the presence or absence of a protein adhering to a medical device, and further, the amount of adhesion, and a step of immersing the medical device in an alkaline solution and extracting the protein; And a step of bringing the extract obtained in the protein extraction step into contact with the protein stain.

  Examples of the alkaline solution include those obtained by dissolving an alkaline agent such as sodium hydroxide, potassium hydroxide, sodium carbonate, sodium hydrogen carbonate, potassium carbonate, potassium hydrogen carbonate, sodium phosphate, potassium phosphate, etc. in a solvent. These can be used alone or in admixture of two or more. Among these, sodium hydroxide and potassium hydroxide are preferably used from the viewpoint of satisfactory protein extraction.

  Examples of the solvent for the alkaline solution include tap water, distilled water, ion exchange water, RO water, and the like, and these can be used alone or in admixture of two or more. The concentration is preferably 0.05 to 2N, more preferably 0.1 to 1N, and particularly preferably 0.15 to 0.75N from the viewpoint of satisfactorily extracting the protein.

  The medical instrument is not particularly limited as long as it is used for surgery, examination, treatment, and the like, and examples thereof include a lever, a forceps, a scissors, a suction tube, an endoscope, a catheter, and an injection needle. In the present invention, the “medical device” includes not only those used in surgery, examination, treatment, etc., but also those that may have proteins attached thereto.

  The extraction temperature is preferably 25 to 90 ° C, more preferably 30 to 80 ° C, and particularly preferably 40 to 70 ° C. Moreover, as extraction time, Preferably it is 20 minutes or more, More preferably, it is 20-60 minutes.

  The amount of the alkaline solution used is not particularly limited as long as it is adjusted according to the size, surface area, usage status, etc. of the medical device to be detected.

  Here, “immersing a medical device in an alkaline solution” means immersing the entire medical device in an alkaline solution. Specifically, a predetermined amount of the alkaline solution is injected into an alkali-resistant container or bag. In addition, a mode in which a medical instrument is placed and shaken is included in this, but it is not particularly limited as long as it is an operation capable of immersing the entire medical instrument in an alkaline solution.

  The protein extraction step may be performed at the stage where the used medical device has been cleaned, or may be performed at the stage where sterilization has been completed. Even if interference substances such as cleaning agents used for cleaning medical devices and sterilizing disinfectants used before cleaning remain in the medical devices, the presence or amount of proteins attached to the medical devices The effect on detection is not a problem.

  The protein detection method of the present invention includes a step of bringing the extract obtained in the protein extraction step into contact with the protein stain after performing the above-described protein extraction step.

  The protein stain is not particularly limited as long as it forms a color by being stoichiometrically bound to the protein. For example, Coomassie Brilliant Blue R-250, Coomassie Brilliant Blue G-250, Amide Black 10B, Acid Examples include acidic dyes such as orange 12, buffalo black, orange G, and bromophenol blue; basic dyes such as safranin O; chelate dyes such as bishiconic acid; fluorescent dyes such as eosin Y, and the like. Among these, Coomassie Brilliant Blue G-250, which is particularly excellent in protein binding properties, is preferably used from the viewpoint of improving detection accuracy. This Coomassie brilliant blue G-250 changes from red (465 nm) to blue (595 nm) when bound to a protein, but the binding to the protein is equilibrated in about 2 minutes, and the binding is about room temperature. Since a stable dispersion state is maintained for 1 hour, there is an advantage that the absorbance measurement described later can be performed quickly and accurately.

  In the contacting step, the protein stain is preferably used in a state dissolved in a solvent such as water or alcohol. In the case of using an acidic dye as the protein stain, this protein stain solution is preferably adjusted to pH 0.5 to 4 by adding an acid component such as acetic acid, phosphoric acid or sulfuric acid, and the protein stain solution is basic. When using a pigment | dye, it is preferable to add weak acid components, such as an acetic acid, and to make it pH 3-8. The protein stain solution may be a commercially available product, for example, Coomassie protein assay reagent kit (trade name) manufactured by PIERCE, which is a solution of the above-mentioned Coomassie Brilliant Blue G-250.

  The amount of the protein stain used may be adjusted according to the size, surface area, usage status of the medical device to be detected, and the type and amount of the protein to be detected, and is particularly limited. is not.

  Here, as an aspect of “contacting the extract with the protein stain”, specifically, a predetermined amount of the protein stain is added to a part or all of the extract obtained in the extraction step described above. Although an aspect etc. are mentioned, it will not specifically limit if it is operation which can make a predetermined amount of extract and protein dyeing agent contact.

  In this way, it is possible to determine whether or not the protein has adhered to the medical device by examining whether or not the contact liquid obtained by bringing the extract into contact with the protein stain is colored. Further, the amount of protein attached to the medical device can be estimated from the degree of coloration of the contact liquid, but can also be easily detected by measuring the absorbance of the contact liquid.

  The method for detecting the amount of attached protein by measuring the absorbance was prepared in advance by measuring the absorbance in the absorption wavelength region peculiar to the protein stain while keeping the extraction conditions, the contact condition between the extract and the protein stain, etc. constant. The amount of attached protein is detected by comparing with the correlation (for example, calibration curve, correlation formula, etc.) between the absorbance measured with the same protein stain and the protein amount.

  Here, the detection limit of the amount of attached protein varies depending on the type and amount of the protein stain used, but by making the type and amount of protein stain constant, the amount of protein attached to the medical device becomes the reference value. It is possible to easily determine whether or not it exceeds.

  In the present invention, when a protein adhering to a medical instrument is detected in a medical institution or the like, the alkaline instrument for immersing the medical instrument to extract the protein and the extracted solution after contact with the alkaline solution are extracted. It can also be supplied as a detection kit comprising a protein stain. The detection kit may be provided with a detection instrument such as a bag made of vinyl or the like used in the protein extraction step, a colorimeter tube, a simple spectrophotometer for measuring absorbance.

  Hereinafter, the method for detecting a protein according to the present invention will be described by taking as an example the case where Coomassie Brilliant Blue G-250 (having an absorption maximum at 595 nm when bound to a protein) is used as a protein stain.

(Create a calibration curve)
A 2 mg / ml bovine serum albumin solution (manufactured by Wako Pure Chemical Industries, Ltd.) was diluted with a 0.2N sodium hydroxide aqueous solution so as to have each concentration shown in Table 1, and each diluted solution was obtained. Next, 100 mg of Coomassie Brilliant Blue G-250 was dissolved in 50 ml of 95% ethanol, 100 ml of 85% (w / v) phosphoric acid was added to this solution, water was further added to make the total volume 1 l, and pH 1.43 A Coomassie Brilliant Blue G-250 solution at (25 ° C.) was prepared. 1 ml of each of the obtained dilutions was added to 3 ml of the thus obtained Coomassie Brilliant Blue G-250 solution, reacted at 25 ° C. for 20 minutes, and the absorbance of the obtained contact liquid at 595 nm. Was measured. Table 1 shows the results, and FIG. 1 shows a calibration curve created from the measurement results of Table 1.

  As shown in FIG. 1, the prepared calibration curve showed good linearity.

  Based on the calibration curve obtained above, the following examination was performed on the extraction conditions with the alkaline solution.

(About extraction concentration and extraction time)
1.0N sodium hydroxide (manufactured by Wako Pure Chemical Industries, Ltd.) is diluted with distilled water to prepare an alkaline solution having a concentration shown in Table 2, and 10 ml of the obtained alkaline solution and a stainless steel plate (15 × 50 mm) Detergent evaluation indicator [manufactured by Pereg, trade name: TOSI] (hereinafter referred to as “TOSI”) with pseudo blood (purified bovine blood: albumin, hemoglobin, fibrinogen, thrombin) attached to a polyethylene bag (Dimensions: 240 × 340 mm, thickness: 0.04 mm, with a chuck), and this bag was immersed in a constant temperature water bath [manufactured by TAITEC, product number: SM-05R] maintained at 50 ° C. Then, TOSI was immersed and shaken in an alkaline solution to extract proteins. The extraction time is as shown in Table 2.

  1 ml of the extract thus obtained was added to 3 ml of Coomassie Brilliant Blue G-250 solution and reacted at 25 ° C. for 20 minutes in the same manner as in the above-described calibration curve preparation example. The operation of measuring the absorbance at 595 nm was repeated 5 times for each condition. Table 2 shows the average value of the protein masses obtained from the results of the five absorbance measurements.

  As shown in Table 2, when the concentration of the alkaline solution (sodium hydroxide aqueous solution) was 0.2 N, the highest protein extraction amount was shown. In addition, when the concentration of the alkaline solution was 0.2 N, the extraction amount of protein was confirmed to increase as the extraction time increased until the extraction time was 30 minutes, but 30 minutes and 60 minutes were compared. Then, a big difference was not seen in the amount of protein extraction.

(About extraction temperature)
10 ml of an alkaline solution (0.2N aqueous sodium hydroxide solution) and TOSI were put in a polyethylene bag, and the bag was immersed in a thermostatic water bath maintained at each temperature (25, 30, 50, 70 ° C.). Then, TOSI was immersed and shaken in an alkaline solution for 30 minutes to extract proteins.

  As described above, 1 ml of the extract thus obtained was added to 3 ml of Coomassie Brilliant Blue G-250 solution, reacted at 25 ° C. for 20 minutes, and the absorbance of the obtained contact liquid at 595 nm was measured. This operation was repeated three times for each condition. Table 3 shows the average value of protein mass obtained from the results of the absorbance measurement performed three times.

  As shown in Table 3, when the extraction temperature was 50 ° C. and 70 ° C., there was no significant difference in the amount of protein extracted, but at the extraction temperature of 50 ° C., the highest protein extraction amount was obtained. Indicated.

(About the number of extractions)
Place 10 ml of alkaline solution (0.2N aqueous sodium hydroxide) and TOSI in a polyethylene bag, immerse this bag in a constant temperature water bath maintained at 50 ° C., and immerse the TOSI in an alkaline solution for 30 minutes. The protein was extracted. The TOSI from which the protein was extracted in this manner was extracted a second time under the same conditions as the first extraction condition.

  As described above, 1 ml each of the first extract and the second extract thus obtained was added to 3 ml of Coomassie Brilliant Blue G-250 solution and reacted at 25 ° C. for 20 minutes. The absorbance of the obtained contact solution was measured at 595 nm, and the protein mass obtained from the result of the absorbance measurement is shown in Table 4.

  As shown in Table 4, in all (Nos. 1 to 5), the amount of protein extracted in the second extraction was 10 μg or less. From the above, it can be said that one extraction is sufficient, and it can be said that almost all proteins attached to the detection target can be extracted by one extraction operation.

  Next, the detection accuracy was evaluated by the following method.

  After applying 25 μl of 5, 10, 20, 30, 40, 50 μg / 25 μl of bovine serum albumin solution to a stainless steel plate (15 × 50 mm), each was dried for 24 hours to prepare 5 each. The test piece thus obtained and 10 ml of an alkaline solution (0.2N sodium hydroxide aqueous solution) are put in a polyethylene bag, and this bag is immersed in a constant temperature water bath maintained at 50 ° C., and the test piece is placed in an alkaline solution. In the solution, the protein was extracted by immersion and shaking for 30 minutes.

  As described above, 1 ml of the extract thus obtained was added to 3 ml of Coomassie Brilliant Blue G-250 solution, reacted at 25 ° C. for 20 minutes, and then the absorbance of the obtained contact liquid at 595 nm was measured. Table 5 shows the amount of protein obtained from the results of the absorbance measurement. Table 5 also shows the average value, standard deviation, and coefficient of variation obtained from the obtained values.

  As shown in Table 5, when the coating amount of albumin was 5 μg, the coefficient of variation was slightly high, 20.05%, but when the coating amount of albumin was 10, 20, 30, 40, 50 μg Both the standard deviation and coefficient of variation showed good values.

  In addition, if the rinsing at the time of cleaning of the medical device is insufficient, and assuming that the disinfectant used before cleaning remains, the interfering substances such as the cleaning agent and the disinfectant remaining on the medical device are detected accurately. The following methods were used to evaluate the effects on

  Distilled water was used so that 5 μg of bovine serum albumin was contained and the concentrations of cleaning agents S1 to S2 and disinfecting agents D1 to D4 shown below were 0.01, 0.1, and 1.0% by weight. As described above, 1 ml of the prepared sample was added to 3 ml of Coomassie Brilliant Blue G-250 solution, reacted at 25 ° C. for 20 minutes, and then the absorbance of the obtained contact liquid at 595 nm was measured. The protein concentration in each condition was determined. The results are shown in Table 6.

Details of the cleaning agent and disinfectant used in the evaluation are as follows.
(Washing soap)
S1: Enzyme-containing detergent [Inuimedex Co., Ltd., trade name: Medipol EX-1]
S2: Enzyme, surfactant-containing detergent [Inuimedex Co., Ltd., trade name: Medipol EX-2]
S3: Detergent containing alkaline surfactant [Inuimedex Co., Ltd., trade name: Medipol F]
S4: Detergent containing no alkaline surfactant [Inuimedex Co., Ltd., trade name: Medipol ZT]
(disinfectant)
D1: Mixture of 15% alkyldiaminoethylglycine (Nippon Yushi Co., Ltd., trade name: Nissan Anon # 300) and distilled water in a weight ratio of 0.3: 99.7 D2: Chlorhexidine gluconate 2% liquid [Product name: Hibiscol, manufactured by Saraya Co., Ltd.]
D3: Popidone iodine 10% solution [Meiji Seika Co., Ltd., trade name: Isodine solution]
D4: glutaraldehyde 3.5% solution [manufactured by Johnson & Johnson, trade name: Sydex Plus 28]

  As shown in Table 6, when 1% by weight of the cleaning agent S3, the cleaning agent S4, the disinfectant D2, and the disinfectant D3 was contained, the value was higher than the control value. However, even if the rinsing at the time of cleaning the medical device is insufficient, it is difficult to assume a case where the residual concentration of an interfering substance such as a cleaning agent or a disinfectant reaches about 1% by weight. Therefore, the influence of interference substances such as cleaning agents and disinfectants on the detection method of the present invention seems to be no problem.

(Measurement of surgical instruments 1)
After applying 0.5 ml of human blood to a scissors (total length: about 15 cm), as shown in Table 7, washing was performed under various conditions. The scissors after washing and 10 ml of an alkaline solution (0.2N sodium hydroxide aqueous solution) are put in a polyethylene bag, the bag is immersed in a constant temperature bath maintained at 50 ° C., and the scissors are placed in an alkaline solution. Protein (human blood) was extracted by soaking and shaking for a minute. As described above, 1 ml of the extract thus obtained was added to 3 ml of Coomassie Brilliant Blue G-250 solution, reacted at 25 ° C. for 20 minutes, and the absorbance of the obtained contact liquid at 595 nm was measured. went. Table 7 shows protein amounts obtained from the results of the absorbance measurement.

Processing 1 and processing 2 shown in Table 7 are as follows.
Treatment 1: A blood coagulation inhibitor [manufactured by Inuimedex Co., Ltd., trade name: Medipol PS] is sprayed.
Treatment 2: An enzyme-based surfactant-containing detergent (manufactured by Inui Medex Co., Ltd., Medipol EX-1) and tap water mixed in a weight ratio of 0.5: 99.5 to an ultrasonic cleaner Put in [Sharp Co., Ltd., product number: MU-624], immerse the scissors in the cleaning solution at 40 ° C., and perform ultrasonic cleaning for 10 minutes.
Washing condition 4 shown in Table 7 quantifies proteins adhering before washing without performing treatments 1 and 2 on a scissors coated with 0.5 ml of human blood.

  As the protein mass measurement results in Table 7 show, the results clearly reflecting the washing conditions were obtained. From this result, it was found that a good deposit removal effect can be obtained by performing the treatment 1 (spraying a blood coagulation inhibitor), particularly immediately after blood adhesion.

(Measurement of surgical instruments 2)
Apply 0.5 ml of human blood to the lumen of the suction fistula (total length: about 12 cm, tip diameter: about 3 mm), leave it for 12 hours, and then wash it under various conditions as shown in Table 8 It was. The suction pipe after washing and 10 ml of alkaline solution (0.2N aqueous sodium hydroxide solution) are put in a polyethylene bag, and the bag is immersed in a constant temperature water bath maintained at 50 ° C. Protein extraction was performed by immersion and shaking in an alkaline solution for 30 minutes. As described above, 1 ml of the extract thus obtained was added to 3 ml of Coomassie Brilliant Blue G-250 solution, reacted at 25 ° C. for 20 minutes, and the absorbance of the obtained contact liquid at 595 nm was measured. Table 8 shows the amount of protein obtained from the results of the absorbance measurement.

Processing 3 and processing 4 shown in Table 8 are as shown below.
Treatment 3: Enzymatic surfactant-containing detergent (trade name: Medipol EX-1 manufactured by Inuimedex Co., Ltd.) and tap water mixed at a weight ratio of 0.5: 99.5 at 40 ° C. In this, it is immersed for 30 minutes.
Treatment 4: A cleaning solution obtained by mixing an alkaline cleaning agent [Medipol F, manufactured by Inuimedix Co., Ltd.] and tap water at a weight ratio of 0.5: 99.5 is placed in an ultrasonic cleaner, The suction soot tube is immersed in the cleaning solution thus prepared, and ultrasonic cleaning is performed for 10 minutes.

  As shown in Table 8, a result that clearly reflected the cleaning conditions was also obtained for the suction fistula.

(Measurement of surgical instruments 3)
Actually, the hemostatic forceps (total length: about 14 cm) used in the surgery were washed under various conditions as shown in Table 9. The hemostatic forceps after washing and 10 ml of alkaline solution (0.2N sodium hydroxide aqueous solution) are put in a polyethylene bag, and the bag is immersed in a constant temperature water bath maintained at 50 ° C. In the solution, the protein was extracted by immersion and shaking for 30 minutes. As described above, 1 ml of the extract thus obtained was added to 3 ml of Coomassie Brilliant Blue G-250 solution, reacted at 25 ° C. for 20 minutes, and the absorbance of the obtained contact liquid at 595 nm was measured. Table 9 shows the amount of protein obtained from the results of the absorbance measurement.

In addition, the process 5-the process 7 shown in Table 9 are as showing below.
Process 5: A blood coagulation inhibitor [manufactured by Inuimedex Co., Ltd., trade name: Medipol PS] is sprayed.
Treatment 6: Brushing treatment 7: A cleaning liquid obtained by mixing an alkaline detergent [manufactured by Inuimedex Co., Ltd., Medipol F] and tap water in a weight ratio of 0.5: 99.5 is placed in an ultrasonic cleaner. Then, the suction tube is immersed in the cleaning liquid at 40 ° C. and ultrasonic cleaning is performed for 15 minutes.

  As shown in Table 9, the results reflected in the cleaning conditions were obtained. Although the hemostatic forceps have relatively little blood adhesion, the results shown in Table 9 were also demonstrated.

  From the results of the surgical instrument measurements 1 to 3, it can be said that the detection method of the present embodiment sufficiently has the detection accuracy required in the medical field.

It is a graph which shows the calibration curve of protein concentration-absorbance in the case where Coomassie Brilliant Blue G-250 is used as a protein stain.

Claims (6)

Immersing the medical device in an alkaline solution and extracting the protein;
A step of bringing the extract obtained in the protein extraction step into contact with a protein stain;
A method for detecting a protein comprising the steps of:   The protein detection method according to claim 1, wherein the alkaline solution is a solution of sodium hydroxide and / or potassium hydroxide.   The protein detection method according to claim 1 or 2, wherein the concentration of the alkaline solution is 0.05 to 2N.   The method for detecting a protein according to any one of claims 1 to 3, wherein the protein extraction step is performed at 25 to 90 ° C.   The protein detection method according to any one of claims 1 to 4, wherein the protein stain is Coomassie Brilliant Blue G-250.   The protein detection according to any one of claims 1 to 5, further comprising a step of measuring the absorbance of the contact liquid obtained by bringing the extract obtained in the extraction step into contact with the protein stain. Method.

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