JP6507445B2 - Method for detecting and quantifying proteins - Google Patents

Description

  The present invention relates to a method for detecting and quantifying proteins.

In recent years, the problem of nosocomial infection has been increasing, and reducing the risk of infection has become an essential issue in medical institutions.
Since medical instruments are generally expensive, cleaning and recycling is performed as much as possible. Medical devices to which protein stains such as blood, body fluids, tissue fragments, etc., in which bacteria or viruses that may be infectious agents may be present, are usually washed (step 1) and disinfected to reduce the risk of infection. Cleaning and recycling are performed through the two steps of (second step).

Examples of the method of cleaning (first step) include immersion cleaning, manual cleaning, and machine cleaning using an ultrasonic cleaner, washer disinfector, or the like. In addition, endoscope-specific cleaning machines and the like according to the structure of a medical instrument such as an endoscope are also used.
However, even after the above cleaning, there may be cases where protein stains such as blood, body fluid, or tissue fragments remain in the medical device. In that case, the effect of the disinfection (the second step) is not sufficiently exerted and the possibility of infection is increased. For this reason, it is necessary to confirm the cleanliness of the medical device after cleaning.

  As a method of confirming the cleanliness of the medical device after washing, for example, in Patent Document 1, there is a step of immersing the medical device in an alkaline solution to extract a protein, and an extract obtained in the protein extraction step; There is disclosed a method of detecting a protein comprising the steps of contacting with a protein stain. In this detection method, a solution of sodium hydroxide and / or potassium hydroxide is used as the alkaline solution, the concentration of the alkaline aqueous solution is 0.05 to 2 N, and the protein extraction step is performed at 25 to 90 ° C. As coomassie brilliant blue G-250 is used.

  In Patent Document 2, a medical device is immersed in an extract solution which is pure water or an alkaline solution having a concentration of 20 mM or less, and a process of extracting a protein by irradiating an ultrasonic wave and extracting a protein obtained by the ultrasonic wave irradiation The step of reacting the liquid and the fluorescent reagent, the step of measuring the amount of fluorescence by irradiating the reacted solution with excitation light, and the step of measuring the amount of fluorescence, and based on the amount of fluorescence measured in the step of measuring fluorescence, There is disclosed a method of detecting a protein, comprising the steps of: determining a concentration of the extract solution.

Unexamined-Japanese-Patent No. 2006-145271 JP 2012-63298 A

  However, in the case of the method described in Patent Document 1, the alkaline solution has a weak ability to extract protein stains, and the alkaline solution causes the remaining protein to be denatured to promote firm adhesion to a medical device, thereby further extracting the protein stains. Problems such as making it difficult to do so or corroding medical devices. Moreover, in the immersion method which does not give a physical stimulus, there existed a problem that extraction power was weak. Furthermore, since the color of coomassie brilliant blue G-250, which is a protein stain, varies depending on the type of protein, the degree of coloration varies depending on the type or ratio of protein in the protein stain, and the protein mass can not be measured accurately. was there. In addition, the color of coomassie brilliant blue G-250 is easily affected by the surfactant, and if the extract solution contains the surfactant, an accurate detection amount can not be measured. Therefore, there is a problem that it is difficult to incorporate a surfactant effective for protein extraction into the extract.

  In the case of the method described in Patent Document 2, pure water or an alkaline solution is weak in osmotic action or dispersive action, and an alkaline solution causes denaturation of residual protein to promote firm adhesion to a medical device, and thus protein stains. There is a problem that it is difficult to extract the There is also a problem that the alkaline solution corrodes the device. Further, the detection method using the fluorescent reagent described in Patent Document 2 has a problem that complicated operation such as dilution is required so that the protein mass of the extract solution falls within the quantitative range because the quantitative range is extremely narrow. there were. Furthermore, substances other than proteins, including alkaline substances, may react with the above-mentioned fluorescent reagent, resulting in the problem that the amount of protein can not be measured accurately.

  The present invention has been made in view of the above circumstances, and an object thereof is to provide a method of sufficiently extracting a protein attached to a test object and detecting the extracted protein conveniently and accurately. .

The present invention has the following aspects.
[1] A method for detecting and quantifying a protein attached to an object to be examined, which comprises contacting an extract containing a surfactant with the object to be examined and performing a physical stimulation operation to obtain a protein extract A method for detecting and quantifying a protein, comprising the steps of: contacting the protein extract with a protein detection solution containing bicinchoninic acid and copper (II); and detecting and measuring the absorbance of the obtained contact solution.
[2] The method for detecting and quantifying a protein according to [1], wherein the physical stimulation operation is ultrasonic irradiation.
[3] The method for detecting and quantifying a protein according to [1] or [2], wherein the test object is a device that has been washed after use.
[4] The method according to any one of [1] to [3], wherein the surfactant is at least one selected from the group consisting of anionic surfactants, nonionic surfactants, and amphoteric surfactants. Method for detecting and quantifying proteins.
[5] The method for detecting and quantifying a protein according to any one of [1] to [4], wherein the concentration of the surfactant is 0.01 to 10% by mass in 100% by mass of the extract.
[6] The method for detecting and quantifying a protein according to any one of [1] to [5], wherein in the protein extraction step, the temperature of the extract when performing physical stimulation operation is 20 to 70 ° C.

  According to the method for detecting and quantifying a protein of the present invention, the protein attached to the test object can be sufficiently extracted, and the extracted protein can be detected and quantified easily and accurately.

It is a top view which shows the state which apply | coated the pseudo | simulation protein contamination liquid on the slide glass surface. It is a front view which shows the instrument for evaluation used in the Example.

Hereinafter, the present invention will be described in detail.
The method for detecting and quantifying a protein according to the present invention is a method for detecting and quantifying a protein attached to a test subject, and has a protein extraction step and a protein detection and determination step described below.

<Inspection object>
The object to be inspected is not particularly limited as long as it has a possibility that protein may be attached thereto, and examples include an appliance, a bed, a desk, a wall, a door, a door knob, a window, a floor and the like.
As instruments, for example, medical instruments used for surgery, examination, treatment, etc., instruments for food used for manufacturing and processing of food, and instruments as described above which were cleaned after use, or washed and disinfected. And the above-mentioned devices. Among these, from the viewpoint of confirming whether or not the instruments that have been cleaned after use can be reused, or from the viewpoint of confirming the appropriateness of the method of cleaning the instruments, the instruments that have been cleaned, cleaned or disinfected after use It is suitable.
Specific examples of the medical instrument include forceps, forceps, scissors, a suction tube, an endoscope, a catheter, an injection needle and the like.

<Protein extraction process>
The protein extraction step is a step of bringing a test object into contact with an extract containing a surfactant and performing a physical stimulation operation to obtain a protein extract.
When the test object is a device, for example, after the device is placed in a container for extraction, the protein extraction step adds an amount of an extract solution that can contact the entire device or the protein-adhered portion of the device without leakage, It can be done by giving a physical stimulus.

The container for extraction used in the protein extraction step is not particularly limited, and a dedicated container molded according to the shape, size, etc. of the device to be detected may be used, or the shape, size of the device A box-like, tubular, bag-like or the like container may be used which can accommodate the entire device or the protein-adhesion portion of the device regardless of the case etc. It is possible to reduce the amount of use of the extract solution, as a result the concentration of the extracted protein can be increased, and from the viewpoint that a trace amount of protein can also be detected, the shape of the device as an extraction container It is preferable to use a dedicated container molded according to the size and the like.
Although it does not specifically limit as a material of the container for extraction, Polyethylene, a polypropylene, polyester, nylon, polyvinyl chloride, glass, metals etc. are mentioned.

  When it is difficult to place the test object in a container for extraction, such as a bed or a wall, the protein extraction step is a physical stimulus such as a swab with a liquid extract or a swab product such as a commercially available swab product. Perform the wiping operation on the surface of the inspection object, install the wiping device that has been wiped out in a container for extraction containing an amount of extractable liquid that the whole wiping device can contact, and wipe the container for extraction etc. Can.

The extract used in the protein extraction step contains a surfactant.
Conventionally, water or an alkaline solution was used as an extract. However, water and alkaline solutions have weak permeability and dispersivity, etc., and alkaline solutions cause denaturation of proteins, so water and alkaline solutions extract strong protein stains remaining after machine cleaning etc. Was inadequate for
On the other hand, surfactants have high wetting power, dispersing power, penetrating power and the like, and are effective in extracting strong protein stains remaining after machine cleaning and the like.

  The surfactant is not particularly limited as long as it has strong detergency particularly against protein stains and is hardly affected by corrosion on the test object, and anionic surfactant, cationic surfactant, Nonionic surfactants and amphoteric surfactants can be mentioned. One or more of these can be used. Among these, anionic surfactants, nonionic surfactants, and amphoteric surfactants are preferable, and anionic surfactants and nonionic surfactants are more preferable, from the viewpoint of high protein extraction efficiency and less corrosion of the test object. .

  The anionic surfactant is not particularly limited, and examples thereof include soaps having 10 to 22 carbon atoms, alkyl benzene sulfonates having 14 to 24 carbon atoms, higher alcohol sulfate esters having 10 to 22 carbon atoms, and 10 carbon atoms of an alkyl group. -22, polyoxyethylene alkyl ether sulfate having 1 to 10 repeating number of oxyethylene units of polyoxyethylene group, 1 to 22 carbon α-sulfo fatty acid ester, 8 to 18 carbon α-olefin sulfone An acid salt, a C10-C22 alkanesulfonic acid salt, a C10-C22 monoalkyl phosphoric acid ester salt etc. are mentioned. Among them, soaps having 10 to 22 carbon atoms, alkyl benzene sulfonates having 14 to 24 carbon atoms, and higher alcohol sulfuric acid esters having 10 to 22 carbon atoms from the viewpoint of high protein extraction efficiency and low corrosion resistance of the test object. A salt, an alkyl group having 10 to 22 carbon atoms, and a polyoxyethylene alkyl ether sulfate having a repeating number of 1 to 10 of a polyoxyethylene oxyethylene unit, an α-olefin sulfonate having 8 to 18 carbon atoms, carbon Alkanesulfonates having a number of 10 to 22 are preferable, and higher alcohol sulfates having 10 to 22 carbon atoms, α-olefin sulfonates having 8 to 18 carbon atoms, and alkanesulfonates having 10 to 22 carbons are more preferable. And higher alcohol sulfuric ester salts having 10 to 22 carbon atoms are particularly preferable. One or more of these can be used.

  The amphoteric surfactant is not particularly limited, and examples thereof include alkylamino fatty acid salts, alkyl betaines, alkylamine oxides and the like. One or more of these can be used.

  The nonionic surfactant is not particularly limited. For example, higher alcohol alkylene oxide adducts, alkylphenol alkylene oxide adducts, fatty acid alkylene oxide adducts, polyhydric alcohol fatty acid ester alkylene oxide adducts, higher alkylamine alkylene oxide adducts, Examples thereof include fatty acid amide alkylene oxide adducts, polyalkylene glycol types of alkylene oxide adducts of polyoxypropylene, glycerol fatty acid esters, pentaerythritol fatty acid esters, sorbitol fatty acid esters, polyhydric alcohol types of sucrose fatty acid esters, and the like. The higher alcohol mentioned here is usually a linear or branched unsaturated or saturated higher alcohol having 8 to 22 carbon atoms. The alkylphenol is usually a linear or branched unsaturated or saturated alkylphenol having 6 to 22 carbon atoms. The fatty acid is usually an unsaturated or saturated fatty acid having 10 to 22 carbon atoms. The polyhydric alcohol is usually a polyhydric alcohol having 3 to 12 carbon atoms. The higher alkylamine is usually a linear or branched unsaturated or saturated higher alkylamine having 8 to 22 carbon atoms. Specific examples of the alkylene oxide include ethylene oxide, propylene oxide, 1,2-, 2,3-, 1,3- and 1,4-butylene oxide, and ethylene oxide and propylene oxide are preferable. The alkylene oxides may be the same or different, and when they are different, block addition, random addition or alternation addition may be used. 1-80 are preferable, as for the addition mole number of alkylene oxide, 2-60 are more preferable, and 5-40 are especially preferable. Among these, higher alcohol alkylene oxide adducts, alkylphenol alkylene oxide adducts, fatty acid alkylene oxide adducts, polyhydric alcohol fatty acid ester alkylene oxide adducts, from the viewpoint of high protein extraction efficiency and less corrosion of test objects. Higher alkylamine alkylene oxide adducts, fatty acid amide alkylene oxide adducts, polyalkylene glycol type of alkylene oxide adducts of polyoxypropylene; glycerol fatty acid esters are preferred, higher alcohol alkylene oxide adducts, alkylphenol alkylene oxide adducts, fatty acid alkylene Oxide adducts, polyhydric alcohol fatty acid ester alkylene oxide adducts, higher alkylamine alkylene oxa De adducts, fatty acid amide alkylene oxide adducts are more preferred. One or more of these can be used.

  The concentration of the surfactant is preferably 0.01 to 10% by mass, and more preferably 0.05 to 5% by mass in 100% by mass of the extract, from the viewpoint of enhancing the extraction efficiency of protein and being economical.

  As a solvent for diluting the surfactant, water is preferable, and specifically, tap water, ion exchanged water, distilled water, RO water and the like can be mentioned. One or more of these can be used.

  The amount of extraction liquid used may be adjusted according to the size, surface area, use condition, etc. of the test object to be detected, and is not particularly limited.

The physical stimulation operation in the protein extraction step is not particularly limited, but ultrasonic irradiation, shaking by hand or machine, brushing, wiping, etc. may be mentioned. Among these, ultrasonic irradiation is preferable from the viewpoint of improving the extraction efficiency by the physical stimulation effect by the elastic vibration wave.
The operating frequency at the time of ultrasonic irradiation is not particularly limited, but from the viewpoint of enhancing the extraction efficiency of protein, 20 to 1000 kHz is preferable, and 20 to 100 kHz is more preferable.
The ultrasonic wave irradiation apparatus is not particularly limited, and examples thereof include a horn type and a standing wave type.

The temperature of the extract at the time of performing the physical stimulation operation is preferably 20 to 70 ° C., and more preferably 30 to 60 ° C. from the viewpoint of enhancing the extraction efficiency of protein.
In addition, the time of the physical stimulation operation is not particularly limited, but it is preferably 1 to 120 minutes, and more preferably 5 to 60 minutes, from the viewpoint of enhancing protein extraction efficiency and being economical.

<Protein detection and quantification process>
The protein detection and determination step is a step of bringing the protein extract obtained in the protein extraction step into contact with a protein detection solution containing bicinchoninic acid and copper (II), and measuring the absorbance of the obtained contact solution.
Hereinafter, a method for detecting and quantifying proteins using a protein detection solution containing bicinchoninic acid and copper (II) is referred to as "BCA method".

The BCA method utilizes the fact that the protein reduces copper (II) to copper (I) in an alkaline environment, and that copper (I) and bicinchoninic acid form a complex that develops a blue-violet color with a maximum absorption wavelength of 562 nm. ing. This series of reactions is very simple because it is completed by bringing the protein extract and the protein detection solution into contact at, for example, 30 to 70 ° C. for 10 to 60 minutes.
In addition, the BCA method is less susceptible to the type and concentration of surfactant, the type and concentration of alkali, and the type of protein, and has the advantage of being able to accurately measure the amount of protein.

The absorbance of the contact solution is measured using a spectrophotometer at a blue-violet maximum absorption wavelength of 562 nm where copper (I) and bicinchoninic acid form a complex to form a color. Then, based on the measurement result of absorbance, the amount of protein is quantified.
The determination of the amount of protein by the measurement of absorbance is to determine the amount of protein by using a calibration curve or a correlation equation prepared in advance using absorbance with respect to a protein solution having a known concentration. The protein mass can be calculated by substituting the value of the absorbance of the contact solution of the protein extract and the protein detection solution into the calibration curve or the correlation equation.
The calibration curve or correlation equation is preferably prepared so that a trace amount of protein remaining on the test object after washing can be detected and quantified, and a protein solution with a protein concentration of 0 to 200 μg / mL is used It is preferable to create a calibration curve or correlation equation. Thus, the protein attached to the test object can be simply detected and quantified.

  The protein extract obtained in the protein extraction step may be diluted or concentrated in advance to adjust the protein concentration appropriately so that it falls within the range of the calibration curve or correlation equation to be used.

  In addition, as a protein detection solution containing bicinchoninic acid and copper (II), from the viewpoint of excellent stability, use a two-component protein detection solution of an alkaline solution containing bicinchoninic acid and a solution containing copper (II). Is preferred. Specific examples of the two-component protein detection solution include a two-component type of reagent A solution containing bicinchoninic acid shown in Table 1 and a reagent B solution containing copper (II) shown in Table 2.

  As a protein detection solution containing bicinchoninic acid and copper (II), a commercially available protein detection reagent for BCA method can be used. For example, the reagent A solution includes Pierce proteomics related product "BCA Protein Assay Reagent A" manufactured by Thermo Fisher Scientific Co., Ltd., etc. As the reagent B solution, Pierce proteomics related product manufactured by Thermo Fisher Scientific Co., Ltd. "BCA Protein Assay Reagent B" etc. are mentioned.

<Function effect>
The method for detecting and quantifying a protein according to the present invention described above extracts a protein attached to an object to be examined by physical stimulation using a liquid extract containing a surfactant and detects and quantifies this by the BCA method. The protein attached to the object can be sufficiently extracted, and the extracted protein can be detected easily and accurately.

  In the present invention, when detecting and quantifying the protein attached to the test object, it can also be supplied as a detection and quantification kit comprising the extract and the protein detection solution. In addition, this detection and quantification kit may be equipped with a detection and measurement instrument such as a specific container used for protein extraction, a colorimetric tube, a simplified spectrophotometer for absorbance measurement, and the like.

  Hereinafter, the present invention will be described in more detail by way of examples and comparative examples, but the present invention is not limited by the following examples.

<Production of evaluation instruments>
Heparinized sheep blood (made by Japan Biotest Laboratories Co., Ltd.) and a protamine sulfate (made by Nacalai Tesque, Inc.) solution adjusted to a concentration of 1 mass% with ion-exchanged water, heparinized sheep blood: protamine sulfate solution = It mixed so that it might be set to 10: 1 (volume ratio), and simulated protein contamination liquid was prepared.

As shown in FIG. 1, a gap of 5 mm is created from one end on a surface of a slide glass (76 mm long × 25 mm wide × 1.3 mm thick) 11 so that 50 μL of a simulated protein contamination solution becomes 25 mm × 25 mm And dried for 24 hours at room temperature to obtain a slide glass to which the pseudo protein contaminants were fixed.
Next, as shown in FIG. 2, a stainless steel plate (5 mm long × 25 mm wide × 1 mm thick) 12 is placed on the surface of the slide glass 11 on the side coated with the simulated protein contaminated liquid X and the other end side After mounting the slide glass 13 to which the pseudo protein contamination liquid was not applied from above, both ends of the slide glasses 11 and 13 were sandwiched and fixed by the clip 14 to obtain an evaluation tool 10.
In addition, when it measured by BCA method, 8350 micrograms of protein were contained in 50 microliters of pseudo-protein contamination liquids.

<Creation of calibration curve for BCA method>
A bovine serum albumin solution (manufactured by Thermo Fisher Scientific Co., Ltd.) is appropriately diluted with ion-exchanged water, and each protein concentration (0, 0.25, 0.5, 1, 5, 50, 100, 150, 200 μg / mL) Dilutions of
After adding 1 mL of each diluted solution prepared above to 2 mL of the protein detection solution prepared by mixing 50 mL of the reagent A solution shown in Table 1 and 1 mL of the reagent B solution shown in Table 2, it is reacted at 60 ° C. for 30 minutes And left for 5 minutes at 15 ° C. to obtain each contact solution. The absorbance at 562 nm of each contact solution obtained was measured with a spectrophotometer ("Lambda 650S" manufactured by PerkinElmer Japan Co., Ltd.), and each absorbance was plotted against the protein concentration in each diluted solution to calibrate the BCA method. I made a line. The absorbance at a protein concentration of 0.25, 0.5, 1, 5, 50, 100, 150, 200 μg / mL was obtained linearly.

<Creation of calibration curve for CBB method>
Bovine serum albumin solution (Thermo Fisher Scientific Co., Ltd.) is appropriately diluted with ion-exchanged water to dilute each protein concentration (0, 0.25, 0.5, 1, 2.5, 5, 25 μg / mL) I got a liquid.
1 mL of each dilution prepared above was added to 3 mL of Coomassie Protein Assay Reagent (CBB reagent (manufactured by Thermo Fisher Scientific Co., Ltd.)) and stirred, and then left at 25 ° C. for 20 minutes to obtain each contact solution. The The absorbance at 595 nm of each contact solution obtained was measured with a spectrophotometer ("Lambda 650S" manufactured by PerkinElmer Japan Co., Ltd.), and each absorbance was plotted against the protein concentration in each diluted solution to calibrate the CBB method. I made a line. The absorbance at a protein concentration of 0, 0.25, 0.5, 1, 2.5, 5, 25 μg / mL was obtained linearly.

"Example 1"
Sodium dodecyl sulfate was dissolved in ion exchange water to a concentration of 0.1% by mass to prepare an extract.
Separately, 50 mL of the reagent A solution shown in Table 1 and 1 mL of the reagent B solution shown in Table 2 were mixed to prepare a protein detection solution.

Put 100 mL of extract in a 200 mL beaker, immerse the evaluation instrument in it, and apply ultrasonic wave irradiation at a temperature of 50 ° C for 10 minutes with an ultrasonic cleaner ("ASU-10" manufactured by As One Corporation) for evaluation A protein extract was obtained by extracting a pseudoprotein contaminant fixed to the device.
After adding 2 mL of the protein detection solution to 1 mL of the obtained protein extract (diluted with ion exchanged water to be 200 μg / mL or less if the protein content in the protein extract is 200 μg / mL or more) and stirring The mixture was allowed to react at 60 ° C. for 30 minutes and then left at 15 ° C. for 5 minutes to obtain a contact solution.
The absorbance at 562 nm of the obtained contact solution was measured with a spectrophotometer ("Lambda 650S" manufactured by PerkinElmer Japan Co., Ltd.), and substituted into the equation of the calibration curve for the BCA method to calculate the amount of protein extracted. Moreover, the extraction rate was calculated | required from the following formula. The results are shown in Table 3.
Extraction ratio (%) = 100- [8350 (μg) -extracted protein mass (μg)] × 100/8350 (μg)

"Examples 2 to 12 and Comparative Examples 1 to 3"
The same treatment as in Example 1 was performed except that the extract having the composition shown in Tables 3 to 5 was used. The results are shown in Tables 3-5.

"Comparative Examples 4 to 8"
Using the extract of the composition shown in Table 6, immerse the evaluation device in a 200 mL beaker containing 100 mL of the extract and leave it for 10 minutes at a temperature of 50 ° C. to extract the pseudoprotein contamination fixed on the evaluation device The protein extract was obtained.
The protein detection of the obtained protein extract was performed in the same manner as in Example 1. The results are shown in Table 6.

The "remainder" of ion exchange water in Tables 3-6 is an amount needed to make the total amount of the extract liquid 100% by mass.
Moreover, the symbol in Tables 3-6 is as follows.
Activator 1: Polyoxyethylene (6 moles) Polyoxypropylene (2.5 moles) Monoalkyl (C12-13) Ether Activator 2: Polyoxyethylene (8.5 moles) Polyoxypropylene (2.5 Mol) monoalkyl (C12-13) ether activator 3: polyoxyethylene (14 mol) polyoxypropylene (2.5 moles) monoalkyl (C12-13) ether activator 4: polyoxyalkylene (9 mol ) Alkyl (C10-16 moles) Ether · Activator 5: Polyoxyethylene (11 moles) Tallow hardened alkylamine · Activator 6: Polyoxyethylene (20 moles) Sorbitan monolaurate Activator 7: Polyoxyethylene ( 10 mol) Octyl phenyl ether, activator 8: 3-[(3-cholamidopropyl) dimethylammo O] -1-propane sulfonate-active agent 9: dodecyl dimethyl (3-sulfopropyl) ammonium Hiro Doki Sid inner salt

As is clear from Tables 3 to 6, in each of the examples in which proteins were extracted by ultrasonic irradiation using an extract containing a surfactant, the extraction efficiency was high, and the extraction rates were all 98% or more.
On the other hand, in the case of Comparative Examples 1 to 3 in which proteins were extracted by ultrasonication using ion-exchanged water or an aqueous sodium hydroxide solution as the extract, the extraction ratio of protein was lower than that in each example and was 95% or less. there were.
Although the extract containing the surfactant was used, in the case of Comparative Examples 4 to 8 in which the protein was extracted only by immersion without ultrasonic irradiation, the extraction efficiency was extremely reduced and the extraction rate was less than 80%. The
From these results, it could be confirmed that ultrasonic irradiation using a surfactant was extremely effective for protein extraction.

"Examples 13 to 19"
Sodium dodecyl sulfate was dissolved in ion exchange water to a concentration of 1% by mass to prepare an extract.
The same treatment as in Example 1 was performed using the obtained extract, except that ultrasonic irradiation was performed at the temperature shown in Table 7 for 10 minutes. The results are shown in Table 7. The results of Example 2 are also shown in Table 7.

  As apparent from Table 7, in each of the examples, the protein attached to the evaluation device could be sufficiently extracted, and the extracted protein could be detected simply and accurately. In particular, when the temperature of the extract solution at the time of performing physical stimulation operation is 20 to 70 ° C., the extraction rate of protein is 95% or more, and it has been confirmed that the extraction temperature is more effective.

"Example 20"
According to the composition shown in Table 8, mock protein extracts (1) to (9) were prepared.
Separately, 50 mL of the reagent A solution shown in Table 1 and 1 mL of the reagent B solution shown in Table 2 were mixed to prepare a protein detection solution.
2 mL of the protein detection solution was added to 1 mL of each simulated protein extract and stirred, then reacted at 60 ° C. for 30 minutes, and left at 15 ° C. for 5 minutes to obtain a contact solution.
The absorbance at 562 nm of the obtained contact solution was measured with a spectrophotometer ("Lambda 650S" manufactured by PerkinElmer Japan Co., Ltd.), and substituted into the equation of the calibration curve for the BCA method to calculate the protein mass. The results are shown in Table 11.

"Example 21"
The same treatment as in Example 20 was performed except that the pseudoprotein extracts (10) to (18) having the compositions shown in Table 9 were used. The results are shown in Table 11.

"Example 22"
The same treatment as in Example 20 was performed except that the pseudoprotein extracts (19) to (27) having the compositions shown in Table 10 were used. The results are shown in Table 11.

"Comparative Example 9"
According to the composition shown in Table 8, mock protein extracts (1) to (9) were prepared.
After adding 3 mL of Coomassie Protein Assay Reagent (CBB reagent (manufactured by Thermo Fisher Scientific Co., Ltd.) as a protein detection solution) to 1 mL of each simulated protein extract and stirring, the mixture is left at 25 ° C. for 20 minutes to obtain a contact solution. The
The absorbance at 595 nm of the obtained contact solution was measured with a spectrophotometer ("Lambda 650S" manufactured by PerkinElmer Japan Co., Ltd.), and substituted into the equation of the calibration curve for the CBB method to calculate the protein mass. The results are shown in Table 12.
If the measured value is high due to the influence of the alkaline compound or surfactant contained in the pseudoprotein extract, use a solution obtained by diluting the pseudoprotein extract with ion-exchanged water. The absorbance was measured.

"Comparative Example 10"
The same treatment as in Comparative Example 9 was performed except that the pseudoprotein extracts (10) to (18) having the compositions shown in Table 9 were used. The results are shown in Table 12.

"Comparative example 11"
The same treatment as in Comparative Example 9 was performed except that the pseudoprotein extracts (19) to (27) having the compositions shown in Table 10 were used. The results are shown in Table 12.

The "remainder" of ion exchange water in Tables 8 to 10 refers to the amount necessary to make the total amount of the pseudoprotein extract liquid 100% by mass.
Moreover, the abbreviations in Tables 8 to 12 are as follows.
Activator 2: Polyoxyethylene (8.5 moles) polyoxypropylene (2.5 moles) monoalkyl (C12-13) ether Activator 5: Polyoxyethylene (11 moles) tallow hardened alkylamine

As apparent from Tables 11 and 12, it is confirmed that quantification of protein by the BCA method is less susceptible to the effects of alkali, anionic surfactant, and nonionic surfactant, and that a protein mass almost equivalent to the amount of protein added is detected. It was done (Examples 20 to 22).
On the other hand, quantification of protein by the CBB method was influenced by alkali, anionic surfactant and nonionic surfactant, and a protein mass different from the amount of protein added was detected (Comparative Examples 9 to 11).
From these results, when a surfactant is used as a cleaning agent for the inspection object, even if there is a concern that the surfactant is attached to the extract due to insufficient rinsing, using the BCA method is accurate. It has been confirmed that it is possible to use any surfactant which has high extraction efficiency as an extracting agent. On the other hand, when the CBB method is used, if there is a concern that the surfactant adheres to the extract due to insufficient rinsing or the like, accurate measurement is difficult, and the surfactant has high extraction efficiency as an extractant. It is also difficult to use an agent.

  According to the method for detecting and quantifying a protein of the present invention, the protein attached to the test object can be sufficiently extracted, and the extracted protein can be detected and quantified easily and accurately. Therefore, the method for detecting and quantifying a protein of the present invention can be particularly suitably used to evaluate the cleanliness of an object to be examined such as an instrument reused in a medical institution or the like, and to evaluate the performance of a medical washing machine or the like.

10 Evaluation Equipment 11 Slide Glass 12 Stainless Plate 13 Slide Glass 14 Clip X Simulated Protein Contamination Solution

Claims (5)

A method for detecting and quantifying a protein attached to a test object, comprising :
A protein extraction step of subjecting the test object to ultrasonic irradiation in a state in which the test object is immersed in an extract containing a surfactant, and obtaining a protein extract;
A protein detection and determination step of bringing the protein extract into contact with a protein detection solution containing bicinchoninic acid and copper (II), and measuring the absorbance of the obtained contact solution;
A method for detecting and quantifying proteins, which has The method for detecting and quantifying a protein according to claim 1 , wherein the test object is a device that has been washed after use. The method for detecting and quantifying a protein according to claim 1 or 2 , wherein the surfactant is at least one selected from the group consisting of an anionic surfactant, a nonionic surfactant, and an amphoteric surfactant. The method for detecting and quantifying a protein according to any one of claims 1 to 3 , wherein the concentration of the surfactant is 0.01 to 10% by mass in 100% by mass of the extract. The method for detecting and quantifying a protein according to any one of claims 1 to 4 , wherein in the protein extraction step, the temperature of the extract when performing physical stimulation operation is 20 to 70 ° C.

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