JP2012063231A - Protein extraction method, protein detection method, and protein detection apparatus - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a protein extraction method, a protein detection method, and a protein detection apparatus that can precisely extract and measure protein that remains in a device with high sensitivity.SOLUTION: A method for extracting protein attached to a device uses alkali solution having density equal to or less than 20 mM as extraction liquid. The protein detection method includes the steps of: reacting protein solution with a fluorescence reagent; radiating excitation light onto reacted solution to measure a fluorescence amount; calculating density of the protein solution on the basis of the fluorescence amount measured in the previous step.

Description

  The present invention relates to a method for extracting a protein adhering to various medical instruments used in a medical institution or the like, various manufacturing instruments used in the field of food production, and the like, a protein detection method, and a protein detection apparatus.

  Along with the increase in the number of examinations and treatments in medical institutions and inspection institutions such as hospitals, a large number of infection accidents due to examinations and treatments have been reported both in Japan and overseas. Many of these infection cases are caused by insufficient cleaning and disinfection of medical instruments such as endoscopes, tubular catheters, forceps, and scissors. Establishment is required. Also in the food manufacturing field, in order to prevent the occurrence of damage such as food poisoning caused by dirt attached to the equipment and equipment used, the hygiene aspects of the equipment and equipment used in the processing and production of food There is a strong demand for management.

  In particular, in the medical field, medical instruments such as surgical instruments and endoscopes are sterilized after being cleaned and removed using a medical washer such as a washer disinfector, and reused. However, if there is a cleaning residue in the cleaning process, sufficient sterilization cannot be performed and the risk of infection increases. For this reason, the cleaning process is managed by measuring the residual protein, which is a cleaning residue after the cleaning by the cleaning machine, and confirming the performance of the medical cleaning machine. Conventionally, as a method for detecting this residual protein, as shown in Patent Document 1, a coomassie brilliant blue G- is used in the case where the washed protein is immersed in an alkaline aqueous solution and the residual protein adhering to the device is eluted. A method is known in which a protein is stained with 250, amide black 10B, or the like, and the color difference is measured visually or with an absorptiometer.

  However, the method disclosed in Patent Document 1 can quantitatively evaluate the amount of protein, but the detection sensitivity is at most about 1 μg / ml, and sufficient detection sensitivity, for example, a protein concentration of 1 μg / ml or less is measured. It was difficult to do. Moreover, in the dyeing | staining using amide black 10B, it becomes visual determination and cannot perform quantitative evaluation. For this reason, in order to reduce the risk of nosocomial infection as much as possible, there is a problem that high-sensitivity inspection cannot be performed even though there is an increasing need for improving the performance of the washing machine.

  On the other hand, as a method for detecting proteins with high sensitivity, a method of detecting by reacting with a protein using a fluorescent reagent and measuring the amount of fluorescence (hereinafter referred to as a fluorescence method) is generally known. For example, as described in Non-Patent Document 1, the fluorescence method can detect a trace amount of protein of 10 ng / ml to 100 ng / ml.

JP 2006-145271 A

Biotechniques, Vol. 34, no. 4 (2003) 850-861

  However, although it is excellent in that a protein dissolved in a protein solution can be detected with high sensitivity in a general fluorescence method, the above-mentioned non-patent literature describes how to prepare a protein solution or how to It is not described whether the protein is extracted into the extract. For this reason, in special applications such as measuring the amount of residual protein adhering to the instrument using the protein solution extracted from the instrument after washing, such as sodium hydroxide contained in the alkaline aqueous solution used for protein extraction It does not provide any solution for the problem that there is a measurement interfering substance and it is difficult to correctly measure the amount of residual protein. That is, even if the fluorescence method disclosed in this document is applied as it is, the fluorescent reagent reacts with an alkaline aqueous solution, so that it is difficult to accurately measure the protein mass. In addition, in an aqueous solution having strong alkalinity, the protein is denatured, which is a problem in detecting the protein with high sensitivity.

  The present invention has been made in view of the above-mentioned problems, and its object is to provide a protein extraction method for accurately extracting the protein remaining in the instrument, and a protein detection method capable of measuring the extracted protein with high sensitivity. And providing a protein detection apparatus.

  The protein extraction method according to the present invention is a method for extracting a protein adhering to an instrument after washing, and is characterized by using an alkaline solution having a concentration of 20 mM or less as an extract.

  The concentration of the alkaline solution is more preferably 10 mM or less.

  The protein detection method according to the present invention is characterized in that the instrument is after washing.

  The protein detection method according to the present invention is characterized in that a protein is detected using a protein solution obtained by using any of the protein extraction methods described above.

  The protein detection method according to the present invention includes a reaction step of reacting the protein solution with a fluorescent reagent, a fluorescence measurement step of measuring the amount of fluorescence by irradiating the reacted solution with excitation light, and the fluorescence measurement step. And a quantitative step of calculating the concentration of the protein solution based on the fluorescence amount measured in (1).

  The protein detection method according to the present invention is characterized in that the fluorescent reagent is nano orange.

  In addition, the protein detection method according to the present invention is characterized in that the accuracy of the washing is measured by detecting the protein adhering to the washed instrument using a fluorescence method.

  The protein detection apparatus according to the present invention is characterized by measuring the amount of protein adhering to the instrument using any one of the protein detection methods described above.

  ADVANTAGE OF THE INVENTION According to this invention, the protein extraction method which extracts accurately the protein which remained in the apparatus after washing | cleaning, and the protein detection method and apparatus which can measure the extracted protein with high sensitivity are realizable.

It is a flowchart which shows the protein detection process of this invention. It is a graph which shows the relationship between a sodium hydroxide density | concentration and fluorescence amount. It is a graph which shows an example of the calibration curve in the fluorescence method of this invention. It is a figure which shows schematic structure of the protein detection apparatus of this invention.

  Embodiments of the present invention will be described below. In the drawings of the present invention, the same reference numerals represent the same or corresponding parts.

  The protein detection method of the present invention is shown in FIG. 1 as a flowchart showing the protein detection step. The process includes an extraction process 101 for extracting residual protein from the cleaned instrument, a reaction process 102 for mixing the extracted protein solution with a fluorescent reagent, a fluorescence measurement process 103 for measuring the fluorescence level, and converting the fluorescence level into a protein concentration. This includes steps such as the quantification step 104. Hereinafter, each step will be described in detail.

(Extraction process)
In the protein extraction step 101, an alkaline solution is used as the extract. As the alkaline solution, an aqueous solution of sodium hydroxide, potassium hydroxide, potassium carbonate, sodium phosphate or the like is used. When an alkaline solution is used as the extract, if the alkali concentration is high, the extract contains a large amount of substances other than protein. Substances other than this protein react with the fluorescent reagent and emit fluorescence, or the protein and fluorescence. There is a possibility of inhibiting the reaction with the reagent. On the other hand, if the alkali concentration is extremely low, there is a possibility that the residual protein adhering to the instrument cannot be sufficiently extracted. Therefore, it is required to adjust to an appropriate concentration in consideration of these. Therefore, the following measurement was performed to investigate the concentration of an alkaline solution suitable for protein extraction.

  First, sodium hydroxide aqueous solutions having different concentrations and containing no protein were prepared, and then nano oranges (manufactured by CSL Behring Co., Ltd.) were reacted with each other to measure the amount of fluorescence. The fluorescence was measured using a fluorescence plate reader (ARVO MX 142, manufactured by Perkin Elmer), with an excitation wavelength of 470 nm and a fluorescence wavelength of 570 nm. The plate used was a 96 well plate (model number 3632) manufactured by Corning.

  FIG. 2 is a graph showing the relationship between the concentration of an aqueous sodium hydroxide solution and the amount of fluorescence measured by the above method. As a result, when the concentration exceeds 20 mM (hereinafter, mol / l is expressed as M) even though the protein is not contained, the amount of fluorescence increases rapidly, which affects the protein measurement. It has been found. For this reason, it is desirable to use a solution having a concentration of 20 mM or less as the extract. More desirably, a solution having a concentration of 10 mM or less is preferably used. In addition, this time, the concentration and the amount of fluorescence were shown using a sodium hydroxide aqueous solution. In addition to sodium hydroxide, in the alkaline solution such as potassium hydroxide described above, if the concentration is 20 mM or less, Since it is hardly affected by the fluorescent reagent, it can be used as a protein extraction solution.

Furthermore, in order to investigate in detail, it experimented about the density | concentration of the alkaline solution as an extract. As the extraction liquid and the extraction temperature, evaluation was performed over time under the conditions A to C shown below. Moreover, the following samples 1 to 3 were used as samples to be evaluated, and whether or not extraction was possible was determined from mass changes before and after extraction. The amount of the extract was 10 ml, and the device was immersed in the extract by putting the device and the extract in a polyethylene bag. A water bath, Thomasstat T-22S (manufactured by TOHMAS KAGAKA) was used for heating. In addition, although distilled water, RO water, deionized water, etc. can be used as the pure water of the condition C, in this experiment, milli Q water (ultra pure water made by the ultra pure water device manufactured by Millipore) Water).
〔conditions〕
Condition A: A sample is immersed in a sodium hydroxide solution at a concentration of 5 mM at 50 ° C. Condition B: A sample is immersed in a sodium hydroxide solution at a concentration of 200 mM at 50 ° C. Condition C: The sample is immersed in pure water at 50 ° C. [Sample]
1: 60 μl of ovine blood (mixed with heparin-supplemented amniotic blood manufactured by Japan Biological Materials Center and 1% protamine sulfate aqueous solution manufactured by Wako Pure Chemicals at a ratio of 10: 1) applied to a stainless steel plate and naturally dried for 24 hours : Indicator for cleaning evaluation (manufactured by Pereg, trade name: TOSI: stainless steel base material coated with pseudo-contamination)
3: Sample 2 cleaning evaluation indicator heated at 130 ° C. for 30 minutes

Table 1 shows the extraction results under each condition. The circles in the table indicate that almost all extraction is possible, the triangles indicate less than 10% residual, and the crosses indicate 90% or more residuals.
<< Results under Condition A >>
In sample 1, almost all proteins could be extracted even in a short time. In Samples 2 and 3, most of the pseudo-contaminated matter was extracted although white residue was slightly left on the base material. The residue is fibrin in the pseudo-contaminant, and since water-soluble proteins such as albumin contained in the pseudo-contaminant have been removed, the water-insoluble substance remains without being dissolved due to the weak alkalinity of the extract. It is thought that.
<< Result under Condition B >>
When the immersion time was 30 minutes, extraction was possible. When the immersion time was less than 10 minutes, most of the protein remained in the instrument. Furthermore, the protein solution extracted in Sample 1 was changed from red to green, and a white suspension was observed. Probably, when the concentration of the extract is 200 mM, the alkalinity is strong and the extracted protein may be denatured, and the accuracy of quantifying the protein from the solution decreases. In sample 2, the contaminants peeled off from the substrate, but the contaminants were floating in the extract as a membrane. This is thought to be because the surface of the contaminant is denatured and is difficult to dissolve in the extract, and it is difficult to measure the protein concentration of the solution.
<< Result under Condition C >>
Even when the immersion time was 30 minutes, in Samples 1 to 3, a large amount of white residue remained on the base material. Compared with condition A, there are many residuals and some red residues are included, and it is considered that the protein is not sufficiently extracted.

  From the above experimental results and the results shown in FIG. 2, when the alkali concentration of the extract is higher than 20 mM, it takes time to extract the protein, and the extract is highly alkaline, so the protein is denatured. It has been found that such a problem tends to occur and is not preferable. In particular, when a protein is detected by a fluorescence method to be described later, the amount of protein cannot be measured accurately because the fluorescent reagent reacts with an alkaline aqueous solution, which is not preferable. From these results, it is preferable to use a 20 mM or less aqueous sodium hydroxide solution as the extract.

  Thus, a protein solution extracted with an extract having an alkali concentration of 20 mM or less is not easily denatured by a protein dissolved in the extract and is hardly affected by the alkali solute dissolved in the extract. Can be measured very accurately. In addition, when this washing is a washing method that does not use an alkaline detergent, such as washing with water, ultrasonic washing, or washing with ozone water, it is particularly effective in terms of extracting residual proteins that are easily soluble in alkali. It is believed that there is.

  Next, a method for detecting a protein from the protein solution obtained by the above method will be described below. In the present embodiment, a detection method using a fluorescence method will be described as an example. Since the fluorescence method is particularly effective as a method for detecting proteins with high sensitivity, it is suitable for the detection of a minute amount of protein such as residual protein adhering to a cleaned instrument, but the detection method is limited to this. It is not a thing.

(Reaction process)
Next, the reaction process 102 in which the protein solution extracted in the extraction process 101 is mixed with a fluorescent reagent and reacted is described. As the fluorescent reagent, a reagent that does not react with a specific protein but can detect the total protein is preferable. For example, nano orange or CBQCA (manufactured by Invitrogen) is preferable because of high detection sensitivity.

  For example, when nano orange is used, nano orange is mixed with the protein solution extracted in the extraction step 101, heated at 93 ° C. for 10 minutes, and cooled at room temperature to complete the reaction step 102. About reaction conditions, what is necessary is just to select suitably the conditions suitable for the fluorescent reagent to be used.

(Fluorescence measurement process)
Next, the fluorescence measurement process 103 will be described. In this step, the extracted protein solution and the fluorescent reagent are mixed, and the reaction mixture is irradiated with excitation light, and the amount of fluorescence emitted from the mixture is measured. The measurement is the same as the procedure described in the extraction step, and a general plate reader, fluorescence microscope, fluorescence spectrometer, or the like can be used. As the wavelength of excitation light or fluorescence, a wavelength suitable for the fluorescent reagent to be used is selected and used. For example, when nano orange is used, the excitation wavelength is around 470 nm and the fluorescence wavelength is around 570 nm, and spectroscopy is performed using a filter suitable for this.

(Quantitative process)
Finally, the quantitative process 104 for converting the fluorescence amount into the protein concentration of the solution will be described. Here, the concentration of the protein solution is calculated based on the amount of fluorescence measured in the fluorescence measurement step 103. Specifically, a calibration curve is prepared in advance with a protein solution with a known concentration, and the protein concentration of the solution is calculated using the detected fluorescence amount with this calibration curve. A protein such as BSA (bovine serum albumin) can be used to prepare a calibration curve.

  FIG. 3 shows the relationship between the BSA concentration and the fluorescence amount when nano orange is used as the fluorescent reagent. As the BSA solvent, the same one as the extract is used. FIG. 3 is a graph when sodium hydroxide having a concentration of 5 mM is used as a solvent. Detection was possible up to a BSA concentration of about 0.1 μg / ml or less. The amount of protein remaining in the instrument can be calculated by adding the amount of the extract to the detected protein concentration. For example, when the detected protein concentration is 0.1 μg / ml and the amount of the extract is 10 ml, the residual protein mass is 1 μg / instrument. In the detection method using the conventional absorbance, the detection sensitivity is about 10 μg / instrument, and the detection can be performed with high sensitivity by using the fluorescence method.

  In order to further increase the sensitivity, the protein solution extracted in the extraction step 101 is preferably concentrated. For example, when the amount of the extract is 10 ml, the actual measurement sensitivity is improved 10 times by concentrating to 1 ml. As a concentration method, an ultrafiltration method is simple, and it can be appropriately concentrated by selecting a filter using the difference between the molecular weight of protein and the molecular weight of sodium hydroxide.

  As shown above, by using an alkaline solution of an appropriate concentration as the extract, protein can be extracted from the instrument with almost no residue in the protein extraction process, and further, protein denaturation and solute during protein detection It is possible to obtain a protein solution that does not interfere with measurement. Moreover, it becomes possible to quantify the amount of residual protein with high sensitivity by detecting the extracted protein using a fluorescence method.

  Below, the protein detection apparatus 1 using the detection method of this invention is demonstrated. The protein detection apparatus 1 of the present invention includes a protein extraction unit 11 from a tool, a fluorescent reagent mixing unit 12, and a fluorescence measurement unit 13, and the above-described steps are performed in each unit.

  FIG. 4 is a schematic diagram of the protein detection apparatus 1. Residual protein adhering to the device 2 to be inspected is first extracted by the protein extraction unit 11. The instrument 2 is immersed in the extract 3 in the bathtub 4 and protein is extracted. When the protein extraction is completed, the first valve 5 is opened, and the protein solution is conveyed to the reagent mixing section through the flow path 6. Next, in the reagent mixing unit 12, the reagent in the reagent container 7 is adjusted to an appropriate amount by the second valve 8 and mixed with the protein solution. Next, the mixed solution is conveyed to the fluorescence measurement unit 13 by the third valve 9, the fluorescence amount is measured by the fluorescence measurement optical system 10, and the protein concentration is calculated by the analysis unit (not shown). Further, the solution after the measurement is discharged from a discharge unit (not shown).

  As described above, the protein detection apparatus 1 in which each process is integrated makes it possible to easily detect a protein and to reduce the size, so that measurement can be performed at any place in a medical institution or a food production line. Is possible.

  Although the above embodiment is a description related to a medical instrument, the present invention is not limited to this, and can be widely applied to instruments attached with proteins such as an instrument used in a food production line. Further, the present invention is not limited to the above-described embodiments, and various modifications can be made within the scope shown in the claims, and the embodiments can be obtained by appropriately combining technical means disclosed in different embodiments. The form is also included in the technical scope of the present invention.

  The protein extraction method, detection method, and detection apparatus according to the present invention can be suitably used for, for example, evaluating the cleaning accuracy of medical instruments, evaluating the performance of medical cleaning machines, and detecting contaminants in production lines of food factories.

DESCRIPTION OF SYMBOLS 1 Protein detection apparatus 2 Instrument 3 Extract liquid 4 Bath 5 1st valve 6 Flow path 7 Reagent container 8 2nd valve 9 3rd valve 10 Fluorescence measurement optical system 11 Protein extraction part 12 Reagent mixing part 13 Fluorescence measurement part

Claims (8)

A method for extracting protein adhering to a device,
A protein extraction method characterized by using an alkaline solution having a concentration of 20 mM or less as an extract.   A protein extraction method, wherein the concentration of the alkaline solution is 10 mM or less.   The protein extraction method according to claim 1 or 2, wherein the instrument is after washing.   A protein detection method comprising: detecting a protein using a protein solution obtained by using the protein extraction method according to any one of claims 1 to 3. The protein detection method comprises a reaction step of reacting the protein solution with a fluorescent reagent;
A fluorescence measurement step of irradiating the reacted solution with excitation light to measure the amount of fluorescence;
The protein detection method according to claim 4, further comprising a quantification step of calculating the concentration of the protein solution based on the fluorescence amount measured in the fluorescence measurement step.   The protein detection method according to claim 5, wherein the fluorescent reagent is nano orange.   A method for detecting a protein comprising measuring the accuracy of the washing by detecting a protein adhering to the instrument after washing using a fluorescence method.   A protein detection apparatus that measures the amount of protein adhering to the instrument using the protein detection method according to any one of claims 4 to 7.