JP2020180890A - Method of detecting protein - Google Patents

Abstract

To provide a method of detecting protein in an object sample through easy operation, but precisely with high sensitivity, according to the present invention.SOLUTION: The present invention relates to a method of detecting protein that comprises: a coloring process of coloring protein in an object of measurement; a filtering process of filtering the protein out of the object of measurement through a filter; and a detecting process of detecting the coloring of the protein of the object of measurement captured on the filter in the filtering process, and that further comprises, before the detecting process: a protein denaturing process of denaturing the protein of the object of measurement captured on the filter; and a decoloring process of adding a decoloring agent to the filter.SELECTED DRAWING: None

Description

The present invention relates to a method for detecting a protein, a method for measuring a concentration of a protein, and a protein detection kit.

In the food industry, it is necessary to grasp the amount of allergen in the rinse water after cleaning the manufacturing equipment, etc., in order to prevent the allergen contained in one food product from being mixed with another food product. Since allergens are often proteins, it is common to measure the amount of protein in the rinse water.
In recent years, consumer demand for low-calorie foods and zero-calorie foods has increased, and foods labeled as zero protein on the nutrition label have become widely distributed in the market. Therefore, there is a need for a method that can easily determine at the time of manufacture whether or not the food contains protein.

In this way, there is a great need to detect and measure the protein contained in the target sample. Until now, it has been common to detect and measure proteins in a target sample by an enzyme-linked immunosorbent assay (ELISA method) or the like. However, the ELISA method is a method for detecting a specific protein using an antibody, and the proteins that can be detected and measured are limited, and lacks versatility.

To solve the above problem, for example, Patent Document 1 discloses a protein analysis method that combines a dot blotting method and a fluorescent staining method.

International Publication No. 2004/011948

However, in Patent Document 1, a special fluorescence image analyzer for carrying out the fluorescence staining method is required, and 4 to 5 hours are required from sample preparation to image analysis, which makes it difficult to detect proteins quickly. there were. Therefore, for example, it is difficult to adopt it as a method for checking the degree of cleaning of rinse water in a manufacturing facility or the like and detecting a protein in a food, which is carried out every day in a food manufacturing factory.

Therefore, it is an object of the present invention to provide a method for detecting a protein in a target sample, a method for measuring a protein concentration, and a protein detection kit with high accuracy and high sensitivity while being simple to operate. To do.

The present inventors have conducted intensive studies to solve the above problems. As a result, the protein to be measured contained in the target sample is colored and filtered with a filter to detect the protein, and the protein to be measured captured by the filter is denatured before the protein is detected. It was also found that by adding a decolorizing agent to the filter, the protein in the target sample can be detected with high accuracy and high sensitivity while being easy to operate.

Further, according to the above method, the present inventors more linearly correlate the correlation between the concentration of the protein to be measured contained in the target sample and the coloration color value of the detected protein to be measured ( It was found that it can be approximated to a linear function) and the concentration of the protein to be measured can be measured more accurately.

That is, the present invention is as follows.
[1] A coloration process for coloring the protein to be measured, and
A filtration process that filters the protein to be measured with a filter,
A detection step for detecting the coloration of the protein to be measured captured by the filter by the filtration step, and a detection step for detecting the coloration of the protein to be measured.
It is a protein detection method that includes
Before the detection step,
A protein denaturation step that denatures the protein to be measured captured by the filter,
The decolorization process of adding a decolorizing agent to the filter and
A method for detecting a protein, which is characterized by further comprising.
[2] The detection method according to [1], wherein the coloration step, the filtration step, the protein denaturation step, the decolorization step, and the detection step are carried out in this order.
[3] The detection method according to the above [1], wherein the filtration step, the coloration step, the protein denaturation step, the decolorization step, and the detection step are carried out in this order.
[4] In any one of the above [1] to [3], in the coloring step, a coloring reagent whose absorption wavelength is shifted by binding to the protein to be measured is used to color the protein to be measured. The detection method described.
[5] The detection method according to any one of [1] to [3] above, wherein in the coloration step, the protein to be measured is colored by the Bradford method.
[6] The detection method according to any one of [1] to [5] above, wherein in the filtration step, the protein to be measured is filtered with a glass filter.
[7] The detection method according to any one of [1] to [6] above, wherein in the protein denaturing step, the protein to be measured captured in the filter is denatured by adding an acid to the filter.
[8] The detection method according to the above [7], wherein the acid is at least one acid selected from the group consisting of acetic acid, phosphoric acid, formic acid, citric acid, hydrochloric acid, and lactic acid.
[9] The detection method according to [7] above, wherein the acid is acetic acid.
[10] The detection method according to any one of [1] to [9] above, wherein the decolorizing agent added to the filter in the decoloring step contains an organic solvent.
[11] The detection method according to the above [10], wherein the organic solvent contains at least one alcohol selected from the group consisting of ethanol, methanol and isopropanol.
[12] The detection method according to the above [10], wherein the organic solvent contains ethanol.
[13] In the protein denaturing step, acetic acid is added to the filter to denature the protein to be measured captured in the filter, and in the decolorization step, ethanol is added to the filter. 12] The detection method according to any one of.
[14] Any one of the above [1] to [13], wherein the protein to be measured is a protein contained in rinse water used for washing manufacturing equipment of a food factory, or a protein contained in food or beverage. The detection method according to 1.
[15] The proteins contained in the rinse water used for cleaning the manufacturing equipment of the food factory are milk, eggs, wheat, buckwheat, peanuts, shrimp, crab, abalone, squid, salmon roe, orange, cashew nuts, kiwifruit, and beef. The detection method according to the above [14], which is a protein contained in salmon roe, sesame, salmon roe, buckwheat, soybean, chicken, banana, pork, matsutake, thigh, yamaimo, apple, or gelatin.
[16] The proteins contained in food or beverage are milk, egg, wheat, buckwheat, peanut, shrimp, crab, abalone, squid, salmon roe, orange, cashew nut, kiwifruit, beef, walnut, sesame, salmon, mackerel, soybean. The detection method according to the above [14], which is a protein contained in chicken, banana, pork, matsutake, thigh, yam, apple, or gelatin.
[17] The detection method according to any one of [1] to [16], wherein the time required for detection is one hour or less.
[18] A coloration step for coloring the protein to be measured, and
A filtration process that filters the protein to be measured with a filter,
A protein denaturing step of denaturing the protein to be measured captured by the filter by the filtration step, and a protein denaturing step.
The decolorization process of adding a decolorizing agent to the filter and
A detection process that detects the coloration of the protein to be measured captured by the filter,
With respect to the coloration of the protein to be measured detected in the detection step, the color value measuring step for measuring the color value and the color value measuring step
It is a method for measuring the concentration of protein, which comprises
Using a plurality of reference samples whose color values and the concentration of the protein to be measured are known, a calibration curve showing the correspondence between the concentration of the protein to be measured in each reference sample and the color value is created. A method for measuring a protein concentration, which comprises measuring the concentration of a protein to be measured from the color value measured in the color value measuring step.
[19] A coloration step of coloring the protein to be measured by the Bradford method, and
A filtration process that filters the protein to be measured with a filter,
A protein denaturing step of denaturing the protein to be measured captured by the filter by the filtration step, and a protein denaturing step.
The decolorization process of adding a decolorizing agent to the filter and
A detection process that detects the coloration of the protein to be measured captured by the filter,
Regarding the color development of the protein to be measured detected in the detection step, a color value measuring step for measuring the b value defined in the L ^* a ^* b ^* color system conforming to the CIE standard, and a color value measuring step.
It is a method for measuring the concentration of protein, which comprises
Using a plurality of reference samples for which the concentration of the protein to be measured and the b value defined in the L ^* a ^* b ^* color system conforming to the CIE standard are known, the measurement target in each reference sample is used. By creating a calibration curve showing the correspondence between the protein concentration and the b value, the concentration of the protein to be measured is measured from the b value measured in the color value measuring step. Concentration measurement method.
[20] A protein detection kit containing a filter for capturing a protein to be measured, a coloring reagent for coloring the protein to be measured, a protein denaturing agent for denaturing the protein to be measured, and a decolorizing agent.
[21] Further, a color determination paper is provided.
A method for measuring the concentration of a protein to be measured by comparing the coloration determination paper with the coloration state of the protein to be measured captured by the filter.
The protein detection kit according to the above [20].

According to the protein detection method according to the embodiment of the present invention, the protein in the target sample can be detected by a simple operation. In addition, it is possible to reduce the coloration that is not derived from the protein contained in the target sample, and it is possible to detect the protein in the target sample with high accuracy and high sensitivity.

Further, according to the protein concentration measuring method according to the embodiment of the present invention, the correlation between the concentration of the protein to be measured contained in the target sample and the color color value of the detected protein to be measured is correlated. The relationship can be approximated more linearly (linear function). Therefore, the protein concentration in the target sample can be measured easily and accurately.

Further, according to the protein detection kit according to the embodiment of the present invention, the above-mentioned protein detection method and concentration measurement method can be easily carried out.

It is a figure which shows the color reaction paper used at the time of the detection of a protein. It is a figure for demonstrating one Embodiment of the detection kit of this invention. It is a figure for demonstrating another embodiment of the detection kit of this invention. It is a figure for demonstrating another embodiment of the detection kit of this invention. It is a graph which shows the relationship between the b value of Comparative Example 3 and Example 9 measured in Test Example 3 and the concentration of sodium caseinate in a sample. It is a graph which shows the relationship between the b value of Comparative Example 4 and Example 10 measured in Test Example 3 and the concentration of sodium caseinate in a sample. It is a graph which shows the relationship between the b value of Comparative Example 5 and Example 11 measured in Test Example 3 and the concentration of sodium caseinate in a sample. It is a graph which shows the relationship between the b value of Comparative Example 6 and Example 12 measured in Test Example 3 and the concentration of sodium caseinate in a sample. It is a graph which shows the relationship between the b value of Comparative Example 7 and Example 13 measured in Test Example 3 and the concentration of sodium caseinate in a sample. It is a graph which shows the relationship between the b value of Comparative Example 8 and Example 14 measured in Test Example 4 and the concentration of sodium caseinate in a sample.

Hereinafter, embodiments of the present invention will be described in detail. The present invention is not limited to the embodiments described below.

<Protein detection method>
A protein detection method according to an embodiment of the present invention is characterized by including, at least, a protein denaturation step and a decolorization step in addition to the above-mentioned coloring step, filtration step, and detection step. Each step will be described below.

[Coloring process]
The coloration step in the present invention is a step of coloring the protein to be measured. The method for coloring the protein is not particularly limited, and any method can be adopted as long as the protein to be measured can be detected in the detection step described later.
When carrying out this step, pretreatment of the target sample is carried out, such as extracting the target sample by dissolving, suspending, or dispersing it in a buffer solution or water in advance according to the coloration method to be adopted. You may.

When the coloration step in the present invention is carried out before the filtration step, the following methods can be exemplified as the coloration method. That is, a dye that binds to a protein to form a complex is added to the target sample. As a result, the protein contained in the target sample and the above-mentioned dye are combined to form a protein-dye complex, whereby the protein to be measured is colored.

Further, when the coloration step in the present invention is carried out after the filtration step, the following methods can be exemplified as the coloration method. That is, a dye that binds to the protein to form a complex is added to the protein captured by the filter in the filtration step. As a result, the protein to be measured is colored by forming a protein-dye complex on the filter.

Various coloration conditions such as the amount of the dye added, the time of contact with the protein after the dye is added, the temperature, and the pH when forming the protein-dye complex are not particularly limited and are selected. It can be set appropriately according to the coloring method. Further, as long as it does not interfere with the color reaction of the protein, the color reaction may be carried out in combination with any other additive.

Among them, a method of coloring the protein to be measured by using a color-developing reagent having a dye whose absorption wavelength is shifted by binding to the protein to be measured is preferable. According to this method, the dye bound to the protein and the dye not bound to the protein are colored in different colors, so that the two can be distinguished by the difference in the coloration. As a result, for example, even when a dye that is not bound to a protein is captured by the filter, the protein to be measured can be detected more accurately by detecting only the coloration of the dye that is not bound to the protein. can do.

Examples of such a coloring method include the Bradford method, the BCA (Bicinchoninic Acid) method, the Lowry method, and the like. Of these, the Bradford method is preferable from the viewpoint that the color reaction can be easily confirmed with the naked eye and the protein to be measured can be detected with higher sensitivity and accuracy.

In the Bradford method, when the acid dye Coomassie Brilliant Blue (CBB) is combined with the protein to be measured to form a protein-CBB complex, the absorption wavelength is 465 nm to 595 nm (brown to blue). It is a method of coloring proteins by utilizing the shift to. Since this color change occurs in proportion to the amount of protein, measuring the absorbance at 595 nm makes it possible to quantify or semi-quantify the protein concentration in the sample as described later. The Bradford method can be carried out using, for example, Takara Bradford Protein Assay Kit (manufactured by Takara Bio Inc.).

[Filtration process]
The filtration step in the present invention is a step of filtering the protein to be measured with a filter and concentrating the protein to be measured on the filter by capturing the protein to be measured by the filter. If the sample is solid, or if it is liquid but highly viscous, extract the target sample by dissolving, suspending, or dispersing it in a buffer solution or water in advance before performing the filtration treatment. Pretreatment of the target sample may be carried out, such as leaving.

In the present invention, the filter used for the filtration treatment refers to a structure having porosity and allowing a liquid to pass through. The filter is not particularly limited as long as it can capture and concentrate the protein to be measured. For example, those capable of physically capturing and concentrating the protein to be measured; those capable of chemically adsorbing the protein to be measured; those capable of electrically adsorbing the protein to be measured can be mentioned. Specific examples thereof include glass filters, syringe filters, membrane filters, filter papers and the like. In the filtration step of the present invention, it is particularly preferable to use a glass filter from the viewpoint of the effect of the present invention.

The retained particle size of the filter in the present invention can be appropriately adjusted according to the type of sample and the type of protein to be measured. The holding particle size of the filter is, for example, preferably 0.1 μm or more, more preferably 0.2 μm or more, and further preferably 0.3 μm or more. The holding particle size of the filter is, for example, preferably 1.6 μm or less, more preferably 1.4 μm or less, and further preferably 1.2 μm or less. When the retained particle size of the filter is within the above range, the colored protein can be adsorbed satisfactorily. The holding particle size of the filter means a particle size that can hold 90% or more when the 7-kind powder-dispersed water specified in JIS Z 8901 is naturally filtered.

The filtration diameter of the filter in the present invention can be appropriately adjusted according to the type of the liquid sample and the type of the protein to be measured. The filtration diameter of the filter is, for example, preferably 3 mm or more, more preferably 5 mm or more, and even more preferably 7 mm or more. The filtration diameter of the filter is, for example, preferably 40 mm or less, more preferably 35 mm or less, and even more preferably 33 mm or less. When the filtration diameter of the filter is within the above range, a protein aqueous solution having a concentration on the order of ppm or more can be measured with a color difference meter or the like. The filtration diameter of the filter means the diameter of the glass filter in the range where the sample is actually filtered.

The amount and time of passing the sample through the filter can be appropriately adjusted according to the concentration of protein in the sample and the flow rate of passing the sample. For example, the flow rate is preferably 10 ml to 100 ml. The liquid passing time is usually in the range of 3 seconds to 40 minutes.

The filtration method in the present invention is not particularly limited, and examples thereof include natural filtration, vacuum filtration, pressure filtration, and centrifugal filtration. Among them, from the viewpoint that low-concentration protein can be easily detected and measured, it is preferable to perform vacuum filtration by suction filtration. Further, a method in which a filter (syringe filter) is fixed to the tip of a syringe and a blanger (presser) is pushed in to perform filtration is also preferable from the viewpoint of easy filtration.

[Protein denaturation process, decolorization process]
The protein denaturing step in the present invention is a step of denaturing the protein to be measured captured by the filter by the above filtration step. Further, the decolorization step in the present invention is a step of adding a decolorizing agent to the filter. All of the above steps are carried out before the detection step described later. In the present invention, by providing both the protein denaturing step and the decoloring step, it is possible to detect the protein to be measured with higher accuracy as follows.

In the coloration step, there is a possibility that a complex in which a substance other than the protein contained in the sample and the dye are non-specifically bonded is slightly formed. In addition, the dye that has not been bound to the protein may be captured by the filter by itself and retained by the filter. In such a case, the filter may have a slight coloration that is not derived from the protein, and the detection accuracy of the protein present in the measurement sample is lowered.

For example, when factory water, particularly rinse water used for cleaning manufacturing equipment of a food factory, is used as a target sample, the components of tap water and groundwater used for the rinse differ depending on the region. Therefore, when non-specific coloration caused by these components occurs, the degree of coloration may differ depending on the measurement sample. Even if the difference is small, if a low concentration of protein is detected, the effect cannot be ignored and the accuracy of the measurement result may be reduced.
In addition, when foods such as foods and beverages are used as measurement samples, since the samples contain many components other than proteins, non-specific coloration due to these components may occur. In addition, many foods such as foods and beverages are originally colored, and when detecting the coloration of the protein to be measured, the color exhibited by the target sample itself may affect the measurement result.

On the other hand, since the present invention includes a protein denaturing step, by denaturing the protein to be measured, the three-dimensional structure of the protein can be changed and the protein can be firmly adsorbed on the filter. Therefore, even when a dye that is not bound to a protein is captured by the filter, it is possible to selectively and firmly adsorb the "protein-dye complex" in which the dye is bound to the protein to the filter. Be done.

As a result, for example, by providing a step of washing the filter after the protein denaturing step, it is possible to preferentially remove the dye that is not bound to the protein from the filter. Therefore, the protein to be measured can be detected with higher accuracy.
Further, since the present invention includes a decolorization step, the protein to be measured can be detected with higher accuracy. This is because the dye (protein-dye complex) strongly adsorbed on the filter by the above protein denaturation step is difficult to be decolorized in the decolorization step, whereas the dye captured by the filter (dye not bound to protein). It is presumed that this is because the color is preferentially decolorized because the adsorption force to the filter is weak. The present invention is not limited to the mechanism described above.

As described above, the present invention can accurately detect the coloration caused by the protein to be measured by including both the protein denaturation step and the decolorization step.

The method for denaturing the protein to be measured is not particularly limited as long as the effect of the present invention is not impaired. For example, a method of heating a protein, a method of adding a protein denaturing agent, and the like can be mentioned.
Examples of the method of heating the protein include a method of heating the filter in which the protein to be measured is captured at 60 ° C. or higher. The heating means is not particularly limited.
As a method for adding a protein denaturing agent, for example, a protein denaturing agent such as an acid or a chaotropic agent can be used.
The acid may be any organic acid or inorganic acid, and examples thereof include acetic acid, phosphoric acid, formic acid, citric acid, hydrochloric acid, and lactic acid.
Examples of the chaotropic agent include urea, formamide, guanidine and the like.

Preferably, it is a method of denaturing the protein to be measured captured by the filter by adding an acid to the filter. The acid is preferably at least one acid selected from the group consisting of acetic acid, phosphoric acid, formic acid, citric acid, hydrochloric acid, and lactic acid. Further, as shown in Examples described later, acetic acid is more preferable from the viewpoint that the S / N ratio between the target sample and the negative control sample can be particularly improved. The pH of the acid is preferably in the range of 2 to 5.

In the decolorization step of the present invention, the method of adding the decolorizing agent to the filter is not particularly limited. As the decolorizing agent to be used, a conventionally known method can be appropriately adopted depending on the coloration method adopted in the coloration step.
Examples of the decolorizing agent include a decolorizing agent containing an organic solvent such as alcohol. From the viewpoint of decolorization efficiency, the organic solvent preferably contains at least one alcohol selected from the group consisting of ethanol, methanol and isopropanol. From the viewpoint of safety and decolorization efficiency, it is preferable to contain ethanol.

The conditions for adding the decolorizing agent to the filter can be appropriately set depending on the type of the target sample, the coloring method, the type of the decolorizing agent, and the like. For example, the pH is 2 to 5 and the temperature is 4 to 80 ° C.

The protein denaturation step and the decolorization step may be carried out in one step by combining both steps, or may be carried out in separate steps. When both steps are combined in one step, for example, a reagent containing both the above-mentioned protein denaturing agent and decolorizing agent is added to a filter in which the protein to be measured is captured. Specific examples of such a reagent include a reagent containing an acid and an alcohol, and particularly preferably a reagent containing acetic acid and ethanol. As a result, protein denaturation and decolorization reaction can be performed at the same time, which is preferable from the viewpoint of ease of operation.

[Detection process]
The detection step in the present invention is a step of detecting the protein to be measured captured by the filter after color development. In the detection step, each of the above steps captures and concentrates the protein to be measured that is colored on the filter, so that the protein to be measured can be detected.

The detection of the protein to be measured can be carried out by an optimum method according to the coloration method. For example, L ^* a ^* b ^* color system, L ^* c ^* h ^* color system, L ^* u ^* v ^* color system, Hunter Lab color system, XYZ (Yxy) color system, Munsell color system, etc. Therefore, it is possible to measure the color value in which the degree of color development is quantified. Alternatively, the color value may not be obtained, and the determination may be made with the naked eye using a color determination paper.

A color difference meter can also be used to measure the color value. Alternatively, for example, the color value may be obtained by comparing the coloration determination paper prepared for each of various color values with the actual coloration state of the protein.

In the present invention, it is particularly preferable that the Bradford method is adopted as the color reaction and the color value is the b value defined in the L ^* a ^* b ^* color system conforming to the CIE standard. The L ^* a ^* b ^* color system is also used in JIS Z8729.

The b value can be measured by a color difference meter. Further, for example, the b value of the protein to be measured may be obtained by comparing the coloration determination paper prepared for each of various b values with the actual coloration state of the protein. FIG. 1 is a diagram showing an example of a color determination paper. The color-developing papers pseudo-show filter papers colored from colorless to dark blue as in (a) to (d), and are assigned b values (not shown). The number of coloration determination papers is not limited, and the larger the number, the more b values are assigned. Therefore, the b value can be obtained more accurately, which is preferable.

The protein detection method of the present invention can be applied not only to a target sample but also to a negative control sample. According to the method of the present invention, as a result of the above-mentioned non-specific coloration being reduced not only for the target sample but also for the negative control sample, the difference between the coloration of the target sample and the coloration of the negative control sample You will be able to judge more clearly with the naked eye. Therefore, by comparing the coloration of the target sample with the coloration of the negative control sample, it is possible to visually determine whether or not the target sample contains a protein.

Further, since the S / N ratio between the target sample and the negative control sample can be improved, more accurate measurement becomes possible by comparing the coloration of both. The S / N ratio is determined by, for example, measuring the b value of the target sample and the negative control sample as shown in Examples described later, and dividing the b value of the obtained target sample by the b value of the negative control sample. Can be sought. The S / N ratio is preferably 6.3 or more, more preferably 7 or more, still more preferably 10 or more.

The negative control sample can be appropriately selected and used according to the type of the target sample. For example, when the measurement sample is the above-mentioned rinse water, tap water or groundwater used for the rinse water itself is used as a negative control target, and the coloring step, the filtration step, the protein modification step, the decolorization step, and the same as the target sample are performed. It is preferable to carry out the detection step. As a result, it is possible to compare the above-mentioned measurement sample with reduced nonspecific coloration and the negative control sample, and more accurate measurement becomes possible.
Further, for example, when the measurement sample is a food such as food or beverage, it is preferable to use a sample of the same type that has been confirmed not to contain protein as a negative control sample.

In addition, the present invention may include a cleaning step of cleaning the filter prior to the detection step. Any method can be carried out in the cleaning step. This cleans, for example, extra components other than the protein trapped in the filter.

As one embodiment of the protein detection method according to the present invention, the color development step, the filtration step, the protein denaturation step, the decolorization step, and the detection step are carried out in this order. In this aspect, the filtration step is performed after the coloration step. Therefore, the sample is subjected to a color treatment, and the protein colored by the subsequent filtration step is captured by the filter. Specifically, a dye that binds to a protein to form a complex is added to the sample, and the protein contained in the sample is bound to the above dye to form a protein-dye complex, whereby the measurement target is measured. Colors the protein. The sample is then filtered through a filter to capture the protein-dye complex.
As described above, by carrying out the filtration step after the coloration step, there is an advantage that a lower concentration of protein can be measured.

Further, as another embodiment of the detection method of the present invention, a filtration step, a coloration step, a protein denaturation step, a decolorization step, and a detection step are carried out in this order. In this aspect, the coloration step is carried out after the filtration step. Therefore, the sample is filtered with a filter to capture the protein to be measured by the filter, and the protein captured by the filter is colored by the subsequent coloration step.
As described above, by carrying out the coloration step after the filtration step, there is an advantage that the range of selection of the protein coloring method is widened.

The sample applied to the present invention is not particularly limited as long as it is a sample that may contain a protein, and examples thereof include foods, beverages, environmental samples, and biological samples. The sample in the present invention may be in a liquid state or in a solid state. In addition, pretreatment may be performed such as dissolving, suspending, or dispersing the sample in a buffer solution or the like to extract the sample.
Examples of foods include foods such as animals and plants, processed foods, seasonings and the like.
Examples of beverages include juices, alcoholic beverages, and carbonated beverages.
Examples of environmental samples include soil, rocks, seawater, freshwater, domestic wastewater, domestic water, factory wastewater, and factory water.
Examples of biological samples include sputum, nasal aspirate, nasal lavage fluid, nasal swab, nasal discharge, pharyngeal swab, rinsing fluid, saliva, whole blood, serum, plasma, sweat, urine, cells, feces and the like. ..

Since the present invention can detect a trace amount of protein in a sample, it is possible to use a sample that is not originally supposed to contain a protein or a sample that contains a very small amount of protein. is there. From this point of view, for example, foods such as foods and beverages whose nutrition label has 0 protein, and rinse water used for cleaning the manufacturing equipment of a food factory among factory wastewater are suitable.

When the sample applied to the present invention contains a substance that interferes with the detection of protein, necessary treatment may be appropriately performed according to the type of the interfering substance to remove the interfering substance. For example, in the case of a sample having a high chlorine concentration such as factory water, it is preferable to add an appropriate neutralizing agent in advance. Examples of the neutralizing agent include sodium ascorbate, sodium thiosulfate and the like.

The type of protein to be measured that can be detected in the present invention is not particularly limited. For example, eggs, milk, wheat, buckwheat, peanuts, shrimp, crabs, abalone, squid, how much, oranges, cashew nuts, kiwifruit, beef, walnuts, sesame, salmon, mackerel, soybeans, chicken, bananas, pork, matsutake, etc. Examples include chicken, egg, and protein contained in gelatin. In addition, it is possible to detect various allergens designated in Japan or abroad.

Compared with conventional methods such as ELISA method, turbidity method, and fluorescence staining method, the detection method of the present invention requires less labor and cost, and can detect proteins in a target sample very simply and quickly. Specifically, according to the detection method of the present invention, the time required for detection can be set to 1 hour or less. The time required for detection refers to the time from when the test instrument is installed until the detection result is obtained. Further, by using the kit described later, it is possible to further shorten the time required for detection.

Although the method for detecting a protein, which is one embodiment of the present invention, has been described above, the method is not limited to the above-described embodiment, and the embodiment other than the above-described embodiment shall be used as long as the effect of the present invention is exhibited. Does not prevent.

<Protein concentration measurement method>
In the present invention, in addition to the coloration step, the filtration step, the protein modification step, the decolorization step, and the detection step in the above detection method, the color for measuring the color value of the coloration state of the protein to be measured detected in the detection step. By providing a value measuring step, a method for measuring the concentration of a protein to be measured in a sample is provided.

There is a certain correlation between the concentration of the protein to be measured in the sample and the color color value of the protein to be measured detected in the detection step described above. Specifically, there is a linear (linear function) relationship between the concentration of the protein to be measured and the color value of the coloration of the protein to be measured.

Therefore, using a plurality of reference samples in which the concentration of the protein to be measured and its color value are known, a calibration curve showing the correspondence between the concentration of the protein to be measured in each reference sample and the above color value. By preparing in advance, it is possible to measure the concentration of the protein to be measured from the color value obtained by the measurement.

In particular, in the present invention, by providing both the protein denaturation step and the decolorization step, the protein to be measured can be detected with high accuracy. Therefore, the concentration of the protein to be measured and the measurement target to be detected can be detected. The correlation between the denaturing color value of the protein and the color value is closer to a more linear (linear function). Therefore, the concentration of the protein to be measured can be measured more accurately.

The measurement of the color value can be performed by the method described above in the method for detecting a protein. When the above color value is measured to measure the protein concentration, the protein concentration may be quantitatively measured using a color difference meter, or the protein concentration may be measured semi-quantitatively using a color determination paper. May be measured.

According to the concentration measuring method of the present invention, although it depends on the type of protein to be measured, even if the concentration is about 0.25 ppb, the measured color value can be applied to the calibration curve in the sample. The protein concentration to be measured can be measured. Preferably, for example, 0.25 ppb or more, 0.5 ppb or more, 1 ppb or more, 2 ppb or more, 5 ppb or more, 10 ppb or more, and the like. The upper limit is not particularly limited, but is, for example, 2000 ppm or less.

Although the method for measuring the concentration of protein, which is one embodiment of the present invention, has been described above, the method is not limited to the above-described embodiment, and the embodiment other than the above-described embodiment is used as long as the effect of the present invention is exhibited. It doesn't prevent you from doing that.

<Protein detection kit>
The protein detection kit of the present invention contains at least a filter that captures the protein to be measured, a coloring reagent that colors the protein to be measured, a protein denaturing agent that denatures the protein to be measured, and a decolorizing agent. It includes. The detection kit of the present invention can be used when carrying out the above-mentioned method for detecting a protein of the present invention and the method for measuring a concentration thereof.

As the filter, color reagent, and decolorizing agent in the protein detection kit, those detailed above can be used. In addition, the kit may include a color determination paper. According to the kit of the present invention, it is possible to detect a protein in a target sample by a simple operation even in a situation where laboratory equipment, materials and the like are not available. In addition, it is possible to reduce the coloration that is not derived from the protein contained in the target sample, and it is possible to detect the protein in the target sample with high accuracy and high sensitivity.

As shown in FIG. 2, the protein detection kit 10 according to an embodiment of the present invention includes a filter 12 for filtering, capturing, and concentrating a protein to be measured, a sample introduction container 11, and a presentation used in a coloration step. A color reagent 13 and a decolorizing agent (not shown) are provided. In addition, a color determination paper (not shown) for measuring the concentration of the protein to be measured may be provided. Hereinafter, how to use the detection kit 10 will be described.

First, the sample is introduced into the sample introduction container 11. Depending on the type of sample, pretreatment may be carried out as appropriate, such as dissolving, suspending, or dispersing in a buffer solution or water to extract the sample. As shown in FIG. 2, the sample introduction container 11 stores the coloring reagent 13 in advance so that the sample is introduced into the sample introduction container 11 and the sample is in contact with the coloring reagent. Alternatively, the sample may be filled in the sample introduction container 11 and then the color-developing reagent 13 may be added to the sample in the sample introduction container 11. Alternatively, at this point, the color-developing reagent may not be used and may be used in the color-developing step after filtration described later.

Subsequently, the filter 12 is fixed near the opening at the top of the sample introduction container 11. After that, the sample is passed through the filter 12 by an arbitrary method and filtered to capture and concentrate the protein to be measured by the filter 12. If the color reagent 13 has not been applied to the sample at this point, the color reagent 13 is applied to the protein to be measured that has been captured and concentrated on the filter 12.

After the above filtration treatment, a protein denaturing agent is added to the filter 12, and the protein trapped in the filter 12 is strongly adsorbed by the filter 12. After that, a decolorizing agent is added to the filter 12. It is considered that this makes it possible to preferentially decolorize the dye that is not bound to the protein from the filter 12. By using a reagent containing a protein denaturing agent and a decolorizing agent, protein denaturation and decolorization may be performed at the same time.

Finally, the coloration of the protein to be measured captured and concentrated by the filter 12 is confirmed, and the protein is detected. The coloration can be confirmed by using a color difference meter, a coloration determination paper, or the like as described above. Further, as described above, the concentration of the protein to be measured may be measured using a calibration curve.

The detection kit 20, which is another embodiment of the present invention, is an embodiment configured so that a protein can be detected in a plurality of samples. As shown in FIG. 3, the detection kit 20 includes a sample introduction container 30, a filter 31, a color-developing reagent (not shown), a decolorizing agent (not shown), a filtrate storage container 40, and a pressure reducing port. 41 and a filtrate discharge port 42 are provided. Although only one sample introduction container 30 is shown in FIG. 3, a plurality of sample introduction containers 30 may be provided depending on the number of target samples.
Hereinafter, how to use the detection kit 20 will be described. The following describes the case where there is one sample, but when there are a plurality of samples, the same operation may be performed according to the quantity.

First, the sample is introduced into the sample introduction container 30 with the sample introduction container 30 removed from the filtrate storage container 40. This can be done by the method described in the method of using the detection kit 10 above.

Subsequently, a filter fixture (not shown) is provided near the opening at the top of the sample introduction container 30 as needed, and the filter is installed there. Use a filter fixture that does not interfere with filtration by the filter. After that, as shown in FIG. 3, the sample introduction container 30 is connected to the filtrate storage container 40 to perform the filtration process. The filtration process can be carried out in the same manner as in the detection kit 10, but if necessary, the pressure reducing port 41 of the filtrate storage container 40 and the aspirator (not shown) are connected to reduce the pressure inside the filtrate storage container 40. By doing so, the protein to be measured contained in the sample may be captured by the filter and concentrated.

The filtrate is discharged into the filtrate storage container 40 from the filtrate outlet 32. The filtrate discharge port 42 is normally closed, and when the amount of the filtrate stored in the filtrate storage container 40 increases, the filtrate discharge port 42 is appropriately opened and the filtrate is discharged from the filtrate storage container 40. Discharge to the outside.

The application of the protein denaturing agent and the decolorizing agent after the filtration treatment, or the detection and concentration measurement of the protein to be measured can be carried out by the same method described in the method of using the detection kit 10.

Further, as another embodiment of the detection kit of the present invention, in the detection kit 20, the detection kit 50 in which the syringe 60 shown in FIG. 4 is used instead of the sample introduction container 30 and the like can be mentioned. The syringe 60 includes a blanger (presser) 61, a syringe filter 62, a filtrate outlet 63, an outer cylinder 64, and a gasket 65. The method of use is almost the same as that of the detection kit 20 except that the target sample is filled in the outer cylinder and the syringe filter 62 is used for filtering by pushing the blanger (pressor) 61. it can.

The protein detection kit according to one embodiment of the present invention has been described above, but the present invention is not limited to the above-described embodiment, and the embodiment other than the above-described embodiment is used as long as the effect of the present invention is exhibited. It doesn't prevent you from doing that.

Hereinafter, the present invention will be described in more detail based on Examples. The present invention is not limited to the following examples.

(Test Example 1)
In this test, casein sodium, which is a milk allergen, diluted with various waters (purified water, tap water (collected in Hachioji), production water (collected in Kyoto), groundwater (collected in Kagawa)) was used as the target sample. The protein was detected by the method of the present invention. Further, the same test was carried out using only the above-mentioned various types of water as a negative control sample.

[Example 1]
Example 1-1: Preparation of target sample As a target sample, sodium casein was diluted with purified water to prepare a 1 ppm aqueous solution of sodium casein. In addition, 30 mg of sodium ascorbate was added to neutralize free chlorine in the aqueous solution.
Example 1-2: Preparation of negative control sample A negative control sample was prepared in the same manner as the target sample except that purified water was used instead of the 1 ppm aqueous casein sodium solution.
Measurement:
To each of the above prepared samples, 1 mL of Coomassie Brilliant Blue G-250 staining solution (manufactured by TAKARA) of Bradford's reagent was added, stirred, and then allowed to stand at room temperature (25 ° C.) for 5 minutes (coloring step). 20 mL of each sample was passed through a glass filter "GF-75" (manufactured by ADVANTEC, holding particle diameter: 0.3 μm, filtration diameter: 30 mm) over 3 seconds (filtration step). Then, 100 mL of 5% acetic acid (pH 2.4) 8% ethanol as a protein denaturing agent and a decolorizing agent was passed through a glass filter over 40 seconds (protein denaturing and decolorizing step). Then, 100 mL of ion-exchanged water was passed through a glass filter over 40 seconds.
The blue coloration on the filter was measured with a color difference meter (CR-5, manufactured by Konica Minolta), and the obtained b value was determined. The measured values are shown in Table 1.

[Example 2]
Example 2-1: Preparation of target sample A target sample was prepared in the same manner as in Example 1-1, except that sodium caseinate was diluted with tap water (collected in Hachioji).
Example 2-2: Preparation of negative control sample A negative control sample was prepared in the same manner as in Example 1-2, except that tap water (collected in Hachioji) was used instead of purified water.
Measurement:
The measurement was carried out in the same manner as in Example 1 to determine the b value. The measured values are shown in Table 1.

[Example 3]
Example 3-1: Preparation of target sample A target sample was prepared in the same manner as in Example 1-1, except that sodium caseinate was diluted with production water (collected in Kyoto).
Example 3-2: Preparation of negative control sample A negative control sample was prepared in the same manner as in Example 1-2, except that production water (collected in Kyoto) was used instead of purified water.
Measurement:
The measurement was carried out in the same manner as in Example 1 to determine the b value. The measured values are shown in Table 1.

[Example 4]
Example 4-1: Preparation of target sample A target sample was prepared in the same manner as in Example 1-1, except that sodium caseinate was diluted with groundwater (collected in Kagawa).
Example 4-2: Preparation of negative control sample A negative control sample was prepared in the same manner as in Example 1-2, except that groundwater (collected in Kagawa) was used instead of purified water.
Measurement:
The measurement was carried out in the same manner as in Example 1 to determine the b value. The measured values are shown in Table 1.

[Comparative Example 1]
A target sample (Comparative Example 1-1) was prepared in the same manner as in Example 1-1, except that the protein denaturation and bleaching steps were not performed before the measurement, and also in the same manner as in Example 1-2. A negative target sample (Comparative Example 1-2) was prepared and the b value was measured. The measured values are shown in Table 1.

[Comparative Example 2]
A target sample (Comparative Example 2-1) was prepared in the same manner as in Example 2-1 except that the protein denaturation and bleaching steps were not performed before the measurement, and also in the same manner as in Example 2-2. A negative target sample (Comparative Example 2-2) was prepared and the b value was measured. The measured values are shown in Table 1.

[Comparative Example 3]
A target sample (Comparative Example 3-1) was prepared in the same manner as in Example 3-1 except that the protein denaturation and bleaching steps were not performed before the measurement, and also in the same manner as in Example 3-2. A negative target sample (Comparative Example 3-2) was prepared and the b value was measured. The measured values are shown in Table 1.

[Comparative Example 4]
A target sample (Comparative Example 4-1) was prepared in the same manner as in Example 4-1 except that the protein denaturation and bleaching steps were not performed before the measurement, and also in the same manner as in Example 4-2. A negative target sample (Comparative Example 4-2) was prepared and the b value was measured. The measured values are shown in Table 1.

As shown in Table 1, the b value of each of the target samples was smaller than that of the negative control sample (the absolute negative value of the b value was large), and the colored blue color became darker. It was shown that the protein (sodium casein) in the target sample can be detected.
In addition, in both the target sample and the negative control sample, the b value was increased by carrying out the protein denaturation step and the decolorization step. In addition, since the b value of the negative control sample approached 0 and almost no coloring was observed, it became possible to visually determine the detection of the protein in the target sample.
From the above results, it was shown that the method of the present invention can accurately detect the protein to be measured while reducing the influence of the background other than the protein contained in the target sample.

(Test Example 2)
In this test, the same test as in Test Example 1 was carried out by variously changing the protein denaturing agent and the decolorizing agent of Test Example 1.

[Example 3]
A target sample was prepared in the same manner as in Example 2-1 except that the protein denaturing agent and the decolorizing agent were changed to 5% phosphoric acid (pH 2.4) and 8% ethanol, and the same as in Example 2-2. A negative control sample was prepared and the b value was measured. In addition, the ratio (S / N ratio) of the b value in the target sample and the b value in the negative control sample was determined. The results are shown in Table 2.

[Example 4]
A target sample was prepared in the same manner as in Example 2-1 except that the protein denaturing agent and the decolorizing agent were changed to 5% formic acid (pH 2.4) and 8% ethanol, and negative as in Example 2-2. A control sample was prepared and the b value was measured. In addition, the S / N ratio of the target sample and the negative control sample was determined. The results are shown in Table 2.

[Example 5]
A target sample was prepared in the same manner as in Example 2-1 except that the protein denaturing agent and the decolorizing agent were changed to 5% citric acid (pH 2.4) and 8% ethanol, and the same as in Example 2-2. A negative control sample was prepared and the b value was measured. In addition, the S / N ratio of the target sample and the negative control sample was determined. The results are shown in Table 2.

[Example 6]
A target sample was prepared in the same manner as in Example 2-1 except that the protein denaturing agent and the decolorizing agent were changed to 5% hydrochloric acid (pH 2.4) and 8% ethanol, and negative as in Example 2-2. A control sample was prepared and the b value was measured. In addition, the S / N ratio of the target sample and the negative control sample was determined. The results are shown in Table 2.

[Example 7]
A target sample was prepared in the same manner as in Example 2-1 except that the protein denaturing agent and the decolorizing agent were changed to 5% lactic acid (pH 2.4) and 8% ethanol, and negative as in Example 2-2. A control sample was prepared and the b value was measured. In addition, the S / N ratio of the target sample and the negative control sample was determined. The results are shown in Table 2.

[Example 8]
A target sample was prepared in the same manner as in Example 2-1 except that the protein denaturing agent and the decolorizing agent were changed to 5% acetic acid (pH 2.3) and 7% methanol, and negative as in Example 2-2. A control sample was prepared and the b value was measured. In addition, the S / N ratio of the target sample and the negative control sample was determined. The results are shown in Table 2.

As shown in Table 2, in each sample, the b value of the target sample was smaller than that of the negative control sample (the absolute negative value of the b value was large), and the colored blue color became darker. It was shown that the protein (sodium casein) in the target sample can be detected.
In addition, the b value was increased by carrying out the protein denaturation and decolorization steps in both the target sample and the negative control sample. Among them, Example 2 in which acetic acid and ethanol were combined as a protein denaturing agent and a decolorizing agent showed the largest increase in b value.
In addition, the S / N ratio between the target sample and the negative control sample was improved by carrying out the protein denaturation and decolorization steps.
From the above results, it was shown that the method of the present invention can accurately detect the protein to be measured while reducing the influence of the background other than the protein contained in the target sample.

(Test Example 3)
In Test Example 3, various drinking waters were used as target samples, and the method of the present invention was carried out in the same manner as in Test Example 1.
[Example 9]
Example 1- of Test Example 1 except that a sample in which sodium caseinate was added to a predetermined concentration (0 ppm, 0.5 ppm, 1 ppm) was used as a target sample in mineral water (transparent color). The same process as in 1 was performed to obtain the b value. In addition, the L value and the a value were also obtained. The results are shown in Table 3 and FIG.

[Example 10]
The same treatment as in Example 9 except that a sample in which sodium caseinate was added to a soft drink (transparent color) to a predetermined concentration (0 ppm, 0.5 ppm, 1 ppm) was used as the target sample. Was performed, and the b value was obtained. In addition, the L value and the a value were also obtained. The results are shown in Table 3 and FIG.

[Example 11]
As a target sample, the same as in Example 9 except that a sample in which sodium caseinate was added to a fruit mixed juice (clear brown) to a predetermined concentration (0 ppm, 0.5 ppm, 1 ppm) was used. Processing was performed and the b value was obtained. In addition, the L value and the a value were also obtained. The results are shown in Table 3 and FIG.

[Example 12]
The same treatment as in Example 9 except that a sample in which sodium caseinate was added to a predetermined concentration (0 ppm, 0.5 ppm, 1 ppm) was used as a target sample in green tea (translucent green). Was performed, and the b value was obtained. In addition, the L value and the a value were also obtained. The results are shown in Table 3 and FIG.

[Example 13]
As the target sample, the same treatment as in Example 9 was carried out except that a sample in which sodium caseinate was added to apple juice (clear brown) to a predetermined concentration (0 ppm, 0.5 ppm, 1 ppm) was used. The b value was obtained. In addition, the L value and the a value were also obtained. The results are shown in Table 3 and FIG.

[Comparative Example 3]
The same treatment as in Example 9 was carried out except that the protein denaturation and decolorization steps were not carried out, and the b value was determined. In addition, the L value and the a value were also obtained. The results are shown in Table 3 and FIG.

[Comparative Example 4]
The same treatment as in Example 10 was carried out except that the protein denaturation and decolorization steps were not carried out, and the b value was determined. In addition, the L value and the a value were also obtained. The results are shown in Table 3 and FIG.

[Comparative Example 5]
The same treatment as in Example 11 was carried out except that the protein denaturation and decolorization steps were not carried out, and the b value was determined. In addition, the L value and the a value were also obtained. The results are shown in Table 3 and FIG.

[Comparative Example 6]
The same treatment as in Example 12 was carried out except that the protein denaturation and decolorization steps were not carried out, and the b value was determined. In addition, the L value and the a value were also obtained. The results are shown in Table 3 and FIG.

[Comparative Example 7]
The same treatment as in Example 13 was carried out except that the protein denaturation and decolorization steps were not carried out, and the b value was determined. In addition, the L value and the a value were also obtained. The results are shown in Table 3 and FIG.

As shown in Table 3, the b value is smaller in the order of 1 ppm, 0.5 ppm, and 0 ppm (the absolute negative value of the b value is larger) in each sample, and it is possible to detect the protein contained in the beverage. Was shown to be.
Moreover, in all the samples, the b value was increased by carrying out the protein denaturation and decolorization steps.
Further, as shown in FIG. 5, in the examples in which the protein denaturation and decolorization steps were performed, the value of R ² was closer to 1 as compared with the comparative example in which those steps were not performed, and the concentration and b value of the target sample were obtained. The result is that the linear correlation between and is high.
From the above results, it was shown that the present invention can measure the concentration to be measured more accurately by including the protein denaturation and decolorization steps.

(Test Example 4)
In this test, the method of the present invention was carried out in the same manner as in Test Example 1 using an aqueous casein sodium solution as a target sample.
[Example 14]
As a target sample, test examples except that sodium casein was diluted with purified water and 0 ppm, 0.1 ppm, 0.25 ppm, 0.5 ppm, 1 ppm, 1.5 ppm, and 2 ppm aqueous casein sodium solution were used. The same process as in Example 1-1 of 1 was carried out to determine the b value. The same test was performed a total of 3 times, and the average value of b values (n = 3) is shown in Table 4 and FIG.

[Comparative Example 8]
The same treatment as in Example 14 was carried out except that the protein denaturation and decolorization steps were not carried out, and the b value was determined. The same test was performed a total of 3 times, and the average value of b values (n = 3) is shown in Table 4 and FIG.

As shown in Table 4, in each sample, the b value was smaller (the absolute negative value of the b value was larger) in descending order of concentration, indicating that protein could be detected.
Further, as shown in FIG. 10, in the examples in which the protein denaturation and decolorization steps were performed, the value of R ² was closer to 1 as compared with the comparative example in which those steps were not performed, and the concentration and b value of the target sample were obtained. The result is that the linear correlation between and is high.
From the above results, it was shown that the present invention can measure the concentration to be measured more accurately by including the protein denaturation and decolorization steps.

According to the present invention, it is possible to detect a protein in a target sample with high accuracy and high sensitivity while being easy to operate. In particular, in the food industry, in order to grasp the amount of allergen remaining in the manufacturing equipment at a food factory, the amount of protein contained in the rinse water after cleaning the equipment can be grasped, or in foods such as foods and beverages. The detection method of the present invention is very useful for grasping whether or not a protein is contained, and its industrial utility is extremely high.

10, 20, 50 Detection kit 11, 30 Sample introduction container 12, 31 Filter 13 Coloring reagent 32, 63 Filtrate outlet 40, 70 Filtrate storage container 41 Decompression port 42, 71 Filtrate discharge port 60 Syringe 61 Blancer ( Oshiko)
62 Syringe filter 64 Outer cylinder 65 Gasket

Claims (21)

The coloring process that colors the protein to be measured and
A filtration process that filters the protein to be measured with a filter,
A detection step for detecting the coloration of the protein to be measured captured by the filter by the filtration step, and a detection step for detecting the coloration of the protein to be measured.
It is a protein detection method that includes
Before the detection step,
A protein denaturation step that denatures the protein to be measured captured by the filter,
The decolorization process of adding a decolorizing agent to the filter and
A method for detecting a protein, which is characterized by further comprising. The detection method according to claim 1, wherein the coloration step, the filtration step, the protein denaturation step, the decolorization step, and the detection step are carried out in this order. The detection method according to claim 1, wherein the filtration step, the color development step, the protein denaturation step, the decolorization step, and the detection step are carried out in this order. The detection method according to any one of claims 1 to 3, wherein in the coloring step, a coloring reagent whose absorption wavelength shifts due to binding to the protein to be measured is used to color the protein to be measured. The detection method according to any one of claims 1 to 3, wherein in the coloration step, the protein to be measured is colored by the Bradford method. The detection method according to any one of claims 1 to 5, wherein in the filtration step, the protein to be measured is filtered with a glass filter. The detection method according to any one of claims 1 to 6, wherein in the protein denaturing step, the protein to be measured captured by the filter is denatured by adding an acid to the filter. The detection method according to claim 7, wherein the acid is at least one acid selected from the group consisting of acetic acid, phosphoric acid, formic acid, citric acid, hydrochloric acid, and lactic acid. The detection method according to claim 7, wherein the acid is acetic acid. The detection method according to any one of claims 1 to 9, wherein in the decolorization step, the decolorizing agent added to the filter contains an organic solvent. The detection method according to claim 10, wherein the organic solvent contains at least one alcohol selected from the group consisting of ethanol, methanol, and isopropanol. The detection method according to claim 10, wherein the organic solvent contains ethanol. Any one of claims 1 to 12, wherein in the protein denaturing step, acetic acid is added to the filter to denature the protein to be measured captured in the filter, and in the decolorization step, ethanol is added to the filter. The detection method described in the section. The detection according to any one of claims 1 to 13, wherein the protein to be measured is a protein contained in rinse water used for cleaning a manufacturing facility of a food factory, or a protein contained in food or beverage. Method. The proteins contained in the rinse water used to clean the manufacturing equipment of the food factory are milk, eggs, wheat, buckwheat, peanuts, shrimp, crabs, abalone, squid, salmon roe, oranges, cashew nuts, kiwifruit, beef, walnuts, etc. The detection method according to claim 14, wherein the protein is contained in sesame, salmon roe, buckwheat, soybean, chicken, banana, pork, matsutake, thigh, yamaimo, apple, or gelatin. Proteins in food or beverages include milk, eggs, wheat, buckwheat, peanuts, shrimp, crabs, abalone, squid, salmon roe, oranges, cashew nuts, kiwifruit, beef, walnuts, sesame, salmon, mackerel, soybeans, chicken, The detection method according to claim 14, wherein the protein is contained in banana, pork, matsutake, thigh, yam, apple, or gelatin. The detection method according to any one of claims 1 to 16, wherein the time required for detection is one hour or less. The coloring process to color the protein to be measured and
A filtration process that filters the protein to be measured with a filter,
A protein denaturing step of denaturing the protein to be measured captured by the filter by the filtration step, and a protein denaturing step.
The decolorization process of adding a decolorizing agent to the filter and
A detection process that detects the coloration of the protein to be measured captured by the filter,
With respect to the coloration of the protein to be measured detected in the detection step, the color value measuring step for measuring the color value and the color value measuring step
It is a method for measuring the concentration of protein, which comprises
Using a plurality of reference samples whose color values and the concentration of the protein to be measured are known, a calibration curve showing the correspondence between the concentration of the protein to be measured in each reference sample and the color value is created. A method for measuring the concentration of a protein, which comprises measuring the concentration of a protein to be measured from the color value measured in the color value measuring step. The coloration process of coloring the protein to be measured by the Bradford method,
A filtration process that filters the protein to be measured with a filter,
A protein denaturing step of denaturing the protein to be measured captured by the filter by the filtration step, and a protein denaturing step.
The decolorization process of adding a decolorizing agent to the filter and
A detection process that detects the coloration of the protein to be measured captured by the filter,
Regarding the color development of the protein to be measured detected in the detection step, a color value measuring step for measuring the b value defined in the L ^* a ^* b ^* color system conforming to the CIE standard, and a color value measuring step.
It is a method for measuring the concentration of protein, which comprises
Using a plurality of reference samples for which the concentration of the protein to be measured and the b value defined in the L ^* a ^* b ^* color system conforming to the CIE standard are known, the measurement target in each reference sample is used. By creating a calibration curve showing the correspondence between the protein concentration and the b value, the concentration of the protein to be measured is measured from the b value measured in the color value measuring step. Concentration measurement method. A protein detection kit containing a filter that captures the protein to be measured, a coloring reagent that colors the protein to be measured, a protein denaturing agent that denatures the protein to be measured, and a decolorizing agent. In addition, it is equipped with a coloration judgment paper.
A method for measuring the concentration of a protein to be measured by comparing the coloration determination paper with the coloration state of the protein to be measured captured by the filter.
The protein detection kit according to claim 20.

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