JP2009047590A - Concentration measuring immunochromatographic device and concentration measuring method using it - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a concentration measuring immunochromatographic device capable of reducing the irregularity of a measuring value due to a change in external environment, and a concentration measuring method using it. <P>SOLUTION: The concentration measuring immunochromatographic device of this invention has a membrane part, the base part for holding the membrane part and the measuring element installed on the base part or membrane part to measure the physical quantity of a factor which exerts an effect on the reaction brought about until a coloration substance is fixed after a sample is added. A change in external environment at the time of reaction is measured as a physical quantity by using the concentration measuring immunochromatographic device; the correction of a measuring value can be performed at the time of measurement using the physical quantity and measuring precision can be enhanced. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to an immunochromatography device for concentration measurement and a concentration measurement method thereof.

  As a qualitative or quantitative method for trace substances contained in biological samples such as blood and urine, an immunological measurement method is widely used because of its good sensitivity. Among these methods, the so-called immunochromatography method using chromatography is easy to operate and requires a short time for the assay, so it is widely used in many situations, such as clinical tests in hospitals and laboratory tests in laboratories. ing. However, due to large variations in measurement, the main purpose of use is to determine the presence or absence of the substance to be measured.

  Various proposals have been made to calculate the concentration of a substance to be measured using an immunochromatography method.

  Patent Document 1 below proposes a substance to be measured that is immobilized at a plurality of positions at a concentration that gradually increases from upstream to downstream when the substance to be measured flows through an immunochromatography device. At this time, the density is visually determined from the plurality of color patterns (number of color bands, etc.).

  In Patent Document 2 below, in order to identify a substance to be measured present at a low concentration, the light reflected from the illumination light from the illumination means LED 44 is detected by a CMOS imaging sensor, and image analysis is performed. There has been proposed an apparatus for dividing a region and a control region and outputting a result.

In Patent Document 3 below, the light beam irradiated to the chromatographic test piece is kept in a state of being irradiated between the labeled reagent holding part and the reagent immobilizing part, and the change in absorbance due to elution of the labeled reagent is observed. A chromatographic measurement device is disclosed that detects and automatically starts measurement after a certain time from the detection.
Japanese Patent No. 3519451 Japanese translation of PCT publication No. 2002-520617 Japanese Patent Laid-Open No. 2003-4743

  However, in conventional immunochromatographic devices for concentration measurement, the coloration level varies depending on the difference in influencing factors of the sample reaction, such as the temperature during reaction and the variation in reaction time. There were cases where the tolerance was exceeded. In the immunochromatography device for concentration measurement, the reaction itself is not performed in a temperature-controlled room with a measuring instrument because of its easy operation, and may be performed in various external environments. The present invention has been made in view of the above circumstances, and provides a concentration measurement immunochromatography device capable of reducing variations in measurement values due to changes in the external environment and a concentration measurement method using the immunochromatography device. Let it be an issue.

  As a result of intensive studies by the present inventors, a measurement element for measuring a physical quantity that is an influence factor of a reaction is attached to an immunochromatography device for concentration measurement, and the measurement value is easily corrected during measurement by detecting the physical quantity at the time of reaction. It was found that an immunochromatography device for concentration measurement and a concentration measurement method capable of improving measurement accuracy can be provided.

  That is, in order to solve the above-mentioned problem, the immunochromatography device for concentration measurement of the present invention is a concentration measurement immunochromatography device used for measuring the concentration of a substance to be measured by antigen-antibody reaction. A membrane addition portion, a membrane portion having a color formation portion that is formed apart from the sample addition portion and that is formed with a color development material that indicates the concentration of the substance to be measured, and a base portion that holds the membrane portion; And a measurement element that measures a physical quantity of a factor that affects a reaction that occurs between the addition of a sample and the immobilization of a colored substance after the sample is added.

  Since the measurement element for measuring the physical quantity of the factor affecting the reaction is installed in the immunochromatographic device, the physical quantity of each immunochromatographic device at the time of the reaction can be measured easily and accurately. Further, the density can be easily corrected using the physical quantity. Therefore, by using the immunochromatography device for concentration measurement of the present invention, the concentration can be measured with high accuracy.

  As physical quantities of factors that influence the reaction, temperature and / or reaction time can be mentioned. Examples of the reaction that occurs after the sample is added and before the colored substance is immobilized include an antigen-antibody reaction and an enzyme reaction. Reactions using such antibodies and enzymes are susceptible to temperature. In addition, since the reaction is performed while the sample moves through the membrane from the sample addition part to the coloration part by capillary action, it is affected by the reaction time and humidity.

  In particular, the measurement element may be a temperature measurement element. The measured temperature may be at least one of the reaction temperature during the reaction and the temperature of the environment during the reaction. When the temperature of the sample to be added is significantly different from the temperature of the environment, it is preferable that the temperature of the sample can be measured as the reaction temperature. However, since the sample is basically very small, when it is added to the sample addition section, it becomes almost the same as the external environment temperature. In addition, in the antigen-antibody reaction and enzyme reaction mentioned as reactions, the temperature of the environment during the reaction and the reaction temperature during the reaction are considered to be almost the same, and the temperature of the environment during the reaction is measured and regarded as the same as the reaction temperature. I can do it.

  The temperature measuring element includes a temperature sensing unit, a temperature display unit that displays the temperature sensed by the temperature sensing unit, a temperature recording unit that records the temperature sensed by the temperature sensing unit, and a temperature that transmits the temperature sensed by the temperature sensing unit. It is preferable to have at least one of the transmission units. In particular, a temperature measuring element having a temperature recording part is desirable. By having the temperature recording part, the temperature change during the reaction can be accurately known during the measurement.

  Further, the coloring substance may be colored by an enzymatic reaction. Since the measurement using a reaction colored by an enzyme reaction is particularly used for measuring a very small amount of a substance to be measured, the concentration measurement tends to vary. Therefore, the effect of the present invention is great.

  The antigen-antibody reaction may be a competitive reaction. The competitive reaction is a reaction that utilizes the fact that the antigen to be measured and the unlabeled antigen are competitively bound to a certain amount of antibody. A competitive reaction is a reaction used when measuring a substance to be measured having a low molecular weight to which the sandwich method (non-competitive reaction) widely used in immune reactions cannot be applied. Moreover, since the measurement using a competitive reaction is suitably used for measuring a trace amount substance, the concentration measurement tends to vary. Therefore, the effect of the present invention is great.

  Further, the concentration measurement method using the concentration measurement immunochromatography device of the present invention is a concentration measurement method for measuring the concentration of the substance to be measured using the concentration measurement immunochromatography device, and the sample containing the substance to be measured is added to the sample. A sample addition process to be added to the part, a reaction process in which the sample moves from the sample addition part through the membrane part, and a colored substance is fixed to the coloration part by an immune reaction or an enzyme reaction, and a reaction in the reaction process. The measurement step for measuring the physical quantity of the influential factor, the coloration measurement step for optically measuring the coloration degree of the colored portion after the reaction step, and the coloration degree measured in the coloration degree measurement step It has a concentration conversion step for converting the concentration of the measurement substance, and a correction step for correcting the concentration converted in the concentration conversion step by the physical quantity measured in the measurement step.

  Concentration correction can be performed using physical quantities of factors that affect the reaction in the reaction process of individual immunochromatographic devices, and the concentration can be measured with high accuracy.

  The reaction step is a step of performing an immune reaction and an enzyme reaction, the immune reaction is a competitive reaction, the enzyme reaction is to generate a color substance, and the correction step is to correct the concentration by temperature as a physical quantity. I can do it. By using a competitive reaction, the concentration of a low molecular weight trace substance can be measured. Moreover, it becomes possible to measure to a much smaller concentration by generating a colored substance by an enzymatic reaction. Further, the correction can be performed with higher accuracy by correcting the concentration with the temperature having a large influence on each reaction.

  The immunochromatography device for concentration measurement according to the present invention as described above is provided with a measurement element for measuring a physical quantity of a factor that affects the reaction in the membrane part or the base part holding the membrane part, so that each individual element at the time of reaction is installed. The physical quantity of the immunochromatographic device can be accurately measured. Therefore, when measuring the concentration after the reaction, the concentration can be easily corrected by the physical quantity measured during the reaction. Therefore, by using the immunochromatography device for concentration measurement of the present invention, it is possible to perform concentration measurement with little variation regardless of the external environment during the reaction.

  In particular, by using the measurement element as a temperature measurement element, it is possible to easily perform concentration correction using temperature, which is a factor that affects the reaction more.

  Further, by using the concentration measurement method using the concentration measurement immunochromatography device of the present invention, it is possible to obtain a highly accurate concentration measurement method with little variation regardless of the external environment.

  Hereinafter, the immunochromatography device for concentration measurement of the present invention and the concentration measurement method using the same will be described in detail.

<Immunochromatography device for concentration measurement>
The immunochromatographic device for concentration measurement according to the first embodiment of the present invention includes a membrane part, a base part that holds the membrane part, and a measurement element installed on the membrane part or the base part.

  The membrane part has a sample addition part and a coloration part formed away from the sample addition part. The material of the membrane part may be any material as long as it can hold antibodies and the substance to be measured can move by capillary action. The membrane portion can be formed of fine powder such as nylon, cellulose and derivatives thereof, fibrous material, filter paper, or the like. The shape of the membrane part is not particularly limited. A strip-shaped membrane is preferably used as the membrane. For example, a strip-shaped nitrocellulose membrane can be used as the membrane portion.

  The sample addition part may be a part of the membrane part, or may be provided with a member different from the membrane part in the membrane part. In the case of another member as well as the membrane portion, the material may be any material as long as the substance to be measured can move by capillary action. In the case of a separate member from the membrane part, in addition to the material of the membrane part, a water-absorbing pad made of a water-absorbing sponge, a water-absorbing nonwoven fabric, filter paper or the like can be used. The sample can be spotted on the membrane part or the water absorbing pad. Alternatively, the sample may be dropped on and fixed to the membrane portion or the water-absorbing pad in advance, and then the movement of the sample may be started by flowing a developing solution or the like.

  The colored portion is a portion where the colored substance is captured in some form and exhibits a color. The colored portion is formed away from the sample adding portion, and is formed in a moving place where the sample moves through the membrane portion by capillary action. The colored portion may be a part of the membrane portion or may be provided with a member different from the membrane portion. In the case of another member as well as the membrane part, the material can be any material as long as the substance to be measured can move by capillary action. Water-absorbing pads made of a conductive nonwoven fabric, filter paper, etc. can be used. There may be a plurality of colored portions depending on the measurement method of the substance to be measured. The shape of the colored portion is not particularly limited as long as it is a shape suitably used when measuring the color of the colored substance. Since the coloring degree can be measured with high sensitivity by accumulating the coloring substances, the shape of the coloring part is preferably a line or a band. This can be formed by carrying an antibody or the like for capturing the coloring substance on the colored portion in a line shape or a belt shape.

  The base part for holding the membrane part is not particularly limited as long as it can maintain the form of the membrane part and does not need to touch the membrane part directly during handling. It may be in the shape of a plate or tray that holds the lower part of the membrane part, or in the shape of a container that covers the whole. In the case of a container shape that covers the whole, an opening, a lid, or the like needs to be formed at a location that covers the sample addition portion so that the sample can be added to the sample addition portion. In addition, it is necessary to prevent the measurement from being disturbed when measuring the coloration degree of the portion covering the colored portion. Specifically, it is necessary that a portion covering the colored portion is opened, or a lid or the like is formed and opened at the time of measurement, or covered with a transparent material that does not interfere with the measurement.

  The measurement element is installed in the membrane part or the base part. Therefore, it is preferable that the size be set at the membrane part or the base part. When installed on the membrane part, it must be installed in a place that does not hinder the movement of the sample. Moreover, when installing in a base, it is necessary to install in the place which does not become obstructed when adding a sample, moving a sample, and measuring a colored part. Moreover, when the base part is the container shape which covers the whole, it may be installed in the inner side of a container and may be installed in the container outer side.

  The measurement element measures the physical quantity of a factor that affects the reaction that occurs from when the sample is added until the colored substance is immobilized. Factors affecting the reaction include temperature, reaction time, humidity, and the like. Regarding the temperature, the temperature of at least one of the environmental temperatures during the reaction may be measured even during the reaction. The antigen-antibody reaction is greatly affected by minute changes in temperature because the antibody is made of protein. In addition, since the substance to be measured is used in the form of a solution such as a buffer solution or water, the degree of evaporation of the solution changes depending on conditions such as temperature, reaction time, and humidity, and the concentration of the substance to be measured changes. Therefore, physical quantities such as temperature, reaction time, and humidity can be measured, and the measurement results can be corrected based on the physical quantities.

  When a reaction time measurement element is used as the measurement element, a commercially available timer (for example, an IC timer) can be used. As the measurement element, one that can measure and display, record, or transmit data for each reaction time from the addition of the sample to the fixation of the colored substance is preferable.

  When a temperature measurement element is used as a measurement element, the temperature detection unit, the temperature display unit that displays the temperature detected by the temperature detection unit, the temperature recording unit that records the temperature detected by the temperature detection unit, and the temperature detection unit What has at least one of the temperature transmission parts which transmit temperature is preferable. The temperature is preferably not the temperature at the time of concentration measurement, but the temperature from when the sample is added until the colored substance is immobilized. In order to use the reaction temperature for correction, it is preferable to have at least one of a temperature display unit, a temperature recording unit, or a temperature transmission unit so that the reaction temperature can be displayed, recorded, or transmitted.

  For example, a temperature measuring element having a temperature sensing unit and a temperature display unit may be used. In this case, it is necessary for the user to manually record the temperature while viewing the temperature display on the temperature display section. Moreover, the temperature measurement element which has a temperature sensing part and a temperature transmission part may be sufficient. In this case, another device capable of recording temperature or displaying temperature may be attached to the destination of the temperature data. In the case of a temperature measuring element having a temperature sensing part and a temperature recording part, it is necessary that the recorded temperature data can be taken out by a personal computer or the like.

  Further, a temperature measurement element having a temperature sensing unit, a temperature recording unit, and a temperature transmission unit can be used. For example, a temperature data logger “Thermocron G type” (trade name) manufactured by KN Laboratories can be suitably used as a temperature measurement element having a temperature recording section as a commercially available product. "Thermocron G type" (trade name) is a small button battery type temperature logger with a diameter of 17mm, connected to a personal computer, set the temperature recording conditions, disconnect the personal computer and automatically measure the temperature at the measurement location, Measurement data can be collected by connecting to the PC again.

  Moreover, it is good also as a temperature measurement element which has a temperature sensing part, a temperature recording part, a temperature transmission part, and a temperature display part.

  The temperature measuring element described above can be made by itself according to the purpose, or a commercially available product can be used.

  The substance to be measured to be measured by the immunochromatography device of the present invention is not particularly limited. Fields to which such immunochromatography devices are applied are diverse, and can be used, for example, for various virus tests, biological hormone tests, blood proteins, blood drugs, environmental hormones, dioxins, agricultural chemicals, antibiotics and the like.

  Here, the measurement principle of the immunochromatographic device will be described. The immunoassay method is a method of measuring a substance using an antigen-antibody reaction. The immunoassay method is roughly classified into a non-competitive binding method (mainly sandwich binding method) and a competitive binding method.

  In the sandwich binding method, which is a non-competitive binding method, the substance to be measured is bound to the antibody immobilized on the membrane part while moving through the membrane part by capillary action. Then, the labeled antibody labeled is bound to the substance to be measured bonded to the antibody. In other words, the substance to be measured is sandwiched between two antibodies and is called the sandwich binding method. The amount of the substance to be measured can be quantified by measuring the labeled antibody labeled. However, this method requires a high molecular weight so that the substance to be measured can be bound to the immobilized or labeled antibody in a one-to-one relationship with another immobilized or labeled antibody. In addition, this measurement method is not suitable when the amount of the substance to be measured is very small.

  The competitive binding method is a method using competitive binding of a substance to be measured and a pseudo substance of the substance to be measured to a certain amount of antibody. By using this method, it is possible to measure a very small amount of a substance to be measured. Further, it can be suitably applied to the measurement of a low molecular weight substance to which the sandwich bonding method cannot be applied.

  In addition, there are various methods for coloring in the coloring portion. The labeling substance to be colored may be bound to the antibody in advance, or the enzyme is bound to the antibody, and the captured antibody is reacted with a substrate capable of producing the colored substance by an enzyme substrate reaction. Coloring substances can also be produced. In this case, the produced color substance remains on the spot, and the substance to be measured can be quantified by measuring the color substance.

  The method of reacting a color substance with an antibody in advance is not suitable for measuring a very small amount of a substance to be measured because it is difficult to measure the color unless the color substance is accumulated to some extent. In addition, a method using an enzyme substrate reaction can affect an antigen-antibody reaction because the molecular weight of the enzyme is generally large, but a trace amount of a substance to be measured can be measured with high sensitivity.

  These coloring substances, antibodies, enzymes, substrates, etc. are to be measured before the substance to be measured is introduced into the sample addition part and the coloring substance related to the concentration of the substance to be measured is captured in the coloring part. What is necessary is just to arrange | position so that a measurement substance may be contacted. For example, these substances may be fixed to the membrane part, may be pre-absorbed to the membrane part, may be mixed with the substance to be measured, or later after the substance to be measured is fixed. It may be added.

  As the coloring substance, a coloring substance that develops itself can be used. For example, gold colloidal particles can be used as the coloring substance. In this case, colloidal gold particles can be labeled with an antibody to produce and use a colloidal gold labeled antibody. The colloidal gold particles are colored red by accumulation.

  When enzyme substrate reaction is used, alkaline phosphatase, peroxidase, β-galactosidase, etc. that are frequently used in enzyme immunoassay can be used as the labeling enzyme. By using a substrate on which each enzyme acts to generate a colored substance, the colored substance can be generated. The colored substance becomes insoluble and precipitates, and color development is observed by being physically fixed to the colored portion. In the case of a luminescent substrate, luminescence is recognized by a chemical reaction.

<Concentration measurement method using immunochromatography device for concentration measurement>
The concentration measurement method of the present invention uses the above-described immunochromatographic device for concentration measurement, and comprises a sample addition step, a reaction step, a measurement step, a coloration measurement step, a concentration conversion step, and a correction step. Have.

  In the sample addition step, a sample containing a substance to be measured is added to the sample addition portion, and in the reaction step, the sample moves from the sample addition portion to the membrane portion, and accordingly, the colored substance is generated by an immune reaction and / or an enzyme reaction. It is a process fixed to the coloring part. The measuring step measures a physical quantity of a factor that affects the reaction in the reaction step.

  The sample addition process, reaction process, and measurement process are the same as those described in the above immunochromatography device for concentration measurement. The reaction process is a process in which a colored substance is fixed to the colored part by an immune reaction and / or an enzyme reaction. In particular, the immune reaction is a competitive reaction, and the enzyme reaction is to generate a colored substance by an enzyme substrate reaction. It is possible to measure a substance having a lower molecular weight and a lower concentration.

  In the coloration degree measuring step, the coloration degree of the colored portion is optically measured after the reaction step. A colored substance is supported and colored in the colored portion. In the optical measurement, the coloration degree can be measured by applying light to the colored portion with a lamp or LED and detecting the reflected light using a photodiode or the like. There are no particular limitations on the instrument for optical measurement. When using a plurality of colored portions, the coloration degree of the plurality of colored portions is measured.

  A density | concentration conversion process converts the density | concentration of a to-be-measured substance from the coloration degree measured at the coloration degree measurement process. A calibration curve of the coloration degree according to the concentration of the substance to be measured is prepared in advance, and the concentration can be converted from the optical quantity measured in the coloration degree measurement step.

  The correction process corrects the density converted in the density conversion process by the physical quantity measured in the measurement process. In the measurement process, physical quantities of factors that affect the reaction during the reaction process are measured during the reaction process. Therefore, the physical quantities of the external environment during the reaction are used regardless of the environmental conditions when measuring the coloration degree. Correction can be performed, and accurate correction can be performed.

  Based on the above embodiment, an immunochromatography device for concentration measurement used in a method of using a competitive reaction as an immune reaction and generating a colored substance by an enzyme substrate reaction was prepared and the concentration was measured.

  Embodiments of the present invention will be described below with reference to the accompanying drawings. It also serves as an explanation of the concentration measurement method using the immunochromatography device for concentration measurement of the present invention. Further, the dimensions described below are not limited to the dimensions described.

  FIG. 1 is a schematic plan view showing a first embodiment of an immunochromatography device for concentration measurement of the present invention. FIG. 2 is a schematic plan view showing a second embodiment of the immunochromatography device for concentration measurement of the present invention. FIG. 3 is a schematic plan view showing a third embodiment of the immunochromatography device for concentration measurement of the present invention. FIG. 4 is a schematic plan view showing a fourth embodiment of the immunochromatography device for concentration measurement of the present invention. FIG. 5 is a schematic cross-sectional explanatory diagram of a second embodiment of the immunochromatography device for concentration measurement shown in FIG.

  In addition, since 1st embodiment-4th embodiment differ only in the measurement element 6, description other than the measurement element 6 shall be the same, and each description is abbreviate | omitted.

  As shown in FIGS. 1, 2, 3, 4, and 5, the membrane 2 has a strip shape with a width of 5 mm, a width of 6 cm, and a thickness of 1 mm, and is accommodated in a case 5 having a width of 2 cm, a width of 7 cm, and a thickness of 5 mm. The case 5 can be divided into two upper and lower parts in the thickness direction, and is a container that covers the membrane 2 by accommodating the membrane 2 inside and fitting the two upper and lower parts. The sample addition part 1, the second coloration part 3, and the first coloration part 4 are formed on the membrane 2 in this order at intervals from the end in the longitudinal direction. The distance from the sample addition part 1 to the second color part 3 is about 2 cm, and the distance from the second color part 3 to the first color part 4 is about 1 cm. The sample addition part 1 has a substantially circular shape with an upper surface of about 2 mm in diameter, and the second coloration part 3 and the first coloration part 4 each have a belt-like shape with a width of about 1 mm. The second color part 3 and the first color part 4 are colored after the reaction.

  The case 5 is a resin container that covers the entire membrane 2 and holds the membrane 2 inside by fitting the case upper part 5a and the case lower part 5b. The case 5 has a substantially circular sample addition opening 7 having a diameter of about 2 mm on the upper surface side covering the sample addition portion 1 and a width of about 5 mm and a length of about 15 mm on the upper surface side covering the second coloration portion 3 and the first coloration portion 4. The rectangular measurement opening 8 is opened.

  The measurement element 6 described in FIG. 1 is a temperature measurement element. The measuring element 6 shown in FIG. 1 includes a temperature sensing unit 6a, a temperature recording unit 6b, a temperature display unit 6c, and a temperature transmission unit 6d. The measuring element 6 shown in FIG. 1 is installed on the upper surface of the case 5 facing upward. The measuring element 6 may be installed so long as it does not fall from the case 5 during movement, and may be attached with an adhesive or an adhesive. May be. In the case of the measurement element 6 shown in FIG. 1, the temperature sensed by the temperature sensing unit 6a is recorded in the temperature recording unit 6b such as an IC chip, the temperature is displayed by the temperature display unit 6c, and the recorded temperature data is recorded. The temperature data can be transmitted to other devices and used by the temperature transmitter 6d such as a USB cable connection port that can be captured by a personal computer or the like.

  The measuring element 6 shown in FIG. 2 is similarly a temperature measuring element. The measurement element 6 shown in FIG. 2 is different from the measurement element 6 shown in FIG. 1 in that there is no temperature display portion 6c. In the measurement element 6 shown in FIG. 2, the temperature display is performed by another device to which data is transmitted. A temperature data logger “Thermocron G type” (trade name) manufactured by KN Laboratories can be suitably used as the measurement element 6 in FIG.

  The measurement element 6 described in FIG. 3 is similarly a temperature measurement element. The measurement element 6 illustrated in FIG. 3 includes a temperature sensing unit 6a and a temperature transmission unit 6d. As shown in FIG. 3, a temperature sensing unit 6 a and a temperature transmission unit 6 d that is a sensor cable are attached on the case 5, and a temperature recording unit 10 b and a temperature display unit in another measuring device 10 provided close to the case 5. 10c. A commercially available temperature terminal can be suitably used as the measuring element 6 shown in FIG.

  Similarly, the measurement element 6 shown in FIG. 4 is a temperature measurement element. The measurement element 6 illustrated in FIG. 4 includes a temperature sensing unit 6a and a temperature display unit 6c. In this case, a commercially available small thermometer can be used. In this case, the user must manually record the temperature.

  Moreover, although the measurement element 6 of 1st Embodiment, 2nd Embodiment, 3rd Embodiment, and 4th Embodiment demonstrated in FIG.1, FIG.2, FIG.3 and FIG.4 was demonstrated as a temperature measurement element, it is temperature sensing. The part may be used as a time measuring element by making it a time sensing part that measures each reaction time from the addition of the sample until the colored substance is fixed.

  Hereinafter, how the sample moves, the reaction occurs, and the colored substance is fixed will be described with reference to FIG. FIG. 6 is a schematic explanatory view illustrating the flow of a sample of one example when performing a competitive reaction. The second color part and the first color part are abbreviated as T2 and T1 as necessary.

  A substance to be measured and a modified antibody are added to the sample addition unit 1 as a sample. The substance to be measured and the modified antibody may be mixed and put, or the antibody to be modified may be supported on the sample addition unit 1 and the substance to be measured may be added thereto. At this time, the modified antibody is in excess of the substance to be measured. Therefore, the modified antibody binds to the substance to be measured, and the excess modified antibody remains alone in the sample.

  The sample dropped on the sample addition part moves in the direction of the arrow Y1 shown in FIG. 6 by capillary action and reaches the second coloration part T2. In the second colored portion T2, the T2 fixing substance is fixed in advance. In the case of a competitive reaction, a pseudo substance of the substance to be measured is immobilized on the second colored portion T2, and an excessively modified antibody that is not bound to the substance to be measured binds to the immobilized pseudo substance here. And fixed. Subsequently, a modified antibody or the like that has not been fixed in the second coloring portion and bound to the substance to be measured moves in the direction of the arrow Y1 shown in FIG. 6 and reaches the first coloring portion T1. In the first colored portion T1, the T1 fixing substance is fixed. The T1 fixing substance is an antibody that binds to the modified antibody. For this reason, the modified antibody bound to the substance to be measured is fixed at the first colored portion T1.

  And the coloration degree of the 2nd coloring part T2 and the 1st coloring part T1 can be optically measured, and the density | concentration of a to-be-measured substance can be converted from the ratio of the intensity | strength.

  The test examples are described below. Concentration measurement was performed using the immunochromatography device for concentration measurement.

  In this test, vitamin C (Japanese Pharmacopoeia) diluted in physiological saline was used as the substance to be measured. The immunochromatographic device will be described with reference to FIG.

(1) Creation of immunochromatography device A nitrocellulose membrane for immunochromatography (manufactured by Nihon Millipore Corporation) having a width of 50 cm, a length of 6 cm, and a thickness of 1 mm was prepared. First, vitamin C-BSA (manufactured by Biogenesis), which is a pseudo substance of vitamin C, was applied at a concentration of 0.7 mg / ml at a position corresponding to the second colored portion 3 shown in FIG. 3 perpendicular to the width direction. As a coating method, the vitamin C-BSA solution was repeatedly applied so as to draw a line perpendicular to the width direction. Similarly, an anti-mouse antibody (manufactured by Rockland) was applied at a concentration of 0.4 mg / ml to the portion corresponding to the first colored portion 4 shown in FIG. After coating, the nitrocellulose membrane was dried and fixed by drying at 50 ° C. for 1 hour, and cut every 5 mm in width to obtain a membrane 2 having a width of 5 mm, a length of 6 cm and a thickness of 1 mm.

  The produced membrane 2 was placed in the case 5. The case 5 is made of polypropylene and has a rectangular parallelepiped shape with a width of 2 cm and a length of 7 cm and a thickness of 5 mm, and can be divided into two in the vertical direction. Further, an opening 7 having a diameter of about 2 mm for sample addition and a rectangular opening 8 having a width of 5 mm and a length of 15 mm are formed on the upper surface of the case 5. A portion of the membrane 2 opened by the sample addition opening 7 is referred to as a sample addition portion 1.

  The measurement element 6 was a temperature terminal (manufactured by Keyence Corporation) as described in the third embodiment of FIG. The temperature terminal is connected to a temperature recording device (manufactured by Keyence Corporation). The temperature recording device (manufactured by Keyence Co., Ltd.) was installed close to the immunochromatography device, and the temperature terminal was attached to the upper case of the immunochromatography device with an adhesive tape.

(2) Preparation of enzyme-labeled antibody AP-labeled anti-vitamin C antibody was prepared using an alkaline phosphatase labeling kit (manufactured by Dojindo) on vitamin C antibody (manufactured by abcam).

(3) Concentration measurement (3-1) Examination of reaction time Measure the time from adding a substrate after adding a sample (antigen-antibody reaction time) and the time from putting the substrate to measurement (enzyme substrate reaction time) with a timer, The coloration of each colored portion at each reaction time was observed.

  The AP-labeled anti-vitamin C antibody prepared above is diluted 50,000 times with PBS buffer solution, and 10 μl of 0.015, 0.125, 1.0 ppm vitamin C dilutions to be measured with respect to 100 μl of the diluted solution. Were mixed and reacted at 25 ° C. for 30 minutes. 75 μl of the mixed solution after the reaction was taken as a sample and dropped into the sample addition part of the immunochromatography device. 20 minutes after sample addition, BCIP / NBT (5-Bromo-4-Chloro-3'-Indolylphosphatase-p-Toluidine salt / Nitro-Blue Tetrazolium Chloride) (manufactured by Nacalai Tesque) is attached as a substrate. 75 μl of the solution diluted 1/100 with the buffer solution was taken and added dropwise to the sample addition part. Each operation was performed in a constant temperature room at 25 ° C. The temperature was measured by attaching the terminal of a temperature recording device (manufactured by Keyence Corporation) to the upper part of the case of the immunochromatography device.

  1, 2, 3, 4, 5, 6, 7, 10, 15, 20, 30, 45, 60, 90 minutes after the addition of BCIP / NBT, the second coloring section 3 and the first presentation The coloration degree of the color part 4 was measured with an immuno server (model number: AIS-GP100) manufactured by Aisin Seiki Co., Ltd.

  In the second color part 3 and the first color part 4, purple color was recognized in a line shape. For the measurement of color development, two green LEDs that irradiate through a filter with a wavelength of 500 nm to 600 mnm are arranged from an oblique 45 ° direction as a light source, and are detected by a photodiode arranged above the measurement object. It was.

  The results are shown in Table 1 and FIG. The intensity was set to the value of the photodiode voltage value of the second color portion T2 / the voltage value of the first color portion T1. It was confirmed that purple coloration occurred 2 minutes after adding the substrate. In addition, it was found that the color was clearly developed 5 minutes after the substrate was added, and increased exponentially until 45 minutes.

  Thus, the coloration degree varies depending on the difference in the antigen-antibody reaction time or the enzyme substrate reaction time. Therefore, it is possible to perform density correction based on the reaction time.

(3-2) Preparation of calibration curve at a temperature of 25 ° C. The AP-labeled anti-vitamin C antibody prepared above is diluted 50,000 times with a PBS buffer solution and shown in Table 2 as a substance to be measured with respect to 100 μl of the diluted solution. 10 μl of each diluted vitamin C dilution was mixed and reacted at 25 ° C. for 30 minutes. 75 μl of the mixed solution after the reaction was taken as a sample and dropped into the sample addition part of the immunochromatography device. 20 minutes later, 75 μl of BCIP / NBT (manufactured by Nacalai Tesque) as a substrate diluted to 1/100 with the attached dilution buffer was dropped into the sample addition section. Each operation was performed in a constant temperature room at 25 ° C. The temperature was measured with a temperature recording device (manufactured by Keyence Corporation).

  Twenty minutes after adding BCIP / NBT, the coloration degree of the second color part 3 and the first color part 4 was measured with an immuno server (model number: AIS-GP100) manufactured by Aisin Seiki Co., Ltd.

  In the second color part 3 and the first color part 4, purple color was recognized in a line shape. The measurement of color development was performed by detecting two photodiodes disposed above the object to be measured by using two green LEDs that irradiate through a filter with a wavelength of 500 nm to 600 mnm as a light source. .

  A calibration curve was created based on the measured values. A calibration curve was prepared by taking the concentration of vitamin C on the X axis and the intensity on the Y axis. The intensity was set to the value of the photodiode voltage value of the second color portion T2 / the voltage value of the first color portion T1.

  FIG. 8 shows a calibration curve of the measured intensity with respect to the concentration at 25 ° C.

(3-3) Measurement (temperature condition change)
Next, the same process as the above process was performed under 20 ° C., 22 ° C., 28 ° C., and 30 ° C. conditions. The temperature was measured in the same manner as described above. Moreover, the coloration degree of the 2nd coloring part 3 and the 1st coloring part 4 was measured with the immuno server (model number: AIS-GP100) by Aisin Seiki Co., Ltd. similarly to the above. The measured intensity was converted into a concentration according to the calibration curve shown in FIG. The results are shown in Table 3.

  As can be seen from Table 3, when the concentration is converted by a calibration curve obtained at 25 ° C., it can be seen that the converted concentration greatly deviates from the actual concentration even when the temperature is several degrees.

(3-4) Correction (temperature)
Next, from the results in Table 3, the converted concentration was plotted against the sample concentration to obtain an approximate expression. FIG. 9 is a graph showing the relationship between the converted concentration and the sample concentration. The slope of the approximate expression was calculated from FIG. 9, and the temperature correction formula: y = −4.4971Ln (x) +15.608 was determined from the temperature and this slope. A correction coefficient for each temperature was calculated from this temperature correction formula, and the corrected concentration was calculated by dividing the converted concentration converted from the calibration curve at 25 ° C. by the correction coefficient.

  A comparison was made between the corrected concentration converted based on this temperature correction formula and the converted concentration not using the temperature correction formula. Two sample concentrations were examined, 0.25 ppm and 1 ppm.

  Table 4 shows the results without temperature correction.

  Next, Table 5 shows the result of the correction density after correction by the correction coefficient calculated based on the temperature correction formula.

  When the results of Table 4 and Table 5 are compared, the CV that shows variation before and after correction was around 60%, which was greatly reduced to 16% or less after correction. In this way, by providing a measurement element that can measure the external environment during the reaction in the immunochromatographic device, accuracy can be reduced with little variation regardless of the external environment during the reaction by performing correction based on the measured physical quantity. A good concentration measurement was possible.

1 is a schematic plan view showing a first embodiment of an immunochromatography device for concentration measurement of the present invention. It is a schematic plan view which shows 2nd embodiment of the immunochromatography device for density | concentration measurement of this invention. It is a schematic plan view which shows 3rd embodiment of the immunochromatography device for density | concentration measurement of this invention. It is a schematic plan view which shows 4th embodiment of the immunochromatography device for density | concentration measurement of this invention. It is a schematic cross section explanatory drawing of the immunochromatography device for density | concentration measurement of FIG. 1B. It is a schematic explanatory drawing explaining the flow of the sample of one Example at the time of performing competitive reaction. It is a graph which shows the relationship between reaction time and measured intensity. It is a graph which shows the calibration curve of the measured intensity with respect to the density | concentration in 25 degreeC. It is a graph which shows the relationship between conversion density | concentration and sample density | concentration.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 ... Sample addition part, 2 ... Membrane, 3 ... 2nd coloring part, 4 ... 1st coloring part, 5 ... Case,
6 ... Measurement element, 6a ... Temperature sensing unit, 6b ... Temperature recording unit, 6c ... Temperature display unit, 6d ... Temperature transmission unit, 7 ... Opening, 8 ... Opening .

Claims (8)

In an immunochromatography device for concentration measurement used to measure the concentration of a substance to be measured by antigen-antibody reaction,
A membrane part comprising: a sample addition part for adding a sample containing the substance to be measured; and a coloration part formed away from the sample addition part and to which a color substance indicating the concentration of the substance to be measured is fixed When,
A base for holding the membrane part;
A measuring element that is installed in the base or the membrane, and measures a physical quantity of a factor that affects a reaction that occurs until the colored substance is immobilized after the sample is added;
An immunochromatographic device for concentration measurement, comprising:   The immunochromatographic device for concentration measurement according to claim 1, wherein the physical quantity of the factor affecting the reaction is temperature and / or reaction time.   The physical quantity of the factor that affects the reaction is temperature, and the measurement element is the reaction temperature of the reaction or the environment during the reaction that occurs from when the sample is added to when the colored substance is immobilized. The immunochromatography device for concentration measurement according to claim 2, which is a temperature measurement element for measuring at least one of temperatures. The temperature measuring element is
A temperature sensor;
At least one of a temperature display unit that displays the temperature sensed by the temperature sensing unit, a temperature recording unit that records the temperature sensed by the temperature sensing unit, and a temperature transmission unit that transmits the temperature sensed by the temperature sensing unit; ,
The immunochromatographic device for concentration measurement according to claim 3, comprising:   The immunochromatographic device for concentration measurement according to any one of claims 1 to 4, wherein the colored substance is colored by an enzymatic reaction.   The immunochromatographic device for concentration measurement according to any one of claims 1 to 5, wherein the antigen-antibody reaction is a competitive reaction. A concentration measurement method for measuring the concentration of a substance to be measured using the immunochromatography device for concentration measurement according to any one of claims 1 to 6,
A sample addition step of adding a sample containing the substance to be measured to the sample addition section;
A reaction step in which the sample moves from the sample addition portion in the membrane portion, and the coloring substance is fixed to the coloration portion by an immune reaction and / or an enzyme reaction accompanying therewith,
A measuring step for measuring a physical quantity of a factor that affects the reaction in the reaction step;
A coloration measurement step for optically measuring the coloration degree of the colored portion after the reaction step;
A concentration conversion step for converting the concentration of the substance to be measured from the coloration degree measured in the coloration degree measurement step;
A correction step of correcting the concentration converted in the concentration conversion step by the physical quantity measured in the measurement step;
A concentration measurement method using an immunochromatography device for concentration measurement having   The reaction step is a step of performing the immune reaction and the enzyme reaction, the immune reaction is a competitive reaction, the enzyme reaction generates the color substance, and the correction step includes a temperature as the physical quantity. The concentration measurement method using the immunochromatography device for concentration measurement according to claim 7, wherein the concentration is corrected by:

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