JP2009145079A - Strip for chromatography analysis - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To measure a substance to be detected with high precision by stably detecting the labeled reagent in the reference reagent fixing part of a strip for chromatography analysis to certainly judge the result of a test. <P>SOLUTION: The strip for chromatography analysis for detecting the substance to be detected in a liquid sample is equipped with a labeled reagent holding part by which the labeled reagent specifically bonded to the substance to be detected is held, a capturing reagent fixing part on which the capturing reagent specifically bonded to the substance to be detected is fixed, a reference reagent fixing part on which the reference reagent specifically bonded to the labeled reagent is fixed and a sodium chloride holding part by which sodium chloride is held. The labeled reagent holding part, the capturing reagent fixing part and the reference reagent fixing part are arranged on the strip for the chromatography analysis in its longitudinal direction so that the liquid sample may be moved through the strip for the chromatography analysis in this order by a capillary phenomenon. The sodium chloride holding part is provided on the upstream side from the end part on the downstream side in the moving direction of the liquid sample of the reference reagent fixing part. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a strip for chromatographic analysis.

  BACKGROUND ART A chromatographic analysis strip for detecting a substance to be detected in a liquid sample is a small assay device used for a lateral flow type membrane assay, and is widely used in the medical field. In particular, the strip for chromatographic analysis enables simple diagnosis in small hospitals and patient homes that do not have specialized clinical laboratory equipment, etc., diagnosis of infectious diseases due to viruses, bacteria, etc., diagnosis of allergies such as hay fever It is effective in determining the presence or absence of pregnancy, and measuring clinical parameters in urinalysis and blood tests.

  As a strip for chromatographic analysis, devices described in Patent Documents 1 to 3 are known, and an immunochromatographic test strip (Patent Document 1), an immunochromatographic test strip (Patent Document 2), and a lateral flow membrane, respectively. It is called an assay device (Patent Document 3).

  For example, in the immunochromatographic test strip described in Patent Document 1, when a liquid sample containing a substance to be detected is added to an absorption pad (collecting member), the liquid sample specifically binds to the substance to be detected by capillary action. It moves to the display carrier fiber block (holding member) holding the display carrier (labeling reagent) on which the affinity substance is immobilized, and the detected substance and the display carrier form a conjugate to detect the porous membrane. The display moves to the member (detection member), and this conjugate is captured by the immobilized substance (capture reagent) fixed to the determination region (capture reagent fixing part), and the detected substance constitutes the conjugate The detection mechanism is based on the nature of the carrier.

  As a labeling reagent used in a chromatographic analysis strip, an antibody or an antigen that specifically binds to a substance to be detected is immobilized on a display carrier such as a dye, radioactive substance, enzyme, colloidal gold, or colored latex particle. However, as a capture reagent, an antibody or an antigen that specifically binds to a substance to be detected is generally used.

  In addition, in the chromatographic analysis strip, the control region (control reagent immobilization part) on which the control reagent that specifically binds to the labeling reagent but does not bind to the substance to be detected is immobilized from the judgment area (capture reagent immobilization part). It is provided on the downstream side in the moving direction of the liquid sample, and is used to determine whether the test is possible (Patent Document 4). Specifically, since the control reagent fixing part is provided downstream of the capture reagent fixing part in the moving direction of the liquid sample, the labeling reagent is captured in the control reagent fixing part and the control line is detected. Means that the amount of liquid sample and labeling reagent required for the measurement have passed through the capture reagent fixing part and the test has been completed, but the labeling reagent is not captured in the control reagent fixing part and the control line is not detected. Even if the detection target substance is detected at the capture reagent fixing part, the amount of liquid sample and labeling reagent necessary for the measurement may not pass therethrough, so the test is not possible. Is considered.

  More recently, a chromatographic analysis strip has been reported in which positive or negative judgment can be made by comparing the intensity of the color detected at the control reagent fixing part and the color detected at the capture reagent fixing part (patent) Reference 5).

JP 2003-121445 A JP 2004-161685 A JP 2004-245831 A JP 2006-194785 A JP 2006-265138 A Millipore, “Lateral Flow Test Strip Development Guide”, Lit. No. TB500JA00 2002/12, p. 26 and 28

  However, when a reaction reagent such as a capture reagent or a control reagent is immobilized on a porous nitrocellulose membrane or the like as a detection member, the components contained in the buffer in which the reaction reagent is dissolved is a problem with the chromatographic analysis strip. It may become. In other words, the reaction reagent is dissolved in a buffer and applied to the detection member, and after application, it is dried by heating, blowing air, etc., but at this time, the salt concentration in the buffer increases, and chemicals are generated during evaporation of moisture. Since the interaction occurs, the binding ability of the capture reagent to the substance to be detected may decrease, or the solid residue remaining on the detection member may change the flow characteristics of the detection member.

  For this reason, in a membrane assay using a strip for chromatographic analysis, it is said that the use of a buffer containing sodium chloride should be avoided when immobilizing a capture reagent, a control reagent, etc. on a detection member. Patent Document 1).

  In addition, the amount of liquid sample that can be added to the chromatographic analysis strip is limited, so that the detected substance is present only in a minute amount in the liquid sample, or the liquid sample is viscous and needs to be diluted. However, there is a problem that sufficient sensitivity cannot be obtained for detecting a substance to be detected. In such a case, it is conceivable to immobilize a large amount of the capture reagent on the detection part of the detection member, but this increases the possibility of non-specific adsorption and false positives. There is a current situation.

  In addition, for accurate positive / negative determination, stable detection of the labeling reagent in the control reagent fixing part is necessary, but depending on the liquid sample or buffer used for detection by the chromatographic analysis strip, the control reagent It may affect the detection sensitivity of the labeling reagent in the fixed part, and the labeling reagent may not be detected.

  Therefore, the present invention enables stable detection of a labeling reagent in a control reagent fixing portion of a chromatographic analysis strip, and enables highly accurate measurement of a substance to be detected by reliably determining the success or failure of a test. It is an object.

  In order to achieve the above object, a chromatographic analysis strip for detecting a substance to be detected in a liquid sample, comprising a labeling reagent holding part holding a labeling reagent that specifically binds to the substance to be detected, A capture reagent immobilization section on which a capture reagent that specifically binds to the detection substance is immobilized, a control reagent immobilization section on which a control reagent that specifically binds to the labeling reagent is immobilized, and sodium chloride are retained. A sodium chloride holding part, and a labeling reagent holding part, a capture reagent fixing part, and a control reagent fixing part are chromatographed so that the liquid sample moves in this order in the chromatographic analysis strip by capillary action. Arranged in the longitudinal direction of the strip for analysis, the sodium chloride holding part is upstream of the downstream end in the direction of movement of the liquid sample of the control reagent fixing part. It is provided, to provide a strip for chromatographic analysis.

  As a result of diligent efforts, the present inventors can detect a substance to be detected with high accuracy by including sodium chloride in the reaction system when detecting a substance to be detected in a liquid sample using a chromatographic analysis strip. I found. However, when a protein such as an antibody is immobilized on a detection member such as a nitrocellulose membrane, it is not preferable to add sodium chloride to the buffer for dissolving the protein. It has been devised that a sodium chloride holding part for holding sodium chloride alone is provided upstream from the end part. As a result, it was found that the detection sensitivity of the control reagent fixing part in the chromatographic analysis strip is stable, and the substance to be detected can be measured with high accuracy.

  Using the above chromatographic analysis strip improves the detection sensitivity of the labeling reagent even when using a liquid sample or developing solution that is difficult to detect with a chromatographic analysis strip without a sodium chloride holder. In addition, in the control reagent fixing part, it is possible to stably detect the labeling reagent (control line), and it is possible to increase the certainty of determining whether the test substance is positive or negative.

  The strip for chromatography analysis includes a holding member including the labeling reagent holding unit, and a detection member including the capture reagent fixing unit and the control reagent fixing unit. The holding member and the detection member include the liquid sample. The sodium chloride holding part is arranged in the longitudinal direction of the chromatographic analysis strip so as to move in this order by capillary action in these members, and the sodium chloride holding part is located at the downstream side of the reference reagent fixing part in the moving direction. It is preferable to be provided on the upstream detection member or holding member.

  The chromatographic analysis strip includes a collecting member to which a liquid sample is added, a holding member having the labeling reagent holding unit, a detection member having the capture reagent fixing unit and the control reagent fixing unit, and the liquid An absorption member that sucks up a sample and a liquid-impermeable support member are provided, and the sampling member, the holding member, the detection member, and the absorption member are moved in this order by capillary action in the liquid sample. In this way, the sodium chloride holding part is arranged on the support member in the longitudinal direction of the chromatographic analysis strip, and the sodium chloride holding part is a detection member, a holding member or a sample upstream of the downstream end part in the moving direction of the control reagent fixing part. More preferably, it is provided on the member.

  By using the chromatography analysis strip, the analysis ability of the substance to be detected is improved, the certainty of positive or negative judgment is further increased, and at the same time, the substance to be detected can be measured with high accuracy.

  Furthermore, it is preferable that the sampling member and the holding member share a single fiber base material.

  By integrating the collection member and the holding member, the strength of the chromatographic analysis strip can be increased, and the liquid sample can be easily moved from the collection member to the holding member.

  The sodium chloride holding part is preferably provided on the upstream side of the upstream end part in the moving direction of the control reagent fixing part, and is 2 from the upstream end part in the moving direction of the chromatographic analysis strip. More preferably, it is within 5 cm, more preferably within 2 cm, and particularly preferably within 1.5 cm.

  In this case, the detection sensitivity of the labeling reagent can be further increased, and the sodium chloride holding part is separated from the capture reagent fixing part, so avoid contact between the capture reagent and sodium chloride until the membrane assay is actually started. And denaturation of the capture reagent by sodium chloride can be prevented. This is also true for the control reagent, and the binding force between the control reagent and the labeling reagent can be increased, and denaturation of the control reagent by sodium chloride can be prevented. For the purpose of enhancing the detection sensitivity of the control reagent, the sodium chloride holding part is located on the detection member upstream of the downstream end part in the moving direction of the capture reagent fixing part and on the downstream side in the moving direction of the control reagent fixing part. It may be on the detection member from the end portion to the downstream end portion in the moving direction of the capture reagent fixing portion, or both.

  According to the present invention, even when a liquid sample or a developing solution that is difficult to detect with a chromatographic analysis strip without a sodium chloride holding part is used, the detection sensitivity of the labeling reagent is improved and the control reagent is fixed. In this part, the labeling reagent (control line) can be stably detected, and the positive or negative judgment of the test substance can be improved. Further, according to the present invention, even when only an antibody having a low affinity can be used as a capture reagent, the sodium chloride holding part is provided upstream from the downstream end in the liquid sample movement direction of the capture reagent fixing part. As a result, the detection sensitivity of the detected substance and the labeling reagent can be improved, and the chromatographic strip that has not been produced because only antibodies having a low affinity can be used is brought into a state in which the detected substance can be detected. Can be provided.

  Hereinafter, the best mode for carrying out the present invention will be described in detail with reference to the drawings as necessary. However, the present invention is not limited to the following embodiments, and the dimensional ratios in the drawings are not limited to the illustrated dimensional ratios. In the drawings, the same elements are denoted by the same reference numerals, and redundant description is omitted. Further, unless otherwise specified, “%” represents “weight to volume percentage (w / v%)”.

(Strip for chromatographic analysis)
The strip for chromatographic analysis of the present invention is a strip for chromatographic analysis that detects a substance to be detected in a liquid sample, and includes a labeling reagent holding part that holds a labeling reagent that specifically binds to the substance to be detected. , Capture reagent immobilization part on which a capture reagent that specifically binds to the target substance is immobilized, control reagent immobilization part on which a control reagent that specifically binds to the labeling reagent is immobilized, and sodium chloride A sodium chloride holding part, and a labeling reagent holding part, a capture reagent fixing part, and a control reagent fixing part are arranged so that the liquid sample moves in this order through the chromatographic analysis strip by capillary action. Arranged in the longitudinal direction of the strip for chromatographic analysis, the sodium chloride holding part is located below the movement direction of the liquid sample in the control reagent fixing part. It is characterized in that provided upstream from the end on the side.

  The “chromatography analysis strip” is a small assay device used for a lateral flow type membrane assay, and is simply called a test strip because the assay device has a strip shape. For example, an immunochromatographic test strip (Patent Document 1), an immunochromatography test strip (Patent Document 2), and a lateral flow membrane assay device (Patent Document 3) described in Background Art are included.

  The strip for chromatography analysis includes a holding member including the labeling reagent holding unit, and a detection member including the capture reagent fixing unit and the control reagent fixing unit. The holding member and the detection member include the liquid sample. The sodium chloride holding part is arranged in the longitudinal direction of the chromatographic analysis strip so as to move in this order by capillary action in these members, and the sodium chloride holding part is located at the downstream side of the reference reagent fixing part in the moving direction. It is preferable to be provided on the upstream detection member or holding member.

The chromatographic analysis strip includes a collecting member to which a liquid sample is added, a holding member having the labeling reagent holding unit, a detection member having the capture reagent fixing unit and the control reagent fixing unit, and the liquid An absorption member that sucks up a sample and a liquid-impermeable support member are provided, and the sampling member, the holding member, the detection member, and the absorption member are moved in this order by capillary action in the liquid sample. In this way, the sodium chloride holding part is arranged on the support member in the longitudinal direction of the chromatographic analysis strip, and the sodium chloride holding part is a detection member, a holding member or a sample upstream of the downstream end part in the moving direction of the control reagent fixing part. More preferably, it is provided on the member.
FIG. 1 is a plan view showing a first embodiment of the chromatographic analysis strip of the present invention, and FIG. 2 is a side end view showing the first embodiment of the chromatographic analysis strip of the present invention. .

  The chromatographic analysis strip 1 shown in FIGS. 1 and 2 is provided with a labeling reagent holding part 12a, a sodium chloride holding part 14a, a capture reagent fixing part 14b, and a control reagent fixing part 14c on the chromatographic member 5. The labeling reagent holding part 12a, the capture reagent fixing part 14b and the control reagent fixing part 14c are arranged in the longitudinal direction of the chromatography analysis strip 1 in this order.

  The sodium chloride holding part 14a is provided in the chromatographic member 5 upstream from the end of the control reagent fixing part 14c on the downstream side in the moving direction of the liquid sample (hereinafter simply referred to as “downstream side”).

  The chromatographic member 5 includes a filter paper or a porous chromatographic member, and examples of the chromatographic member include a nitrocellulose membrane and a nitrocellulose membrane containing cellulose acetate.

  When detecting a substance to be detected in a liquid sample using the chromatographic analysis strip 1, first, at the end of the chromatographic member 5 in the moving direction of the liquid sample (hereinafter simply referred to as “upstream side”). The liquid sample is added to the chromatographic analysis strip 1 by dropping the liquid sample or by directly contacting the body fluid (eg, tear fluid, blood, etc.) of the subject animal. The added liquid sample will move downstream by capillary action.

  The labeling reagent holder 12a holds a labeling reagent that specifically binds to the substance to be detected. As a labeling reagent, an affinity substance that specifically binds to a substance to be detected (for example, a substance that specifically binds to the substance to be detected, for example, a dye, a radioactive substance, an enzyme, a gold colloid, or a colored latex particle). Antibodies, antigens or aptamers that are immobilized). As a labeling reagent, a colloidal gold immobilized with an antibody that specifically binds to the substance to be detected is not necessary to determine the presence or absence of the substance to be detected. Is particularly preferred.

  The labeling reagent is eluted in the liquid sample that has moved downstream due to capillary action, and in the process of moving the liquid sample further downstream, it binds to the substance to be detected in the liquid sample, (Hereinafter simply referred to as “substance to be detected”). The detected substance conjugate formed in the labeling reagent holding unit 12a moves further downstream by capillary action.

  The sodium chloride holding part 14a is provided in the chromatographic member 5 upstream from the downstream end of the control reagent fixing part 14c, but is provided upstream from the upstream end of the control reagent fixing part 14c. Preferably, it is in a region within 2.5 cm from the upstream end of the chromatographic analysis strip 1, more preferably in a region within 1.5 cm, and a region within 1.0 cm It is particularly preferable that

  A sodium chloride solution is applied to the sodium chloride holding portion 14a in a line perpendicular to the longitudinal direction of the chromatographic analysis strip 1, and is held in an area applied by drying. When the sodium chloride holding part 14a is provided in the capture reagent fixing part 14b, the sodium chloride solution and the capture reagent described below may be mixed and the mixed solution applied, and the sodium chloride holding part 14a is a control. When provided in the reagent fixing portion 14c, a sodium chloride solution and a control reagent described below may be mixed and the mixed solution applied.

  The concentration of the sodium chloride solution to be applied is preferably 3 to 30%, and more preferably 3 to 9%. Moreover, it is preferable that the sodium chloride holding | maintenance part 14a hold | maintains 90-900 microgram / cm of sodium chloride, and it is still more preferable that 90-270 microgram / cm is hold | maintained.

  A capture reagent that specifically binds to the substance to be detected is fixed to the capture reagent fixing portion 14b in a line perpendicular to the longitudinal direction of the chromatographic analysis strip 1.

  Examples of the capture reagent include an antibody that specifically binds to the substance to be detected (for example, an antibody that the labeling reagent has) or an antigen, but a substance that can specifically bind to the substance to be detected and can be immobilized on the detection member 14. If there is no particular limitation.

  As the capture reagent, for example, a solution in which an amount of capture reagent necessary for detection of the detection target substance is dissolved is applied to the capture reagent fixing part 14b, and the capture reagent is fixed at 30 to 60 ° C for 15 to 60 minutes (preferably at 50 ° C for 30 minutes). ) It can be fixed on the chromatographic member 5 by drying.

  The detected substance conjugate that has moved to the capture reagent fixing portion 14b due to the capillary phenomenon is captured by the capture reagent fixed to the capture reagent fixing portion 14b, and the properties of the display carrier contained in the detected substance conjugate are changed. If the detected substance conjugate is detected by using it, the presence or absence of the detected substance can be determined. For example, when a gold colloid in which an antibody that specifically binds to a detection substance is immobilized is used as a labeling reagent, when the detection substance conjugate is captured, a red line is formed on the capture reagent fixing portion 14b. Therefore, if this line is visually confirmed, it can be determined that the substance to be detected is present in the liquid sample.

  A control reagent that specifically binds to the labeling reagent held by the holding member 12 is fixed to the control reagent fixing portion 14c in a line perpendicular to the longitudinal direction of the chromatographic analysis strip 1.

  Examples of the control reagent include an antibody against a labeling reagent, for example, an antibody against an antibody against a substance to be detected that constitutes the labeling reagent.

  For the control reagent, for example, a solution in which an amount of the control reagent necessary for detection of the labeling reagent is dissolved is applied to the control reagent fixing part 14c, and the control reagent is fixed at 30 to 60 ° C. for 15 to 60 minutes (preferably at 50 ° C. for 30 minutes). It can be fixed on the chromatographic member 5 by drying.

  The labeling reagent that has passed through the capture reagent fixing part 14b due to capillary action is captured by the control reagent fixed to the control reagent fixing part 14c, and the labeling reagent is removed using the properties of the display carrier contained in the labeling reagent. If it detects, it can be determined whether the to-be-detected substance conjugate | bonded_body passed the capture reagent fixing | fixed part 14b. For example, when a gold colloid in which an antibody that specifically binds to the substance to be detected is immobilized is used as the labeling reagent, if the labeling reagent is captured by the control reagent immobilization part 14c, Since a red line appears on this line, if this line is visually confirmed, it can be confirmed that the detected substance conjugate has passed through the capture reagent fixing part 14b and the membrane assay has been performed correctly.

The chromatographic analysis strip 1 according to the first embodiment can be manufactured, for example, by a manufacturing method including the following steps (1) to (5).
(1) The labeling reagent holding part 12a is provided by holding the labeling reagent downstream from the end of the chromatographic member 5 to which the liquid sample is added.
(2) On the downstream side of the labeling reagent holding unit 12a, a capture reagent and a control reagent are sequentially applied in a linear shape, and a capture reagent fixing unit 14b and a control reagent fixing unit 14c are provided.
(3) The chromatographic member 5 is dried at 30 to 60 ° C. for 15 to 60 minutes, and the capture reagent and the control reagent are fixed on the chromatographic member 5.
(4) A sodium chloride solution is applied to the control reagent fixing part 14c or the chromatographic member 5 upstream from the downstream end of the control reagent fixing part 14c, and a sodium chloride holding part 14a is provided.
(5) If necessary, the chromatographic member 5 is cut into a strip having a desired width.

  FIG. 3 is a plan view showing a second embodiment of the chromatographic analysis strip of the present invention, and FIG. 4 is a side end view showing the second embodiment of the chromatographic analysis strip of the present invention. .

  The chromatographic analysis strip 1 shown in FIGS. 3 and 4 includes a sampling member 10, a holding member 12, a detection member 14, an absorption member 16, and a support member 18, and the sampling member 10 and the holding member. 12, the detection member 14 and the absorption member 16 are provided on the support member 18 so that the liquid sample added to the collection member 10 moves in this order by capillary action on the support member 18. Are arranged in the longitudinal direction.

  The holding member 12 is provided with a labeling reagent holding part 12a, and the detection member 14 is provided with a capture reagent fixing part 14b and a control reagent fixing part 14c. The capture reagent fixing part 14b and the control reagent fixing part 14c are arranged in the longitudinal direction of the detection member 14 so that the liquid sample added to the chromatographic analysis strip moves in this order.

  The sodium chloride holding part 14a is provided on the detection member 14, the holding member 12 or the collecting member 10 upstream from the downstream end of the control reagent fixing part 14c.

  The collection member 10 is arranged so as to protrude from the support member 18 on the upstream side in the moving direction of the liquid sample added to the collection member 10 and has a structure that facilitates direct collection of the liquid sample. However, it may have a structure that does not protrude from the support member 18 depending on the application.

  The collection member 10 is made of, for example, a fiber base material such as polyester or pulp nonwoven fabric, filter paper, glass fiber, or the like, and plays a role of absorbing the liquid sample and applying the liquid sample to the chromatographic analysis strip 1. .

  When a substance to be detected in a liquid sample is detected using the chromatographic analysis strip 1, first, the liquid sample is dropped on the collection member 10 or the body fluid (eg, tear fluid, blood, etc.) of the subject animal. The liquid sample is added to the collecting member 10 by bringing the sample into direct contact with the collecting member 10. Since a part of the collecting member 10 overlaps with a part of the holding member 12, the liquid sample absorbed by the collecting member 10 moves downstream by capillary action and moves to the holding member 12. Will be.

  A labeling reagent holder 12a provided on the holding member 12 holds a labeling reagent that specifically binds to the substance to be detected. As a labeling reagent, an affinity substance that specifically binds to a substance to be detected (for example, a substance that specifically binds to the substance to be detected, for example, a dye, a radioactive substance, an enzyme, a gold colloid, or a colored latex particle). Antibodies, antigens or aptamers that are immobilized). As a labeling reagent, a colloidal gold immobilized with an antibody that specifically binds to the substance to be detected is not necessary to determine the presence or absence of the substance to be detected. Is particularly preferred.

  The labeling reagent is eluted in the liquid sample that has moved from the collection member 10 by capillary action, and in the process in which the liquid sample moves further downstream, it binds to the substance to be detected in the liquid sample, Form. Since a part of the holding member 12 has a structure that overlaps a part of the detection member 12, the detected substance conjugate formed in the holding member 12 moves further downstream by capillary action, and is detected. It will move to the member 14.

  The detection member 14 is composed of a porous chromatography member, and examples thereof include a nitrocellulose membrane and a nitrocellulose membrane containing cellulose acetate.

  The sodium chloride holding part 14a is provided in the detection member 14, the holding member 12 or the collecting member 10 upstream from the downstream end of the control reagent fixing part 14c, but the upstream end of the control reagent fixing part 14c. It is preferable that it is provided on the upstream side of the part, more preferably in a region within 2.5 cm from the upstream end of the chromatographic analysis strip 1, and further in a region within 1.5 cm. Preferably, it is particularly preferably in a region within 1.0 cm.

  A sodium chloride solution is applied to the sodium chloride holding portion 14a in a line perpendicular to the longitudinal direction of the chromatographic analysis strip 1, and is held in an area applied by drying. When the sodium chloride holding part 14a is provided in the capture reagent fixing part 14b, the sodium chloride solution and the capture reagent described below may be mixed and the mixed solution applied, and the sodium chloride holding part 14a is a control. When provided in the reagent fixing portion 14c, a sodium chloride solution and a control reagent described below may be mixed and the mixed solution applied.

  The concentration of the sodium chloride solution to be applied is preferably 3 to 30%, and more preferably 3 to 9%. Moreover, it is preferable that the sodium chloride holding | maintenance part 14a hold | maintains 90-900 microgram / cm of sodium chloride, and it is still more preferable that 90-270 microgram / cm is hold | maintained.

  A capture reagent that specifically binds to the substance to be detected is fixed to the capture reagent fixing portion 14 b provided on the detection member 14 in a line perpendicular to the longitudinal direction of the chromatography analysis strip 1.

  Examples of the capture reagent include an antibody that specifically binds to the substance to be detected (for example, an antibody that the labeling reagent has) or an antigen, but a substance that can specifically bind to the substance to be detected and can be immobilized on the detection member 14. If there is no particular limitation.

  As the capture reagent, for example, a solution in which an amount of capture reagent necessary for detection of the detection target substance is dissolved is applied to the capture reagent fixing part 14b, and the capture reagent is fixed at 30 to 60 ° C for 15 to 60 minutes (preferably at 50 ° C for 30 minutes). ) It can be fixed on the detection member 14 by drying.

  The detected substance conjugate that has moved to the detection member 14 due to the capillary phenomenon is captured by the capture reagent fixed to the capture reagent fixing portion 14b, and utilizes the properties of the display carrier contained in the detected substance conjugate. By detecting the detected substance conjugate, the presence or absence of the detected substance can be determined. For example, when a gold colloid in which an antibody that specifically binds to a detection substance is immobilized is used as a labeling reagent, when the detection substance conjugate is captured, a red line is formed on the capture reagent fixing portion 14b. Therefore, if this line is visually confirmed, it can be determined that the substance to be detected is present in the liquid sample.

  A control reagent that specifically binds to the labeling reagent held on the holding member 12 is lined perpendicular to the longitudinal direction of the chromatographic analysis strip 1 on the control reagent fixing portion 14c provided on the detection member 14. It is fixed to the shape.

  Examples of the control reagent include an antibody against a labeling reagent, for example, an antibody against an antibody against a substance to be detected that constitutes the labeling reagent.

  For the control reagent, for example, a solution in which an amount of the control reagent necessary for detection of the labeling reagent is dissolved is applied to the control reagent fixing part 14c, and the control reagent is fixed at 30 to 60 ° C. for 15 to 60 minutes (preferably at 50 ° C. for 30 minutes). It can be fixed on the chromatographic member 5 by drying.

  The labeling reagent that has moved to the detection member 14 due to capillary action passes through the capture reagent fixing part 14b and is then captured by the control reagent fixed to the control reagent fixing part 14c. If the labeling reagent is detected by using it, it can be determined whether or not the detected substance conjugate has passed through the capture reagent fixing part 14b. For example, when a gold colloid in which an antibody that specifically binds to the substance to be detected is immobilized is used as the labeling reagent, if the labeling reagent is captured by the control reagent immobilization part 14c, Since a red line appears on this line, if this line is visually confirmed, it can be confirmed that the detected substance conjugate has passed through the capture reagent fixing part 14b and the membrane assay has been performed correctly.

  The absorbing member 16 is not particularly limited as long as it functions to absorb the liquid sample and the labeling reagent that have moved from the detecting member 14 due to capillary action, and examples thereof include cellulose filter paper.

  The support member 18 is not particularly limited as long as it is a liquid-impermeable material such as polyethylene terephthalate, and can be appropriately selected according to the application.

  The chromatographic analysis strip 1 has a sampling member 10, a holding member 12, a detection member 14, and an absorption member 16 so that the liquid sample applied to the sampling member 10 moves in this order by capillary action. 18 can be prepared by fixing with an adhesive or the like, but the downstream end of the sampling member 10 and the surface opposite to the support member 18 at the upstream end of the holding member 12 and the detection member 14 are adhered to each other. Adhering with a tape or the like, and further adhering the downstream end of the detection member 14, the absorption member 16 and the downstream end of the support 18 with an adhesive tape or the like so as to be sandwiched from the downstream side, the support member 18 It is preferable to fix each member on the top. An example of the adhesive tape in this case is a paper adhesive tape.

The chromatographic analysis strip 1 according to the second embodiment can be manufactured by, for example, a manufacturing method including the following steps (1) to (7).
(1) The labeling reagent holding part 12a is provided by holding the labeling reagent on the holding member 12.
(2) The detection member 14 is fixed on the support 18 by adhesion or lamination.
(3) On the detection member 14, a capture reagent and a control reagent are respectively applied in a line, and a capture reagent fixing part 14b and a control reagent fixing part 14c are provided.
(4) The detection member 14 is dried at 30 to 60 ° C. for 15 to 60 minutes, and the capture reagent and the control reagent are fixed on the detection member 14.
(5) A sodium chloride solution is applied to the control reagent fixing part 14c or the detection member 14, the holding member 12 or the sampling member 10 upstream from the downstream end of the control reagent fixing part 14c, and the sodium chloride holding part 14a is provided.
(6) The collecting member 10, the holding member 12, and the absorbing member 16 are bonded on the support 18.
(7) The multilayer card thus formed is cut into strips having a desired width.

  FIG. 5 is a plan view showing a third embodiment of the chromatographic analysis strip of the present invention, and FIG. 6 is a side end view showing the third embodiment of the chromatographic analysis strip of the present invention. .

  The strip 1 for chromatographic analysis shown in FIGS. 5 and 6 is characterized in that the sampling member 10 and the holding member 12 are integrated to form a single fiber substrate. 4 has the same configuration as the chromatographic analysis strip 1 according to the second embodiment shown in FIG. Since the collection member 10 and the holding member 12 are integrated, there is an advantage that the strength of the chromatographic analysis strip 1 can be increased and the liquid sample can easily move from the collection member 10 to the holding member 12.

  The chromatography analysis strip 1 according to the third embodiment can be manufactured by the steps (1) to (7) shown in the method for manufacturing the chromatography analysis strip 1 according to the second embodiment.

  EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated concretely, this invention is not limited to a following example.

  A half strip having a shape excluding the holding member from the chromatographic analysis strip shown in FIGS. 3 and 4 was produced by the following procedure, and the effect of improving the detection sensitivity of the labeling reagent by providing the sodium chloride holding portion was tested.

(Example 1) Effect of improving the detection sensitivity of the labeling reagent by providing a sodium chloride holding part on the detection member upstream of the control reagent fixing part:
1) Preparation of half strip First, a card-type nitrocellulose membrane (HF180MC100; Millipore) serving as a detection member of the chromatographic analysis strip shown in FIGS. 3 and 4 is prepared, and a sample pad (collecting member) and a conjugate are prepared. The part which affixes a pad (holding member) was cut off.

  Thereafter, 1 mg of an anti-goat IgG antibody solution (0.3 mg / mL; OEMconcepts) is placed at a position 16 mm from the lower end (flow start portion) of the nitrocellulose membrane on the side opposite to the side where the absorbent pad (absorbing member) is applied. The sample was applied linearly with a dispensing device (XYZ3050; BioDot) so as to be 2 μL / cm, and a control reagent fixing part was provided.

  Subsequently, 0.3 mm, 0.9, 3 or 9% sodium chloride solution is linearized at a position of 1.5 mm from the flow start part by a dispensing device (XYZ3050; BioDot) so as to be 3 μL / cm. The sodium chloride holding | maintenance part was provided by apply | coating.

  At that time, in order to produce a control half strip without a sodium chloride holding part, a dispensing device was placed at a position of 1.5 mm from the flow starting part so that distilled water was 3 μL / cm instead of the sodium chloride solution. (XYZ3050; BioDot) coated in a linear form was also prepared.

  Then, the nitrocellulose membrane provided with the control reagent fixing part and the sodium chloride holding part is dried at 50 ° C. for 30 minutes to fix the applied anti-goat IgG antibody on the nitrocellulose film, and subsequently blocking the nitrocellulose film. Blocking was performed by immersing in buffer (1% casein / 50 mM boric acid, pH 8.5) at room temperature for 30 minutes, and washing and stabilizing buffer (1% sucrose / 0.1% sodium cholate / 100 mM Tris, pH 7. After washing in 5), it was naturally dried overnight at room temperature.

  Thereafter, an absorbent pad made of cellulose (Millipore) corresponding to the absorbent member is bonded to the end of the nitrocellulose membrane opposite to the flow start portion, and the flow start portion is adjusted so that the lateral width becomes 1.5 mm. Cut the nitrocellulose membrane from the opposite end in the direction of the flow start, to prepare each half strip and a control half strip provided with sodium chloride holders with different amounts of sodium chloride.

2) Measurement of detection sensitivity In order to detect gold colloid-labeled goat IgG antibody (gold colloid: Tanaka Kikinzoku Co., Ltd .: antibody: Bethyl Co.) in a control reagent immobilization part, a gold colloid-labeled goat IgG antibody solution (OD520 nm = 10) prepared in advance. Was added to each well of a 96-well plate, and 1 μL of distilled water was added thereto for dilution.

  Here, in the usual chromatographic analysis strip, the gold colloid-labeled goat IgG antibody solution is held on the holding member as a labeling reagent, and the liquid sample dropped on the collecting member moves to the holding member by capillary action Thus, the liquid sample and the gold colloid-labeled goat IgG antibody solution are mixed, and the detected substance in the liquid sample and the gold colloid-labeled goat IgG antibody form a conjugate. In the experiment used, the above-mentioned conjugate was formed in the well of a 96-well plate without using a holding member, and the anti-goat IgG antibody immobilized on the control reagent immobilization part directly detected the gold colloid-labeled goat IgG antibody. Since 1 μL of distilled water is added to the liquid sample in the chromatographic analysis strip. Therefore in which labeled reagent is added to correspond to the ratio to be diluted.

  Thereafter, the flow start part of each half strip is immersed in a diluted solution of gold colloid-labeled goat IgG antibody solution in each well of a 96-well plate, and in this state, each half strip is allowed to stand for 7 minutes. The antibody was moved to the control reagent fixing part. Subsequently, the flow start part of each half strip was transferred to a new well containing 30 μL of distilled water and allowed to stand for 7 minutes to completely move the gold colloid-labeled goat IgG antibody to the control reagent fixing part.

  Then, each half strip measured the degree of color development in the control reagent fixing part with an immunochromatography reader (ICA-1000; Hamamatsu Photonics), and examined the relationship with the concentration of the sodium chloride solution applied to the sodium chloride holding part.

  FIG. 7 examined the effect of improving the detection sensitivity by providing a sodium chloride holding part on the detection member upstream of the control reagent fixing part, and the relationship between the concentration of sodium chloride solution applied to the sodium chloride holding part and the detection sensitivity. It is a result.

  As a result, gold colloid-labeled goat IgG antibody was detected in the control reagent immobilization part even in the control half-strip and the half strip provided with a sodium chloride holding part by applying 0.3 or 0.9% sodium chloride solution. However, in the half strip provided with the sodium chloride holding part by applying 3% and 9% sodium chloride solution, the detection sensitivity of the colloidal gold labeled goat IgG antibody in the control reagent fixing part is about 3 to 5 times. It became clear that it increased.

  From the above results, when 3% and 9% sodium chloride solution having a concentration higher than that of physiological saline is applied to the detection member on the upstream side in the moving direction of the liquid sample from the control reagent fixing part, the strip for chromatography analysis It was suggested that the detection sensitivity of the labeling reagent due to was significantly improved, and that the substance to be detected can be measured with high accuracy.

(Example 2) Effect of improving detection sensitivity of labeling reagent by providing sodium chloride holding part in control reagent fixing part:
1) Production of Half Strip As in Example 1, first, a card-type nitrocellulose membrane (HF180MC100; Millipore) serving as a detection member for the chromatographic analysis strip shown in FIGS. 3 and 4 was prepared, and a sample pad was prepared. The part to which the (collecting member) and the conjugate pad (holding member) were attached was cut off.

  Thereafter, the final concentration is 0.3, 0.9, or 9% at a position 16 mm from the lower end (flow start portion) of the nitrocellulose membrane on the side opposite to the side to which the absorbent pad (absorbing member) is applied. An anti-goat IgG antibody solution (0.3 mg / mL; OEMconcepts) supplemented with sodium chloride solution was applied linearly with a dispensing device (XYZ3050; BioDot) to a concentration of 1.2 μL / cm, and a control reagent A fixing part was provided. Thereby, the control reagent fixing part corresponds to the sodium chloride holding part at the same time, and the sodium chloride holding part is provided in the control reagent fixing part.

  At that time, in order to prepare a control half-strip, an anti-goat IgG antibody solution (0.3 mg / mL; OEMconcepts) containing no sodium chloride was similarly dispensed at 1.2 μL / cm. (XYZ3050; BioDot) coated in a linear form was also prepared.

  Thereafter, in the same manner as in Example 1, the above nitrocellulose membrane provided with a control reagent immobilization part was dried at 50 ° C. for 30 minutes to immobilize the applied anti-goat IgG antibody on the nitrocellulose membrane, and subsequently the nitrocellulose membrane. Was immersed in a blocking buffer (1% casein / 50 mM boric acid, pH 8.5) at room temperature for 30 minutes for blocking, and washed and stabilized buffer (1% sucrose / 0.1% sodium cholate / 100 mM Tris, After washing with pH 7.5), it was naturally dried overnight at room temperature.

  Thereafter, an absorbent pad made of cellulose (Millipore) corresponding to the absorbent member is bonded to the end of the nitrocellulose membrane opposite to the flow start portion, and the flow start portion is adjusted so that the lateral width becomes 1.5 mm. The nitrocellulose membrane was cut from the end on the opposite side in the direction of the flow start portion, and each half strip and control half strip were prepared in which a sodium chloride holding portion having a different amount of sodium chloride was provided in the control reagent fixing portion.

2) Measurement of detection sensitivity As in Example 1, colloidal gold labeled goat IgG antibody (gold colloid: Tanaka Kikinzoku Co., Ltd .: antibody: Bethyl Co.) was detected in advance in order to detect the colloidal gold labeled goat IgG antibody. 15 μL of the IgG antibody solution (OD520 nm = 10) was placed in each well of a 96-well plate, and 1 μL of distilled water was added thereto for dilution.

  Thereafter, the flow start part of each half strip is immersed in a diluted solution of gold colloid-labeled goat IgG antibody solution in each well of a 96-well plate, and in this state, each half strip is allowed to stand for 7 minutes. The antibody was moved to the control reagent fixing part. Subsequently, each half strip was transferred to a new well containing 30 μL of distilled water and allowed to stand for 7 minutes to completely move the gold colloid-labeled goat IgG antibody to the control reagent fixing part.

  Thereafter, each half strip was measured with an immunochromatography reader (ICA-1000; Hamamatsu Photonics Co., Ltd.) for the degree of color development in the control reagent fixing part, and the concentration of the sodium chloride solution in the sodium chloride holding part provided in the control reagent fixing part. I investigated the relationship.

  FIG. 8 shows the results of examining the effect of improving the detection sensitivity by providing the sodium chloride holding part in the control reagent fixing part and the relationship between the concentration of the sodium chloride solution applied to the sodium chloride holding part and the detection sensitivity.

  As a result, even in a half strip for control and a half strip provided with a sodium chloride holding part by applying an anti-goat IgG antibody solution containing 0.3 or 0.9% sodium chloride, a colloidal gold colloid is used in the control reagent fixing part. Although the labeled goat IgG antibody could be detected, in the half strip provided with the sodium chloride holding part by applying an anti-goat IgG antibody solution containing 9% sodium chloride to the control reagent fixing part, the gold in the control reagent fixing part It was revealed that the detection sensitivity of the colloid-labeled goat IgG antibody was increased about 2-fold.

  From the above results, when a 9% sodium chloride solution having a concentration higher than that of physiological saline is applied to the control reagent fixing part, the detection sensitivity of the labeling reagent by the chromatographic analysis strip is remarkably improved. It was suggested that highly accurate measurement would be possible.

It is a top view which shows 1st embodiment of the strip for chromatographic analysis which concerns on this invention. 1 is a side end view showing a first embodiment of a chromatographic analysis strip according to the present invention. It is a top view which shows 2nd embodiment of the strip for chromatographic analysis based on this invention. FIG. 6 is a side end view showing a second embodiment of the chromatographic analysis strip according to the present invention. It is a top view which shows 3rd embodiment of the strip for chromatographic analysis based on this invention. FIG. 6 is a side end view showing a third embodiment of the chromatographic analysis strip according to the present invention. It is the result of investigating the improvement effect of the detection sensitivity by providing the sodium chloride holding part in the detection member upstream from the control reagent fixing part, and the relationship between the concentration of the sodium chloride solution applied to the sodium chloride holding part and the detection sensitivity. It is the result which investigated the improvement effect of the detection sensitivity by providing a sodium chloride holding | maintenance part in a control reagent fixing | fixed part, and the relationship between the sodium chloride solution density | concentration apply | coated to this sodium chloride holding | maintenance part, and a detection sensitivity.

Explanation of symbols

  DESCRIPTION OF SYMBOLS 1 ... Strip for chromatographic analysis, 5 ... Chromatographic member, 10 ... Collecting member, 12 ... Holding member, 12a ... Labeling reagent holding part, 14 ... Detection member, 14a ... -Sodium chloride holding part, 14b ... Capture reagent fixing part, 14c ... Control reagent fixing part, 16 ... Absorbing member, 18 ... Supporting member.

Claims (5)

A strip for chromatographic analysis for detecting a substance to be detected in a liquid sample,
A labeling reagent holder that holds a labeling reagent that specifically binds to the substance to be detected;
A capture reagent immobilization part on which a capture reagent that specifically binds to the substance to be detected is immobilized;
A control reagent immobilization part on which a control reagent that specifically binds to the labeling reagent is immobilized;
A sodium chloride holding part in which sodium chloride is held,
The labeling reagent holding part, the capture reagent fixing part, and the control reagent fixing part are arranged in the longitudinal direction of the chromatography analysis strip so that the liquid sample moves through the chromatography analysis strip in this order by capillary action. Arranged in a direction,
The sodium chloride holding part is provided on the upstream side of the downstream end part in the moving direction of the liquid sample of the control reagent fixing part,
Strip for chromatography analysis. A holding member comprising the labeling reagent holding unit;
A detection member comprising the capture reagent immobilization part and the control reagent immobilization part,
The holding member and the detection member are arranged in the longitudinal direction of the chromatographic analysis strip so that the liquid sample moves in this order by capillary action in these members,
The chromatography analysis strip according to claim 1, wherein the sodium chloride holding part is provided on the detection member or the holding member upstream of the downstream end in the moving direction of the control reagent fixing part. A collecting member to which the liquid sample is added;
A holding member comprising the labeling reagent holding unit;
A detection member comprising the capture reagent fixing part and the control reagent fixing part;
An absorbent member for sucking up the liquid sample;
A liquid-impermeable support member,
The sampling member, the holding member, the detecting member and the absorbing member are arranged on the support member so that the liquid sample moves in this order by capillary action on the supporting member. Arranged in the longitudinal direction,
The chromatography according to claim 1, wherein the sodium chloride holding part is provided in the detection member, the holding member, or the collecting member upstream from the downstream end in the moving direction of the control reagent fixing part. Analytical strip.   The chromatographic analysis strip according to claim 3, wherein the collection member and the holding member share a single fiber substrate.   The strip for chromatographic analysis according to any one of claims 1 to 4, wherein the sodium chloride holding part is in a region within 2.5 cm from an upstream end in the moving direction of the strip for chromatographic analysis. .

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