JP3640354B2 - Specific binding analysis method and device used therefor - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
  The present invention relates to a specific binding analysis method for performing qualitative or quantitative analysis of an analyte in a sample.
[0002]
[Prior art]
  In recent years, with the enhancement of domestic and regional medical care and the increase in urgent clinical tests, the development of specific binding analysis methods that enable quick, simple, and accurate measurement is expected, even if you are not a clinical laboratory specialist. It has come to be desired.
  As specific binding analysis methods, there are many known methods such as immunoassay applying antigen-antibody reaction, receptor assay using receptor, and nucleic acid probe assay using hybridization of complementary nucleic acid sequences. Analytical methods are frequently used in a wide range of fields including clinical tests because of their high specificity.
[0003]
  More specifically, a chromatographic analysis method which is one type of immunoassay is mentioned. In this chromatographic analysis method, for example, a liquid sample is brought into contact with a matrix composed of a porous carrier or a fine particle-packed type in which a specific binding substance is insolubilized (immobilized), and the liquid sample penetrates along the matrix by capillary action. The presence or absence of an analysis object in a sample is analyzed by utilizing the outflow by force (Japanese Patent No. 2504923, Japanese Patent No. 2667793, Japanese Patent Publication No. 7-78503, Japanese Patent Laid-Open No. 10-73592 and (Kaihei 8-240591).
[0004]
  Specifically, a specific binding substance labeled with a labeling material that can be arbitrarily detected by the naked eye or an optical method is subjected to a specific binding reaction. Then, the specific binding substance that has undergone specific binding reaction with the analyte is bound to the binding material immobilized on the matrix, and finally the presence or absence of the analyte in the sample is determined according to the amount of label immobilized on the matrix. Is analyzed.
  In this chromatographic analysis method, since the surface area of the carrier in the matrix is large, a large amount of specific binding substances can be insolubilized, and the collision frequency between reaction molecules that can cause specific binding reactions is higher than that in the reaction in the liquid phase. Therefore, it is advantageous in terms of measurement sensitivity and measurement time.
[0005]
  In the above conventional chromatographic analysis method, it is necessary to use a water-absorbing material capable of developing and transporting a liquid sample by capillary action as a matrix material. Examples of the water-absorbing material include glass fiber filter paper, cellulose membrane, nitrocellulose membrane, and nylon membrane. These are porous and have pores having a pore diameter of about 1 to 50 μm.
  In particular, nitrocellulose is excellent because it has the ability to bind to a large amount of a protein such as an antibody without prior sensitization. Furthermore, since nitrocellulose having various pore sizes can be obtained, the flow rate of the sample can be selected by using this nitrocellulose.
[0006]
[Problems to be solved by the invention]
  However, although the matrix material as described above is composed of a fibrous material, it is difficult to produce it by controlling the pore diameter and the hydrophilicity of the surface with good reproducibility. The average value and distribution state of the pore diameter, and the hydrophilicity of the surface of the fibrous material greatly affect the developing movement speed of the sample, that is, the flow velocity. Since the time during which the specific binding reaction occurs largely depends on this flow rate, the measured value also varies depending on the flow rate change.
[0007]
  That is, since the measured value reacts very sensitively to the characteristics of the matrix material, the measurement accuracy depends on the manufacturing accuracy of the matrix material.
  And it is difficult to improve the manufacturing accuracy of this matrix material until sufficient accuracy of quantitative measurement is ensured. Therefore, there is a problem in that a matrix material selection step is required and costs are increased. Moreover, since the range of the hole diameter and the manufacturing accuracy thereof are limited, the selection range of the sample flow rate is also limited.
[0008]
  Accordingly, an object of the present invention is to provide a specific binding analysis method capable of easily controlling the flow rate in a wide range and having high production reproducibility of the flow rate, and a specific binding analysis device used therefor. According to the present invention, it is possible to expand the selection range of the sample flow rate, and furthermore, the flow rate can be reproduced with high accuracy even at the manufacturing level, so that a highly accurate specific binding analysis device can be realized at low cost.
[0009]
[Means for Solving the Problems]
  In order to solve the above problems, the present invention provides a sample spotting portion for spotting a sample containing an analysis object, a space forming portion exhibiting a capillary phenomenon connected to the sample spotting portion, and the space formation A detection unit capable of detecting a signal obtained from a specific binding reaction in the unit, and the spotted sample moves to the detection unit in the space forming unit by capillarity and specific binding Using a specific binding analysis device that generates a reaction and qualifies or quantifies the analyte by detecting a signal derived from the specific binding reaction,By disposing a breathable member in a portion communicating with the external atmosphere of the space forming portion,Suppresses the speed at which the sample passes through the detectorShiThe specific binding analysis method is characterized in that the time during which the specific binding reaction occurs is controlled so that the intensity of the signal with respect to the same sample becomes substantially constant.In addition, you may suppress the passage speed of the said sample by controlling dimensions, such as the length of a breathable member.
[0010]
  Here, when a fluid sample such as a liquid containing an analysis object passes through the space formation part too early due to capillary action, the analysis object does not contribute to the specific binding reaction in the space formation part without fail. It will pass the detection part. Therefore, according to the present invention, the specific binding reaction is generated by the method described below so that the speed at which the sample passes through the detection unit is suppressed and the intensity of the signal with respect to the same sample becomes substantially constant. It is characterized by controlling the running time.
[0011]
  Furthermore, the specific binding analysis method comprises a step A in which the analysis target is bound to a first specific binding substance that specifically binds to the analysis target and is labeled with a detectable labeling material; Step B, which binds the analyte to the second specific binding substance that specifically binds to the substance and is substantially immobilized on the detection part, and originates from the labeling material generated in the detection part It is preferable to include a step C for measuring the intensity of the obtained signal and a step D for qualitatively or quantitatively determining the analyte in the sample based on the intensity of the signal measured in the step C.
[0012]
  In the step C, it is preferable that the first specific binding substance and the second specific binding substance are bound via the analyte.
  In addition, the first specific binding substance is held on a contact surface of the space forming part between the sample spotting part and the detection part, and the sample is spotted and becomes wet. Accordingly, it is preferable that the first specific binding substance is moved on the contact surface and moved to the detection unit.
[0013]
  It is preferred that the signal is color, fluorescence or luminescence.
  Moreover, it is preferable that at least one of the first specific binding substance and the second specific binding substance is an antibody.
  The labeling material is preferably a metal sol, a dye sol, particles containing a fluorescent substance, or colored latex particles.
[0014]
  Further, the present invention provides a sample spotting part for spotting a liquid containing an analysis object, a space forming part connected to the sample spotting part and exhibiting a capillary phenomenon, and a specific binding reaction in the space forming part. A detection unit that can detect the signal obtained from the sample, and the spotted sample moves to the detection unit in the space formation unit by capillary action, and a specific binding reaction occurs, A specific binding analysis device for qualitatively or quantitatively determining the analyte by detecting a signal derived from a specific binding reaction, further comprising a ventilation resistance control means for suppressing a speed at which the sample passes through the detection unit. It also relates to a specific binding analysis device characterized by this.
[0015]
  Preferably, the ventilation resistance control means is a ventilation resistance arranged in a portion communicating with the external atmosphere of the space forming portion.
  The ventilation resistance is preferably a gas permeable member.
  Furthermore, it is preferable that the contact surface of the space forming part with the sample has hydrophilicity.
[0016]
  The space forming part is composed of two flat plates and a spacer for defining a distance between the flat plates, the detection unit is provided on the flat plate, and detects a signal derived from the specific binding reaction via the flat plate. Is preferred.
  The detection unit is preferably provided by immobilizing a matrix carrier having a large surface area on the flat plate and immobilizing a specific binding substance on the matrix carrier.
[0017]
DETAILED DESCRIPTION OF THE INVENTION
  Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings.
  FIG. 1 is a schematic cross-sectional view showing a configuration of a specific binding analysis device according to an embodiment of the present invention. FIG. 2 is a schematic view of the specific binding analysis device viewed from the Z direction shown in FIG.
  This device has, for example, a glass capillary 1 constituting a space forming part having an inner diameter (d1) of 5 mm and a length (L) of 10 mm, an inner diameter (d2) of 0.5 mm, a length (L) of 3 mm, and an outer The glass capillary tube 2 has a diameter of about 5 mm and serves as a ventilation resistance control means.
[0018]
  As shown in FIG. 1, the glass capillary 2 is inserted into the glass capillary 1. Here, the outer side surface of the glass capillary tube 2 and the inner side surface of the glass capillary tube 1 are in close contact with each other, and air cannot substantially pass therethrough. The glass capillary tube 2 and the glass capillary tube 1 are closely bonded with, for example, an adhesive.
  A detection unit 3 is provided inside the glass capillary tube 1, and the detection unit 3 is formed by fixing a second specific binding substance as a binding material on the inner wall of the glass capillary tube 1. The detection unit 3 is located at a distance (Z1) of about 2 mm from the opening 4 (the side where the glass capillary 2 is not inserted) of the glass capillary 1. Z1 indicates the distance from the end of the opening 4 to the center of the detection unit 3. Moreover, the distance (Z2) from the opening part 4 of the glass capillary tube 1 to the opening part in the glass capillary tube 1 of the glass capillary tube 2 is about 7 mm. In the present embodiment, the opening 4 serves as a sample spotting portion.
[0019]
  The specific binding analysis device shown in FIG. 1 is arranged so that the opening 4 faces downward and the length direction of the device is substantially perpendicular to the horizontal direction, and the opening 4 is brought into contact with the sample. . Then, the sample moves in the Z direction by capillary action. That is, the liquid level of the sample rises.
  At this time, the liquid level of the sample moves (rises) until the vertical component of the surface tension of the sample becomes equal to the gravity acting on the liquid column of the raised sample. When the device shown in FIG. 1 was used and an aqueous solution such as urine was used as a sample in a normal atmospheric pressure and room temperature atmosphere, the moving distance was about 6 mm. At this time, it moved 6 mm in about 30 seconds and stopped.
[0020]
  However, in the absence of the glass capillary tube 2, the moving distance was about 6 mm, but the moving speed increased remarkably and stopped still for 2 to 3 seconds. This is for the following reason.
  As the sample moves upward due to capillary action, the air is pushed by the sample, so the air also moves. Here, if the opposite side of the opening 4 of the glass capillary 1 is completely sealed, the pressure in the capillary 1 rises because air is compressed. Then, the difference between the pressure of the compressed air and the atmospheric pressure, that is, the increased pressure is further added to the gravity, so that the sample stops without increasing by 6 mm.
[0021]
  However, if the opposite side of the opening 4 of the glass capillary 1 is not completely sealed, the compressed air is gradually released by the movement of the sample, so that the pressure inside the glass capillary 1 eventually becomes atmospheric pressure. Since the sample is in an equilibrium state, the sample rises 6 mm and stops. However, the time to stand still increases as compared with the case where the opposite side of the opening 4 of the glass capillary 1 is completely open (the state where there is no glass capillary 2). In other words, the moving speed of the sample decreases. The degree of decrease in the moving speed depends on the ventilation resistance on the opposite side of the opening 4 of the glass capillary 1. Since the ventilation resistance changes depending on the presence or absence of the glass capillary 2, the moving speed also changes depending on the presence or absence of the glass capillary 2. Specifically, when there is no glass capillary 2, the ventilation resistance is reduced and the moving speed is increased as compared with the case where there is no glass capillary tube 2.
[0022]
  From these things, it turns out that the moving speed of the sample can be suppressed by controlling the ventilation resistance on the opposite side of the opening 4 of the glass capillary tube 1. Here, the smaller the inner diameter (d2) of the glass capillary 2 is, that is, the smaller the inner cross-sectional area of the internal space (space forming portion) is, the larger the airflow resistance is. The longer the glass capillary 2 is, the longer the airflow resistance is. Becomes larger. For example, when the inner diameter (d2) of the glass capillary 2 is 1.0 mm and the length (L) is 3 mm, the glass capillary 2 rises 6 mm in 7 to 10 seconds and stops. When the inner diameter (d2) of the glass capillary tube 2 was 0.5 mm and the length (L) was 2 mm, the glass capillary tube 2 rose 6 mm in 20 to 25 seconds and stopped.
[0023]
  This moving speed defines the time during which the specific binding reaction between the analyte and the second specific binding substance in the detection unit 3 occurs. That is, if the moving speed at which the sample passes through the detection unit 3 is large, the collision between the second specific binding substance immobilized on the detection unit 3 and the analyte in the sample passing through the detection unit 3 Reduces the time for interaction. Since the specific binding reaction is generated by this interaction, the time during which the specific binding reaction occurs is also reduced, and the amount of the analyte to be bound by the specific binding is reduced even in the same concentration sample. That is, the specific binding amount decreases as the moving speed increases.
[0024]
  This is because, in the case of a capillary as shown in FIG. 1, the diffusion of the analyte in the length direction (Z direction) of the capillary is substantially negligible at the analysis time as shown in the present invention. It is. In other words, under the above conditions, the specific binding amount substantially depends on the moving speed. However, this is based on the premise that the amount of sample passing through the detection unit 3 is constant.
  As described above, by controlling the ventilation resistance on the opposite side of the opening 4 of the glass capillary tube 1, the rate at which the sample passes through the detection unit 3 can be suppressed, and the specific binding amount can be controlled. As a result, the signal intensity can also be controlled, so that sensitivity and concentration range in analysis can be freely set.
[0025]
  Further, the capillary force when the sample moves in the Z direction by capillary action depends on the contact angle between the sample and the inner wall surface of the glass capillary 1 that is the space forming portion. The contact angle of the sample with respect to the inner wall surface of the glass capillary 1 is 0 degree.
  If this contact angle is different, even if the spatial dimension of the space forming portion is exactly the same, the strength of action of the capillary is also different. Therefore, the moving amount (distance) and moving speed of the sample are also different. That is, since the contact angle differs depending on the hydrophilicity (wetability or wettability with an aqueous solution) of the inner wall surface, the amount and speed of movement of the sample can be suppressed by controlling the hydrophilicity of the inner wall. .
[0026]
  Since the hydrophilicity varies depending on the material of the inner wall of the space forming portion, it can be controlled by selecting the material constituting the capillary tube. For example, when the space forming portion is created using polystyrene or the like, the contact angle is different from that in the case of glass, so that it is possible to realize a moving amount and moving speed of a sample different from those of glass.
  Further, by coating the inner wall surface in contact with the sample with a surfactant, the hydrophilicity can be changed, a contact angle different from the original material can be obtained, and the moving amount and moving speed of the sample can be suppressed.
[0027]
  In the specific binding analysis, after the specific binding substance is immobilized on the detection portion, the inner wall surface other than the detection portion of the capillary tube may be coated with a blocking agent. Thereby, it is preferable to reduce non-specific adsorption to the inner wall surface. Blocking is achieved by treatment with a protein (such as bovine serum albumin or milk protein), polyvinyl alcohol or ethanolamine, or combinations thereof. Skimmed milk can also be used. By selecting or combining the blocking agents, the contact angle can be easily changed, and a desired sample movement amount and movement speed can be realized. In this case, the blocking effect and the desired movement amount and movement speed control can be realized simultaneously, which is efficient.
[0028]
  In addition, the inner wall surface can be subjected to plasma irradiation treatment to modify the hydrophilicity of the inner wall surface, thereby suppressing the moving amount and moving speed of the sample.
  As described above, by controlling the contact angle between the inner wall surface of the glass capillary 1 and the sample, the speed and amount of passage of the sample through the detector 3 can be controlled, and the specific binding amount can be controlled. As a result, the signal intensity can also be controlled, so that sensitivity and concentration range in analysis can be freely set.
[0029]
  Further, the moving amount and moving speed of the sample can also be suppressed by controlling the distance Z1 from the opening 4 to the detecting unit 3. The difference between the vertical component of the surface tension of the sample and the gravity acting on the rising sample liquid column acts on the sample liquid level rising due to the capillary phenomenon. Therefore, during the ascent, the force acting on the liquid level of the sample decreases as the amount of the sample contained in the space forming portion increases as the ascent height increases. And when moving to the predetermined height, the moving speed becomes zero.
[0030]
  From these, by controlling the distance (Z1) from the opening 4 of the detection unit 3, the speed at which the sample passes over the detection unit 3 can be suppressed. That is, if the distance Z1 is increased, the passage amount and the passage speed can be reduced, and if the distance Z1 is reduced, the passage amount and the passage speed can be increased.
  As described above, by controlling the distance Z1 between the detection unit 3 and the opening 4, the speed and amount of passage of the sample through the detection unit 3 can be controlled, and the specific binding amount can be controlled. As a result, the signal intensity can also be controlled, so that sensitivity and concentration range in analysis can be freely set.
[0031]
  Here, FIG. 5 shows an exploded perspective view showing a configuration of a specific binding analysis device according to another embodiment of the present invention. As shown in FIG. 5, this specific binding analysis device includes a substrate 16 made of glass or resin, spacers 17 and 18 having a thickness (x direction) made of glass, resin, metal, or the like (x-direction) of about 250 μm, glass fiber filter paper GA200 (Toyo Co., Ltd.) A breathable member 19 having a thickness (x direction) of about 250 μm and a transparent substrate 20 made of glass or resin. Further, on the substrate 16, an anti-hCG monoclonal antibody that can participate in a sandwich reaction with hCG as the second specific binding substance is immobilized to form a detection unit 21.
[0032]
  As shown in FIG. 6, the transparent substrate 20 is overlapped with the substrate 16 with the spacers 17 and 18 interposed therebetween. As a result, the substrate 16, the spacers 17 and 18, and the transparent substrate 20 constitute a space forming portion. And the sample spotting part 22 which can introduce a test sample into this space formation part simultaneously is comprised.
[0033]
  In the specific binding analysis device shown in FIGS. 5 and 6, the ventilation resistance is the thickness of the spacers 17 and 18 (x direction), the density of the glass fiber filter paper in the breathable member 19, the breathable member 19 and the spacer 17 and 18 or the length of the gap (z direction) or the like.
  Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited to these examples.
[0034]
【Example】
<< Example 1 and Comparative Example 1 >>
  In this example, the specific binding analysis device according to the present invention shown in FIG. 1 described above was used, and human chorionic gonadotropin (hCG) in urine was analyzed as an analysis target.
  An anti-hCG monoclonal antibody capable of participating in a sandwich reaction with hCG was used as the first specific binding substance and the second specific binding substance, and gold colloid was used as the labeling material. Here, when colored particles such as gold colloid are used, since the colored particles are fine, the label can be concentrated in a small area or volume, and the label of the first specific binding substance is detected in the detection unit 3. Qualitative or quantitative analysis of hCG could be performed accurately using signals derived from the reaction contributed by the colloidal gold. The inner wall of the glass capillary tube 1 was blocked by passing an aqueous dispersion of skim milk.
[0035]
  First, a mixed solution of urine and anti-hCG monoclonal antibody labeled with colloidal gold was prepared, and this mixed solution was used as a sample. In the sample in this state, hCG as an analysis target was bound to an anti-hCG monoclonal antibody labeled with gold colloid. When this sample was spotted on the opening 4 which is the sample spotting portion, the sample rose by capillary action, and the upper surface of the sample moved about 5 mm in about 30 seconds and stopped.
[0036]
  At this time, the analyte in the sample passing through the detection unit 3 was specifically bound to the second specific binding substance. Thereby, the analysis object was fixed to the detection unit 3 via the second specific binding substance. That is, the anti-hCG monoclonal antibody that is the first specific binding substance labeled with colloidal gold is the anti-hCG monoclonal antibody that is the second specific binding substance immobilized on the detection unit 3 via the analyte hCG. Combined. As a result, the detection unit 3 developed color according to the concentration of the analysis object hCG.
[0037]
  Samples of various concentrations are prepared by adding hCG to the control urine for accuracy control where the hCG concentration is substantially zero, and the degree of coloration by the gold colloid in the detection unit 3 is confirmed based on the principle as described above. did. The hCG concentration of each sample is 0 (IU / L), 30 (IU / L), 100 (IU / L), 300 (IU / L), 1000 (IU / L), 3000 (IU / L) and 10,000 (IU / L). As a result, color development could be confirmed when a sample having an hCG concentration of 300 (IU / L) or higher was used.
[0038]
  Next, a similar experiment was performed when the glass capillary 2 was not inserted. In this case, the sample rose due to capillary action, and the upper surface of the sample moved about 5 mm in about 2-3 seconds and stopped. Color development was confirmed only when a sample having a hCG concentration of 10,000 (IU / L) was used.
[0039]
  As described above, according to the specific binding analysis device according to the present embodiment, by controlling the ventilation resistance on the opposite side of the opening 4 of the glass capillary 1, the speed at which the sample passes through the detection unit 3 is suppressed. It was possible to control the amount of specific binding. In addition, since the signal intensity could be controlled by this, the sensitivity and concentration range in analysis could be set freely.
[0040]
  In this example, the first specific binding substance labeled with colloidal gold and urine were mixed before spotting, but the first specific binding substance was mixed with the opening that was the sample spotting part. 4 and the detection unit 3 may be held in a dry state. As a result, urine can be spotted on the opening 4 as it is and analyzed. In this case, the urine, which is a liquid sample, allows the first specific binding substance held in a dry state to be wet and move freely, and the analyte and the first specific binding substance are bound. In the state, it moves to the detection part 3 and can also make the color development according to a density | concentration similarly.
[0041]
Example 2
  Next, human chorionic gonadotropin (hCG) in urine was analyzed as an analysis object using the specific binding analysis device according to the present invention shown in FIG.
  In FIG. 3, the glass capillary tube 1, the detection unit 3, and the opening 4 that is the sample spotting unit are the same as those in FIG. 1. However, a fibrous breathable member 5 was inserted on the opposite side of the opening 4 of the glass capillary 1 to control the ventilation resistance on the opposite side of the opening 4 of the glass capillary 1. In this example, glass fiber filter paper GA200 (manufactured by Toyo Corporation) was processed into dimensions of 0.5 mm in diameter and 3 mm in length to obtain a breathable member 5. The inner wall of the glass capillary 1 was blocked with skim milk. FIG. 4 is a schematic view of the specific binding analysis device viewed from the Z direction shown in FIG.
[0042]
  Also in this example, a mixed solution of urine and anti-hCG monoclonal antibody labeled with colloidal gold was prepared in the same manner as in Example 1, and this mixed solution was spotted on the opening 4 as a sample. The sample moved 5 mm in about 120 seconds due to capillary action and stopped.
  Similarly to Example 1, hCG is added to the control urine for accuracy control whose hCG concentration is substantially zero to prepare samples of various concentrations, and the color development degree by the gold colloid in the detection unit 3 is adjusted. confirmed. The hCG concentration of each sample is 0 (IU / L), 30 (IU / L), 100 (IU / L), 300 (IU / L), 1000 (IU / L), 3000 (IU / L) and It was set to 10,000 (IU / L). As a result, color development could be confirmed when a sample having an hCG concentration of 100 (IU / L) or more was used.
[0043]
  Further, by changing the length of the air-permeable member 5, the time until the sample rises in the glass capillary tube 1 and stops still, that is, the moving speed can be changed, and the degree of color development for each density can be adjusted. We were able to.
  As described above, according to the specific binding analysis device according to the present embodiment, the sample controls the detection unit 3 by controlling the ventilation resistance on the opposite side of the opening 4 of the glass capillary tube 1 with the breathable member. The passing speed could be suppressed and the amount of specific binding could be controlled. Thereby, since the signal intensity can also be controlled, the sensitivity and concentration range in the analysis can be set freely.
[0044]
Example 3
  In this example, the blocking agent of Example 2 was replaced with bovine serum albumin. When the same experiment as in Example 2 was performed, the sample moved by 4 mm in about 120 seconds due to capillary action and stopped. When a sample having an hCG concentration of 300 (IU / L) or higher was used, color development could be confirmed.
  Further, when the inner wall of the glass capillary 1 was treated with various surfactants, the amount of movement and the movement speed could be changed, and the degree of color development with respect to the density could be controlled.
[0045]
  As described above, according to the specific binding analysis device according to the present embodiment, by controlling the hydrophilicity of the inner wall of the glass capillary 1, it is possible to suppress the speed at which the sample passes through the detection unit 3, The amount of specific binding could be controlled. As a result, the signal intensity can also be controlled, so that the sensitivity and concentration range in the analysis can be freely set.
[0046]
Example 4
  In this example, the same operation was performed using the same specific binding analysis device as in Example 2 except that the dimension of Z1 was 3 mm. The sample moved by 5 mm in about 120 seconds and became stationary due to capillary action as in Example 2. However, since the amount of sample passing through the detection unit 3 and the passage speed are different, the color development characteristic different from that of Example 2 was shown with respect to the concentration.
[0047]
  As described above, according to the specific binding analysis device according to the present embodiment, the speed and amount of passage of the sample through the detection unit 3 are controlled by controlling the distance Z1 between the detection unit 3 and the opening 4. It was possible to control the amount of specific binding. As a result, the signal intensity can also be controlled, so that the sensitivity and concentration range in the analysis can be freely set.
[0048]
Example 5
  In this example, using the specific binding analysis device according to the present invention shown in FIGS. 5 and 6, human chorionic gonadotropin (hCG) in urine was analyzed as an analysis object. An anti-hCG monoclonal antibody capable of participating in a sandwich reaction with hCG was used as the first specific binding substance and the second specific binding substance, and gold colloid was used as the labeling material. Further, after the detection unit 21 was created, the inner walls of the substrate 16 and the transparent substrate 20 were blocked by applying and drying an aqueous dispersion of skim milk.
[0049]
  First, a mixed solution of urine and anti-hCG monoclonal antibody labeled with colloidal gold was prepared, and this mixed solution was used as a sample. In the sample in this state, hCG as an analysis target was bound to an anti-hCG monoclonal antibody labeled with gold colloid. This sample was placed so that the length direction (Z direction) of the specific binding analysis device was substantially perpendicular to the horizontal direction, and was spotted on the opening which is the sample spotting portion 22. After spotting, the sample rose by capillary action and passed through the detection unit 21. Then, after about 2 minutes, the uppermost part of the liquid level stopped without reaching the air-permeable member 19.
[0050]
  In the detection unit 21, the analyte in the spotted sample specifically bound to the second specific binding substance. Thereby, the analysis object was fixed to the detection unit 3 via the second specific binding substance. That is, an anti-hCG monoclonal antibody that is a first specific binding substance labeled with colloidal gold is an anti-hCG monoclonal antibody that is a second specific binding substance immobilized on the detection unit 21 via an analyte hCG. Combined. Thereby, the detection part 21 developed color. When the concentration of the analyte hCG was changed, color was developed according to the concentration. It was also confirmed that the color development characteristics can be controlled by changing the density of the air-permeable member 19.
[0051]
  As described above, according to the specific binding analysis device according to the present embodiment, by controlling the ventilation resistance on the opposite side of the sample spotting portion 22 in the space forming portion, the speed at which the sample passes through the detection portion 21 is increased. The color development characteristics could be controlled. In particular, in the present embodiment, the detection unit 21 can be created by superimposing flat plates, so that the specific binding analysis device can be easily manufactured.
[0052]
Example 6
  In the present embodiment, the detection unit 21 is not directly installed on the flat plate 16, but first, glass fiber or the like is fixed on the flat plate 16, and further participates in the sandwich reaction with the second specific binding substance hCG. The same operation as in Example 5 was performed except that the detection unit 21 was formed by immobilizing the obtained anti-hCG monoclonal antibody on the glass fiber.
  According to this example, an anti-hCG monoclonal antibody that can participate in a sandwich reaction with a larger amount of the second specific binding substance hCG than in Example 5 can be immobilized on the detection unit 21, and the degree of color development. Was able to be enlarged.
[0053]
【The invention's effect】
  As described above, according to the specific binding analysis method of the present invention and the device used therefor, the amount and speed of the sample passing through the detection unit can be suppressed, and the sensitivity and concentration in the analysis can be controlled by controlling the specific binding amount. The range can be set freely. That is, the present invention is extremely effective in practice.
[Brief description of the drawings]
FIG. 1 is a schematic cross-sectional view showing a configuration of a specific binding analysis device according to an embodiment of the present invention.
FIG. 2 is a view of the specific binding analysis device shown in FIG. 1 as viewed from the Z direction.
FIG. 3 is a schematic cross-sectional view showing a configuration of a specific binding analysis device according to another embodiment of the present invention.
4 is a view of the specific binding analysis device shown in FIG. 3 as viewed from the Z direction. FIG.
FIG. 5 is an exploded perspective view showing a configuration of a specific binding analysis device according to another embodiment of the present invention.
6 is a partially transparent perspective view after assembly of the specific binding analysis device shown in FIG.
[Explanation of symbols]
      1 Glass capillary 1
      2 Glass capillary 2
      3 detector
      4 openings
      5 Breathable members

Claims (9)

A sample spotting part for spotting a sample containing an analysis object, a space forming part exhibiting a capillary phenomenon connected to the sample spotting part, and a specific binding reaction in the space forming part. The spotted sample is moved to the detection part in the space forming part by capillary action to cause a specific binding reaction, and is derived from the specific binding reaction. Using a specific binding analysis device that qualifies or quantifies the analyte by detecting a signal,
Furthermore, by arranging a breathable member in a portion of the space forming part that communicates with the external atmosphere, the speed at which the sample passes through the detection unit is suppressed, and the intensity of the signal with respect to the same sample is substantially constant. And controlling the time during which the specific binding reaction occurs. A step A for binding the analyte to the first specifically binding substance that specifically binds to the analyte and is labeled with a detectable labeling material; specifically binds to the analyte; and Step B for binding the second specific binding substance substantially immobilized on the detection unit and the analyte, and measuring the intensity of the signal generated in the detection unit and obtained from the labeling material step C, and based on the intensity of the measured signal in the step C, specific bonding analysis method according to claim 1 Symbol mounting and having a step D of qualitative or quantitative analyte in the sample. 3. The specific binding analysis method according to claim 2 , wherein in the step C, the first specific binding substance and the second specific binding substance are bound via the analysis target. By holding the first specific binding substance on the contact surface of the space forming part between the sample spotting part and the detection part with the sample, the sample is spotted and becomes wet. , specific bonding analysis method according to any one of claims 1 to 3, characterized in that moving the first specific binding substance to the detection unit by mobilization at the contact surface. A sample spotting part for spotting a sample containing an analysis object, a space forming part exhibiting a capillary phenomenon connected to the sample spotting part, and a specific binding reaction in the space forming part. The spotted sample is moved to the detection part in the space forming part by capillary action to cause a specific binding reaction, and is derived from the specific binding reaction. A specific binding analysis device for qualitatively or quantifying the analyte by detecting a signal,
Furthermore, the specific binding analysis device characterized by comprising a ventilation resistance control means for suppressing the speed at which the sample passes through the detection section. The specific binding analysis device according to claim 5, wherein the ventilation resistance control means is a ventilation resistance arranged in a portion communicating with the external atmosphere of the space forming portion. The specific binding analysis device according to claim 6 , wherein the ventilation resistance is a gas permeable member. The space forming part is composed of two flat plates and a spacer for defining a distance between the flat plates, the detection unit is provided on the flat plate, and detects a signal derived from the specific binding reaction via the flat plate. The specific binding analysis device according to claim 5 . 9. The specific binding analysis device according to claim 8 , wherein the detection unit is provided by immobilizing a matrix carrier having a large surface area on the flat plate and immobilizing a specific binding substance on the matrix carrier.

Images