JP2004233114A - Antigen separator, and method and instrument for measuring antigen by using the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an antigen separator for eliminating a problem of foreign matter clogging in antigen separation and securing the amount of treated fluid meeting the need, and to perform the shortening of antigen measurement time and high-accuracy measurement by performing image measurement on antigens separated by using the separator. <P>SOLUTION: This antigen separator is equipped with a specimen inlet 1 for a specimen fluid including antigens, a reaction part 3 used for separating the antigens by antigen/antibody reaction and made by disposing a plurality of antibody beads 2 on a bottom surface of a micro-channel having a rectangular cross-section through which the specimen fluid flows, a specimen-liquid inlet path 4 for causing the inlet to communicate with the reaction part, and an exhaust path 5 for the specimen fluid. The inlet path 4 is substantially equipped with a difference 4a in flow path level from the reaction part so that it is equipped with an antigen condensing function in the vicinity of the bottom surface, and/or, the reaction part 3 is equipped with an electric field generating means 30 for attracting the antigens by an electric attracting force to the bottom surface side where the antibody beads are disposed. An image measuring instrument is provided above the reaction part. <P>COPYRIGHT: (C)2004,JPO&NCIPI

Description

[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to an apparatus for separating antigens such as bacteria and in-vivo proteins in a sample, and a method and apparatus for measuring an antigen using the apparatus. Objects to be separated and measured include all substances that can be measured using antigen-antibody reactions, such as prokaryotes such as bacteria and actinomycetes, eukaryotes such as yeasts and molds, and microorganisms such as lower algae and viruses. And cultured cells derived from animals and plants and cells such as pollen such as cedar and cypress. In addition, immunoanalysis, detection of endocrine disrupting substances (environmental hormones), characteristics of causative substances of atopic dermatitis, etc. are also targeted. The target sample is more often a liquid sample than a gas sample, but also includes, for example, measurement of antigens of a gas sample such as bacteria and fungi floating in the air. The fields of application of the present invention include medicine, food production, water and sewage, environmental analysis, and the like.
[0002]
[Prior art]
For example, detection of microorganisms, tissue cells such as animals and plants in a sample, and the like is an industrially important technique for confirming a sterilized state and detecting abnormalities in the survival state of cells.
[0003]
Conventionally, in order to observe and count microorganisms such as bacteria that cannot be observed with the naked eye present on the test surface, a culture method, that is, pressing a solid plate medium formed on agar or the like against the test surface, A method is generally used in which a microorganism on the test surface is transferred onto an agar plate medium, and the microorganism is cultured as it is on a plate medium under an appropriate environment, and the colonies that appear are measured with the naked eye or a stereoscopic microscope while measuring the colony. Has been used in many ways.
[0004]
The culture method has a drawback that the measurement time is 48 to 72 hours including various preparations. In recent years, a method for measuring an image by labeling an antigen with a fluorescent reagent using an antigen-antibody reaction has been developed. ing. For example, a microorganism collected on a filter is brought into contact with an appropriate staining solution, and the number of colored cells is counted with a microscope or the like, thereby detecting the microorganism without culturing.
[0005]
According to the image measurement method, measurement can be performed in two to three hours. The time required for the measurement is, for example, about 70 minutes for antigen-antibody reaction and magnetic separation of antibody magnetic beads from dust in the sample solution, about 30 minutes for fluorescent staining, and about 30 minutes for microscopic observation. It becomes 3.
[0006]
By the way, as an image analysis method in the measurement method, image analysis is generally performed using a manually focused microscope or an imaging device, and the depth of field is narrow under high-magnification use conditions. In many cases, automatic focusing and automatic analysis are desired. From this viewpoint, a fluorescent image measurement method invented by the present applicant has been filed in Japanese Patent Application No. 2002-30648 regarding a method of performing fluorescent image measurement by performing automatic focusing.
[0007]
FIG. 6 shows an example of an apparatus for performing the method described in Japanese Patent Application No. 2002-30648. According to the apparatus shown in FIG. 6, before the sample 81 is irradiated with the excitation light from the excitation light source 80, the autofocus (AF) light emitted in the fluorescence image measurement wavelength band is changed to the excitation light as shown in the drawing. Irradiation by the light source 82 from the same side as the irradiation side, the degree of focusing is determined from the image information obtained thereby, and at least one of the sample 81 and the light receiving system is driven in accordance with the degree to search for the focal point position. When the focus position is reached, the irradiation of the AF light is stopped, and thereafter, the excitation light is irradiated from the light source 80 to the sample 81, so that the fluorescence image measurement can be performed. According to this apparatus, since the transmitted light is not used, it is possible to measure a sample captured on the surface of the membrane filter.
[0008]
As the AF light source 82, a light emitting diode or a semiconductor laser is preferable. The image of the specimen at the time of the AF light irradiation is captured by the image sensor 87 via the objective lens 85, the dichroic mirror 83, the fluorescent light receiving side filter 84, and the imaging lens 86. As the imaging element, a CCD camera element and a CMOS camera element are preferable. The image obtained by the image sensor 87 is sent to the arithmetic unit 88, where the contrast is evaluated. The evaluation of the contrast is performed by a general AF method, for example, calculating as a luminance difference between adjacent pixels and setting a position where the contrast becomes maximum as a focal point position.
[0009]
In FIG. 6, 89 denotes a stage moving mechanism, 91 denotes a condenser lens for excitation light, 92 denotes a filter, and 93 denotes a fluorescent filter block. Although not shown in FIG. 6, a focusing marker for evaluating the contrast is provided with a pattern (mark) on the surface of a slide glass holding a sample, or a membrane for filtering and capturing the sample. A method of providing a pattern (mark) on the surface of the filter is employed (for details, refer to the aforementioned Japanese Patent Application No. 2002-30648). According to the above method, a simple configuration with a small number of elements does not cause the sample to be quenched by irradiation with excitation light, making it undetectable, and also autofocus even for a sample supplemented on the surface of the membrane filter or a sample whose contrast is unclear. (AF) becomes possible.
[0010]
Further, in the fluorescence image measurement method, it is desirable to eliminate the influence of fluorescent impurities regardless of the properties of the sample, and to improve the measurement accuracy. From this viewpoint, the live cells of the present invention invented by the present applicant are desirable. A counting method and apparatus have been filed in Japanese Patent Application No. 2002-148888.
[0011]
The image measurement device described in Japanese Patent Application No. 2002-148888 performs image measurement based on the difference between the image information of the living cells after the fluorescent labeling and the image information before the fluorescent labeling, and removes the fluorescence error of foreign substances. It has functions, and the above application discloses three methods.
[0012]
That is, the first method is to label living cells such as microorganisms and cell tissues in a sample with a fluorescent reagent, and to count the number of living cells in a method and apparatus for counting living cells. Before performing the method, a fluorescent image (first image) of the sample is obtained, and after the living cells are fluorescently labeled, a fluorescent image (second image) of the sample is obtained. The difference (B-A) in the number of bright points is determined. Further, as a method different from the above, a second method of obtaining a difference image between the first image and the second image and calculating the number of bright points in the difference image, or a method of calculating the number of bright points in the second image Of these, the third method is to determine the number of bright spots whose positions are not included in the dead area associated with each bright spot in the first image (for details, see Japanese Patent Application No. 2002-148888).
[0013]
FIG. 7 is a diagram illustrating, as an example, a method for counting the number of bacteria according to the second method, which utilizes obtaining a difference image between a first image and a second image.
[0014]
As a procedure, for example, bacteria contained in the sample solution are captured on a membrane filter by filtration. Next, a fluorescence image (first image 96 shown in the center of FIG. 7) of the membrane filter capturing the bacteria before fluorescence labeling is obtained. Subsequently, a fluorescent labeling reagent is added onto the membrane filter, and the bacteria are fluorescently labeled. Thereafter, a fluorescence image of the membrane filter (the second image 97 shown on the left side of FIG. 7) is acquired again. Then, a difference image 98 shown on the right side of FIG. 7 is obtained from the second image 97 and the first image 96.
[0015]
The bright spots present in the difference image 98 are the bright spots appearing by the fluorescent labels, and by counting them as the number of bacteria, it is possible to remove the fluorescent error of the foreign matter.
[0016]
By the way, there is also a need for an improved fluorescence image measurement method as described above so that measurement can be performed more easily, in a short time, and with high accuracy. Further, depending on the measurement target, it is desired that measurement can be performed with a small amount of sample. From such a viewpoint, an immunoanalyzer and a method for measuring a small amount of a sample by causing an antigen-antibody reaction in a microchip are known from Patent Document 1.
[0017]
FIG. 8 is a perspective view of the immunological analyzer described in Patent Document 1. According to the description of Patent Document 1, the immunoassay apparatus shown in FIG. 8 has solid microparticles 102 having a diameter of 1 mm or less as a reaction solid phase, and a microchannel reactor having a cross-sectional area larger than the diameter of the solid microparticles 102. 103 and a microchannel separation section 104 having a cross-sectional area smaller than the diameter of the solid fine particles 102, and an introduction section or microchannel inflow sections 105 and 106 for separately leading the antigen and the labeled antibody to the reaction tank section 103. An immunoassay microchip having the above is constructed and analyzed using this.
[0018]
In FIG. 8, a microchannel inflow portion 106 for introducing a first antibody together with a microchannel inflow portion 106 for introducing a labeled antibody as a second antibody into a reaction tank portion 103, and introduction of a buffer solution and a washing solution And microchannel inlets 105, 106, 107, and 108. At the end of each of the microchannel inlets 105, 106, 107, and 108, an antigen, a labeled antibody (second antibody), a first antibody, and a washing solution are injected. Holes 105A, 106A, 107A and 108A are also provided. In the example of FIG. 8, a waste liquid part 104A is provided at an end of the microchannel separation part 104.
[0019]
The solid fine particles 102 serve as a reaction solid phase for an immune antigen-antibody reaction. For example, glass beads or polymer beads such as polystyrene are used. The solid fine particles 102 have a diameter of 1 mm or less, for example, 15 to 85 μm.
[0020]
With the use of the above-described immunoassay microchip, immunoassay can be easily performed with a short reaction time by using a small amount of a sample. As an immunoassay method, the solid fine particles 102 as a reaction solid phase are introduced into the microchannel reaction tank 103, and the antigen and the labeled antibody introduced from the introduction section or the microchannel inflow sections 105 and 106, and if necessary, the microchannel inflow. The antibody introduced from the section 107 is reacted on the solid fine particles 102, and the unreacted substance is separated by the microchannel separation section 104, and can be analyzed by photothermal conversion analysis, fluorescence analysis, or the like.
[0021]
[Patent Document 1]
JP 2001-4628 A (page 3-4, FIG. 1)
[0022]
[Problems to be solved by the invention]
By the way, the invention described in Patent Document 1 also relates to a device for separating an object to be measured, and has the following problems.
[0023]
In the case of the apparatus of Patent Literature 1, specifically, as described in Patent Literature 1, "the size of the microchannel Is 100 μm or more, more preferably 150 μm or more, and the microchannel separation portion 104 is, for example, 10 μm or less in depth and 10 μm or less in width. Does not flow into the microchannel separation unit 104 and is blocked, and only unreacted substances flow into the microchannel separation unit 104 and are separated. Only the reaction products desorbed from are separated. "
As described in Patent Literature 1, the separation device of Patent Literature 1 uses a “dam” method. Therefore, when there is “dust” in the sample liquid, the dust condensed in the portion narrowed to 10 μm or less. There is a problem that measurement is not easy.
[0024]
In addition, when the sample to be measured is relatively homogeneous, highly accurate detection is possible with a very small amount of sample, but as the sample to be measured, for example, a sample solution in which the number of bacteria contained per unit volume is small However, when the distribution in the liquid is not uniform, the amount of the liquid to be measured needs to be a certain amount, and if the amount of the liquid is too small, the measurement accuracy is reduced or the measurement is not performed. Becomes virtually impossible.
[0025]
The present invention has been made in view of the above points, and an object of the present invention is to prevent the separation of antigens in a sample fluid without the clogging of the microchannel with dust in the liquid, and to achieve the separation. Provide an antigen separation device that can be made simple and in a short time and a fluid throughput can be secured as needed. Further, an antigen that can be easily, quickly and accurately measured using this separation device To provide a measuring method and apparatus.
[0026]
[Means for Solving the Problems]
In order to solve the above-mentioned problems, the present invention provides an inlet for a sample fluid containing an antigen such as a bacterium or an in vivo protein, and a plurality of antibodies on a bottom surface in a microchannel having a rectangular cross section through which the sample fluid flows. A carrier is disposed, a reaction section for separating the antigen by an antigen-antibody reaction, a microchannel-shaped sample fluid introduction path communicating between the sample fluid inlet and the reaction section, A discharge path for discharging a sample fluid, wherein the introduction path generates a vector of fluid flow in a direction perpendicular to the bottom surface, and reduces the dispersion concentration of the antigen in the sample fluid near the bottom surface. The reaction section and the flow path step are substantially provided so as to locally increase the pressure (provide an antigen concentration function) (the invention of claim 1).
[0027]
The “rectangular cross section” in the present invention is a polygonal cross section in which one side opposite to the bottom side is parallel, and includes a cross section such as a trapezoid or a hexagon.
[0028]
In order to separate an antigen by an antigen-antibody reaction, it is necessary to concentrate the antigen near an antibody carrier (for example, an antibody bead). For example, it is necessary to concentrate from antibody beads to about 20 to 30 μm, preferably to about 10 μm as in a conventional apparatus. According to the above-described separation apparatus, the solid matter in the liquid is concentrated without damming or restricting the flow path of the sample liquid as in the related art, and only the specific antigen is captured and separated by the antibody beads in the reaction part, and the remaining part is separated. Since dust can be discharged from the reaction section, the problem of dust clogging can be solved, and antigen can be separated easily and in a short time. Further, for example, a processing amount of about 10 mL can be secured in 10 minutes, so that various needs can be met. Details will be described later.
[0029]
The antigen concentrating function of the first aspect of the present invention can also be achieved as in the second aspect of the present invention. That is, in the separation device according to claim 1, the introduction path having the antigen concentrating function is configured such that the sample fluid and the fluid such as air or water are in the same horizontal plane, instead of the introduction path substantially having the flow path step. At the same time, and has a configuration in which the antigen concentration function is provided by layer separation of a fluid layer of air, water or the like perpendicular to the bottom surface and a sample fluid layer. Since the flow velocity of the processing fluid is sufficiently small, the Reynolds number determined by the velocity, viscosity, density, and effective diameter is a condition under which the above-mentioned layer separation is possible, and can be concentrated with a simple structure. Details will be described later.
[0030]
Further, the antigen concentrating function of the first aspect of the present invention can also be achieved as in the third aspect of the present invention. That is, a plurality of antibody carriers are disposed on an inlet of a sample fluid containing an antigen such as a bacterium or an in-vivo protein, and a bottom surface in a microchannel having a rectangular cross section through which the sample fluid flows. An antigen having a reaction section for separating the antigen according to the above, an introduction path for the microchannel-shaped sample fluid communicating the inlet for the sample fluid and the reaction section, and a discharge path for discharging the sample fluid from the reaction section. In the separation device of (1), the reaction section includes an electric field generating means for sucking the antigen by an electric suction force on the bottom surface side on which the antibody carrier is provided.
[0031]
Since the antigen is negatively charged in its natural state, the bottom surface on which the antibody carrier is disposed is used as a positive electrode, and an electric field is generated using the opposite surface as a negative electrode. Is concentrated by suction. The electric field can be formed by an alternating electric field.
[0032]
Furthermore, according to the fourth aspect of the present invention, the concentration function is further improved. That is, in the separation device according to the third aspect, the introduction path has the antigen concentration function according to any one of the first and second aspects.
[0033]
5. The separation device according to claim 1, wherein the introduction path is an injection port for injecting a fluorescent staining reagent into the sample fluid between the introduction port and the reaction unit. 6. (The invention of claim 5). As a result, as described later, the antigen can be measured by observing the fluorescent image.
[0034]
Furthermore, in the separation apparatus according to any one of claims 1 to 5, the separation apparatus main body includes a member including a flow path such as an inlet, an introduction path, a reaction section, and a discharge path of the sample fluid; The light-transmitting lid member is bonded together (the invention of claim 6). The separation device includes various peripheral devices such as a pipe, a sealing device, and a control valve for introducing a sample solution, a fluorescent staining reagent, and the like, and the separation device main body is configured as in the above-described claim 6. Thereby, the main body of the separation device can be made into a chip, and the productivity can be improved and the cost can be reduced. Since the tip can be disposable, the mobility and maintainability of the device are also improved.
[0035]
Further, in order that the sample solution contains various kinds of antigens and to simultaneously separate them, the invention of the following claim 7 is preferable. That is, the separation device according to any one of claims 1 to 6, further comprising a plurality of the reaction units, wherein each reaction unit is an antibody carrier on which various types of antibodies are fixed or an antibody different for each reaction unit. The introduction channel is configured such that the sample fluid flows in series or in parallel through the respective reaction sections, and separates each antigen in the sample fluid containing various types of antigens. Configuration. As described above, by using a multichip provided with a plurality of reaction units, measurement with high versatility and low cost can be performed. Details will be described later.
[0036]
Next, the following inventions 8 to 11 are preferable as an antigen measuring device using the separation device. That is, an antigen measurement device using the antigen separation device according to claim 5, wherein the separation device includes an image measurement device that performs image measurement based on fluorescence image information of the separated antigen ( The invention of claim 8). As a result, the measurement time, which conventionally required 2 to 3 hours, may be significantly reduced to 25 to 30 minutes because there is no magnetic separation operation.
[0037]
As an embodiment of the invention of claim 8, the following inventions of claims 9 to 11 are preferable. That is, in the antigen measuring device according to claim 8, the image measuring device includes a focusing marker and includes a focusing unit that performs autofocus on the antigen to be measured (claim 9). Invention). This operation and effect are as described above.
[0038]
Furthermore, in the antigen measuring device according to the ninth aspect, the focusing marker is a plurality of antibody carriers disposed on the bottom surface of the reaction part (the tenth aspect). When, for example, polystyrene beads are used as the antibody carrier, the beads arranged on the bottom surface can function as focusing markers, so that it is not necessary to provide markers again.
[0039]
Furthermore, in the antigen measuring device according to any one of claims 8 to 10, the image measuring device is configured to perform a measurement based on a difference between image information after fluorescent labeling of the antigen and image information before fluorescent labeling. It has an arithmetic function for performing image measurement and removing the fluorescence error of the contaminant (the invention of claim 11). This operation and effect are as described above.
[0040]
Next, as a method for measuring an antigen, the invention of the following claim 12 or 13 is preferable. That is, a method for measuring an antigen using the antigen measuring device according to any one of claims 8 to 10 includes the following steps (the invention of claim 12).
1) A step of flowing a sample fluid containing an antigen at a predetermined flow rate to the reaction section of the antigen separation device.
2) a step of concentrating the antigen in the reaction section with or without applying an electric field, and adsorbing the antigen to the antibody carrier by an antigen-antibody reaction;
3) a step of injecting a fluorescent reagent into the sample fluid to fluorescently label an antigen;
4) a step of flowing the washing solution to wash the fluorescent reagent;
5) a step of measuring the image of the antigen using a fluorescent image device;
[0041]
In the step 3), when the sample fluid is a gas, it is preferable that the fluorescent staining reagent is injected in the form of a mist.
[0042]
Also, in the method for measuring an antigen using the antigen measuring device according to the eleventh aspect, the fluorescent label according to the step of the third aspect according to the twelfth aspect in addition to the step of the twelfth aspect. The image measurement is also performed before the step of performing the step (b), and the image measurement of the antigen is performed based on the difference between the image information obtained by the image measurement according to the step (5) according to claim 12 and the image information obtained before the fluorescent label ( The invention of claim 13).
[0043]
BEST MODE FOR CARRYING OUT THE INVENTION
With respect to the embodiment of the present invention, an example of measuring bacteria in a liquid sample will be described below with reference to FIGS. First, an antigen separation device will be described.
[0044]
FIG. 1 is a conceptual schematic diagram of a main body of an antigen separation device according to the first, third and fourth aspects of the present invention. FIG. 2 is a schematic configuration diagram of the antigen measuring device, and also shows a perspective cross-sectional view of a specific configuration of the separation device main body. FIG. 2A is an exploded perspective sectional view of a lower member and a lid member, and FIG. 2B is a perspective sectional view of an assembled state.
[0045]
1 and 2, 1 is an inlet for a sample solution, 2 is an antibody bead made of, for example, polystyrene, 3 is a reaction section, 4 is a sample solution introduction path, 5 is a sample solution discharge path, and 6 is a fluorescent staining reagent. Reference numeral 7 denotes a sample liquid flow path including the inlet, the introduction path, the reaction section, and the discharge path. In FIG. 1, reference numeral 30 denotes an electric field generating means, and in FIG. 2, 8 denotes a molded member, 9 denotes a cover member, 10 denotes a fluorescent staining reagent, and 20 denotes an image measuring device.
[0046]
The operation of separating the antigen will be described with reference to FIG. The sample liquid introduced into the introduction path 4 from the introduction port 1 contains, for example, bacteria 1a and dust 1b as antigens. In the course of the introduction of this sample solution into the reaction section 3, the bacteria 1a in the sample solution receive a vertical flow vector as shown by the arrow V at the flow path step 4a of the introduction path 4, It is concentrated below the reaction section 3 together with the dust 1b. The depth of the channel of the reaction section 3 is, for example, 100 μm, and the length is 3000 μm. The depth (h in the figure) of the portion to be concentrated is, for example, 20 μm.
[0047]
Due to the concentration, the bacteria 1a are easily captured by the antibody beads 2 by the antigen-antibody reaction, only the dust 1b is discharged from the discharge channel 5, and the bacteria 1a as the antigen is easily separated from the sample solution and the dust. . In addition, since the sample liquid can flow smoothly in the flow paths other than the part to be concentrated, the processing amount can be secured as needed.
[0048]
The flow path step 4a shows a step where the introduction path 4 bends at a right angle, but the introduction path 4 and the reaction section 3 may be connected with a smooth curve without dead space, The flow path may have various shapes as long as the flow path has a substantial step to receive the flow vector. FIG. 1 shows an example in which a step is also provided in the discharge path 5, but this step can be omitted.
[0049]
In addition, as described above, in addition to the flow path step 4a, the electric field generating means 30 generates an electric field in which the bottom surface on which the antibody beads are disposed is a positive electrode and the opposing surface is a negative electrode. The bacteria 1a as a negatively charged antigen in the state can be sucked into the antibody beads 2 and concentrated. The power supply voltage for generating an electric field is, for example, several tens of volts.
[0050]
Next, based on FIG. 2, the configuration of the main body of the antigen separation device and the relationship with the fluorescence image measurement will be described. First, the configuration of the separation device main body will be described with reference to FIG. FIG. 3 is a sectional perspective exploded view (a), a sectional perspective view (b), and a sectional view (c) of FIG.
[0051]
As described above, the main body of the separation device is made of a resin-integrated molded member 8 including a flow path 7 such as the sample liquid inlet 1, the inlet 4, the reactor 3, and the outlet 5, and a glass or light-transmissive resin. And the lid member 9 is bonded. FIG. 2 shows an example in which an inlet 6 for a fluorescent staining reagent 10 is provided in the introduction path 4. In addition, an example is shown in which the lid member 9 is provided with a protruding portion 9a for closing a part of the step portion of the discharge path 5, but when the step of the discharge path 5 is omitted, the protruding portion 9a is unnecessary. .
[0052]
Next, the procedure for measuring the antigen will be described with reference to FIG. In FIG. 2, an image measuring device 20 is the same device as that shown in FIG. 6, and the bacteria 1a as an antigen captured by the antibody beads 2 are observed through, for example, a glass lid member 9. It is arranged to be able to. A specific example of the measurement procedure will be described later, and a basic procedure will be described first with respect to an example of applying an electric field.
[0053]
First, a sample solution containing the bacterium 1a as an antigen is introduced into the reaction section 3 at a predetermined flow rate. Then, an electric field is applied to concentrate the antigen, and the bacterium 1a is adsorbed on the antibody beads 2 by an antigen-antibody reaction. Subsequently, a fluorescent staining reagent 10 is injected to fluorescently label the antigen. Then, after flowing the washing solution to wash the fluorescent staining reagent, image measurement is performed by the image measurement device 20 to enable counting of bacteria. At the time of image measurement, air may be introduced into the reaction section to discharge the sample liquid, but measurement can be performed with the liquid filled. Further, since the fluorescence output increases proportionally with the increase in the volume concentration of the bacterium, the volume concentration of the bacterium can be counted based on data calibrated in advance at the standard concentration.
[0054]
In addition, in order to prevent the fluorescence output error of foreign substances such as dust, as described above, image measurement is also performed before the step of fluorescent labeling, and based on the difference between the image information before and after the fluorescent labeling, the image of the antigen is determined. It is desirable to make measurements. In measurement, it is desirable to perform autofocus on the antigen, and this specific method will be described later.
[0055]
Next, a specific example of a method of performing image measurement by performing fluorescent labeling will be described. As a specific fluorescent reagent, for example, when targeting microorganisms in general, a fluorescent nucleobase analog, a fluorescent stain for staining nucleic acid, a staining solution for staining protein, an environment used for structural analysis of proteins, etc. Aqueous fluorescent probes, staining solutions used for analysis of cell membranes and membrane potential, staining solutions used for labeling fluorescent antibodies, and, for aerobic bacteria, staining solutions that develop color by cell respiration, etc. In the case of eukaryotic microorganisms, a stain for mitochondria, a stain for the Golgi apparatus, a stain for the endoplasmic reticulum, a stain for reacting with intracellular esterase, and its modifying compounds, etc. When cells are targeted, a staining solution used for observing bone tissue, a staining solution that is a nerve cell tracer, and the like can be applied. By staining with these, a fluorescent image can be observed with a fluorescent microscope.
[0056]
Next, an example of obtaining a fluorescence observation image of an antigen such as a microorganism and a cell stained with fluorescence by the method described above will be described below. That is, a reagent that fluoresces by esterase activity, for example, carboxyfluorescein diacetate (hereinafter abbreviated as CFDA) is reacted with the separated antigens of microorganisms and cells. When acquiring a fluorescence image of an antigen that has been fluorescently stained with CFDA, first, an antibody bead as a marker is irradiated with autofocus light that emits CFDA fluorescence wavelength light (for example, 500 to 550 nm).
[0057]
The depth of focus of the microscope is, for example, about 10 μm, and the antibody beads and the antigen captured by the beads are, for example, about 1 μm, respectively. Is also in focus. Therefore, after focusing on the antibody beads, the focused antigen measurement surface is irradiated with light having a wavelength (for example, 450 to 500 nm) capable of exciting CFDA to acquire a fluorescent image of the antigen. From the obtained fluorescent images, antigens such as microorganisms and cells can be recognized and detected.
[0058]
Next, FIG. 4 will be described. FIG. 4 relates to the invention of claim 2, and is a conceptual explanatory diagram in a case where the antigen is concentrated by, for example, layer separation of an air layer and a sample liquid layer. (A) is a schematic plan view, and (b) is an explanatory view of a layer separation state. In FIG. 4, reference numeral 1 denotes a sample liquid inlet, and 11 denotes an air inlet. The sample liquid and air are simultaneously introduced in the same horizontal plane, and flow into the reaction unit 3 via the introduction path 4. As a result, as shown in FIG. 3 (b), the air is separated into an air layer 40a and a sample liquid layer 40b, and is passed through the reaction section 3 in this state. An antigen-antibody reaction will be performed. In FIG. 4, reference numeral 5 denotes an outlet, and reference numeral 6 denotes an inlet for a fluorescent staining reagent.
[0059]
The optimum state of the above-mentioned layer separation is determined by the flow rate ratio of the air and the sample liquid on the condition that the flow state of the sample liquid satisfies the condition not more than the critical Reynolds number for maintaining the laminar flow. The optimum conditions are determined by preliminary experiments from the viewpoint of obtaining an optimum concentration state.
[0060]
Next, FIG. 5 will be described. FIG. 5 is a conceptual explanatory diagram of a configuration in the case of individually separating each antigen in a sample fluid containing various types of antigens according to the invention of claim 7. (A) is a schematic diagram when four reaction units are configured in series, and (b) is a schematic diagram when configured in parallel. In FIG. 5, reference numeral 1 denotes a sample fluid inlet, 4 denotes a sample fluid inlet, 5 denotes a sample fluid outlet, and 6 denotes a fluorescent dye reagent inlet. The four reaction parts are indicated by 3a to 3d, respectively.
[0061]
Each of the reaction sections 3a to 3d includes beads on which various types of antibodies are fixed or beads on which different antibodies are fixed for each reaction section. According to the above configuration, in each of the series configuration and the parallel configuration, each antigen in the sample fluid containing various types of antigens can be individually separated. However, in consideration of the uniformity of distribution of the sample fluid to each reaction section, the serial configuration is preferable in terms of measurement accuracy, and the structure is simple.
[0062]
【The invention's effect】
As described above, according to the present invention, a plurality of antibody carriers are provided at the inlet of a sample fluid containing an antigen such as a bacterium or an in vivo protein, and on the bottom surface of a microchannel having a rectangular cross section through which the sample fluid flows. Disposed, a reaction section for separating the antigen by an antigen-antibody reaction, a microchannel-shaped sample fluid introduction path communicating between the sample fluid inlet and the reaction section, and a sample fluid from the reaction section. And a discharge path for discharging the antigen, wherein the introduction path generates a fluid flow vector in a direction perpendicular to the bottom surface, and locally adjusts the dispersion concentration of the antigen in the sample fluid near the bottom surface. The reaction section and the flow path step, or an antigen concentration function by layer separation of an air layer and a sample fluid, and / or Parts are antigen, the antibody bearing member disposed the bottom side, since those having an electric field generating means for attracting by an electric attraction,
It is possible to provide an antigen separation apparatus which can solve the problem of dust clogging at the time of antigen separation, can perform separation easily and in a short time, and can secure a throughput of fluid as needed.
[0063]
In addition, by performing image measurement based on the fluorescence image information of the antigen separated by the separation device, the measurement time of the antigen, which conventionally required 2 to 3 hours, is reduced to 25 to 30 minutes. Highly accurate measurement is possible.
[0064]
Furthermore, as described above, by forming the separation device into one chip with a molding resin and providing a plurality of reaction parts to form a multi-chip, highly versatile and low-cost measurement can be realized.
[Brief description of the drawings]
FIG. 1 is a conceptual schematic diagram of an embodiment of the main body of the antigen separation device of the present invention.
FIG. 2 is a schematic configuration diagram of an embodiment of an antigen measuring device according to the present invention.
FIG. 3 is an exploded cross-sectional perspective view, a cross-sectional perspective view, and a cross-sectional view of the main body of the antigen separation device of the present invention.
FIG. 4 is a conceptual explanatory diagram in the case where antigens are concentrated by layer separation of an air layer and a sample liquid layer according to the present invention.
FIG. 5 is a conceptual explanatory diagram of a configuration in the case where individual antigens in a sample fluid containing various types of antigens are individually separated according to the present invention.
FIG. 6 is a diagram showing an example of a configuration of a fluorescence image measurement device that performs autofocus described in Japanese Patent Application No. 2002-30648.
FIG. 7 is a diagram showing a method for measuring the number of bacteria using a difference image.
FIG. 8 is a perspective view of an immune analyzer described in Patent Document 1.
[Explanation of symbols]
1: inlet, 1a: bacteria, 1b: dust, 2: antibody beads, 3, 3a to 3d: reaction section, 4: introduction path, 4a: stepped section, 5: discharge path, 6: injection port of fluorescent staining reagent , 7: flow path, 8: molded member, 9: lid member, 10: fluorescent staining reagent, 11: air inlet, 20: image measuring device, 30: electric field generating means.

Claims (13)

A plurality of antibody carriers are disposed on the bottom of a microchannel having a rectangular cross section through which the sample fluid flows, and an inlet for a sample fluid containing antigens such as bacteria and in vivo proteins. Separation of antigen comprising a reaction part for separating antigen, an introduction path for the microchannel-shaped sample fluid communicating the inlet for the sample fluid and the reaction part, and a discharge path for discharging the sample fluid from the reaction part In the apparatus, the introduction path generates a fluid flow vector in a direction perpendicular to the bottom surface, and locally increases the dispersion concentration of the antigen in the sample fluid near the bottom surface (provides an antigen concentration function). As described above, the antigen separation apparatus substantially comprises the reaction section and a flow path step. 2. The separation device according to claim 1, wherein the introduction path having the antigen concentrating function includes a sample fluid and a fluid such as air or water simultaneously in the same horizontal plane, instead of the one having the flow path step. An antigen separation apparatus characterized in that the antigen separation function is provided by separating the sample fluid layer from a fluid layer such as air or water perpendicular to the bottom surface. A plurality of antibody carriers are disposed on the bottom of a microchannel having a rectangular cross section through which the sample fluid flows, and an inlet for a sample fluid containing antigens such as bacteria and in vivo proteins. Separation of antigen comprising a reaction part for separating antigen, an introduction path for the microchannel-shaped sample fluid communicating the inlet for the sample fluid and the reaction part, and a discharge path for discharging the sample fluid from the reaction part In the apparatus, the reaction unit may include an electric field generating means for suctioning the antigen by an electric suction force on a bottom surface on which the antibody carrier is provided. The antigen separation device according to claim 3, wherein the introduction path has the antigen concentration function according to any one of Claims 1 and 2. 5. The separation device according to claim 1, wherein the introduction path includes an inlet for injecting a fluorescent staining reagent into the sample fluid, between the inlet and the reaction unit. 6. A device for separating an antigen, comprising: The separation device according to any one of claims 1 to 5, wherein the separation device main body includes a member including flow paths such as an inlet, an introduction path, a reaction section, and a discharge path of the sample fluid; An antigen separation device, which is configured to be bonded to a lid member. The separation apparatus according to any one of claims 1 to 6, further comprising a plurality of the reaction units, wherein each reaction unit has an antibody carrier on which various types of antibodies are fixed or a different antibody fixed for each reaction unit. Having an antibody carrier, the introduction path is configured such that the sample fluid flows in series or in parallel with each of the reaction sections, and each antigen in the sample fluid containing various types of antigens is separately separated; and An antigen separation device, comprising: An antigen measurement device using the antigen separation device according to claim 5, wherein the separation device includes an image measurement device that performs image measurement based on fluorescence image information of the separated antigen. Measuring device. 9. The antigen measurement device according to claim 8, wherein the image measurement device has a focusing marker, and includes a focusing unit that performs autofocus on an antigen to be measured. . 10. The antigen measuring device according to claim 9, wherein the focusing marker is a plurality of antibody carriers disposed on a bottom surface of the reaction section. The antigen measuring device according to any one of claims 8 to 10, wherein the image measuring device performs image measurement based on a difference between image information after fluorescent labeling of the antigen and image information before fluorescent labeling. An antigen measuring apparatus, which has an arithmetic function for removing fluorescence errors of impurities. An antigen measuring method using the antigen measuring device according to any one of claims 8 to 10, wherein the method includes the following steps.
1) A step of flowing a sample fluid containing an antigen at a predetermined flow rate to the reaction section of the antigen separation device.
2) a step of concentrating the antigen in the reaction section with or without applying an electric field, and adsorbing the antigen to the antibody carrier by an antigen-antibody reaction;
3) a step of injecting a fluorescent reagent into the sample fluid to fluorescently label an antigen;
4) a step of flowing the washing solution to wash the fluorescent reagent;
5) a step of measuring the image of the antigen using a fluorescent image device; A method for measuring an antigen using the apparatus for measuring an antigen according to claim 11, wherein the step of fluorescently labeling according to the step of 3) according to claim 12 in addition to the step of claim 12. The image measurement is also performed in the former stage, and the image measurement of the antigen is performed based on the difference between the image information obtained by the image measurement in the step (5) according to claim 12 and the image information obtained in the former stage of the fluorescent labeling. Method for measuring the amount of antigen.

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