JP4171974B2 - Antigen separation apparatus and antigen measurement method and apparatus using the same - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
  The present invention relates to an apparatus for separating antigens such as bacteria and biological proteins in a sample, and an antigen measuring method and apparatus using the same. Separation / measurement targets include everything that can be measured using antigen-antibody reactions. Prokaryotes such as bacteria and actinomycetes, eukaryotes such as yeast and mold, lower algae, microorganisms such as viruses And cultured cells derived from animals and plants and cells such as pollen such as cedar and cypress. In addition, immunological analysis, detection of endocrine disrupting substances (environmental hormones), and characteristics of causative substances of atopic dermatitis are also targeted. The target sample is more often a liquid sample than a gas sample, but includes, for example, measurement of an antigen of a gas sample such as bacteria or mold floating in the air. Fields of application of the present invention include medical care, food production, water and sewage, environmental analysis, and the like.
[0002]
[Prior art]
  For example, detection of tissue cells such as microorganisms and animals and plants in a sample is an extremely important technology in the industry for confirming the sterilization 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 on the test surface, the culture method, that is, by pressing a solid plate medium shaped by 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 colony that appears by culturing the microorganism as it is on a plate medium in an appropriate environment is measured with the naked eye or a stereomicroscope. Is used.
[0004]
  Since the culture method has a drawback that it takes 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, the microorganisms collected on the filter are 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 microorganisms without culturing.
[0005]
  According to the image measurement method, measurement is possible in 2 to 3 hours. The breakdown of the time required for measurement is, for example, about 70 minutes for antigen-antibody reaction and magnetic separation of antibody magnetic beads and dust in the sample solution, about 30 minutes for fluorescent staining, and about 30 minutes for microscopic observation. 3
[0006]
  By the way, as an image analysis method in the measurement method, image analysis using a manually focused microscope, an imaging device, or the like is generally performed, and focusing is performed because a depth of field is narrow under a high-magnification use condition. Therefore, automatic focusing and automatic analysis are desired. From this point of view, a fluorescence image measurement method invented by the present applicant has been filed in Japanese Patent Application No. 2002-30648 regarding a method of performing fluorescence image measurement by performing automatic focusing.
[0007]
  Figure5These show an example of the apparatus which implements the method described in the said Japanese Patent Application No. 2002-30648. Figure5According to the apparatus shown in FIG. 1, 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 irradiated on the excitation light irradiation side as shown in the figure. Is irradiated from the same side as the light source 82, the degree of focus is determined from the image information obtained by this, and at least one of the specimen 81 and the light receiving system is driven according to the degree to search for the in-focus position. When reaching the position, the irradiation of the AF light is stopped, and thereafter, the fluorescence image measurement can be performed by irradiating the specimen 81 with the excitation light from the light source 80. According to the present apparatus, since 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 suitable. The image of the specimen at the time of AF light irradiation is captured by the image sensor 87 via the objective lens 85, the dichroic mirror 83, the fluorescence receiving side filter 84, and the imaging lens 86. As the image sensor, a CCD camera element or a CMOS camera element is suitable. The image obtained by the image sensor 87 is sent to the calculation unit 88, where the contrast is evaluated. The contrast is evaluated by, for example, a general AF method in which the brightness difference between adjacent pixels is calculated, and the position where the contrast is maximized is the in-focus position.
[0009]
  Figure5, 89 is a stage moving mechanism, 91 is a condensing lens for excitation light, 92 is a filter, and 93 is a fluorescent filter block. Further, as a focusing marker for performing the contrast evaluation, FIG.5Although not shown in the figure, a pattern (mark) is applied to the surface of the slide glass holding the specimen, or a method of applying a pattern (mark) to the surface of the membrane filter for collecting and filtering the specimen is adopted. And Japanese Patent Application No. 2002-30648). According to the above method, with a simple configuration with a small number of elements, the sample is not extinguished due to excitation light irradiation and cannot be detected, and auto-focusing is performed even for samples captured on the membrane filter surface or samples with poor contrast (AF) is possible.
[0010]
  Further, in the fluorescence image measurement method, it is desirable to eliminate the influence of fluorescent contaminants regardless of the properties of the sample, and to improve the measurement accuracy. From this viewpoint, the living cell invented by the applicant of the present application is desirable. A counting method and apparatus have been filed by Japanese Patent Application No. 2002-148884.
[0011]
  The image measuring apparatus described in Japanese Patent Application No. 2002-14884 performs image measurement based on a difference between image information after fluorescent labeling of live cells and image information before fluorescent labeling, and removes fluorescence errors of impurities. The above application discloses three types of methods.
[0012]
  That is, the first method is to label live cells with a fluorescent labeling method and apparatus for measuring the number of live cells by labeling live cells such as microorganisms and cell tissues in a sample with a fluorescent reagent. A fluorescent image of the sample (first image) is acquired, and after the live cells are fluorescently labeled, a fluorescent image of the sample (second image) is acquired, and the first image and the second image The difference (B−A) in the number of bright spots is determined. Further, as a method different from the above, the second image is obtained by obtaining a difference image between the first image and the second image, and the number of bright spots in the difference image is obtained. Among these, the position is a third method for obtaining the number of bright spots that are not included in the dead area associated with each bright spot in the first image (for details, refer to Japanese Patent Application No. 2002-148884).
[0013]
  Figure6These are figures which show the measuring method of the bacteria count which concerns on said 2nd method using calculating | requiring a difference image between a 1st image and a 2nd image as an example.
[0014]
  As a procedure, for example, bacteria contained in the sample solution are captured on a membrane filter by filtration. Next, the fluorescence image before the fluorescent labeling of the membrane filter that captured the bacteria (Fig.6A first image 96) shown in the central part of FIG. Subsequently, a fluorescent labeling reagent is added onto the membrane filter to fluorescently label the bacteria. Then, again, the fluorescence image of the membrane filter (Fig.6A second image 97) shown on the left side of FIG. The second image 97 and the first image 96 are used to6The difference image 98 shown on the right side is obtained.
[0015]
  The bright spot present in the difference image 98 is a bright spot that appears due to the fluorescent label, and by counting this as the number of bacteria, the fluorescence error of impurities can be removed.
[0016]
  Incidentally, there is a demand for an improved fluorescent image measurement method as described above that can perform measurement with high accuracy in a simpler and shorter time. Further, depending on the measurement target, it is desired to enable measurement with a very small amount of sample. From this point of view, Patent Document 1 discloses an immunoassay apparatus and method for measuring a minute sample by causing an antigen-antibody reaction in a microchip.
[0017]
  Figure7These show the perspective view of the immunoassay apparatus described in patent document 1. FIG. Figure7According to the description of Patent Document 1, the immunoanalyzer shown in FIG. 1 has a microchannel reaction vessel 103 having a cross-sectional area larger than the diameter of the solid microparticles 102 together with the solid microparticles 102 having a diameter of 1 mm or less as a reaction solid phase. And a microchannel separation section 104 having a cross-sectional area smaller than the diameter of the solid microparticle 102, and has an introduction section or a microchannel inflow section 105, 106 that guides the antigen and the labeled antibody separately to the reaction tank section 103. An immunoassay microchip is constructed and used for analysis.
[0018]
  Figure7In order to introduce a microchannel inflow portion 107 for introducing the first antibody and a buffer solution or a washing solution together with the microchannel inflow portion 106 for introducing the labeled antibody as the second antibody into the reaction vessel portion 103. The microchannel inflow portions 108 are provided at the end of each of the microchannel inflow portions 105, 106, 107, and 108, and injection holes for the antigen, the labeled antibody (second antibody), the first antibody, and the washing liquid are provided. 105A, 106A, 107A, and 108A are also provided. Also figure7In this example, a waste liquid portion 104 </ b> A is provided at the end of the microchannel separation portion 104.
[0019]
  The solid microparticles 102 serve as a reaction solid phase for immune antigen-antibody reaction, and for example, glass beads or polymer beads such as polystyrene are used. The solid fine particles 102 having a diameter of 1 mm or less, for example, 15 to 85 μm are used.
[0020]
  By the use of a small amount of sample or the like, the immunoassay microchip can easily perform an immunoassay with a short reaction time. As an immunoassay method, solid microparticles 102 as a reaction solid phase are introduced into a microchannel reaction vessel 103, and an antigen and a labeled antibody introduced from the introduction part or the microchannel inflow parts 105 and 106, and if necessary, into the microchannel. The antibody introduced from the unit 107 is reacted on the solid microparticles 102, and unreacted substances are separated by the microchannel separation unit 104 and can be analyzed by photothermal conversion analysis or fluorescence analysis.
[0021]
[Patent Document 1]
          Japanese Unexamined Patent Publication No. 2001-4628 (page 3-4, FIG. 1)
[0022]
[Problems to be solved by the invention]
  By the way, the invention described in the above-mentioned Patent Document 1 also has the following problems mainly related to the measuring object separation apparatus.
[0023]
  In the case of the apparatus of Patent Document 1, specifically, as described in Patent Document 1 above, “when the size of the microchannel reaction vessel 103 is a hemispherical hole, for example, its radius And the microchannel separator 104 has a depth of 10 μm or less and a width of 10 μm or less, for example, so that the solid microparticles 102 as a reaction solid phase can be obtained. Does not flow into the microchannel separation section 104, and is clogged, and only unreacted substances flow into the microchannel separation section 104 and are separated. Only the reaction product desorbed from is separated. "
  As described in Patent Document 1, since the separation device of Patent Document 1 is a “damping method”, when there is “dust” in the sample liquid, the portion clogged to 10 μm or less is filled with dust. There is a problem that it is easy to measure.
[0024]
  In addition, when the sample to be measured is relatively homogeneous, high-precision detection is possible with a very small amount of sample, but as the sample to be measured, for example, a sample solution containing a small number of bacteria per unit volume However, when the distribution in the liquid is not uniform, a certain amount of processing amount of the liquid to be measured is necessary, and if the processing amount is too small, the measurement accuracy is lowered or the measurement is performed. Is 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 clogging of dust in the liquid in the microchannel during the separation of the antigen in the sample fluid, and the separation. Providing an antigen separation device that can be performed simply and in a short time, and can secure a fluid throughput as required, and further, using this separation device, an antigen that can be measured easily and in a short time with high accuracy It is in providing the measuring method and apparatus of this.
[0026]
[Means for Solving the Problems]
  In order to solve the aforementioned problems, the present invention provides a plurality of antibodies on 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. A reaction part that disposes the antigen by antigen-antibody reaction, a microfluid-shaped sample fluid introduction path that communicates with the sample fluid introduction port and the reaction part, and a reaction part. In the antigen separation apparatus comprising a discharge path for discharging a sample fluid, the introduction path generates a fluid flow vector in a direction perpendicular to the bottom surface, and the dispersion concentration of the antigen in the sample fluid is determined in the vicinity of the bottom surface. The reaction part and the flow path step are substantially provided so as to be locally high (provided with an antigen concentration function) (invention of claim 1).
[0027]
  The “rectangular cross section” in the present invention is a polygonal cross section in which one side facing the base is parallel, and includes a cross-sectional shape such as a trapezoid or a hexagon.
[0028]
  In order to separate the antigen by the antigen-antibody reaction, it is necessary to concentrate the antigen in the vicinity of the antibody carrier (for example, antibody beads). It is necessary to concentrate the antibody beads to, for example, about 20 to 30 μm, preferably about 10 μm as in the conventional apparatus. According to the separation apparatus, the solid matter in the liquid is concentrated without clogging or squeezing the flow path of the sample liquid as in the prior art, and only the specific antigen is captured and separated by the antibody beads in the reaction section, and the rest Since dust can be discharged from the reaction part, the problem of dust clogging can be solved, and antigens can be separated easily and in a short time. Moreover, since a processing amount of about 10 mL can be ensured in 10 minutes, for example, various needs can be met. Details will be described later.
[0029]
  AlsoThe antigen concentration function of the invention of claim 1 is as follows.2Like the invention ofThen, the concentration function is further improved.. That is,Claim 1In this separation apparatus, the reaction section includes an electric field generating means for sucking the antigen by an electric suction force on the bottom surface side where the antibody carrier is disposed.
[0030]
  Since the antigen is negatively charged in a natural state, the antigen is generated on the side of the antibody carrier by generating an electric field with the bottom surface on which the antibody carrier is disposed as the positive electrode and the opposite surface as the negative electrode. To be concentrated. The electric field can also be formed by an alternating electric field.
[0031]
  In addition, the claim 1Or 2In the separation apparatus according to claim 1, the introduction path includes an injection port for injecting a fluorescent staining reagent into the sample fluid between the introduction port and the reaction unit.3Invention). Thereby, as will be described later, the antigen can be measured by observing the fluorescence image.
[0032]
  Furthermore, the claims 1 to3In the separation apparatus according to any one of the above, the separation apparatus main body is formed by attaching a member including a flow path such as an inlet, an introduction path, a reaction section, and a discharge path of the sample fluid and a light-transmitting lid member. Combined configuration (claims)4Invention). The separation device includes various peripheral devices such as piping for introducing a sample solution, a fluorescent staining reagent, etc., a sealing device, and a control valve.4By constituting like this invention, the separation device main body can be made into a chip, and mass productivity can be improved and cost can be reduced. Since the chip can be disposable, the mobility and maintainability of the apparatus are also improved.
[0033]
  In addition, the sample solution contains many types of antigens, and in order to separate them simultaneously, the following claims5The invention is preferred. That is, the claims 1 to4The separation apparatus according to any one of the above, wherein a plurality of the reaction parts are provided, and each reaction part has an antibody carrier to which various types of antibodies are immobilized, or an antibody carrier to which different antibodies are immobilized for each reaction part. The introduction path is configured so that the sample fluid flows through the reaction units in series or in parallel, and each antigen in the sample fluid containing many kinds of antigens is individually separated. As described above, by using a multichip having a plurality of reaction units, it is possible to perform measurement with high diversity and low cost. Details will be described later.
[0034]
  Next, as an antigen measurement device using the separation device, the following claim6Or9The invention is preferred. That is, the claim3An antigen measurement apparatus using the antigen separation apparatus according to claim 1, wherein the separation apparatus includes an image measurement apparatus that performs image measurement based on fluorescence image information of the separated antigen.6Invention). As a result, the measurement time which has conventionally taken 2 to 3 hours can be greatly reduced to 25 to 30 minutes due to the absence of magnetic separation operation.
[0035]
  Claims6As an embodiment of the present invention, the following claims are provided.7~9The invention is preferred. That is, the claim6In the antigen measurement device according to claim 1, the image measurement device includes a focusing marker and includes a focusing unit that autofocuses the antigen to be measured.7Invention). This effect is as described above.
[0036]
  And claims7In the antigen measurement apparatus according to claim 1, the focusing marker is a plurality of antibody carriers arranged on the bottom surface of the reaction part (claim).8Invention). For example, when polystyrene beads are used as the antibody carrier, the beads arranged on the bottom surface can function as a focusing marker, so there is no need to provide a marker again.
[0037]
  Furthermore, the claim6Or8The antigen measurement apparatus according to any one of claims 1 to 3, wherein the image measurement apparatus performs image measurement based on a difference between image information after the fluorescent labeling of the antigen and image information before the fluorescent labeling, and fluorescence of impurities. An arithmetic function for removing errors is provided (claims)9Invention). This effect is as described above.
[0038]
  Next, the antigen measurement method includes the following claims:10Or11The invention is preferred. That is, the claim6Or8An antigen measurement method using the antigen measurement apparatus according to claim 1, comprising the following steps:10Invention).
1) A step of passing a sample fluid containing an antigen at a predetermined flow rate through the reaction part 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 fluorescently labeling the antigen by injecting a fluorescent reagent into the sample fluid.
4) A step of washing the fluorescent reagent by flowing a washing solution.
5) A step of measuring an antigen image using a fluorescence imaging apparatus.
[0039]
  In the above step 3), when the sample fluid is a gas, the fluorescent staining reagent is preferably injected in the form of a mist.
[0040]
  Also, the claim9An antigen measurement method using the antigen measurement apparatus according to claim 1, wherein10In addition to the steps described in claim10The image measurement is also performed before the fluorescent labeling step according to the step 3) described in claim 1,10The antigen image measurement is performed based on the difference between the image information obtained by the image measurement according to the step 5) described above and the image information of the previous stage of the fluorescent labeling.12Invention).
[0041]
DETAILED DESCRIPTION OF THE INVENTION
  Regarding examples of the present invention, examples of measuring bacteria in a liquid sample will be described below with reference to FIGS. First, an antigen separation apparatus will be described.
[0042]
  FIG. 1 claims1 and 2It is a notional schematic diagram of the main body of an antigen separation apparatus according to the invention. FIG. 2 is a schematic configuration diagram of the antigen measuring apparatus, and also shows a perspective sectional view of a specific configuration of the separation apparatus main body. FIG. 2A shows an exploded perspective cross-sectional view of the lower body member and the lid member, and FIG. 2B shows an assembled perspective cross-sectional view.
[0043]
  1 and 2, 1 is a sample solution introduction port, 2 is a polystyrene antibody bead, 3 is a reaction part, 4 is a sample solution introduction channel, 5 is a sample solution discharge channel, and 6 is a fluorescent staining reagent. , 7 denotes a flow path for the sample solution including the introduction port, the introduction channel, the reaction part, the discharge channel, and the like. In FIG. 1, 30 is an electric field generating means, and in FIG. 2, 8 is a molding member, 9 is a lid member, 10 is a fluorescent staining reagent, and 20 is an image measuring device.
[0044]
  The antigen separation operation will be described with reference to FIG. The sample solution introduced from the introduction port 1 into the introduction path 4 includes, for example, bacteria 1a and dust 1b as antigens. In the process of introducing the sample liquid into the reaction unit 3, the vertical flow vector as indicated by the arrow V is received at the flow path step 4a of the introduction path 4, and the bacteria 1a in the sample liquid is It concentrates below the reaction part 3 with the waste 1b. The channel depth of the reaction unit 3 is, for example, 100 μm, and the length is 3000 μm. Further, the depth (h in the drawing) of the portion to be concentrated is, for example, 20 μm.
[0045]
  By the above concentration, the bacteria 1a are easily captured by the antibody beads 2 by the antigen-antibody reaction, and only the dust 1b is discharged from the discharge path 5, and the bacteria 1a as the antigen is easily separated from the sample solution and the dust. . Further, since the sample solution can flow smoothly in the flow path other than the portion to be concentrated, the processing amount can be ensured as necessary.
[0046]
  Although the flow path step 4a is illustrated as a step where the introduction path 4 bends at a right angle, the introduction path 4 and the reaction unit 3 may be connected by a smooth curve without a dead space, and the vertical direction As long as the flow path configuration has a substantial step to receive the flow vector, various shapes can be taken. Moreover, in FIG. 1, although the example which provided the level | step difference also in the discharge path 5 is shown, this level | step difference can be abbreviate | omitted.
[0047]
  Further, as described above, in addition to the flow path level difference 4a, the electric field generating means 30 generates an electric field having the bottom surface side where the antibody beads are disposed as a positive electrode and the opposite surface as a negative electrode. Bacteria 1a as a negatively charged antigen in a state can be sucked and concentrated on the side of the antibody bead 2. Note that the power supply voltage for generating the electric field is, for example, a few tens of volts.
[0048]
  Next, based on FIG. 2, the structure of the antigen separation apparatus main body 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 cross-sectional perspective exploded view (a view), a cross-sectional perspective view (b view), and a cross-sectional view (c view) focusing only on the separation device main body.
[0049]
  As described above, the main body of the separation device includes the resin integrated molding member 8 including the introduction port 1 for the sample liquid, the introduction path 4, the reaction part 3, the discharge path 5, and the like, and glass or light-transmitting resin. The lid member 9 is bonded. FIG. 2 shows an example in which the inlet 6 for the fluorescent staining reagent 10 is provided in the introduction path 4. Moreover, although the example which provided the protrusion part 9a which block | closes a part of level | step-difference part of the discharge path 5 is shown in the cover member 9, when the level | step difference of the discharge path 5 is abbreviate | omitted, the said protrusion part 9a is unnecessary. .
[0050]
  Next, an antigen measurement procedure will be described with reference to FIG. In FIG. 2, the image measuring device 20 is5The apparatus is the same as that shown in FIG. 1 and is arranged so that the bacteria 1a as the antigen captured by the antibody beads 2 can be observed through a glass lid member 9, for example. A specific example of the measurement procedure will be described later. First, a basic procedure will be described with respect to an example of applying an electric field.
[0051]
  First, after introducing a sample solution containing bacteria 1a as an antigen into the reaction unit 3 at a predetermined flow rate, an electric field is applied to concentrate the antigens, and the bacteria 1a are adsorbed to the antibody beads 2 by an antigen-antibody reaction. Subsequently, the fluorescent staining reagent 10 is injected to fluorescently label the antigen. Thereafter, a washing solution is passed through to wash the fluorescent staining reagent, and then image measurement is performed by the image measuring device 20 so that bacteria can be counted. At the time of image measurement, air may be introduced into the reaction part to discharge the sample liquid, but the measurement can be performed in a state where the liquid is filled. Further, since the fluorescence output increases proportionally with the increase in the volume concentration of the bacteria, it is possible to count the volume concentration of the bacteria based on data previously calibrated at the standard concentration.
[0052]
  In addition, in order to prevent the fluorescence output error of contaminants such as dust, as described above, image measurement is also performed before the fluorescent labeling process, and the image of the antigen is based on the difference in image information before and after the fluorescent labeling. It is desirable to perform measurement. Further, in the measurement, it is desirable to autofocus on the antigen, but this specific method will be described later.
[0053]
  Next, a specific example of a method for performing image measurement by performing fluorescent labeling will be described. As specific fluorescent reagents, for example, when targeting microorganisms in general, fluorescent nucleobase analogs, fluorescent stains for staining nucleic acids, staining solutions for staining proteins, environments used for structural analysis of proteins, etc. Fluorescent probes, stains used for analysis of cell membranes and membrane potentials, stains used for fluorescent antibody labeling, and stains that develop color by respiration of cells when targeting aerobic bacteria. For eukaryotic microorganisms, stains that stain mitochondria, stains that stain the Golgi apparatus, stains that stain the endoplasmic reticulum, stains that react with intracellular esterases and their modifying compounds, and higher animals In the case of targeting cells, a staining solution used for observation of bone tissue, a staining solution that is a nerve cell tracer, and the like can be applied. By staining with these, a fluorescence image can be observed with a fluorescence microscope.
[0054]
  Next, examples for obtaining fluorescence observation images of antigens such as microorganisms and cells fluorescently stained by the method as 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 antigens such as separated microorganisms and cells. When acquiring a fluorescent image of an antigen fluorescently stained with CFDA, first, autofocus light that emits CFDA fluorescence wavelength light (for example, 500 to 550 nm) is irradiated onto an antibody bead as a marker.
[0055]
  The depth of focus of the microscope is about 10 μm, for example, and the antibody beads and the antigen captured on the beads are about 1 μm, for example. It is also in focus. Therefore, after focusing on the antibody beads, the focused antigen measurement surface is irradiated with light having a wavelength capable of exciting CFDA (for example, 450 to 500 nm) to obtain a fluorescence image of the antigen. From the obtained fluorescence image, antigens such as microorganisms and cells can be recognized and detected.
[0056]
  Next, figure4Is described. Figure4Claims5It is a conceptual explanatory drawing of the structure in the case of isolate | separating each antigen in the sample fluid containing many types of antigens separately in connection with this invention. (A) is a schematic diagram in the case where four reaction parts are configured in series, and (b) is a schematic diagram in the case where they are configured in parallel. Figure4In the figure, 1 is a sample fluid inlet, 4 is a sample fluid inlet, 5 is a sample fluid outlet, and 6 is a fluorescent staining reagent inlet. The four reaction parts are indicated by 3a to 3d, respectively.
[0057]
  Each of the reaction units 3a to 3d includes beads to which various types of antibodies are immobilized, or beads to which different antibodies are immobilized for each reaction unit. With the above configuration, each antigen in the sample fluid containing many types of antigens can be separated separately in both cases of the serial configuration and the parallel configuration. However, considering the uniformity of the distribution of the sample fluid to each reaction part, the series configuration is preferable in terms of measurement accuracy, and the structure is simple.
[0058]
【The invention's effect】
  As described above, according to the present invention, a plurality of antibody carriers are provided on the inlet of a sample fluid containing antigens such as bacteria and in vivo proteins and on the bottom surface of a microchannel having a rectangular cross section through which the sample fluid flows. A reaction portion that separates the antigen by antigen-antibody reaction, a microfluid-shaped sample fluid introduction path that communicates the inlet of the sample fluid and the reaction portion, and a sample fluid from the reaction portion. In the antigen separation apparatus comprising the discharge passage, the introduction passage generates a fluid flow vector in a direction perpendicular to the bottom surface, and the dispersion concentration of the antigen in the sample fluid is locally determined in the vicinity of the bottom surface. The reaction part and the flow path step are substantially provided so as to be higher (having an antigen concentration function).RumoBecause it was
  It is possible to provide an antigen separation apparatus that eliminates the problem of dust clogging during antigen separation, can be easily separated in a short time, and can secure a fluid throughput as required.
[0059]
  In addition, by performing image measurement based on the fluorescence image information of the antigen separated by the separation device, the antigen measurement time, which has conventionally taken 2-3 hours, is reduced to 25-30 minutes. High-precision measurement is possible.
[0060]
  Furthermore, as described above, the separation device is made into one chip with a molded resin, and a plurality of reaction parts are provided to make a multi-chip, so that measurement with high diversity and low cost can be realized.
[Brief description of the drawings]
FIG. 1 is a conceptual schematic diagram of an embodiment of an antigen separating apparatus main body of the present invention.
FIG. 2 is a schematic configuration diagram of an embodiment of an antigen measurement apparatus of the present invention.
FIG. 3 is a cross-sectional perspective exploded view, a cross-sectional perspective view, and a cross-sectional view of the antigen separation device body of the present invention.
FIG. 4 relates to the present invention,Conceptual explanatory diagram of the configuration when each antigen in a sample fluid containing multiple types of antigens is individually separated
[Figure 5]The figure which shows an example of a structure of the fluorescence image measuring device which performs autofocus described in Japanese Patent Application No. 2002-30648
[Fig. 6]The figure which shows the measurement method of the number of bacteria with the difference image
[Fig. 7]Perspective view of immunoassay device described in Patent Document 1
[Explanation of symbols]
  1: introduction port, 1a: bacteria, 1b: dust, 2: antibody beads, 3, 3a to 3d: reaction part, 4: introduction path, 4a: step part, 5: discharge path, 6: injection port for fluorescent staining reagent , 7: flow path, 8: molded member, 9: lid member, 10: fluorescent staining reagent20: Image measuring device, 30: Electric field generating means.

Claims (11)

  A plurality of antibody carriers are disposed on the inlet of a sample fluid containing an antigen such as a bacterium or in vivo protein and on the bottom surface of a microchannel having a rectangular cross section through which the sample fluid flows, and the antigen-antibody reaction causes Separation of an antigen comprising a reaction part for separating an antigen, an introduction path for the sample fluid in the form of a microchannel communicating the introduction port of 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 vector of fluid flow in a direction perpendicular to the bottom surface, and locally increases the concentration of the antigen in the sample fluid near the bottom surface (having an antigen concentration function). As described above, an antigen separation apparatus comprising substantially the reaction section and a flow path step. 2. The separation apparatus according to claim 1 , wherein the reaction unit includes an electric field generating means for sucking an antigen by an electric suction force on a bottom surface side where the antibody carrier is disposed. Antigen separator. 3. The antigen according to claim 1, wherein the introduction path includes an injection port for injecting a fluorescent staining reagent into the sample fluid between the introduction port and the reaction unit. Separation equipment. The separation apparatus according to any one of claims 1 to 3 , wherein 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 for the sample fluid; An antigen separating apparatus comprising a lid member and a structure bonded thereto. In the separation device according to any one of claims 1 to 4, wherein a plurality of reaction sections, each reaction unit was fixed many types of different antibodies antibody antibody carrier or each reaction section to fix the respective An antibody carrier, and the introduction path is configured such that a sample fluid flows in each reaction part in series or in parallel, and each antigen in a sample fluid containing a plurality of types of antigens is individually separated; An antigen separation apparatus characterized by comprising: An antigen measurement apparatus using the antigen separation apparatus according to claim 3 , wherein the separation apparatus includes an image measurement apparatus that performs image measurement based on fluorescence image information of the separated antigen. Measuring device. The antigen measurement apparatus according to claim 6 , wherein the image measurement apparatus includes a focusing marker, and includes a focusing unit that performs autofocus on the antigen to be measured. . 8. The antigen measurement apparatus according to claim 7 , wherein the focusing marker is a plurality of antibody carriers arranged on the bottom surface of the reaction part. The measuring device of the antigen according to any one of claims 6 to 8, wherein the image measuring apparatus performs image measurement based on a difference between the image information and fluorescent labels previous image information after fluorescence labeling of the antigen An antigen measurement apparatus comprising an arithmetic function for removing fluorescence errors of impurities. An antigen measurement method using the antigen measurement apparatus according to any one of claims 6 to 8 , comprising the following steps.
1) A step of passing a sample fluid containing an antigen at a predetermined flow rate through the reaction part of the antigen separation device.
2) A step of concentrating the antigen in the reaction part with or without applying an electric field and adsorbing the antigen to the antibody carrier by an antigen / antibody reaction.
3) A step of fluorescently labeling the antigen by injecting a fluorescent reagent into the sample fluid.
4) A step of washing the fluorescent reagent by flowing a washing solution.
5) A step of measuring an antigen image using a fluorescence imaging apparatus. A method of measuring antigens using the measurement device of the antigen according to claim 9, in addition to the process described in claim 10, the step of fluorescently labeled according to the step 3) according to claim 10 Image measurement is also performed in the previous stage, and antigen image measurement is performed based on the difference between the image information obtained by the image measurement according to the step 5) of claim 10 and the image information in the previous stage of the fluorescent labeling. Measuring method of antigen.

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