JP2015197399A - Method, apparatus, and kit for immunoassay - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method of measuring a target antigen-antibody reactive substance contained in a sample with higher sensitivity compared to a moving sample method.SOLUTION: An immunoassay apparatus includes; a reaction plate 1 having a film-like body 2 which is made of a material capable of moving liquid by capillary action and comprises a sample holding portion 3 that holds a sample S containing a target substance T and a comparison sample holding portion 4 that holds a comparison sample S' containing a prescribed amount of the target substance T; developing liquid supply means 10 for supplying developing liquid W to the sample S and comparison sample S' held by the film-like body 2; marker carrier supply means 11 for supplying, together with the developing liquid W, a marker carrier M, which exhibits antigen-antibody reaction specifically with the target substance T, to the sample S and comparison sample S' held by the film-like body 2; and measurement tools 12, one of each being provided for the sample holding portion 3 and comparison sample holding portion 4, for measuring accumulated amounts of the marker carrier M associated with antigen-antibody reaction by the sample S and comparison sample S'.

Description

  The present invention relates to an immunoassay method and apparatus for measuring a measurement target substance contained in a specimen by an antigen-antibody reaction, and an immunoassay kit.

Conventionally, as this kind of immunoassay method, for example, those described in Patent Documents 1 and 2 are already known.
In Patent Document 1, an antigenic substance in a test sample is adsorbed on insoluble carrier particles, and an antibody or antibody complex that specifically reacts with the antigenic substance is reacted to selectively aggregate insoluble carrier particles. An aggregation immunoassay method in which the antibody or antibody complex does not substantially react with the antigenic substance present in the liquid phase in a native state, but does not react with the antigenic substance immobilized on the insoluble carrier particles. Are disclosed which react specifically.
In Patent Document 2, a test sample is directly applied to a membrane carrier and chromatographed toward a capture site formed of a first substance that can immunologically react with a specimen, and the capture site is obtained. After passing the sample, the developing solvent containing the second substance that can immunologically react with the specimen is chromatographed on the membrane carrier toward the capture site in the direction opposite to the chromatographic development direction of the test sample. An immunochromatographic measurement method is disclosed in which the test sample is developed and passed through the capture site twice.

Japanese Patent No. 2677753 (means for solving the problems) Japanese Patent No. 4909692 (the best mode for carrying out the invention, FIG. 1)

  A technical problem to be solved by the present invention is to measure a measurement target substance capable of antigen-antibody reaction contained in a sample with higher sensitivity than a method of moving a sample.

The invention according to claim 1 holds in advance a specimen in which a measurement target substance is contained in a part of a film-like body in which a liquid can move by capillary action and a comparison specimen in which a predetermined amount of the measurement target substance is contained. Thereafter, a developing solution is added to the membranous body, and the developing solution contains an insoluble label carrier that specifically reacts with the substance to be measured for antigen-antibody reaction. An antigen-antibody reaction is performed between the measurement target substance contained in the sample and the labeled carrier that moves together with the developing solution, and then the amount of the labeled carrier that accompanies the antigen-antibody reaction of the sample and the comparative sample is measured. An immunoassay method characterized by that.
The invention according to claim 2 is the immunoassay method according to claim 1, wherein the measurement target substance is hemoglobin A1c.
The invention according to claim 3 is the immunoassay method according to claim 1, wherein the labeling carrier is fluorescent latex.
The invention according to claim 4 is the immunoassay method according to claim 3, wherein the substance to be measured is hemoglobin A1c, the insoluble label carrier is a first fluorescent latex that reacts with hemoglobin A1c, and hemoglobin The amount of hemoglobin A1c or hemoglobin accumulated is measured by irradiating light containing at least light having a wavelength corresponding to the first fluorescent latex and the second fluorescent latex. It is an immunoassay method characterized by enabling.

  The invention according to claim 5 includes a sample holding unit that has a film-like body made of a material to which a liquid can move by capillary action, holds a sample containing a substance to be measured in the film-like body, and is determined in advance. A reaction plate provided with at least a comparison sample holding unit for holding a comparison sample containing a specified amount of a measurement target substance, and a development for supplying a developing solution to the sample and the comparison sample held in the film-like body Liquid supply means; label carrier supply means for supplying an insoluble label carrier that specifically undergoes an antigen-antibody reaction with the measurement target substance to the specimen and comparison specimen held in the film-like body together with the developing liquid; and A measuring instrument that is provided corresponding to the sample holding unit and the comparison sample holding unit and that measures an accumulation amount of the label carrier accompanying the antigen-antibody reaction of the sample and the comparison sample. Immunoassay It is the location.

The invention according to claim 6 is an immunoassay kit including a reaction plate used at the time of immunoassay, wherein the reaction plate includes a film-like body made of a material to which a liquid can move by capillary action, A specimen holder provided on a part of the film-like body for holding a specimen containing the substance to be measured, and a contrast provided on a part of the membrane-like body and containing a predetermined amount of the substance to be measured. A comparison sample holding unit that holds a sample for comparison, a developing solution supply unit that is provided in a part of the film-like body and supplies a developing solution to the sample held in the film-like body and the comparison sample; A label carrier that is provided in a part of the membrane and supplies an insoluble label carrier that specifically undergoes an antigen-antibody reaction with the measurement target substance together with the developing solution to the specimen and the comparative specimen held on the membrane Provided in the supply unit and a part of the film-like body; A immunoassay kit characterized by comprising a developing liquid absorbing portion for absorbing excess developing solution that has passed through the sample holding portion and the comparison specimens holding portion.
The invention according to claim 7 is the immunoassay kit according to claim 6, wherein the label carrier supply unit solid-phases the label carrier in advance on the membranous body and adds the label carrier in the developing solution after the addition. An immunoassay kit, wherein a labeled carrier is mixed and supplied.
The invention according to claim 8 is the immunoassay kit according to claim 6, wherein the sample holder and the comparison sample holder are sequentially arranged between the developing solution supply unit and the developing solution absorber. It is a kit for immunoassay characterized by the above.
The invention according to claim 9 is the immunoassay kit according to claim 6, wherein the comparison sample holder holds a comparison sample selected within the allowable range of the measurement target substance of the sample. The immunoassay kit is characterized by having a sample holding unit for comparison and a second sample holding unit for comparison that holds a sample for comparison selected outside the allowable range of the measurement target substance of the sample.
The invention according to claim 10 is an immunoassay kit comprising the reaction plate according to any one of claims 6 to 9 and a sample collection device for collecting a sample to be measured by the reaction plate.

According to the first aspect of the present invention, it is possible to measure with high sensitivity the measurement target substance capable of antigen-antibody reaction contained in the sample, compared to the method of moving the sample.
According to the second aspect of the present invention, hemoglobin A1c as a measurement target substance capable of antigen-antibody reaction contained in a specimen can be measured with high sensitivity.
According to the invention of claim 3, the measurement target substance capable of antigen-antibody reaction contained in the specimen can be measured with a fluorescent label with high sensitivity.
According to the invention of claim 4, hemoglobin A1c as a measurement target substance capable of antigen-antibody reaction contained in a specimen can be measured with a fluorescent label with high sensitivity.
According to the invention which concerns on Claim 5, compared with the system which moves a test substance, the immunoassay apparatus which can measure with high sensitivity the measuring object substance which can be reacted with the antigen antibody contained in a test substance can be provided.
The invention according to claim 6 can provide an immunoassay kit capable of measuring a measurement target substance capable of antigen-antibody reaction contained in a sample with higher sensitivity than the method of moving a sample. .
According to the invention which concerns on Claim 7, compared with the aspect which does not have this structure, a label | marker support | carrier can be quantitatively supplied with respect to a test substance and a test substance for a comparison.
According to the eighth aspect of the present invention, the label carrier can be efficiently moved in one direction together with the developing solution with respect to the sample holding unit and the comparison sample holding unit, as compared with the aspect without this configuration. it can.
According to the invention which concerns on Claim 9, compared with the aspect which does not have this structure, it is possible to correct | amend the measurement precision of the measuring object substance of a sample to a higher thing.
According to the invention of claim 10, it is possible to provide an immunoassay kit capable of collecting a specimen and capable of measuring a measurement target component of the collected specimen.

It is explanatory drawing which shows the outline | summary of embodiment of the immunoassay method and its apparatus with which this invention was applied. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a perspective explanatory view showing an overall configuration of an immunoassay device according to a first embodiment. FIG. 3 is an explanatory plan view of FIG. 2. (A) is explanatory drawing which shows the reaction plate which is one element of the kit for immunoassay, (b) is explanatory drawing which shows each function part of a reaction plate typically. (A) is a perspective view showing an example of a blood collection instrument that is an element of an immunoassay kit, (b) is a cross-sectional explanatory view thereof, (c) is an explanatory view showing a blood collector main body of the blood collection instrument, (d) is It is perspective explanatory drawing of the blood-collector main body of (c). (A) is explanatory drawing which shows the detail of the measuring instrument of the immunoassay apparatus which concerns on Embodiment 1, (b) is BB sectional drawing in (a). 3 is an explanatory diagram illustrating a measurement principle of a measurement instrument used in Embodiment 1. FIG. 4 is a flowchart showing an HbA1c measurement control process used in the first embodiment. It is a flowchart which shows the detail of the HbA1c calculation process shown in FIG. (A)-(c) is explanatory drawing which shows typically the reaction process of the reaction plate used in Embodiment 1. FIG. (A) is explanatory drawing which shows the solid-phase principle of the test substance to a reaction plate, (b) (c) is explanatory drawing which shows typically the coupling | bonding operation process of Hb, HbA1c, and fluorescent latex in a test substance. 3 is an explanatory diagram illustrating a measurement principle by a measurement instrument used in Embodiment 1. FIG. (A)-(g) is explanatory drawing which shows typically the measurement process by the measuring instrument used in Embodiment 1. FIG. (A) is explanatory drawing which shows the deformation | transformation form of the reaction plate used with the immunoassay apparatus which concerns on Embodiment 2, (b) is explanatory drawing which shows typically the reaction process of the reaction plate shown to (a). (A) is explanatory drawing which shows the reaction plate used with the immunoassay apparatus which concerns on a comparison form, (b) is explanatory drawing which shows the reaction process of the reaction plate shown to (a). (A) is the graph which computed the average value of the green component with respect to the sample with which HbA1c is known, and the red component, when producing the calibration curve used by HbA1c measurement control processing by a measuring instrument, (b) is a graph of (a) It is explanatory drawing which shows an example of the calibration curve by which the relationship between a HbA1c value and M value (ratio of a red component / green component) is described based on a result.

Outline of Embodiment FIG. 1 shows an outline of an embodiment of an immunoassay method and apparatus to which the present invention is applied.
In the figure, the immunoassay method according to the present embodiment includes a specimen S in which a measurement target substance T is contained in a part of a membrane-like body 2 to which a liquid can move by capillary action, and a predetermined amount of the measurement target substance. After holding the comparison sample S ′ containing T in advance, a developing solution W is added to the membrane 2, and an insoluble labeling carrier M that specifically reacts with the substance to be measured T for antigen-antibody reaction is added to the developing solution W. And the antigen-antibody reaction between the measurement target substance T contained in the specimen S held in the membrane-like body 2 and the contrast specimen S ′ and the label carrier M that moves together with the developing solution W, and then the specimen The accumulation amount of the label carrier M accompanying the antigen-antibody reaction of S and the sample for comparison S ′ is measured.

In such technical means, the membranous body 2 only needs to be made of a material that allows liquid to move by capillary action, and holds the specimen holding portion 3 that holds the specimen S and the comparative specimen S ′. It is necessary to have the comparison specimen holding part 4 (4a, 4b in this example) and the developing liquid W addition part.
Here, since the developing solution W moves through the membrane-like body 2 by capillary action, the developing solution W may be added to a part of the membrane-like body 2.
Further, the label carrier M may be mixed in advance in the developing liquid W, or may be mixed on the film-like body 2 with the developing liquid W added to the film-like body 2.
Furthermore, as a measurement method, any method may be selected as long as it is a method that enables measurement of the amount of label carrier M accumulated due to antigen-antibody reaction.
In the immunoassay method of this example, the measurement target substance T contained in the sample S is immobilized on a part of the membrane-like body 2, and the sample S or the labeled carrier M that has moved together with the developing solution W is immobilized. The principle of antigen-antibody reaction is used according to the amount of the substance T to be measured in the comparison sample S ′.

Next, a typical aspect or a preferable aspect of the immunoassay method according to the present embodiment will be described.
In this example, glycohemoglobin (hemoglobin A1c) is mentioned as a representative example of the substance T to be measured. However, the present invention is not limited to this, and a virus such as influenza virus can be used as long as it can be measured by an immunoassay. You can select as appropriate.
A typical example of the label carrier M includes fluorescent latex, but is not limited thereto, and colored latex or the like can also be used.
Furthermore, as a preferable measurement method of hemoglobin A1c as the measurement target substance T, the insoluble label carrier M includes a first fluorescent latex that reacts with HbA1c (hereinafter abbreviated as HbA1c as necessary), and hemoglobin (hereinafter referred to as necessary). And a second fluorescent latex that reacts with HbA1c or a second fluorescent latex that reacts with light having at least a wavelength corresponding to the first fluorescent latex and the second fluorescent latex. The thing which can measure the accumulation amount of Hb is mentioned.
In this embodiment, the irradiation light only needs to include light having a wavelength corresponding to the first fluorescent latex and the second latex, and an individual light source may be used, but from the viewpoint of simplifying the apparatus configuration. In this case, an LED including light in a predetermined wavelength range is preferable.

  In addition, as shown in FIG. 1, an immunoassay apparatus embodying such an immunoassay method has a membrane-like body 2 made of a material to which a liquid can move by capillary action. A sample holding unit 3 that holds a sample S containing the measurement target substance T in the body 2 and a comparison sample holding unit 4 that holds a comparison sample S ′ containing a predetermined amount of the measurement target substance T (this example) Then, the reaction plate 1 provided with at least 4a, 4b), the developing solution supply means 10 for supplying the developing solution W to the sample S and the comparison sample S ′ held in the film-like body 2, and the membrane-like body 2 Label carrier supply means 11 for supplying the insoluble label carrier M that specifically reacts with the measurement target substance T to the antigen-antibody reaction to the specimen S and the specimen for comparison S ′ together with the developing solution W, the specimen holder 3 and the contrast Corresponding to the specimen holding section 4 (4a, 4b) Vignetting, a measurement instrument 12 for measuring the integrated quantity of labeled carrier M due to antigen-antibody reaction of the specimen S and the comparison specimens S ', include those having a.

In such technical means, the reaction plate 1 has a predetermined film-like body 2, and the film-like body 2 is provided with a specimen holder 3 and a contrast specimen holder 4 (4a, 4b in this example). What is necessary is just to provide at least.
The developing solution supply means 10 may be appropriately selected as a developing solution container as long as it is a functional element that supplies the developing solution W that moves to the membrane 2 by capillary action.
Further, the label carrier supply means 11 may be provided with the label carrier M mixed in advance in the developing liquid W and provided also as the developing liquid supply means 10 or provided separately from the developing liquid supply means 10. A mode in which the label carrier M is separately supplied to the membrane-like body 2 or a mode in which the label carrier M is solid-phased on a part of the membrane-like body 2 through which the developing solution W passes and the labeling carrier M is mixed with the supplied developing solution W. It may be selected as appropriate.
The measuring instrument 12 may be appropriately selected as long as it can measure the accumulation amount of the label carrier M accompanying the antigen-antibody reaction according to the type of the label carrier M.

As this type of immunoassay device, it is preferable to prepare an immunoassay kit in consideration of the convenience of a user such as a doctor.
This type of immunoassay kit includes a reaction plate 1 used at the time of immunoassay. The reaction plate 1 is composed of a membrane-like body 2 made of a material capable of moving a liquid by capillary action, and this membrane-like body. A specimen holding unit 3 that is provided in a part of the body 2 and holds a specimen S containing the measurement target substance T, and a predetermined amount of the measurement target substance T that is provided in a part of the film-like body 2 is included. The comparison sample holding unit 4 (4a, 4b) that holds the comparison sample S ′ and the sample S and the comparison sample S ′ that are provided in a part of the film-like body 2 and are held on the film-like body 2 The developing solution supply unit 5 for supplying the developing solution W and the sample S and the sample for comparison S ′, which are provided in a part of the membrane 2 and are held in the membrane 2, are specific to the measurement target substance T. A label carrier supply unit 6 for supplying an insoluble label carrier M that reacts with an antigen and an antibody together with a developing solution W; Provided in a part, a developing liquid absorbing unit 7 for absorbing the developing solution W of surplus that has passed through the sample holding portion 3 and the comparison specimens holder 4 (4a, 4b), include those having a.
As a reaction plate 1 of such an immunoassay kit, a sample holder 3 and a contrast sample holder 4 are provided on the membrane 2, and a developing solution is supplied to the membrane 2 by a developing solution supply unit 5. Typically, W can be supplied, the label carrier M can be supplied by the label carrier supply unit 6, and the developing solution absorbing unit 7 is provided for processing the excess developing solution W. Here, the developing solution supply unit 5 and the label carrier supply unit 6 may be provided in different parts. However, in a mode in which the developing solution W and the label carrier M are mixed in advance, they may be provided in the same part. .

Next, typical or preferred embodiments of the immunoassay kit will be described.
First, as a typical embodiment of the label carrier supply unit 6, there is a method in which the label carrier M is solid-phased in advance on the film-like body 2, and the label carrier M is mixed and supplied in the developing solution W after being added. is there.
Moreover, as a preferable aspect of the sample holding part 3 and the comparison sample holding part 4, an aspect in which the sample holding part 3 and the developing liquid absorption part 7 are sequentially arranged side by side is exemplified. In this embodiment, the labeling carrier M may be supplied together with the developing solution W from one side of the membrane-like body 2, and the labeling carrier M together with the developing solution W supplied in this way is formed on the membrane-like body 2 by capillary action. It moves sequentially toward the other side.
Furthermore, as a preferable aspect of the comparison sample holding unit 4, a first comparison sample holding unit 4a that holds the comparison sample S ′ selected within the allowable range of the measurement target substance T of the sample S, and the sample S And a second comparison specimen holding unit 4b that holds the comparison specimen S ′ selected outside the allowable range of the measurement target substance T.
In this aspect, by grasping the antigen-antibody reaction for each of the first comparison sample and the second comparison sample, the variation in the amount of retention of the sample S is corrected, and the measurement target substance T of the sample S is changed. It is possible to measure.
Furthermore, as a typical embodiment of the kit for immunoassay, an embodiment having only the reaction plate 1 may be used, but a sample collection device (not shown) for collecting the sample S to be measured on the reaction plate 1 is further included. The one that was made.
The present embodiment is a combination of the reaction plate 1 and a sample collection device. For example, as a sample collection device, blood as a sample S of a user can be collected, and blood as a collected sample S can be diluted. By doing so, the user purchases the immunoassay kit so that the user can collect the sample S and use the reaction plate 1 on the spot to inspect the measurement target substance T in the sample. Is possible.

Hereinafter, the present invention will be described in detail based on embodiments shown in the accompanying drawings.
Embodiment 1
2 and 3 show the overall configuration of the immunoassay apparatus according to Embodiment 1. FIG.
-Overall configuration of immunoassay device-
In the figure, the immunoassay device 20 measures HbA1c (%) from the reaction plate 21 that causes an antigen-antibody reaction with respect to HbA1c (corresponding to hemoglobin A1c) contained in the collected blood, and the reaction result of this reaction plate 21. Measuring instrument 22.
Here, HbA1c is a test for examining how much sugar is bound to Hb (corresponding to hemoglobin), which is a protein in blood erythrocytes, and knows the average high blood sugar level in the past 1 to 2 months. It is an indicator that can. In general, the blood glucose level itself changes from moment to moment, and variation occurs in a single test, whereas the HbA1c level is not affected by the previous meal, and the average level of the blood glucose level is grasped. It is an effective index to do.

-Reaction plate-
In the present embodiment, the reaction plate 21 has an elongated resin plate body 30 extending along one direction as shown in FIGS. 2 to 4A and 4B. And a plurality of groove portions 31a are integrally arranged on the surface as a grip portion 31 that can be gripped by the plate main body 30 on one end side of the plate main body 30. The plate main body 30 adjacent to the grip portion 31 is formed with a shallow recess 32 surrounded by the grip portion 31 and the surrounding wall portion.
And in the recessed part 32 of the plate main body 30, the developing solution pad 33 which can supply the developing solution W in order from the holding part 31 side, HbA1c and Hb which are measurement object substances contained in the blood as a specimen are respectively specific. A conjugate pad 34 on which a label carrier M that reacts with an antigen-antibody in advance is solid-phased, a membrane 35 corresponding to a membrane-like body in which the developing solution W and the label carrier M move along the longitudinal direction while penetrating by capillary action, And the absorption pad 36 which absorbs the excessive developing liquid W is provided.

<Development pad>
The developing solution pad 33 is fixed to the recess 32 of the reaction plate 21 using an adhesive sheet (not shown). And as the developing solution pad 33, it is possible to use a sheet or film made of porous synthetic resin such as porous polyethylene or polypropylene, cellulose paper such as filter paper, woven cloth or non-woven cloth such as cotton cloth. it can.
Here, the developing solution W may be appropriately selected as long as it can be used in the immunochromatography measurement method. For example, water, physiological saline, a buffer solution, or the like is used as an aqueous solvent. Examples of the buffer include Tris buffer, phosphate buffer, and HEPES (2-hydroxypiperazine-N′-2-ethanesulfonic acid) buffer. Furthermore, the developing solution W may contain various additives such as a pH adjuster (for example, an acidic agent such as hydrochloric acid or an alkaline agent such as sodium hydroxide), a surfactant, a preservative, and an inorganic salt as necessary. Is also possible.
<Absorption pad>
The absorbent pad 36 may be any material that can absorb and retain excess developing liquid W, and filter paper, cotton cloth, or nonwoven fabric made of porous synthetic resin can be used. The absorption pad 36 is fixed to the recess 32 of the reaction plate 21 using an adhesive sheet (not shown), and one end thereof is disposed so as to cover one end of the membrane 35.

<Conjugate pad>
The conjugate pad 34 is made of substantially the same material as the developing solution pad 33 and is fixed to the recess 32 of the reaction plate 21 using an adhesive sheet or the like not shown. Then, one end of the conjugate pad 34 is in contact with the developing solution pad 33 and the other end is disposed so as to cover one end of the membrane 35.
As shown in FIGS. 4 (a) and 4 (b), the conjugate pad 34 is preliminarily solid-phased with a label carrier M that specifically reacts with HbA1c and Hb for antigen-antibody reaction. The labeling carrier M of this example includes a first fluorescent latex M1 that fluoresces with a green component of Anti-Hb sensitization that specifically reacts with Hb for antigen-antibody reaction, and an Anti-HbA1c-sensitivity that reacts specifically with HbA1c for antigen-antibody reaction. A second fluorescent latex M2 that fluoresces with the red component of the product is used.
Each of the fluorescent latexes M1 and M2 is solid-phased on the conjugate pad 34 via the semi-solid body 341, and when the developing solution W added to the developing solution pad 33 moves to the conjugate pad 34 by capillary action. In addition, they move from the semi-solid body 341 and move sequentially toward the membrane 35 together with the developing solution W.

<Membrane>
As the membrane 35, a cellulose film, a nylon film, a glass fiber film, etc. may be appropriately selected and used as long as the developing solution W moves sequentially in the longitudinal direction by capillary action together with the fluorescent latexes M1 and M2. Is possible.
In this example, a sample holding part 40 that dilutes and holds the blood as the sample S at a predetermined ratio is provided substantially at the center of the membrane 35, and the developing liquid W is more than the sample holding part 40 in the membrane 35. In the upstream side of the moving direction, the first blood that is diluted and held at a predetermined ratio is the blood as the comparative sample C1 selected within the allowable range of the measurement target substance of the sample S (that is, within the normal range). A first control unit 41 is provided as a sample holding unit for comparison, and on the downstream side of the membrane 35 in the movement direction of the developing solution W from the sample holding unit 40, the sample S is outside the allowable range of the measurement target substance (that is, A second control section 42 is provided as a second comparison specimen holding section that holds the blood as the comparison specimen C2 selected as the abnormal range) diluted at a predetermined ratio.
Here, the sample holding unit 40 applies a predetermined amount (for example, 1 to 2 μl) of blood diluted as a sample S at a predetermined ratio (for example, 1000 times), to a predetermined temperature (for example, 37 ° C.). The blood as the specimen S applied with a constant temperature block (not shown) that has been constant temperature is dried and held on the membrane 35 in a thermally denatured state (see FIG. 11A). Reference numeral 401 denotes a solid phase body that firmly solidifies a measurement target substance (Hb, HbA1c) in blood as the specimen S.
In addition, the first control unit 41 applies a predetermined amount (for example, 1 to 2 μl) of blood diluted as a comparison sample C1 in the normal range at a predetermined ratio (for example, 1000 times). The blood C1 is dried by heating to a predetermined temperature (for example, 37 ° C.) and held on the membrane 35 in a state of being heat-denatured.
On the other hand, the second control unit 42 applies a predetermined amount (for example, 1 to 2 μl) of a diluted sample of blood as the comparison sample C2 in the abnormal range at a predetermined ratio (for example, 1000 times). The blood C2 is dried by heating to a predetermined temperature (for example, 37 ° C.) and held on the membrane 35 in a heat-denatured state.

-Immunoassay kit-
In the present embodiment, the immunoassay kit 25 is a combination of one or a plurality of reaction plates 21 and a blood collection device 50 that collects blood as the sample S, as shown in FIGS. It is provided to the user as a set of consumables.
For this reason, if the user purchases the measuring instrument 22 having a long life in advance when using the immunoassay device 20, the user may purchase the immunoassay kit 25 as needed.
-Blood collection device-
In this example, as shown in FIGS. 5 (a) and 5 (b), the blood collection device 50 includes a blood collection body 51 that collects blood as the specimen S, and blood collected by the blood collection body 51 in diluted water. A dilution container 57 for dilution, and is made of a synthetic resin such as polypropylene.
<Blood collector body>
In this example, the blood collection device main body 51 has a cylindrical knob 52 as shown in FIGS. 5C and 5D, and a hook-shaped flange 53 is provided at one end of the knob 52. , A triangular protruding plate 54 having a diameter portion of the knob 52 as one side is formed, and a rectangular blood collection receiving piece 55 is provided at the tip of the protruding plate 54. A cut groove 56 capable of holding blood is formed. Here, the cut groove 56 is formed by cutting according to the blood collection volume to be collected. For example, when a blood collection volume of 1 μl is required, the width dimension, the depth dimension are 0.5 mm, and the length dimension is It is set to 4 mm.

<Dilution container>
The dilution container 57 has a cylindrical container body 57a with one end opened, and a hollow conical container protrusion 57b is formed on the other end of the container body 57a, and the tip of the container protrusion 57b is formed. An injection hole 57c is formed in the container body 57 while a flange-like flange portion 57d is formed on one end side of the container body 57a. A cap 58 for opening and closing the injection hole 57c is detachably attached to the tip of the container projection 57b.
In this example, the blood collection device main body 51 is fitted into the container body 57a of the dilution container 57, and the flange portion 53 of the blood collection device main body 51 is supported by the flange portion 57d of the container body 57a.

<How to use blood collection device>
(1) Processing at the time of blood collection If the blood collection device main body 51 of the blood collection device 50 is removed from the dilution container 57 and blood as the sample S is brought into contact with the blood collection piece 55 of the blood collection device main body 51, the cut is caused by capillary action. A predetermined amount (1 ml in this example) of blood is collected in the groove 56.
(2) Blood Dilution Treatment After injecting a predetermined amount (for example, 1 cc) of diluted water into the dilution container 57 with the cap 58 in place, the blood collector main body 51 is fitted into the dilution container 57. In this state, the blood held in the cut groove 56 of the blood collector main body 51 is immersed in the diluted water, and the collected blood is diluted to a predetermined ratio with the diluted water.
(3) Addition process to reaction plate When the blood dilution process is completed, the cap 58 of the dilution container 57 is removed, and then the injection hole 57c of the blood collection device 50 is disposed above the sample holder 40 of the reaction plate 21, In this state, the dilution container 57 is deformed from the outside, and the blood as the diluted sample S in the dilution container 57 is dropped from the injection hole 57c as shown in FIG. Blood as the specimen S may be added to the unit 40.
In addition, what is necessary is just to perform a predetermined | prescribed drying process as mentioned above, in order to make the sample holding | maintenance part 40 hold | maintain the blood as the sample S after blood addition.

-Measuring instrument-
As shown in FIGS. 2, 3, 6 (a), (b) and FIG. 7, the measuring instrument 22 has a substantially bowl-shaped instrument housing 60, and the upper surface of the instrument housing 60 is gently inclined. The display unit 62 is formed on the upper side of the inclined part 61, and various operation buttons 63 are installed on the lower side of the inclined part 61. Reference numeral 64 denotes a name plate of the measuring instrument 22 provided near the center of the inclined portion 61.
In addition, an insertion port 65 for the reaction plate 21 is provided on the side wall of the instrument housing 60 located on the lower side of the inclined portion 61.
A guide receiving portion 66 is provided in the instrument housing 60 to guide the reaction plate 21 inserted from the insertion port 65 to a predetermined position. A position detector (not shown) for detecting whether or not the plate 21 is set at a normal position is provided.
In addition, a measurement opening 67 is opened in a portion of the guide receiving unit 66 where the sample holding unit 40 and the first and second control units 41 and 42 of the membrane 35 of the reaction plate 21 are exposed.
Furthermore, in the present embodiment, a support cover 68 having a substantially trapezoidal cross section is provided in the instrument housing 60 so as to face the measurement opening 67 and cover the measurement opening 67 from the upper side. An LED 70 that irradiates measurement light toward the measurement opening 67 is installed on the side wall, and an imaging device 80 such as a camera is installed on the opposite wall of the support cover 68 that faces the measurement opening 67. .
Here, since LED70 irradiates the light containing the light component of multiple wavelengths, the light component (green component) which makes 1st fluorescence latex M1 (green fluorescence) fluoresce, and 2nd fluorescence latex M2 ( The amount of accumulated first fluorescent latex M1 or second fluorescent latex M2 can be determined by color-separating the red fluorescent light into a fluorescent light component (red component). In this example, the LED 70 is used as a light source. However, a light source that emits light of a predetermined single wavelength may be used individually.
In addition, the imaging device 80 may be appropriately selected as long as it can grasp the accumulated amount of the first fluorescent latex M1 or the second fluorescent latex M2. For example, the optical information of each fluorescent latex M1 or M2 is used for brightness. Processing may be performed by replacing the information.

-Control device-
A control device 100 is built in the instrument housing 60 of the measurement instrument 22. The control device 100 includes a microcomputer system including a CPU, a RAM, a ROM, and an input / output port. For example, an HbA1c measurement control program (see, for example, FIGS. 8 and 9) is preinstalled in the ROM, and the operation buttons 63 and the unillustrated In response to the input signal from the position detector, the CPU executes the above-described HbA1c measurement control program, and sends predetermined control signals to the LED 70, the imaging device 80, and the display 62.

-Reaction process by reaction plate (Fig. 10, Fig. 11)-
First, the reaction process by the reaction plate 21 will be described.
As described above, the user collects blood as the sample S using the blood collection device 50 and dilutes the blood to a predetermined ratio with the diluent.
Thereafter, the user adds the blood as the specimen S to the specimen holding part 40 of the membrane 35 of the reaction plate 21 so that the blood is dried in a constant temperature block not shown.
Then, as shown in FIG. 11A, the measurement target substance (Hb, HbA1c) in the blood is held in the specimen holding unit 40 via the solid phase body 401 in a thermally denatured state.
<Supply of developing solution>
In this state, the user supplies the developing solution W to the developing solution pad 33 of the reaction plate 21 as shown in FIG. At this time, as a method for supplying the developing liquid W, a dropper or an automatic supply device may be used.
Then, the developing solution W moves from the developing solution pad 33 to the conjugate pad 34 by capillary action.
<Mixing of each fluorescent latex into the developing solution>
In the conjugate pad 34, as shown in FIG. 10B, each of the fluorescent latexes M1 and M2 is solid-phased via the semi-solid body 341. However, when the developing solution W moves, the developing solution As the W moves, each of the fluorescent latexes M1 and M2 is mixed into the developing solution W and moves to the membrane 35 together with the developing solution W by capillary action.

<Antibody-antibody reaction with each fluorescent latex>
Thereafter, when the developing solution W mixed with each of the fluorescent latexes M1 and M2 moves to the membrane 35, first, as shown in FIG. An antigen-antibody reaction by M2 occurs.
Furthermore, a large amount of each of the fluorescent latexes M1 and M2 is still mixed in the developing solution W that has passed through the first control unit 41. As shown in FIG. When the unit 40 is reached, an antigen-antibody reaction is caused by the fluorescent latexes M1 and M2 in the specimen holding unit 40.
Furthermore, a large amount of each of the fluorescent latexes M1 and M2 is still mixed in the developing solution W that has passed through the specimen holding unit 40. As shown in FIG. When the control unit 42 is reached, an antigen-antibody reaction by the fluorescent latexes M1 and M2 occurs in the second control unit 42.
Thus, in the first control unit 41, the sample holding unit 40, and the second control unit 42, the same antigen-antibody reaction occurs, and as shown in FIGS. 11 (b) and 11 (c), the sample The first fluorescent latex M1 and the second fluorescent latex M2 bind to Hb or HbA1c, which is an antigen contained in S (or the comparison samples C1 and C2), according to the corresponding antigen amount.
Here, the first control unit 41 obtains the result of the antigen-antibody reaction in the comparison sample C1 (corresponding to the sample S within the normal range), and the sample holding unit 40 obtains the result of the antigen-antibody reaction in the sample S. The second control unit 42 obtains the result of the antigen-antibody reaction in the comparison sample C2 (corresponding to a sample outside the normal range).

-Measurement processing with measuring instruments-
When the antigen-antibody reaction by the reaction plate 21 is completed, the reaction result needs to be measured by the measuring instrument 22.
First, as shown in FIGS. 2 and 3, the user inserts the reaction plate 21 from the insertion port 65 of the measuring instrument 22 and sets it along the guide receiving portion 66.
In this state, as shown in FIG. 8, the control device 100 determines whether or not the reaction plate 21 is set at the normal position. If the reaction plate 21 is set at the normal position, the operation button 63 is Determine whether it was turned on.
Then, when the operation button 63 is turned on, the control device 100 turns on the LED 70, and the imaging instrument 80 causes the sample point (sample holding unit 40) and the comparison point (first control unit 41, second control unit). The result of antigen-antibody reaction of HbA1c in 42) is imaged.
Thereafter, the control device 100 calculates HbA1c based on the imaging result obtained by the imaging instrument 80 and displays the measurement result on the display 62.

<HbA1c calculation process>
Here, the calculation process of HbA1c will be described in detail based on FIG. 9, FIG. 12, and FIG.
First, as shown in FIG. 9, the control device 100 separates the imaging result for each predetermined fluorescent component (green component / red component).
Then, the control apparatus 100 converts the green components (Hb aggregates) of the comparison samples C1 and C2 and the sample S into pixels, and calculates an average value. Similarly, the red components (HbA1c aggregates) of the comparison samples C1 and C2 and the sample S are converted into pixels, and an average value is calculated.
That is, as shown in FIG. 12, when the imaging device 80 is photographed by irradiating the LED 70, for example, the accumulated amount of the first fluorescent latex M1 by green fluorescence Lx (Lx: abbreviation of latex) is combined with Hb. Hb aggregates are obtained, and the degree of aggregation of these is obtained as lightness information (GN (C1), GN (S), GN (C2)), which is quantified (A (C1), A (S), A (C2)). Further, the amount of the second fluorescent latex M2 accumulated by the red fluorescence Lx binds to HbA1c to form an HbA1c aggregate, and the degree of aggregation is (RD (C1), RD (S), RD (C2)). The brightness information is obtained and digitized (B (C1), B (S), B (C2)) (see FIGS. 13A to 13D).
At this time, the quantification of GN by green fluorescence Lx (A (C1), A (S), A (C2)) does not change much between the three, but the quantification of RD by red fluorescence Lx (B (C1)) , B (S), B (C2)) tend to be B (C1) <B (C2), and it is grasped whether B (S) is closer to B (C1) or B (C2).

Next, as shown in FIG. 9, the control device 100 calculates D = red component (RD) / green component (GN) for the comparison samples C1, C2 and the sample S, and D (C1), D (S ), D (C2) is calculated (see FIG. 13E).
Here, D (C1) or D (C2) for which HbA1c is known is compared with calibration curve data (L) obtained in advance through experiments or the like, and it is determined whether or not the difference ΔU is within the allowable range α. To do. An example of creating calibration curve data (L) will be shown in the examples described later.
When the difference ΔU is within the allowable range α, the obtained D (S) is determined as it is, and when the difference ΔU exceeds the allowable range α, a non-zero correction amount ΔD is determined and obtained. The final D (S) is determined in consideration of the correction amount ΔD for the obtained D (S) (see FIGS. 13F and 13G).
In the present embodiment, the calculation results of the first control unit 41 (contrast sample within the normal range) and the second control unit 42 (contrast sample outside the normal range) are compared with the calibration curve data (L). Therefore, it is possible to grasp the normal state and the abnormal state from the calculation results of the control units 41 and 42, and to correct the calculation result of the sample holding unit 40 more accurately. When a simple calculation result is sufficient, the calculation result of either one of the control units 41 or 42 may be used. When a simple calculation result is sufficient, each control unit 41 or 42 is not provided. It is also possible to do.

In the present embodiment, a mode in which the first fluorescent latex M1 and the second fluorescent latex M2 are preliminarily solid-phased in the developing solution W in the reaction plate 21 is employed. For example, as shown in FIGS. 14 (a) and 14 (b), the first fluorescent latex M1 and the second fluorescent latex M2 are mixed in advance in the developing solution W, or When supplying the developing solution W, the fluorescent latexes M1 and M2 may be mixed.
In this embodiment, the conjugate pad 34 becomes unnecessary, and the developing solution W in which the fluorescent latexes M1 and M2 are mixed may be added to the developing solution pad 33.

◎ Comparison 1
In this embodiment, the sample S is solid-phased on the sample holding unit 40 of the reaction plate 21. However, in evaluating the performance of this, the mode in which the sample S is moved is considered as Comparative Example 1. It is as follows.
FIGS. 15A and 15B show the reaction plate 121 used in the first comparative example.
In the figure, a reaction plate 121 has a long resin plate body 130 extending along one direction, and a grip portion 131 that can be gripped by the plate body 130 is formed on one end side of the plate body 130. A shallow recess 132 is formed in the plate main body 130 adjacent to the grip 131.
In this example, in the concave portion 132 of the plate main body 130, the sample supply pad 133 to which the sample S can be supplied, and Hb and HbA1c, which are measurement target substances contained in the blood as the sample S, are sequentially provided from the gripper 131 side. Conjugate pad 134, specimen S and labeling carrier M on which solid-phased labeling carrier M (specifically M1, M2) that specifically reacts with an antigen-antibody moves in the longitudinal direction while penetrating by capillary action. A membrane 135 corresponding to the film-like body to be performed and an absorption pad 136 for absorbing the surplus specimen S are provided. In this example, the conjugate pad 134 is assigned to a part of the membrane 135.
In this example, on the membrane 135, a first capture 141 that captures an aggregate in which the measurement target substance (Hb, HbA1c) and the label carrier M (M1, M2) in the sample S are bound, and the label carrier M A second capture 142 coupled to only (M1, M2) is provided.
When such a reaction plate 121 according to Comparative Example 1 is used, it is assumed that the measurement target substance in the sample S binds to the label carrier M (M1, M2) of the conjugate pad 134. The specimen S and the label carrier M that have passed through 134 are captured by the first capture 141, and surplus label carrier M is captured by the second capture 142.
Therefore, in the conjugate pad 134 or the first capture 141, the measurement target substance of the specimen S and the label carrier M are bound, and measurement of the measurement target substance may be possible by measuring this. However, the measurement target substance in the sample S is not only captured in the form of being bisected by the conjugate pad 134 and the first capture 141, but the measurement target substance is not captured by the movement of the sample S and is absorbed by the absorption pad 136. There are also concerns about being absorbed.
Therefore, it is understood that the reaction plate 121 of the comparative form 1 has a lower measurement accuracy of the measurement target substance of the sample S than the aspect according to the first embodiment.

This example shows an example of creating calibration curve data used in the immunoassay apparatus according to the first embodiment.
Now, in preparing the calibration curve data (L), samples with known HbA1c (%) (in this example, HbA1c (%) is “1.3”, “4.5”, “8.1”, “ 11.7 ”and“ 15.0 ”), each sample is photographed with the imaging device 80, and the green component of the respective green fluorescence Lx and the red component of the red fluorescence Lx are pixelated to calculate the average value. (See FIG. 16A).
Thereafter, when the ratio D between the green component by the green fluorescence Lx and the red component by the red fluorescence Lx is calculated, the result of FIG. 16B is obtained.
By such processing, calibration curve data (L) can be created. For example, the calibration curve data (L) can be used in the form shown in the first embodiment.

  DESCRIPTION OF SYMBOLS 1 ... Reaction plate, 2 ... Membrane, 3 ... Specimen holding | maintenance part, 4 (4a, 4b) ... Contrast specimen holding | maintenance part, 5 ... Development liquid supply part, 6 ... Label carrier supply part, 7 ... Development liquid absorption part , 10... Development liquid supply means, 11. Label carrier supply means, 12. Measuring instrument, M. Label carrier, S.

Claims (10)

After preliminarily holding a specimen in which a substance to be measured is contained in a part of a film-like body to which a liquid can move by capillary action and a specimen for comparison in which a predetermined amount of the substance to be measured is contained,
In addition to adding a developing solution to the membrane, the developing solution contains an insoluble label carrier that specifically reacts with the measurement target substance with an antigen-antibody,
An antigen-antibody reaction between a measurement target substance contained in the specimen held in the membrane and the contrast specimen and a labeled carrier that moves together with the developing solution,
Thereafter, an immunoassay method is characterized in that the amount of label carrier accumulated in the antigen-antibody reaction of the specimen and the comparative specimen is measured. The immunoassay method according to claim 1,
The immunoassay method, wherein the measurement target substance is hemoglobin A1c. The immunoassay method according to claim 1,
An immunoassay method, wherein the labeling carrier is a fluorescent latex. In the aspect of the immunoassay method according to claim 3, the measurement target substance is hemoglobin A1c.
The insoluble labeling carrier has a first fluorescent latex that reacts with hemoglobin A1c and a second fluorescent latex that reacts with hemoglobin,
An immunoassay method comprising measuring the amount of hemoglobin A1c or hemoglobin accumulated by irradiating light containing at least light having a wavelength corresponding to the first fluorescent latex and the second fluorescent latex. A sample holding unit for holding a sample containing a measurement target substance in a film-like body made of a material capable of moving liquid by capillary action, and a predetermined amount of the measurement target substance A reaction plate provided with at least a comparison sample holder for holding the included comparison sample;
A developing solution supply means for supplying a developing solution to the specimen and the comparative specimen held in the membrane;
A label carrier supply means for supplying an insoluble label carrier that specifically undergoes an antigen-antibody reaction with the measurement target substance together with the developing solution to the specimen and the comparative specimen held in the membrane,
A measuring instrument that is provided corresponding to the sample holding unit and the comparison sample holding unit, and that measures an accumulation amount of a label carrier accompanying an antigen-antibody reaction of the sample and the comparison sample;
An immunoassay device comprising: An immunoassay kit including a reaction plate used for immunoassay,
The reaction plate is
A film-like body made of a material capable of moving liquid by capillary action;
A specimen holding part that is provided in a part of the film-like body and holds a specimen containing a substance to be measured;
A comparison sample holding unit that is provided in a part of the film-like body and holds a comparison sample containing a predetermined amount of a measurement target substance;
A developing liquid supply unit that is provided in a part of the film-like body and supplies a developing liquid to the specimen and the comparative specimen held in the film-like body;
A label provided on a part of the membrane and supplying an insoluble label carrier that specifically reacts with the substance to be measured and antigen-antibody with the developing solution to the sample and the comparative sample held on the membrane A carrier supply unit;
A developing solution absorbing portion that is provided in a part of the film-like body and absorbs an excessive developing solution that has passed through the sample holding portion and the contrast sample holding portion;
An immunoassay kit comprising: The immunoassay kit according to claim 6,
The kit for immunoassay characterized in that the label carrier supply section solid-phases the label carrier in advance on the membranous body, and mixes and supplies the label carrier in a developing solution after addition. The immunoassay kit according to claim 6,
The immunoassay kit, wherein the sample holder and the contrast sample holder are sequentially arranged between the developing solution supply unit and the developing solution absorber. The immunoassay kit according to claim 6,
The comparison sample holding unit is selected outside the allowable range of the first measurement sample holding unit for holding the comparison sample selected within the allowable range of the measurement target substance of the sample, and out of the allowable range of the measurement target substance of the sample A kit for immunoassay, comprising: a second sample-holding unit for comparison that holds a sample for comparison. A reaction plate according to any one of claims 6 to 9,
A sample collection device for collecting a sample to be measured on the reaction plate;
An immunoassay kit comprising:

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