JP2001004628A - Immunoassay and its method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To easily, rapidly execute accurate immunoassay with a trace sample. SOLUTION: This immunoassay method uses an immunity analysis microchip. The microchip includes a solid fine particle 2 of a diameter below 1 mm as a reactive solid phase, a micro channel reaction bath part 3 having a larger cross section than the diameter of the solid fine particle 2, a micro channel separation part 4 having a smaller cross section than the diameter of the solid fine particle 2, and an inlet part or a micro channel inflow parts 5, 6 for separately leading an antigen and a labeled antibody to the reaction bath part 3.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an immunoassay analyzer and an analysis method using the same.

[0002]

2. Description of the Related Art Conventionally, immunoassay methods using various antigen-antibody reactions have been known, and studies on analysis kits as a means for simplifying this analysis have been advanced. . However, in the conventional general method, there are problems such as a complicated process and time required for several days for immunoassay, and in many cases, labeling with a fluorescent dye is required. Therefore, there is a problem that the damage of the sample due to the fluorescent dye label cannot be avoided.

For this reason, conventionally, in the immunoassay method for biopolymers and the like, the reaction / separation / detection can be performed in a short time with high accuracy by simple means, and furthermore, the analysis does not damage the sample. The realization of the method was desired. Under such circumstances, an attempt to integrate a chemical system on a substrate such as glass (hereinafter referred to as a microchip) is expected to bring advantages such as simplification of operation and shortening of analysis time by automation. -TAS has been actively used in recent years. The inventor of the present application is also studying ultra-trace analysis using a thermal lens microscope and the fact that molecular diffusing force is effective for rapid molecular transport as a size effect of a minute space, and is studying its own integration.
However, as for immunoanalysis, it is expected as an important target of μ-TAS using electroosmosis and electrophoresis in the same manner as DNA analysis, and chips of various shapes with advanced structures have been developed. Until now, there have been few reports of cases in which an antigen-antibody reaction was actually performed in a microchip.

Therefore, the invention of this application is to overcome the limitations of the prior art as described above, and to provide a new immunoassay apparatus capable of performing an immunoassay by actually enabling an antigen-antibody reaction in a microchip. It is an object of the present invention to provide an analysis method using the method. The invention of this application uses such a new analyzer and a new analysis method to perform reaction / separation / detection in a short time with high accuracy by simple means, and to prevent damage to the sample.

[0005]

Means for Solving the Problems In order to solve the above-mentioned problems, the invention of the present application firstly discloses a method in which, together with solid fine particles having a diameter of 1 mm or less as a reaction solid phase, the diameter of the solid fine particles is larger than that of the solid fine particles. A microchannel reactor having a vertical cross-sectional area, a microchannel separator having a vertical cross-sectional area smaller than the diameter of the solid fine particles, and an introduction section or a microchannel inflow section for separately leading an antigen and a labeled antibody to the reaction tank section. And a microchip having the following.

[0006] Second, an immunoassay device having a microchannel inflow portion for an antibody different from the labeled antibody is used. Third, solid fine particles are glass beads or polymer beads. An immune analyzer according to claim 1 or 2 is provided. Fourthly, the invention of this application is, in a fourth aspect, an immunoassay method using any one of the first to third microchips, wherein solid fine particles as a reaction solid phase are charged into a microchannel reaction tank. A microchip immunoassay method comprising reacting an antigen and a labeled antibody introduced from a microchannel inflow section on solid fine particles, separating unreacted substances in a microchannel separation section, and analyzing them by photothermal conversion analysis. Fifth, a method of introducing a labeled antibody as a second antibody to a reaction tank together with a first antibody, sixth, a method of using a colloidal gold-labeled antibody as a labeled antibody, and a seventh method. Also provides an analysis method in which the photothermal conversion analysis is an analysis by a thermal lens microscope, fluorescence analysis or chemiluminescence with a high spatial resolution of 2 μm or less.

[0007]

BEST MODE FOR CARRYING OUT THE INVENTION The invention of this application has the features as described above, and embodiments thereof will be described below. First, the immunoassay apparatus of the present invention will be described by way of example with reference to FIG.

For example, as shown in FIG. 1, in the case of the immunoassay microchip of the present invention, a substrate such as glass or silicon, more preferably a substrate (1) such as transparent glass, is used as a reaction solid phase. Along with the solid fine particles (2) having a diameter of 1 mm or less, a microchannel reactor (3) having a vertical sectional area larger than the diameter of the solid fine particles (2), and a vertical sectional area smaller than the diameter of the solid fine particles (2). A microchannel separation section (4), and an introduction section or a microchannel inflow section (5) (6) for separately leading the antigen and the labeled antibody to the reaction tank section.
have.

In the example shown in FIG. 1, the labeled antibody serving as the second antibody is introduced into the reaction vessel section (3) together with the microchannel inflow section (6) and the micro antibody for introducing the first antibody is introduced. A channel inflow section (7) and a microchannel inflow section (8) for introducing a buffer solution or a washing solution, and an end of each of the microchannel inflow sections (5), (6), (7) and (8). Are provided with injection holes (5A), (6A), (7A), and (8A) for an antigen, a labeled antibody (second antibody), a first antibody, and a washing solution.

In the example of FIG. 1, a waste liquid part (4A) is provided at the end of the microchannel separation part (4). The solid fine particles (2) serve as a reaction solid phase for an immune antigen-antibody reaction. For example, glass beads or polymer beads such as polystyrene are used. This solid fine particle (2)
Has a diameter of 1 m in the microchip of the present invention.
m or less, for example, 15 to 85 μm, and further, a diameter of 40 to 85 μm.
Used as 65 μm.

The size of the microchannel reaction tank (3) is determined by its vertical cross-sectional area, more specifically, in the case of a hemispherical or curved hole, the maximum cross-sectional area in the vertical direction is determined by the solid fine particles. 1 is larger than the diameter of (2), while the cross-sectional area of the flow channel of the microchannel separation part (4), that is, FIG.
It is a requirement of the present invention that the cross-sectional area at the α-α position is smaller than the diameter of the solid fine particles (2).

For example, more specifically, when the size of the microchannel reaction tank (3) is a hemispherical hole, the radius thereof is, for example, 100 μm or more, and more preferably 150 μm or more. Is taken into account. In addition, it is considered that the microchannel separation section (4) has a depth of 10 μm or less and a width of 10 μm or less, for example. By doing so, the solid fine particles (2) as the reaction solid phase do not flow into the microchannel separation part (4) but are blocked. Then, only unreacted substances flow into the microchannel separation section (4) and are separated. If necessary, only the reaction product desorbed from the solid fine particles (2) is separated.

The microchannel inflow section (5) for introducing the antigen does not necessarily have to be as shown in FIG. 1, and the antigen may be directly introduced into the reaction tank section (3), or Alternatively, the solid fine particles (2) to which an antigen has been previously adsorbed may be introduced into the microchannel reaction tank. Micro channel inlet (5)
For (6), (7) and (8), for example, a depth of 15
It can be configured to have a width of about 0 μm or less and a width of about 300 μm or less. The cross-sectional area should be large enough to allow antigens and antibodies to flow into the microchannel reaction tank (3).

The immunoassay microchip of the present invention may have the above structure formed on the surface of the substrate (1), or may have a multi-layer structure, for example, as shown in FIG.
As shown in the cross section of the α-α portion in FIG. 1, a protection plate (11) is provided on the surface of the substrate (1) on which the microchannel separation portion 4 and the like are formed, and further, the back surface is further reinforced if necessary. The support plate (12) for this may be laminated. In the case of the structure of the laminate as shown in FIG. 2,
In the protection plate (11), the reaction tank (3), the injection holes (5A) (6A) (7A) (8A), and the waste liquid (4)
It is assumed that an opening is provided at a position corresponding to A).

In any case, the immunoassay microchip of the present invention enables an immunoassay with a short reaction time simply by using a small amount of a sample or the like. As an immunoassay method, in the present invention, the solid fine particles (2) as a reaction solid phase are introduced into the microchannel reaction tank section (3) and introduced from the introduction section or the microchannel inflow sections (5) and (6). The reacted antigen and labeled antibody, and if necessary, the antibody introduced from the microchannel inflow section (7) are reacted on the solid fine particles (2), and the unreacted substances are separated in the microchannel separation section (4). It is possible to analyze by analysis.

As the labeled antibody, typically, a colloidal gold-labeled antibody is exemplified as a suitable one. The reactants are
Only the unreacted substances adsorbed on the solid fine particles (2) are separated in the microchannel separation section (4), or the reaction products are desorbed from the solid fine particles (2) and are transferred to the microchannel separation section (4). Will be separated.

As a typical example of the photothermal conversion analysis, there is an analysis using a thermal lens microscope which has already been proposed by the inventors of the present application. More preferably, 2
This is an analysis with a thermal lens microscope having a high spatial resolution of less than μm. This analysis may be performed on the solid fine particles (2) on which the reaction product is adsorbed, or may be performed on the microchannel separation unit (4). Further, not only analysis using a thermal lens microscope but also fluorescence analysis, chemiluminescence analysis, or the like may be employed.

According to the invention of this application as described above,
Reaction / separation detection can be performed easily, in a short time, and with high accuracy, and does not damage the sample unlike the conventional method. Therefore, examples will be shown below, and the invention of this application will be described in more detail.

[0019]

EXAMPLES (Example 1) slgA known as a local immunity and a stress-related substance was selected as a model sample, and B /
The integration such as F separation operation was studied. That is,
Glass microparticles and polystyrene microparticles having a diameter of 43 to 60 μm to which human slgA had been adsorbed were respectively introduced into the microchannel reaction tank, and blocked to form a reaction solid phase.
Next, the gold colloid-labeled antibody was allowed to flow into the reaction tank through the microchannel inflow portion, and an antigen-antibody reaction was performed. Thereafter, the phosphate buffer was fed through the microchannel inflow portion, and the unreacted residue was separated (B / F separation). A thermal lens microscope was used for detection.

In the experiment, a depth of 100 μm and a width of 200 μm
The microchannel inflow section is formed in a quartz glass substrate, and a finer channel with a depth of 5 μm is processed by FAB (Fast Atom Beam) to form a microchannel separation section. In addition, a reaction tank section having a radius of about 1 mm Was processed into a microchip. This microchip is intended to have a structure in which the channel depth is small only in the FAB processing portion and beads used as a reaction solid tank can be dammed.

Note that this microchip does not employ a multilayer structure. Experiments for optimizing the conditions for carrying out the reaction, such as B / F separation, were carried out with the amount of parc. Next, under optimized conditions, fine particles were introduced into the reaction tank portion having a dam structure in the channel, and a PBS buffer was fed to carry out B / F separation as it was. Thereafter, the labeled gold colloid bound on the fine particles was measured with a thermal lens microscope.
For comparison, fine particles subjected to B / F separation by a commonly used method were introduced into the same chip and measured similarly. As a result, a calibration curve for the signal intensity was obtained with respect to the slgA concentration, and no difference was observed between the beads subjected to the B / F separation in the microchannel and the beads subjected to the B / F separation in the ordinary method. It was confirmed. From the above, it was shown that the immunoassay utilizing micro air conditioning of the microchannel is possible. (Example 2) In Example 1, human slgA was introduced into the reaction tank from the inlet of the microchannel, and was adsorbed on glass microparticles. Then, the reaction was carried out in the same manner, and analysis was performed using a thermal lens microscope.

As a result, it was confirmed that similar B / F separation was possible in a short time with a much smaller amount of sample as compared with the usual analysis of the bulk amount.

[0023]

As described in detail above, according to the description of this application, it is possible to carry out an antigen-antibody reaction in a microchip and to carry out an immunoassay, exceeding the limits of the prior art. With such a new microchip and a new analysis method, the reaction and separation / detection can be performed in a short time with high accuracy by simple means, and furthermore, an analysis without damaging the sample is realized.

[Brief description of the drawings]

FIG. 1 is a perspective view illustrating a microchip of the present invention.

FIG. 2 is a cross-sectional view illustrating a multilayer structure.

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 1 Substrate 2 Solid fine particle 3 Microchannel reaction tank part 4 Microchannel separation part 4A Waste liquid part 5,6,7,8 Microchannel inflow part 5A, 6A, 7A, 8A Injection hole part 11 Protection plate 12 Support plate

Continued on the front page (72) Inventor: Seiichi Sato 3-27-G-2103, Nakadai, Itabashi-ku, Tokyo F-term (reference) 2G045 AA40 FA16 FB03 FB07 FB12 FB13

Claims (7)

[Claims] 1. A microchannel reactor having a vertical cross-sectional area larger than the diameter of a solid fine particle having a diameter of 1 mm or less as a reaction solid phase, and a microchannel having a vertical cross-sectional area smaller than the diameter of the solid fine particle. An immunoanalyzer comprising: a channel separation section; and a microchip having an introduction section or a microchannel inflow section for separately guiding an antigen and a labeled antibody to the reaction tank section. 2. The immunoassay apparatus according to claim 1, further comprising a microchannel inlet for an antibody other than the labeled antibody. 3. The immunoassay apparatus according to claim 1, wherein the solid fine particles are glass beads or polymer beads. 4. An immunoassay method using the apparatus according to claim 1, wherein solid fine particles as a reaction solid phase are charged into a microchannel reaction tank, and the antigen and the antigen introduced from the microchannel inflow section are introduced. A microchip immunoassay method, comprising reacting a labeled antibody on solid fine particles, separating unreacted substances in a microchannel separation part, and analyzing the unreacted substances by photothermal conversion analysis. 5. The analysis method according to claim 4, wherein the labeled antibody as the second antibody is led to the reaction tank together with the first antibody. 6. The analysis method according to claim 4, wherein the labeled antibody is a colloidal gold labeled antibody. 7. The analysis method according to claim 4, wherein the photothermal conversion analysis is an analysis by a thermal lens microscope, fluorescence analysis, or chemiluminescence with a high spatial resolution of 2 μm or less.