JP2003270248A - Antigen detection system - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an antigen detection system that can detect an antigen contained in a biological liquid with extremely high accuracy even when concentration of the antigen in the liquid is low. <P>SOLUTION: This antigen detection system is provided with a detecting section 33 which contains a substrate 33a immobilizing an antibody W5 which is specifically bonded to the antigen contained in the biological liquid, and detects the antigen contained in the biological liquid supplied to the substrate 33a by catching the antigen by means of the antibody W5 immobilized on the substrate 33a by utilizing an antigen-antibody reaction. This system is provided with a circulating section 3 which circulates the biological liquid flowing in a detecting section 33 from the entrance of the section 33 and flows out of the outlet of the section 33, so that the liquid may be made to again flow in the entrance of the section 33 and the antigen contained in the biological liquid may be separately concentrated in the antibody W5 immobilized on the substrate 33a. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an antigen detection system for detecting an antigen in a biological fluid such as saliva, blood, or lymph by an antigen-antibody reaction.

[0002]

2. Description of the Related Art Conventionally, for example, a flow injection method has been used as a method for detecting an antigen in a biological fluid such as saliva, blood, or lymph. This method creates a controlled continuous flow using a metering pump, and performs various reactions or separations such as antigen-antibody reaction and color reaction in the flow, and a detection unit such as a spectrophotometer connected to the end. Is used to rapidly detect antigens and the like in biological fluids.

[0003]

However, since the above-mentioned flow injection method detects the antigen in the biological fluid that is continuously flowing, the detection sensitivity or the concentration of the antigen in the biological fluid is detected. For example, the antigen may be discharged without being detected by the detection unit. as a result,
When the antigen concentration is particularly low, there is a problem that the antigen in the biological liquid cannot be detected accurately.

The present invention has been made in view of the above problems, and provides an antigen detection system capable of detecting an antigen with extremely high accuracy even when the concentration of the antigen in the biological liquid is low. The purpose is to

[0005]

In order to solve the above-mentioned problems, the present invention comprises a substrate on which an antibody that specifically binds an antigen in a biological liquid is immobilized and is supplied to the substrate. In an antigen detection system equipped with a detection unit that captures and detects an antigen in a biological liquid using an antibody immobilized on a substrate by utilizing an antigen-antibody reaction, and flows in from an inlet of the detection unit,
The biological liquid flowing out from the outlet is provided with a circulation unit that circulates so as to flow again into the inlet of the detection unit, so that the antigen on the substrate is separated and concentrated in the antibody on the substrate. Characterize.

According to this structure, since the biological liquid is circulated through the detection portion by providing the circulation portion,
In the detection part, the antigen-antibody reaction between the antigen in the biological liquid and the antibody immobilized on the substrate was insufficient, and the antigen was not specifically bound to the antibody and was discharged from the detection part together with the biological liquid. Even in such a case, the biological liquid containing the antigen that has not been specifically bound can be returned to the detection portion and can be reacted again with the antigen-antibody. By repeating the antigen-antibody reaction in this manner, the antigen in the biological liquid and the antibody immobilized on the substrate can be efficiently reacted. As a result, even if the concentration of the antigen in the biological liquid is low, the antibody immobilized on the substrate can efficiently and specifically bind the antigen in the biological liquid to separate and concentrate it. Therefore, the detection section can directly and accurately detect the antigen in the biological liquid.

[0007] It is also possible to shield the biological liquid flowing inside the detection unit and the circulation unit from the outside air so as not to come into contact with the outside air. This allows
An antigen that easily reacts with the outside air can be accurately detected.

More specifically, as the circulation unit, a circulation passage that connects the inlet and the outlet of the detection unit, and a circulation pump that is interposed in the circulation passage and circulates the biological liquid therein are provided. You may prepare. By using the circulation pump in this way, it is possible to forcibly circulate the biological liquid in the detection portion and the circulation passage that connects the inlet and outlet.

A tube pump, a magnet pump, a plunger pump or the like can be used as the circulation pump.

In the present invention, the circulation part may further include a mixing part for mixing the biological liquid circulating therein with one or more fluids.

Specifically, it further comprises a first supplying means for supplying a regenerating solution that separates the specifically bound antibody and the antigen and regenerates as an antibody to the mixing section, and is supplied from the first supplying means. It is also possible to mix the regeneration solution and the biological liquid circulating in the circulation section in the mixing section.

According to this structure, after the antigen specifically bound to the antibody on the substrate of the detection section is detected, the regenerating solution is supplied to the mixing section using the first supply means and flows through the mixing section. By mixing the regenerating solution with the biological liquid, the specific binding between the antibody and the antigen can be separated. As a result, the antibody on the substrate of the detection part is regenerated and can be used again for detection of the antigen in another biological fluid.

Further, a stabilizing solution for stably presenting the antigen when the antigen is chemically unstable, or an identification solution for identifying the antigen for facilitating the detection of the antigen is supplied to the mixing section. It is also possible to further provide a supply means and mix these solutions with the biological liquid circulating in the circulation section.

According to this configuration, the stabilizing solution or the discriminating solution can be supplied to the mixing section by using the second supplying means, and the stabilizing solution or the discriminating solution can be mixed with the biological liquid flowing therein. Therefore, it is possible to prevent chemically unstable antigens from binding to another substance to cause changes in the antigens and to prevent the antigenicity from disappearing, or to identify the trace amount of antigens for accurate detection. . As a result, it is possible to directly detect a trace amount of chemically unstable antigen without changing it to another substance, and it is also possible to perform a highly accurate trace amount detection.

The above solution is an example, and another type of solution may be mixed depending on the type of the object to be detected or the method of detection.

The present invention may further include a metering unit connected to the circulation unit for measuring the biological liquid supplied to the circulation unit.

According to this configuration, since the amount of the biological liquid supplied to the circulation unit can be accurately measured by providing the measuring unit, the antigen in the biological liquid is measured based on the measurement result. Can be more accurately quantified.

The antigen used in the present invention may be nitric oxide (a kind of antigen) in blood formed in the living body. Nitric oxide in blood is chemically unstable and its concentration is as low as about 100 nmol / L. Therefore, in the present invention, for example, sGC that forms a stable complex with nitric oxide as the above-mentioned stabilizing solution. (Soluble guanylate cyclase)
Is further used as a discrimination solution for the fluorescent indicator (for example,
Fluorescein treated so as to specifically bind to the nitric oxide complex) is used.
The stabilizing solution and the discriminating solution are not limited to these, and are appropriately selected depending on the combination of the antigen and the antibody or the detection method. Here, the above-mentioned "nitrogen monoxide is chemically unstable" means that nitric oxide is changed to a more stable form, that is, nitrogen dioxide (NO2) or nitric oxide (NO3).

The blood containing nitric oxide treated as described above circulates in the circulation part, whereby nitric oxide in the blood is efficiently and specifically bound to the antibody on the substrate of the detection part, and separated and concentrated. be able to.

[0020]

BEST MODE FOR CARRYING OUT THE INVENTION Embodiments of an antigen detection system according to the present invention will be described based on the attached drawings.

FIG. 1 is a system diagram when the antigen detection system according to the present invention is applied to the detection of nitric oxide (a kind of antigen) in blood.

The antigen detection system comprises a syringe 1 containing blood K, and a syringe 1 connected to the syringe 1 in which blood K is contained.
And a weighing unit 2 for introducing and weighing
The measuring unit 2 includes a circulating unit 3 for introducing and circulating the measured blood K and the like. Hereinafter, these components will be described in more detail.

The above-mentioned syringe 1 is composed of a cylinder and a piston, and can collect and store blood K from the inside of a living body. As shown in the drawing, the syringe 1 includes a pipe 11 which will be described later.
It is connected to a metering valve 21 of the metering unit 2 described later via a, and is configured so that the blood K stored in the cylinder can be led to the metering unit 2 described later by pushing the piston. Although the manual syringe 1 is shown in FIG. 1, an automatic syringe 1 such as a syringe pump may be used. In addition, in FIG.
Although the case where one syringe 1 is used is shown, a plurality of syringes 1 may be connected by piping (not shown) and used while switching.

Next, the measuring unit 2 will be described. The metering unit 2 includes a metering valve 21, and the metering valve 21 includes six ports A.
First circuit 201-203 having
And the second circuits 204 to 206 indicated by broken lines can be switched. Metering valve 2 of this type
1 is generally called a rotary valve. The syringe 1 is connected to the port A of the metering valve 21 via a pipe 11a. An inlet and an outlet of a measuring pipe 22 for measuring the blood K are connected to the ports C and F of the measuring valve 21, respectively. The measuring tube 22 has a seamless tube shape so that a substance such as blood K that contaminates the subject is not mixed therein, and secures a preset volume. A buffer pump 23 is connected to the port D of the metering valve 21 via a pipe 23e. The buffer pump 23 has a buffer tank 24 for storing a cleaning solution (buffer solution containing a surfactant) W1. , And is connected via a pipe 24e. Further, the piping 20 is connected to the ports B and E of the metering valve 21.
7, 208 are respectively connected. Although a rotary valve is used as the metering valve 21 here, the metering valve 21 is not limited to this.

Next, the circulation unit 3 will be described. The circulation unit 3 includes a detection unit 33, in which a substrate 33a made of gold, platinum, or the like is built-in.
The antibody W5 is immobilized on the surface of the liquid contacting side. Note that mAb (monoclonal antibody) or the like is used as the antibody W5. Further, the detection unit 33 has
A measurement window 33b made of glass or the like is provided facing the surface of the substrate 33a on the liquid contact side. Further, a laser oscillator 44 and an optical measuring instrument 43 which are provided separately from the detecting unit 33.
, Which is disposed facing the measurement window 33b and is on the surface of the substrate 33a on the liquid contact side (in the present embodiment, the nitric oxide complex (NO + W2) described later is attached with the fluorescence identification solution W3). It is configured to be able to detect.

Further, a spirally wound tubular volume 32 is connected to the inlet of the detector 33, and the volume 32 is connected to the outlet of the mixer 31. The mixing section 3
1 is the port E of the above-mentioned metering valve 21 via the pipe 208.
Connected with. The mixing unit 31 is connected to the outlet of the circulation pump 34 via a pipe 34e, and the inlet of the circulation pump 34 is connected to the outlet of the detector 33 via a pipe 33e.

Furthermore, chemically unstable nitric oxide NO
Is provided with a stabilizing solution supply pump 35 for supplying a stabilizing solution W2 which prevents the nitrogen from changing to nitrogen oxides such as nitrogen dioxide, and its outlet is connected to the mixing section 31 through a pipe 35e to supply the stabilizing solution. A storage tank 36 for storing the stabilizing solution W2 is connected to the inlet of the pump 35 via a pipe 36e. Similarly, a fluorescence identification solution supply pump 37 that supplies a fluorescence identification solution W3 that facilitates the detection of nitric oxide NO is provided, and its outlet is connected to the mixing unit 31 via a pipe 37e, and the fluorescence identification solution supply pump is provided. 37
At the entrance of the storage tank 3 for storing the fluorescence identification solution W3
8 is connected via a pipe 38e. Further, carbon monoxide NO and the antibody W5 immobilized on the above-mentioned substrate 33a.
A regeneration solution supply pump 39 for supplying a regeneration solution W4 for regenerating the antibody W5 by separating specific binding with is provided.
The outlet is connected to the mixing section 31 via a pipe 39e,
A storage tank 40 for storing the regeneration solution W4 is connected to the inlet of the regeneration solution supply pump 39. In the present embodiment, the case where the stabilizing solution W2, the fluorescence identifying solution W3, and the regenerating solution W4 are used as the solution to be mixed is shown. However, these are different depending on the type of the object to be detected. The solution is not limited to the above. Further, in place of the pumps 35, 37, 39, an inert gas such as argon or nitrogen gas is supplied into each of the storage tanks 36, 38, 40 to pressurize the storage tanks 3
6, 38, 40 to the mixing section 31 and the above solution W2, W
It is also possible to supply 3 and W4 respectively.

A pipe 41i is branched and attached to the pipe 33e, and a check valve 41, a pipe 41e, and a drain tank 42 are connected to the downstream side of the pipe 41i. Further, the pipe 207 connected to the above-described metering valve 21 is connected to the pipe 41e, and is configured to allow the fluid discharged from the metering valve 21 to flow to the drain tank 42. Here, the check valve 41 is provided so that the fluid discharged through the pipe 207 or the drainage in the drain tank 42 when the circulation pump 34 is operated does not flow into the detection unit 33. is there.

Next, the operation of the antigen detection system according to this embodiment will be described in the order of steps. (1) Preparation Step The circuit of the metering valve 21 is in the state of the second circuits 204 to 206, and a part (for example, about 0.1 ml) of the blood K stored in the syringe 1 is supplied to the pipe 11a. Then second
Drain tank 42 via circuit 204 and piping 207
To discharge. After that, the buffer pump 23 is activated,
The cleaning solution W1 is sucked from the buffer tank 24 and supplied to the port D of the metering valve 21 via the pipe 23e. The cleaning solution W1 flows through the second circuit 205, the measuring pipe 22, and the second circuit 206, is discharged from the port E of the measuring valve 21, and is supplied to the mixing section 31 via the pipe 208.
The cleaning solution W1 introduced into the mixing unit 31 has a volume 32,
It is discharged to the drain tank 42 via the detection unit 33, the pipe 33e, the pipe 41i, the check valve 41, and the pipe 41e. At the same time, the circulation pump 34 is intermittently operated several times to clean the inside of the circulation pump 34 and the pipe 34e.

Similarly, the stabilizing solution supply pump 35, the fluorescence identification solution supply pump 37, and the regeneration solution supply pump 3
Piping 35 connected to the mixing section 31 by intermittently operating 9 several times
e, 36e, 37e, 38e, 39e, 40e are washed respectively. After these washings are completed, all the pumps 23, 34, 35, 37, 39 described above are stopped.

As described above, the insides of the metering unit 2 and the circulation unit 3 can be cleaned and the air inside can be discharged. As a result, nitric oxide N in the air
Since O can be discharged from the circulation unit 3, nitric oxide NO in blood K can be detected with higher accuracy. (2) Metering process After the above preparation process is completed, the circuit of the metering valve 21 is first
Switch to circuits 201-203. Blood K stored in the syringe 1 is connected to the port A of the metering valve 21 via the pipe 11a.
To introduce. The blood K is supplied to the measuring tube 2 via the first circuit 201.
2 and is introduced into the port C of the metering valve 21. Further, a part of the introduced blood K is discharged to the drain tank 42 via the first circuit 202 and the pipe 207.
Since the volume of the measuring pipe 22 is preset, the measuring pipe 2
2 by filling the blood K with a predetermined amount (for example, 0.
1 ml) can be weighed. (3) Introducing process After the above measuring process is completed, the circuit of the measuring valve 21 is changed to the second circuit.
Switch to circuits 204-206. Buffer pump 23
Is operated to introduce the cleaning solution W1 into the second circuit 205. The cleaning solution W1 is pumped to the mixing section 31 through the second circuit 206 and the pipe 208 together with the blood K in the measuring tube 22. Then, the stabilizing solution supply pump 35 is operated to supply the stabilizing solution W2 to the mixing section 31 through the pipe 35e. After that, the operation of all the pumps 23 and 35 is stopped. Thereby, the blood K is introduced into the circulation unit 3, and the nitric oxide NO in the blood K and the stabilizing solution W2 are combined with each other so that the nitric oxide NO can be stably present.

As the stabilizing solution W2, for example, s
GC (Soluble Guanylyl Cyclase) is used.
Further, for convenience of explanation, hereinafter, a mixture of nitric oxide NO and the stabilizing solution W2 will be referred to as a nitric oxide complex (NO).
+ W2). (4) Separation and Concentration Step After the introduction step is completed, the circulation pump 34 is operated, and the washing solution W1, the blood K, and the nitric oxide complex (NO + W2) are mixed in the mixing section 31, the volume section 32, and the detection section. Three
3 and the pipes 33e and 34e (hereinafter, referred to as the detection unit 33 and the like) are circulated. Then, when these solutions flow through the detection unit 33, the nitric oxide complex (NO + W2) is specifically bound to the antibody W5 immobilized on the liquid contact side surface of the substrate 33a of the detection unit 33, thereby separating and concentrating. To be done.
In the present embodiment, since the solution circulates in the detection unit 33 and the like, even if the solution first introduced into the detection unit 33 does not separate and concentrate the nitric oxide complex (NO + W2). Even if the solution is discharged from the detection unit 33, the discharged solution returns to the detection unit 33 again, and the detection unit 33 separates and concentrates the nitric oxide complex (NO + W2) from the solution. By repeatedly performing such an action in the detection unit 33, the nitric oxide complex (NO + W2) is efficiently and specifically bound to the antibody W5 in the detection unit 33 to separate and concentrate from the solution. be able to. After the separation and concentration of these are completed, the operation of the circulation pump 34 is stopped. (5) Fluorescence discrimination solution introduction step After the above separation and concentration step is completed, the fluorescence discrimination solution supply pump 37 is operated, the fluorescence discrimination solution W3 is sucked from the storage tank 38 through the pipe 38e, and the pipe 37e is introduced into the mixing section 31. A predetermined amount of the fluorescence identification solution W3 (for example,
Supply about 0.1 ml). After that, the operation of the fluorescence identification solution supply pump 37 is stopped.

As the fluorescence identification solution W3, for example,
Another antibody (not shown) that specifically binds to the nitric oxide complex (NO + W2) to which a fluorescent identifier (fluorescein or the like) is added is used. (6) Fluorescent Discriminating Body Addition Step After the above-mentioned fluorescent discriminating solution introducing step is completed, the circulation pump 34 is operated and the fluorescent discriminating solution W3 introduced into the mixing section 31 is detected together with the solution in the circulating section 3 in the detecting section 33. And circulate inside. As a result, the fluorescent discriminant in the fluorescent discriminating solution W3 is added to the separated and concentrated nitric oxide complex (NO + W2). Then, the operation of the circulation pump 34 is stopped. (7) Measurement Preparation Step The cleaning solution W1 is mixed with the mixing unit 3 by the same method as the introduction step described above.
1, and the circulation pump 34 is intermittently operated a plurality of times. By introducing the cleaning solution W1 into the mixing section 31,
The fluorescent discriminant in the fluorescent discriminating solution W3 remaining in the detection unit 33 and the like is discharged to the drain tank 42 through the pipe 41i, the check valve 41, and the pipe 42e. Then, the operation of the buffer pump 23 and the circulation pump 34 is stopped. (9) Measurement step As shown in the figure, the laser oscillator 44 has a short wavelength for the antibody, the nitric oxide conjugate (NO + W2), and the fluorescence discriminating solution W3, which are immobilized on the substrate 33a of the detection unit 33. Laser beam 44a is emitted. As a result, the fluorescent discriminant in the fluorescent discriminating solution W3 is excited and the fluorescence 43b is colored. This colored fluorescence 43b is the optical measuring device 4
Introduced in 3 and its intensity is measured. Then, the concentration of nitric oxide NO is calculated using this measurement result. By doing so, the concentration of nitric oxide NO in blood K can be accurately measured. (10) Regeneration Step of Detection Unit 33 After the measurement of nitric oxide NO in the blood K is completed, the regeneration solution supply pump 39 is operated to suck the regeneration solution W4 from the storage tank 40 through the pipe 40e, and the mixing unit. 31 through a pipe 39e to a predetermined amount of the regeneration solution W4 (for example,
Supply about 0.2 ml). At this time, the circulation pump 34 is intermittently operated a plurality of times to promote introduction of the regeneration solution W4 into the mixing section 31. Then, the operation of the regeneration solution supply pump 39 is stopped. Then, the circulation pump 34 is operated to circulate the regenerated solution W4 together with the solution inside the detection unit 33 and the like. The regenerating solution W4 separates the nitric oxide complex (NO + W2) and the fluorescence discriminating solution W3 from the antibody immobilized on the substrate 33a of the detection unit 33. In this way, the antibody W5 in the detection unit 33 is regenerated so that it can be reused.

As the regenerating solution W4, for example, dilute hydrochloric acid whose concentration is adjusted is used. (11) Final treatment step After the steps (1) to (10) above are completed, the cleaning solution W1 is introduced into the mixing section 31 and the circulation pump 34 is intermittently operated a plurality of times by the same method as the above-mentioned introduction step. To do. By introducing the cleaning solution W1 into the mixing unit 31, the detection unit 3
3 regenerant solution W4 remaining in the pipe 3 i, check valve 4
1 and the discharge to the drain tank 42 through the pipe 42e. After that, the buffer pump 23 and the circulation pump 3
Stop the operation of 4.

In this embodiment, the optical measuring method is used as the detecting method, but other methods such as the surface plasmon effect using the vibration frequency change of the quartz oscillator or the evanescent wave are used. It is also possible to use a measuring method to do so. Further, although the present embodiment shows the case where the present invention is applied to the detection of nitric oxide NO in blood K, it may be applied to the detection of an antigen in another biological liquid.

[0036]

INDUSTRIAL APPLICABILITY The antigen detection system according to the present invention circulates a biological liquid, which is an analyte, so that the antigen present therein and the antibody in the detection part can be specifically utilized by utilizing the antigen-antibody reaction. The antigen can be efficiently separated and concentrated from the biological liquid by binding. Thus, even when the concentration of the antigen in the biological liquid is low, it can be detected with extremely high accuracy.

[Brief description of drawings]

FIG. 1 is a system diagram when the antigen detection system according to the present invention is applied to the detection of nitric oxide (a kind of antigen) in blood.

[Explanation of symbols]

1 syringe 2 Weighing department 3 circulation department 31 mixing section 33 Detector 33a substrate 34 Circulation pump NO Nitric oxide W1 cleaning solution W2 stabilizing solution W3 fluorescence identification solution W4 regenerating solution W5 antibody

   ─────────────────────────────────────────────────── ─── Continued front page    (72) Inventor Shinya Yamamoto             1-22 Misakihonmachi, Hyogo-ku, Kobe-shi, Hyogo             No. Showa Seiki Co., Ltd. (72) Inventor Shingo Tsuyama             1-7 Satsukinonishi, Mihara Town, Minamikawachi District, Osaka Prefecture             No. 16 (72) Inventor Shigeo Takenaka             1 67-10 10 Mochimotocho, Sakai City, Osaka Prefecture 301 (72) Inventor Takeaki Yoshimura             A201, 2-3-1, Kasugaoka, Itami City, Hyogo Prefecture

Claims (5)

[Claims] 1. A substrate on which an antibody that specifically binds to an antigen in a biological liquid is immobilized, and the antigen in the biological liquid supplied to the substrate is utilized by utilizing an antigen-antibody reaction. In an antigen detection system including a detection unit that captures and detects an antibody immobilized on the substrate, the biological liquid that flows in from the inlet of the detection unit and flows out from the outlet of the detection unit enters the inlet of the detection unit. An antigen detection system, characterized in that the antigen detection system is provided with a circulation portion that circulates so as to flow in again, and is configured to separate and concentrate the antigen in the biological liquid to the antibody on the substrate. 2. The antigen detection system according to claim 1, wherein the circulation unit further includes a mixing unit that mixes the biological liquid circulating therein with one or more fluids. Antigen detection system. 3. The antigen detection system according to claim 2, wherein the circulation unit supplies the mixing unit with a regenerating solution that separates the specifically bound antibody and the antigen and regenerates them as the antibody. Supplying means, a stabilizing solution that is stably present when the antigen is chemically unstable, or an identification solution that identifies the antigen to facilitate detection of the antigen is supplied to the mixing section. An antigen detection system further comprising: 4. The antigen detection system according to claim 1, further comprising a measuring unit connected to the circulation unit and configured to measure the biological liquid supplied to the circulation unit. Characteristic antigen detection system. 5. The antigen detection system according to any one of claims 1 to 4, wherein the antigen is nitric oxide formed in a living body.