JPH01221666A - Instrument and method for immunological inspection - Google Patents

Abstract

PURPOSE:To detect the degree of immunity with high accuracy by effecting an antigen-antibody reaction between the antigens subjected to the antigen- antibody reaction with previously fixed antibodies and labeled antibodies, then allowing labeling materials to emit light and measuring the quantity of the emitted light. CONSTITUTION:A soln. to be measured is housed in a housing chamber 1b and the antigen-antibody reaction is effected between the antigens 7 in the soln. to be measured and the previously fixed antibodies 6. The soln. to be measured is then discharged from the housing chamber 1b and a labeling material soln. is housed therein to effect the antigen-antibody reaction between the antigens 7 subjected to the antigen-antibody reaction with the antibodies 6 and the antibodies 8 labeled by the labeling materials 9. The labeling soln. is thereafter discharged and a reference liquid is housed into the chamber so that the labeling material 9 emits light. The emitted light is guided through a light guide 2 and a light leading-out part 3 to a photodetecting part. The photodetecting part converts the quantity of the labeling materials 9 labeling the antibodies 8 accepted in the antibodies 6 to electric signals. A detecting part detects the degree of the immunity in accordance with said electric signals.

Description

[Detailed Description of the Invention] <Industrial Application Field> The present invention relates to an immunoassay device and an immunoassay method, and more specifically, to immunoassays by detecting the presence or absence of antigens or antibodies using optical waveguides. The present invention relates to an immunoassay device and an immunoassay method for conducting an inspection.

<Conventional technology> Conventionally, for the purpose of performing immunological tests, on the surface of an optical waveguide,
By forming a region on which an antigen or an antibody is immobilized, introducing light into the optical waveguide while bringing blood or the like into contact with this region, and determining the light intensity ΔP1 emitted from the optical waveguide, the antigen, Alternatively, a device is provided that detects the presence or absence of antibodies and performs an immunological test (see Patent Application Publication Box 58-
(See Publication No. 501481).

To explain in more detail, when light propagates through an optical waveguide, most of the intensity distribution of the light is confined within the optical waveguide, but some of it is trapped in the cladding surrounding the optical waveguide. (Hereinafter, this light component and the light component that enters the inside of the optical waveguide from outside the optical waveguide and propagates inside the optical waveguide as it is are called evanescent waves.) ).

The amount of the evanescent wave reflected into the optical waveguide changes under the influence of the reflectance of the cladding portion.

That is, if an antigen or an antibody is fixed in advance on a portion corresponding to the cladding portion, and light is made to enter the optical waveguide while blood or the like is in contact with this portion, the blood or the like will be completely absorbed by the antibody or antibody. If the blood does not contain an antigen, the intensity of the emitted light changes due to the reflectance of the part on which the antigen or antibody is immobilized; on the other hand, if the blood etc. contains antibodies or antigens, teeth,
When the antigen and antibody combine, a completely different substance is present in the cladding portion, resulting in an emitted light intensity different from the above intensity. (However, it is known that the intensity of the emitted light is lower when an antigen-antibody reaction is performed than when only an antigen or antibody is used).

Therefore, by detecting the presence or absence of a change in the intensity of the emitted light, it is possible to detect whether or not an antibody or antigen is present.

However, in the immunoassay device with the above configuration, the intensity of the emitted light changes only in a short period of time, and the rate at which the intensity of the emitted light changes is extremely small.
It is not possible to reliably distinguish between changes in the intensity of the emitted light due to changes in the degree of humidity and changes in the intensity of the emitted light due to antigen-antibody reactions, resulting in a significant decrease in the accuracy of the immunological test.

In order to solve this problem, an antigen-antibody reaction corresponding to the degree of immunity is caused by bringing blood, etc. into contact with an area on which antigens or antibodies have been immobilized in advance, and then the area is identified using a fluorescent substance. There has been provided an immunoassay device in which an antigen-antibody reaction is caused by contacting the antigen or antibody, and in this state, light is made to enter an optical waveguide. In this case, regarding the direction of the emitted light, there are two methods: detecting the light intensity caused by the fluorescent light on the side where the incident light is emitted as is, and detecting the light intensity caused by the fluorescent light on the side where the light enters. There is a method of detecting the light intensity caused by fluorescence on the side of the optical waveguide.

Therefore, even in devices that test immunity using antigens or antibodies labeled with fluorescent substances, if the intensity of emitted light affected by evanescent waves is detected, it is possible to detect antigens or antibodies in blood, etc. It is possible to test whether or not it exists, that is, whether or not there is immunity.

<Problems to be Solved by the Invention> In an immunoassay device using an antigen or antibody labeled with the above-mentioned fluorescent substance, the fluorescent substance is introduced into an optical waveguide in order to obtain a fluorescent signal corresponding to an antigen-antibody reaction. Excitation light is required, which not only increases the size of the device as a whole, but also generates excess heat and increases power consumption, and furthermore, it does not significantly improve the accuracy of the immunological test. There are also problems.

To explain in more detail, as shown in Figure 5, there is a difference of several tens of TVs between the peak wavelength of excitation light and the peak wavelength of fluorescence, but compared to the light intensity of fluorescence, there is a difference between the peak wavelength of excitation light and the peak wavelength of fluorescence. Since the light intensity of the excitation light is extremely high, the excitation light intensity corresponding to the peak wavelength of the fluorescent light becomes considerably large, resulting in a large error. Filtering the excitation light may be considered in order to eliminate this inconvenience, but since it is impossible to achieve complete filtering, it may only reduce the degree of error and still be used in immunoassays. Accuracy cannot be improved sufficiently.

Furthermore, it is conceivable to adopt a configuration in which the fluorescence is measured on the excitation light incident side, but in this case, the reflection at the excitation light incidence surface will be in a state that cannot be ignored compared to the intensity level of the fluorescence. Similarly, the accuracy of the immunological test cannot be sufficiently improved.

In addition, the light source that generates the excitation light must output only light in an extremely narrow wavelength range centered on the peak wavelength, and must be able to maintain a stable output light intensity, otherwise the accuracy of the immunoassay will be compromised. Therefore, a normal light source cannot be used, and a Xe flash lamp or the like must be used, and a control device must be provided to stabilize the output light intensity. Since the light source and the optical waveguide are arranged in a state where they can be arranged to a certain extent, the overall size of the immunoassay device increases, and the light source generates heat and increases power consumption. There is also the problem that if the light source or the part where the antibody is fixed is exposed, the accuracy of the immunoassay will be reduced.

Furthermore, if a method of detecting the light intensity caused by fluorescence on the side of the optical waveguide is adopted, the fluorescence emitted from all the antigens or antibodies identified by the fluorescent substance can be used in immunoassays. The problem is that the immunoassay accuracy is greatly reduced because it enters as an optical signal for immunoassay, and therefore contains fluorescent light that has not undergone an antigen-antibody reaction.

<Object of the invention> This invention was made in view of the above problems,
It is an object of the present invention to provide an immunoassay device and an immunoassay method that do not require the introduction of excitation light, have a compact and simple configuration, and can significantly improve the accuracy of an immunoassay.

Means for Solving the Problems> In order to achieve the above object, the immunoassay device of the present invention has a storage chamber for storing a test target solution formed at a predetermined position on the side surface of an optical waveguide, and a storage chamber for storing a solution to be tested. An antigen or antibody labeled with a substance that emits light in the presence of a specific substance is immobilized on the side of the optical waveguide located at the Also provided is a light receiving section that receives evanescent waves propagating through the optical waveguide among the light emitted from the received identification antigen or antibody.

However, it is preferable that the optical waveguide is formed to have a rectangular cross section, and that a storage chamber for accommodating the solution to be tested is formed on the side surface of the optical waveguide.

In addition, in order to achieve the above object, the immunoassay method of the present invention involves reacting a test substance with an antigen or antibody fixed on the side surface of an optical waveguide, and then, as a condition of the presence of a specific substance, An antigen or antibody labeled with a luminescent substance is reacted, and then a specific substance is supplied to cause the labeled substance to emit light, and only the luminescence from the labeled substance located near the optical waveguide is propagated through the optical waveguide. This is a method of immunological testing based on light.

Still another immunoassay method involves reacting an antigen or antibody fixed on the side of an optical waveguide with an antigen or antibody labeled with a substance that emits light in the presence of a test substance and a specific substance, Next, a specific substance is supplied to cause the labeling substance to emit light, and only the light emitted from the labeling substance located near the optical waveguide is propagated through the optical waveguide, and an immunological test is performed based on the propagated light.

In the immunoassay device with the above configuration, an antigen or antibody labeled with a substance that emits light in the presence of a specific substance is attached to the side of the optical waveguide located in the storage chamber that houses the solution to be tested. The antigen or antibody that accepts the test substance as a medium is immobilized, and the accepted identification antigen,
Alternatively, a light receiving part is provided to receive evanescent waves propagated through the optical waveguide among the light emitted by the antibody, so the solution to be tested is placed in the storage chamber and the substance to be tested and the pre-immobilized antigen or antibody are combined. An antigen-antibody reaction corresponding to the amount of the substance to be tested is carried out between the parts, and then, or at the same time, a solution containing an antigen or an antibody deposited with a luminescent substance is placed in the part where the antigen-antibody reaction is carried out. Further, an antigen-antibody reaction can be performed against the target. Thereafter, by putting the specific substance into the storage chamber, the luminescent substance can be caused to emit light without introducing any excitation light.

This light emission occurs near the side of the optical waveguide, so it enters the optical waveguide as an evanescent wave.
It propagates inside the optical waveguide as it is. Therefore, by receiving evanescent waves propagating within the optical waveguide at the light receiving section, it is possible not only to detect the presence of immunity, but also to detect the degree of immunity based on the intensity of the evanescent waves.

If the optical waveguide is formed to have a rectangular cross-section and a storage chamber is formed on the side surface of the optical waveguide to accommodate the test target solution, the area on which the antigen or antibody is immobilized is enlarged. Therefore, the intensity of the evanescent waves propagating through the optical waveguide as a whole can be increased, and the accuracy of the immunological test can be improved.

In the above immunoassay method, after a test substance is reacted with an antigen or antibody fixed on the side of an optical waveguide, an antigen labeled with a substance that emits light under the condition of the presence of a specific substance, or In this method, antibodies are reacted, and then a specific substance is supplied to cause the labeling substance to emit light, and only the light emitted from the labeling substance located near the optical waveguide is propagated through the optical waveguide, and an immunological test is performed based on the propagated light. There is no need to introduce excitation light into the optical waveguide, and even if the intensity of the emitted light is weak, there is no effect of the excitation light, which significantly improves the measurement accuracy of the emitted light intensity, which in turn significantly improves the accuracy of immunoassays. be able to.

In addition, in other immunoassay methods described above, an antigen fixed to the side of an optical waveguide, or an antigen labeled with a substance that emits light in response to the presence of a test target substance and a specific substance, or The labeling substance is made to emit light by reacting the antibody and then supplying a specific substance, and only the luminescence from the labeling substance located near the optical waveguide is propagated through the optical waveguide, and an immunological test is performed based on the propagated light. There is no need to introduce excitation light into the optical waveguide, and even if the intensity of the emitted light is weak, there is no effect of the excitation light, which significantly improves the measurement accuracy of the emitted light intensity, which in turn significantly improves the accuracy of immunoassays. be able to. In addition, antigens or antibodies labeled with a substance that emits light in the presence of the test substance and a specific substance can be reacted simultaneously with pre-immobilized antigens or antibodies, making it suitable for immunological testing. Necessary operations can be simplified.

<Example> Hereinafter, embodiments will be described in detail with reference to the accompanying drawings showing examples.

~ Fig. 1 is a schematic perspective view showing an embodiment of the immunoassay device of the present invention, and Fig. 2 is a central vertical cross-sectional view. A region (1) in which a large number of antibodies (6) are fixed in advance is formed on the upper surface of the optical waveguide formed by the optical waveguide.
3) is guided to a light receiving section (4) having a photomultiplier tube etc., and an electric signal outputted from the light receiving section (4) is supplied to a detecting section (5) having a differential amplifier etc. are doing. In addition, a side wall (la) is provided to surround the area (1).
The housing chamber (1b) is formed by integrally forming the housing chamber (1b).

Although not shown, the antibody (6) performs an antigen-antibody reaction with the antigen (7) (see FIG. 3A) contained in a solution to be measured such as blood, and further includes: An antibody (8) that performs an antigen-antibody reaction with the antigen (7) is labeled with a labeling material (9) that emits light in the presence of a substrate solution (see FIGS. 3B and C).

The labeling substance (9) may be one that emits light by a chemiluminescence mechanism, such as peroxidase, or one that emits light by a bioluminescence mechanism, such as luciferase, ecurion, etc. It may be.

The operation of the immunoassay apparatus having the above configuration is as follows.

First, by storing the solution to be measured in the storage chamber (1b), an antigen-antibody reaction is caused between the antigen (7) in the solution to be measured and the preliminarily immobilized antibody (6).
(See Figure C). The amount of antigen-antibody reaction in this case is determined based on the amount of antigen in the solution to be measured, that is, the degree of immunity.

Next, the solution to be measured is discharged from the storage chamber (1b), and the labeling substance solution is contained in the chamber (1b), whereby the antigen (7) that has undergone an antigen-antibody reaction with the antibody (6) is labeled with the labeling substance (9). An antigen-antibody reaction is carried out with the obtained antibody (see Figure 3C).

Therefore, in this state, among the large number of antibodies (6) immobilized in advance on the region (1), only the antigen (7' ), the antibody (8) labeled with the labeling substance (9) is received.

Thereafter, when the labeling substance solution is discharged from the storage chamber (1b) and the substrate liquid is stored, the labeling substance (9) emits light (see FIG. 3C). Since the light emitted from this labeling substance (9) is generated near the top surface of the optical waveguide, a part of the emitted light is introduced into the optical waveguide (2) and propagates through the optical waveguide (2) as it is. The amount of the labeling substance (9) labeling the antibody (8) that is guided to the light receiving part (4) and received by the antibody (6), i.e.
It is converted into an electrical signal corresponding to the amount of antigen (7) that has undergone an antigen-antibody reaction with the antibody (6).This converted electrical signal is supplied to the detection section 6), and is used to detect the presence of immunity and the presence of immunity. In some cases the degree of immunity can be detected.

As is clear from the above explanation, since no excitation light is introduced into the optical waveguide (2), the light emitted from the light emitting portion (3) is directed against the antibody 0 immobilized on the region (1). Only a part of the luminescence of the labeling substance (9) labeled to the antibody B) bound to the antibody B) via the antibody (7) becomes a part of the luminescence.
) is incident on the light receiving section (4) with an intensity corresponding to the amount of antigen ω that has undergone an antigen-antibody reaction, that is, the degree of immunity.

Therefore, the immunoassay accuracy can be significantly improved, and since the excitation light source is omitted, the entire immunoassay apparatus can be downsized.

Further, it is possible to reliably prevent temperature rise due to heat generation of the light source and increase in power consumption due to energization of the light source.

FIG. 4 is a schematic diagram illustrating an immunological test using another procedure. The difference from the procedure in FIG. 3 is that instead of storing the measurement target solution and labeling substance solution in this order,
As shown in Figure A, the only difference is that the measurement target solution and the labeling substance solution are mixed in advance and the mixed solution is stored in the storage chamber (1b).

Therefore, in the case of this example as well, the antigen (7) that has undergone an antigen-antibody reaction with a large number of previously immobilized antibodies (6) is used as the antibody (8) that has already been labeled with the labeling substance θ). will be accepted. Then, light with an intensity corresponding to the amount of antibody (2) received by the antigen (7), that is, the amount of labeling substance (9), is generated, and a part of this light is propagated through the optical waveguide (2), and the light guide unit Derived from (3), the light receiving part (4)
can generate an electrical signal corresponding to the degree of immunity.

As a result, as in the case where the above procedure is employed, it is possible to conduct an immunological test with high precision, and also to achieve the advantages associated with omitting the antigen for excitation light.

The substrate solution may be determined according to the labeling substance (9); for example, luminol solution is used for peroxidase, luciferin solution is used for luciferase, and calcium solution is used for ecurion. Each solution may be determined separately.

Specific example> When measuring human chorionic gonadotropin (hereinafter abbreviated as hCG) using a luciferase-labeled antibody, ■ immobilize the anti-hCG antibody in region (1), and prepare the ■ holding chamber. (
1b) contains the solution to be measured, hCG antigen and anti-hC
An antigen-antibody reaction is performed with the G antibody, ■ By discharging the measurement target solution and storing a labeling substance solution, an antigen-antibody reaction is performed between the hCG antigen and the anti-hCG antibody labeled with luciferase. By discharging the substance solution and storing the luciferin solution, luminescence was caused by a bioluminescence mechanism, and the luminescence was measured for 5 minutes.

Then, by performing the above n1 constant, 5ng/
It was possible to measure hCG at a concentration of xl.

As is clear from this specific example, since no excitation light is used, low concentrations of hCG can be measured with high sensitivity even with weak emitted light. Therefore, the accuracy of immune testing can be significantly improved.

Note that the present invention is not limited to the above embodiments, and for example, instead of immobilizing the antibody (6) to the region (1), it is possible to immobilize the antigen, or to immobilize the labeling substance. It is possible to use other substances as well as a corresponding substrate liquid, and furthermore, it is possible to appropriately set the size of the storage chamber (1b), and to appropriately set the refractive index of the optical waveguide (1b). In addition, various other design changes can be made without changing the gist of the invention.

<Effects of the Invention> As described above, in the first invention, the luminescent substance is made to emit light without introducing any excitation light, and this emitted light is propagated through the optical waveguide and guided to the light receiving section.
It is possible to test the presence or absence of immunity and, if there is immunity, the degree of immunity with extremely high accuracy, and the entire testing device can be made smaller. Moreover, it is possible to prevent temperature fluctuations, and to reduce consumption. This has the unique effect of reducing power consumption.

The second invention is that the optical waveguide is formed to have a rectangular cross section, and a storage chamber for accommodating the test target solution is formed on the side of the optical waveguide, so that the area for immobilizing the antigen or antibody can be enlarged. This has the unique effect of increasing the intensity of the evanescent waves propagating through the optical waveguide as a whole and improving the accuracy of the immunological test.

The third invention eliminates the need to introduce excitation light into the optical waveguide, and eliminates the influence of excitation light even if the intensity of the emitted light is weak, thereby significantly improving the 111J accuracy of the emitted light intensity, and furthermore, It has the unique effect of significantly improving the accuracy of immunological tests.

The fourth invention is that there is no need to introduce excitation light into the optical waveguide, and even if the intensity of the emitted light is weak, there is no effect of the excitation light, thereby significantly improving the measurement accuracy of the emitted light intensity, and further improving the immune system. Test accuracy can be significantly improved, and antigens or antibodies labeled with substances that emit light in the presence of the test substance and specific substance,
Since it is possible to simultaneously react with pre-fixed antigens or antibodies, it has the unique effect of simplifying the operations necessary for immunological testing.

[Brief explanation of the drawing]

Fig. 1 is a schematic perspective view showing one embodiment of the immunoassay device of the present invention, Fig. 2 is a central vertical cross-sectional view, Fig. 3 is a schematic diagram explaining an immunoassay procedure, and Fig. 4 is an example of another immunoassay. A schematic diagram explaining the procedure, and FIG. 5 is a diagram explaining the relationship between the peak wavelength of excitation light and the peak wavelength of fluorescence. (1b)... Containment chamber, (2)... Optical waveguide, (4)
... Light receiving part, (6) (8) ... Antibody, (7) ...
・Antigen, (9)...Labeling substance patent applicant Daikin Industries, Ltd. Representative Patent attorney Tomoshi Tsugawa Figure 1 Figure 2 Figure 3 (B)

Claims (1)

[Claims] 1. A storage chamber (1b) for accommodating a solution to be tested is formed at a predetermined position on the side surface of the optical waveguide (2), and the optical waveguide (2) is located within the storage chamber (1b). On the side of the plate, there is an antigen labeled with a substance (9) that emits light in the presence of a specific substance, or an antigen or antibody (6) that accepts the test substance (7) as a medium. It is fixed;
Moreover, the immunoassay apparatus is characterized in that it is provided with a light receiving section (4) that receives evanescent waves propagating through the optical waveguide (2) among the light emitted from the received identification antigen or antibody (8). 2. The optical waveguide (2) is formed to have a rectangular cross section, and a storage chamber (1b) for accommodating the solution to be tested is formed on the side surface of the optical waveguide (2). The immunoassay device described. 3. Antigen or antibody (
After reacting the substance to be tested (7) with 6), reacting with an antigen or antibody (8) labeled with a substance (9) that emits light in the presence of the specific substance, and then reacting with the specific substance. By supplying the labeled substance (9), the labeled substance (9) is caused to emit light, and only the emitted light from the labeled substance (9) located near the optical waveguide (2) is propagated through the optical waveguide (2), and an immunological test is performed based on the propagated light. An immunological testing method characterized by: 4. Antigen or antibody (
6) is reacted with the substance to be tested (7) and an antigen or antibody (8) labeled with a substance (9) that emits light in the presence of a specific substance, and then labeled by supplying the specific substance. The substance (9) is made to emit light, only the emitted light from the labeling substance (9) located near the optical waveguide (2) is propagated through the optical waveguide (2), and an immunological test is performed based on the propagated light. Immunology test method.