JPH0746108B2 - Immunoassay device and immunoassay method - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to an immunoassay device and an immunoassay method, and more specifically to an immunoassay by detecting the presence or absence of an antigen or an antibody using an optical waveguide. The present invention relates to an immunoassay device and an immunoassay method for performing an inspection.

<Prior Art> Conventionally, for the purpose of performing an immunological test, the surface of the optical waveguide is
By forming a region on which an antigen or antibody is fixed, and introducing blood into the region while contacting blood or the like, and measuring the intensity of light emitted from the optical waveguide, Provides an apparatus for detecting the presence or absence of an antibody and performing an immunoassay (Patent Application Publication No. 58-501481).
(See the official gazette).

More specifically, when light propagates in an optical waveguide, most of the intensity distribution of light is confined in the optical waveguide, but part of it is in the clad part surrounding the optical waveguide. It is known that the light leaks out and is reflected again inside the optical waveguide (hereinafter, this optical component and the optical component that penetrates the inside of the optical waveguide from the outside of the optical waveguide and propagates inside the optical waveguide as it is is an evanescent wave. Called)).

Then, the amount of reflection of the evanescent wave inside the optical waveguide changes due to the influence of the reflectance of the cladding portion.

That is, if an antigen or an antibody is preliminarily fixed to a portion corresponding to the clad portion and light is incident on the optical waveguide in a state where blood or the like is in contact with this portion, blood or the like is completely antibody, When no antigen is present, the intensity of emitted light changes under the influence of the reflectance of the above-mentioned antigen or antibody-immobilized portion, while when blood or the like has an antibody or antigen, Means that the antigen and the antibody are bound to each other and a completely different substance is present in the clad portion, and the emitted light intensity is different from the above intensity. (However, it is known that the intensity of the emitted light is smaller when the antigen-antibody reaction is carried out than when only the antigen or the antibody is used).

Therefore, whether or not there is an antibody or an antigen can be detected by detecting the presence or absence of a change in the intensity of emitted light.

However, in the immunoassay apparatus having the above-mentioned configuration, the intensity of the emitted light changes only briefly, and the rate of change of the emitted light intensity is extremely small.
It is not possible to reliably distinguish the change in the emitted light intensity due to the change in the degree of wetness and the change in the emitted light intensity due to the antigen-antibody reaction, and the accuracy of the immunological test is significantly reduced.

In order to solve such a problem, an antigen-antibody reaction corresponding to the degree of immunity is performed by bringing blood or the like into contact with an area to which an antigen or an antibody has been immobilized in advance, and then a distinction is made by a fluorescent substance. There is provided an immunoassay device in which an antigen-antibody reaction is further carried out by contacting the obtained antigen or antibody, and light is made incident on the optical waveguide in this state. In this case, regarding the direction of the emitted light, a method of detecting the light intensity caused by the fluorescence on the side where the incident light is emitted as it is and a method of detecting the light intensity caused by the fluorescence on the side where the light is incident are detected. There is a method and a method of detecting the light intensity due to fluorescence on the side of the optical waveguide.

Therefore, even in an apparatus for inspecting immunity using an antigen or antibody labeled with a fluorescent substance, if the intensity of emitted light affected by the evanescent wave is detected, the antigen or antibody will be present in blood or the like. It can be tested for its presence, ie its immunity.

<Problems to be Solved by the Invention> In the immunoassay apparatus using the above-described fluorescent substance-labeled antigen or antibody, the immunoassay is introduced into an optical waveguide in order to obtain a fluorescent signal corresponding to an antigen-antibody reaction. Excitation light is required, and not only does the device become large in size as a whole, but it also has problems such as generation of extra heat and increase in power consumption, and moreover, the accuracy of immunoassay cannot be improved so much. I also have problems.

More specifically, as shown in FIG. 5, there is a difference of several tens of nm between the peak wavelength of the excitation light and the peak wavelength of the fluorescent light. Compared with the light intensity of the fluorescent light, Since the light intensity of the excitation light is extremely large, the intensity of the excitation light corresponding to the peak wavelength of the fluorescent light becomes considerably large, which causes a large error. It may be possible to apply filtering to the excitation light in order to eliminate such inconvenience, but it is not possible to achieve perfect filtering, so the degree of error is only reduced, and the immunoassay still remains. The accuracy cannot be improved sufficiently.

Further, it is possible to adopt a configuration in which fluorescence is measured on the incident side of the excitation light, but in this case, the reflection at the incident surface of the excitation light is in a state that cannot be ignored for the intensity level of the fluorescence. Therefore, similarly, the accuracy of the immunological test cannot be sufficiently improved.

In addition, as a light source for generating the excitation light, if only a light in a remarkably narrow wavelength range centered on the peak wavelength is output and stable output light intensity can not be maintained continuously, the accuracy of the immunoassay is improved. Since it cannot be increased, a normal light source cannot be used, a Xe flash lamp or the like must be used, and a control device for stabilizing the output light intensity must be provided. Further, since the light source and the optical waveguide are arranged in a state of being separated from each other to some extent, the immunoassay apparatus as a whole becomes large in size, and heat generation and power consumption increase in the light source. Or, if it reaches the portion where the antibody is immobilized, there is also a problem that the accuracy of the immunoassay is lowered.

Furthermore, when the method of detecting the light intensity due to the fluorescence on the side of the optical waveguide is adopted, the fluorescence emitted from all of the antigens or antibodies identified by the fluorescent substance is detected by the immunoassay. Since it is incident as an optical signal for this purpose, fluorescence that does not undergo an antigen-antibody reaction is included, and there is a problem that the accuracy of the immunoassay is significantly reduced.

<Objects of the Invention> The present invention has been 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 can reduce the size of the structure and simplify the structure without introducing excitation light and can significantly improve the accuracy of the immunoassay.

<Means for Solving the Problems> In order to achieve the above object, the immunoassay device of the present invention has an accommodation chamber for accommodating a solution to be inspected at a predetermined position on the side surface of the optical waveguide, and the accommodation chamber. An antigen labeled with a substance that emits light under the presence of a specific substance, or an antigen that receives an antibody through the substance to be tested or an antibody is immobilized on the side surface of the optical waveguide located at, and A light receiving unit is provided for receiving an evanescent wave propagating through the optical waveguide among the emitted light of the received identification antigen or antibody.

However, it is preferable that the optical waveguide is formed in a rectangular cross-section, and that a side wall of the optical waveguide has a storage chamber for storing the solution to be inspected.

In order to achieve the above-mentioned object, the immunoassay method of the present invention comprises reacting an antigen or antibody immobilized on the side surface of an optical waveguide with a substance to be inspected, and then conditioned on the presence of a specific substance. An antigen or antibody labeled with a substance that emits light reacts, and then a specific substance is supplied to cause the labeled substance to emit light. Only the emitted light from the labeled substance located near the optical waveguide is propagated through the optical waveguide and propagated. It is a method of performing an immunological test based on light.

Still another immunoassay method is to react an antigen immobilized on the side surface of an optical waveguide, or an antibody labeled with a substance that emits light under the presence of a substance to be inspected and a specific substance, or an antibody, Then, a specific substance is supplied to cause the labeling substance to emit light, and only the emission from the labeling substance located near the optical waveguide is propagated through the optical waveguide, and an immunoassay is performed based on the propagated light.

<Operation> In the immunoassay device having the above configuration, an antigen or an antibody labeled with a substance that emits light on the condition that a specific substance is present is provided on the side surface of the optical waveguide located in the storage chamber that stores the solution to be tested. Is fixed by the antigen or antibody that receives the substance as a test substance, and the received discriminating antigen,
Alternatively, since a light receiving part for receiving the evanescent wave propagating through the optical waveguide among the light emitted from the antibody is provided, the solution to be inspected is put in the accommodation chamber and the substance to be inspected is fixed to the antigen or antibody in advance. Between them, an antigen-antibody reaction corresponding to the amount of the substance to be inspected is performed, and then, or at the same time, a solution containing an antigen or antibody labeled with a luminescent substance is added to the portion subjected to the antigen-antibody reaction. Further, an antigen-antibody reaction can be performed. After that, by inserting the specific substance into the accommodation chamber, the luminescent substance can be made to emit light without introducing any excitation light. Since this light emission is generated in the vicinity of the side surface of the optical waveguide, it enters the optical waveguide as an evanescent wave and propagates as it is in the optical waveguide. Therefore, by receiving the evanescent wave propagating in the optical waveguide at the light receiving portion, it is possible to detect not only immunity but also the degree of immunity based on the intensity of the evanescent wave.

When the optical waveguide is formed in a rectangular cross section and a storage chamber for storing the solution to be tested is formed on the side surface of the optical waveguide, the area for fixing the antigen or the antibody is enlarged. Therefore, the strength of the evanescent wave propagating through the optical waveguide can be increased as a whole, and the immunoassay accuracy can be improved.

In the above immunoassay method, the antigen immobilized on the side surface of the optical waveguide, or the antibody to be inspected is reacted with the antibody, and then the antigen labeled with a substance that emits light under the presence of a specific substance, or Reacts with an antibody, then supplies a specific substance to cause the labeling substance to emit light, and only the emission from the labeling substance located near the optical waveguide is propagated through the optical waveguide, and an immunoassay is performed based on the propagated light. There is no need to introduce excitation light into the optical waveguide, and even if the emitted light intensity is weak, there is no effect of the excitation light, which significantly improves the emission light intensity measurement accuracy, and thus significantly improves the immunoassay accuracy. be able to.

Further, in the other immunoassay methods described above, an antigen immobilized on the side surface of the optical waveguide, or an antigen labeled with a substance that emits light on the condition that the substance to be inspected and the specific substance are present for the antibody, or The antibody is made to react and then the specific substance is supplied to cause the labeling substance to emit light, and only the emission from the labeling substance located near the optical waveguide is propagated through the optical waveguide, and the immunoassay is performed based on the propagated light. Since it is not necessary to introduce excitation light into the waveguide, and even if the emitted light intensity is weak, the influence of the excitation light is completely eliminated, so that the measurement accuracy of the emitted light intensity is significantly improved, which in turn improves the immunoassay accuracy. You can Then, an antigen or antibody labeled with a substance that emits light under the presence of a substance to be tested and a specific substance can simultaneously react with an antigen or antibody that has been fixed in advance. The required operation can be simplified.

<Example> Hereinafter, detailed description will be given with reference to the accompanying drawings illustrating an example.

FIG. 1 is a schematic perspective view showing an embodiment of the immunoassay device of the present invention, and FIG. 2 is a central longitudinal sectional view, which is entirely flat and has a light guiding portion (3) formed on the lower surface of one end. A region (1) formed by immobilizing a large number of antibodies (6) in advance is formed on the upper surface of the optical waveguide (2). Then, the light derived from the light deriving unit (3) is guided to a light receiving unit (4) having a photomultiplier tube or the like, and an electric signal output from the light receiving unit (4) has a differential amplifier or the like. It is supplied to the detector (5). In addition, the side wall (1a) so as to surround the area (1).
The housing chamber (1b) is formed by integrally forming.

Although not shown, the antibody (6) performs an antigen-antibody reaction with the antigen (7) (see FIG. 3A) contained in the solution to be measured such as blood. An antibody (8) that undergoes an antigen-antibody reaction with the above-mentioned antigen (7)
A labeling substance that emits light in the presence of a substrate solution (9)
(See FIG. 3, B and C). The labeling substance (9) emits light by a chemiluminescence mechanism, for example, peroxidase (peroxidase).
Etc., or those that emit light by a bioluminescence mechanism, for example, luciferase, eclion, etc. may be used.

The operation of the immunoassay system with the above configuration is as follows.

First, by storing the solution to be measured in the storage chamber (1b), an antigen-antibody reaction is carried out between the antigen (7) in the solution to be measured and the antibody (6) that has been immobilized in advance (Fig. 3). (See A). 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.

Then, the solution to be measured is discharged from the accommodation chamber (1b) and the labeling substance solution is accommodated, so that the antigen (7) that has undergone an antigen-antibody reaction with the antibody (6) and the labeling substance (9) are labeled. An antigen-antibody reaction is carried out with the antibody (8) (see FIG. 3B).

Therefore, in this state, only the number of antibodies (6) corresponding to the degree of immunity of the solution to be measured out of the large number of antibodies (6) pre-fixed in the region (1) is the antigen (7). The antibody (8) labeled with the labeling substance (9) is received via the above.

After that, when the labeling substance solution is discharged from the storage chamber (1b) and the substrate liquid is stored, the labeling substance (9) emits light (Fig. 3C).
reference). Since the emitted light of the labeling substance (9) is generated in the vicinity of the upper surface of the optical waveguide (2), a part of the emitted light is introduced into the optical waveguide (2) and propagates through the optical waveguide (2) as it is. Then, the amount of the labeling substance (9) labeling the antibody (8) received by the antibody (6) is derived from the propagated light from the light derivation unit (3) and is guided to the light receiving unit (4). That is, it is converted into an electric signal corresponding to the amount of the antigen (7) that has undergone the antigen-antibody reaction with the antibody (6). This converted electric signal is supplied to the detection unit (5), and it is possible to detect the presence or absence of immunity and, if there is immunity, the degree of immunity.

As is clear from the above description, since the excitation light is not introduced into the optical waveguide (2) at all, the light emitted from the light deriving part (3) is the antibody (6) fixed in the region (1). Against the antigen (7), only a part of the luminescence of the labeling substance (9) labeled with the antibody (8) bound via the antigen (7) is generated, and the antigen (7) is reacted with the antibody (6). Of light, that is, light having an intensity corresponding to the degree of immunity is incident on the light receiving section (4).

Therefore, the accuracy of the immunoassay can be remarkably improved, and the immunoassay apparatus as a whole can be miniaturized due to the omission of the light source for excitation light.
Further, it is possible to reliably prevent an increase in temperature due to heat generation of the light source and an increase in power consumption due to energization of the light source.

FIG. 4 is a schematic diagram for explaining an immunoassay according to another procedure. The difference from the procedure in FIG. 3 is that instead of carrying out the accommodation of the measurement target solution and the labeling substance solution in this order,
As shown in FIG. A, the solution to be measured and the labeling substance solution are mixed in advance, and the mixed solution is housed in the housing chamber (1b).

Therefore, also in the case of this example, the antigen (7) that has undergone an antigen-antibody reaction with a large number of antibodies (6) that have been immobilized in advance has the antibody (8) already labeled with the labeling substance (9). )
Will be accepted. Then, light having an intensity corresponding to the amount of the antibody (8) received by the antigen (7), that is, the amount of the labeling substance (9) is generated, and a part of this light is propagated through the optical waveguide (2). By being guided from the light guiding section (3) and guided to the light receiving section (4), an electric signal corresponding to the degree of immunity can be generated.

As a result, as in the case of adopting the above-mentioned procedure, it is possible to perform an immunoassay with high accuracy and also to achieve the advantages associated with omitting the antigen for excitation light.

It should be noted that the above-mentioned substrate liquid may be determined according to the labeling substance (9), and for example, luminol liquid for peroxydose, luciferin liquid for luciferase, and eclion. The calcium solution may be defined respectively.

<Specific example> When human chorionic gonadotropin (hCG) is to be measured using an antibody labeled with luciferase, the anti-hCG antibody is immobilized in the region (1) and the chamber (1b ) Containing the solution to be measured, causing an antigen-antibody reaction between the hCG antigen and the anti-hCG antibody, discharging the solution to be measured and containing the labeling substance solution, the hCG antigen and luciferase were labeled. Antigen h
The antigen-antibody reaction with the CG antibody was performed, the labeling substance solution was discharged, and the luciferin solution was contained therein to cause luminescence by the bioluminescence mechanism, and the luminescence was measured for 5 minutes.

By performing the above measurement, hCG at a concentration of 5 ng / ml could be measured.

As is clear from this specific example, since no excitation light is used at all, low concentration hCG can be measured with high sensitivity even with weak emission light. Therefore, the accuracy of immunoassay can be significantly improved.

The present invention is not limited to the above-mentioned embodiment, and for example, instead of immobilizing the antibody (6) on the region (1), it is possible to immobilize the antigen and the labeling substance. It is possible to use other substances as well as a corresponding substrate liquid, and it is possible to appropriately set the size of the storage chamber (1b) and to appropriately set the refractive index of the optical waveguide (2). Settings can be made, and various other design changes can be made without departing from the scope of the present invention.

<Effects of the Invention> As described above, in the first invention, the luminescent substance is caused to emit light without introducing any excitation light, and the emitted light is propagated through the optical waveguide to be guided to the light receiving portion.
The presence / absence of immunity and, if there is immunity, the degree of immunity can be tested with extremely high accuracy, and the size of the inspection device as a whole can be reduced. Furthermore, temperature fluctuations can be prevented and consumption can be reduced. There is a unique effect that power can be reduced.

In the second invention, the optical waveguide is formed in a rectangular cross section, and the accommodation chamber for accommodating the solution to be inspected is formed on the side surface of the optical waveguide, so that the area for fixing the antigen or the antibody is enlarged. Therefore, it is possible to increase the strength of the evanescent wave propagating through the optical waveguide as a whole, and it is possible to improve the immunoassay accuracy, which is a unique effect.

The third invention eliminates the need to introduce excitation light into the optical waveguide at all, and even if the intensity of emitted light is weak, there is no influence of the excitation light, thereby significantly improving the measurement accuracy of the emitted light intensity, and thus immunity. There is a unique effect that the inspection accuracy can be remarkably improved.

In the fourth invention, it is not necessary to introduce excitation light into the optical waveguide, and even if the intensity of emitted light is weak, the influence of the excitation light is eliminated. The accuracy of the test can be significantly improved, and an antigen or antibody labeled with a substance that emits light under the presence of the test substance and a specific substance
Since it is possible to react with an antigen or antibody that has been fixed in advance at the same time, there is a unique effect that the operation required for an immunoassay can be simplified.

[Brief description of drawings]

FIG. 1 is a schematic perspective view showing an embodiment of the immunoassay device of the present invention, FIG. 2 is a central longitudinal sectional view, FIG. 3 is a schematic diagram illustrating an immunoassay procedure, and FIG. 4 is another immunoassay. FIG. 5 is a schematic diagram illustrating the procedure, and FIG. 5 is a diagram illustrating the relationship between the peak wavelength of excitation light and the peak wavelength of fluorescence. (1b) ... Accommodation chamber, (2) ... Optical waveguide, (4) ... Light receiving part, (6) (8) ... Antibody, (7) ... Antigen, (9) ... Labeling substance

 ─────────────────────────────────────────────────── ─── Continuation of the front page (56) References JP-A 61-191965 (JP, A) JP-A 61-184442 (JP, A) JP-A 62-254038 (JP, A) JP-A 62- 254039 (JP, A) JP-A-62-254040 (JP, A)

Claims (4)

[Claims] 1. A storage chamber (1b) for storing a solution to be inspected is formed at a predetermined position on a side surface of an optical waveguide (2), and a side surface of the optical waveguide (2) located inside the storage chamber (1b). , An antigen or an antibody (8) labeled with a substance (9) that emits light under the presence of a specific substance is a substance to be tested (7)
The antigen or antibody (6) that receives it as a mediator is immobilized, and the discriminating antigen or antibody (8) that has been received
The immunoassay device is provided with a light receiving section (4) for receiving an evanescent wave propagating through the optical waveguide (2) among the light emission of (1). 2. The above-mentioned claims, wherein the optical waveguide (2) is formed in a rectangular cross section, and a storage chamber (1b) for storing a solution to be tested is formed on the side surface of the optical waveguide (2). The immunoassay device according to the first aspect. 3. A substance (9) which emits light under the presence of a specific substance after reacting a substance (7) to be inspected with an antigen or an antibody (6) fixed on the side surface of an optical waveguide (2). ) Is reacted with an antigen or antibody (8) labeled with), and then a specific substance is supplied to cause the labeling substance (9) to emit light, which is emitted from the labeling substance (9) located in the vicinity of the optical waveguide (2). An immunoassay method characterized in that only light emission is propagated through an optical waveguide (2) and an immunoassay is performed based on the propagated light. 4. An antigen or antibody (6) fixed to the side surface of an optical waveguide (2) is labeled with a substance (9) which emits light under the condition that a substance to be inspected (7) and a specific substance are present. The labeling substance (9) is caused to emit light by reacting with an antigen or an antibody (8), and then a specific substance is supplied, and only the light emission from the labeling substance (9) located in the vicinity of the optical waveguide (2) is transmitted to the optical waveguide. (2) An immunoassay method characterized by propagating through and conducting an immunoassay based on the propagated light.