JP2526998B2 - Method and apparatus for detecting reaction start point in fluorescence immunoassay - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a measurement start time detection method and apparatus for fluorescence measurement, and more specifically, by introducing excitation light into an optical waveguide, an antigen antibody As a result of the reaction, the fluorescent dye existing near the surface of the optical waveguide is excited, and the start time of the antigen-antibody reaction is detected in the immunoassay in which the presence or absence of the antigen or antibody is measured based on the fluorescence emitted from the fluorescent dye. Method and apparatus for doing

<Prior Art and Problems to be Solved by the Invention> Conventionally, a radioimmunoassay method and a fluorescent immunoassay method have been provided for measuring the presence or absence of an antigen or an antibody, but labeling of labeled antibody is easy. , And that the introduction of excitation light is easy, the antibody is fixed in advance on the surface of the optical waveguide, and the excitation light is introduced into the optical waveguide, and the immunoassay target solution and fluorescence An optical waveguide type fluorescence immunoassay method has been provided in which labeled antibodies labeled with a dye are injected in this order onto the surface of an optical waveguide.

In the above-mentioned optical waveguide type fluorescence immunoassay method, an antibody corresponding to the amount of the antigen present in the immunoassay target solution among the antibodies previously immobilized on the surface of the optical waveguide by injecting the immunoassay target solution. Only the antigen-antibody reaction takes place.
Then, when a labeled antibody is injected, an antigen-antibody reaction is performed with the antigen that has undergone the antigen-antibody reaction, so that only the labeled antibody corresponding to the amount of the antigen is indirectly bound to the previously fixed antibody. .

Therefore, because the excitation light introduced into the optical waveguide seeps out from the surface of the optical waveguide, the fluorescent dye of the labeled antibody indirectly bound is excited and emits fluorescent light. Since the remaining suspended dye of the labeled antibody is not excited, the degree of immunity can be detected by measuring the intensity of the fluorescence.

In addition, the fluorescence immunoassay method includes an immunoassay method based on the fluorescence intensity in a state of equilibrium due to progress of antigen-antibody reaction (hereinafter, abbreviated as END POINT method) and fluorescence until equilibration. It is classified into an immunoassay method based on the rate of change in intensity (hereinafter abbreviated as RATE ASSAY method).

In the above END POINT method, after the number of antigen-antibody reaction starts
Since the immunoassay is performed based on the fluorescence intensity at the time when the equilibrium state has been reached after 10 minutes have passed, the error in the measurement time does not pose a problem, and the immunoassay can be performed with a fairly high accuracy. .

On the contrary, in the fluorescence measurement using the laser nephelometry, FPIA, and optical waveguide to which the RATE ASSAY method is applied, a predetermined time (for example, 30 seconds, 1 minute after starting the antigen-antibody reaction) Since the immunoassay is carried out based on the fluorescence intensity at the time when the appropriate time) has elapsed, it is necessary to accurately determine the start time of the antigen-antibody reaction. Then, in order to satisfy such a need, it is possible to provide an automatic liquid feeding mechanism, but since the automatic liquid feeding mechanism is remarkably large and expensive, the fluorescent immunoassay device is downsized, Moreover, there is a problem that the cost cannot be reduced. In particular, in a fluorescent immunoassay device in which excitation light is introduced into an optical waveguide, the automatic liquid feeding mechanism is large in size as a whole despite the fact that the entire configuration including the optical system can be downsized, and therefore becomes large in size as a whole. Of.

Further, the present inventor has found that the fluorescence emitted from the fluorescent dye excited by the excitation light introduced into the optical waveguide changes discontinuously at the time of injecting the solution to be measured, that is, the reaction start time. , It was considered to detect the discontinuity of the fluorescence, but the level of the excitation light is significantly higher than that of the fluorescence, and the characteristics of the optical filter for cutting the excitation light are insufficient. , It is difficult to accurately identify the discontinuity point due to the fact that the side surface of the optical waveguide is not sufficiently flattened and the signal difference in the discontinuity is relatively small and susceptible to noise components. It turns out that it has the problem of being.

<Purpose of the invention> The present invention has been made in view of the above problems, by exciting the fluorescence corresponding to the amount of the antigen-antibody reaction by introducing excitation light into the optical waveguide, excited fluorescence It is an object of the present invention to provide a method and an apparatus for accurately detecting the reaction start time point without increasing the size of the whole fluorescence immunoassay apparatus when measuring the presence or absence of an antigen or an antibody based on the above.

<Means for Solving the Problems> In order to achieve the above-mentioned object, the reaction initiation time point detection method of the present invention comprises introducing excitation light into an optical waveguide to cause the antigen-antibody reaction to result in the vicinity of the surface of the optical waveguide. Exciting a fluorescent dye to be present, in the case of measuring the presence or absence of an antigen or antibody based on the fluorescence emitted from the fluorescent dye, in addition to the fluorescent labeled antibody labeled with the fluorescent dye An antigen-antibody reaction is performed by injecting a measurement reagent to which the same fluorescent dye as the photo dye is added, and the fluorescence is detected by detecting the discontinuity point of the detection signal corresponding to the intensity of the emitted fluorescence. This is a method of detecting the reaction start point for immunoassay.

However, the detection signal corresponding to the intensity of the fluorescence emitted isotropically is preferably a time differential signal based on the fluorescence intensity detection signal.

In order to achieve the above object, the reaction start time detection device of the present invention is a measurement in which the same fluorescent dye as the fluorescent dye is added alone in addition to the fluorescently labeled antibody labeled with the fluorescent dye. Target solution, differentiating means for generating a time differential signal based on the electric signal corresponding to the intensity of the emitted fluorescence, which is excited by the excitation light introduced into the optical waveguide, and the time output from the differentiating means And a discontinuity point detecting means for detecting the discontinuity point with the differential signal as an input.

The reaction initiation time point detection method of another invention for achieving the above-mentioned object, by introducing excitation light into the optical waveguide,
As a result of the antigen-antibody reaction, the fluorescent dye existing near the surface of the optical waveguide is excited, and when the presence or absence of the antigen or antibody is measured based on the fluorescent light emitted from the fluorescent dye, the measurement target solution is not injected. In this method, the side surface of the optical waveguide is kept in a highly scattering state, and the point at which the reaction for fluorescent immunoassay is started is detected by detecting the point at which the amount of scattered excitation light sharply decreases with the injection of the measurement reagent.

In a reaction initiation time point detection device of another invention for achieving the above object, a dry layer containing a substrate for carrying out an antigen-antibody reaction is formed on the surface of an optical waveguide into which a solution to be measured is injected. Although various substances can be used as the above-mentioned substrate, for example, proteins such as immobilized antibody and buffer solutions for favorably carrying out the antigen-antibody reaction are preferably dried, and particularly, those More preferably, it is freeze-dried.

A reaction initiation time point detection device of still another invention for achieving the above object, a solid layer having light scattering property and solubility is formed on the surface of an optical waveguide into which a solution to be measured is to be injected. . Various types of solid layers can be used as the soluble solid layer, but for example, a light-scattering solid layer composed of soluble powder, granules, granules or the like is preferable.

<Operation> In the case of the above reaction start point detection method, by introducing excitation light into the optical waveguide, the fluorescent dye existing near the surface of the optical waveguide as a result of the antigen-antibody reaction is excited, and In the case of measuring the presence or absence of an antigen or an antibody based on the emitted fluorescence, in addition to the fluorescent-labeled antibody labeled with the fluorescent dye, the same fluorescent dye as the fluorescent dye is added alone An antigen-antibody reaction is caused by injecting the target solution, and the detection signal corresponding to the intensity of the fluorescence emitted from the fluorescent dye changes discontinuously and rapidly at the time when the antigen-antibody reaction is started. Therefore, the discontinuity can be accurately detected, and the reaction start point for the fluorescent immunoassay can be detected based on the discontinuity.

Therefore, the immunoassay can be performed with high accuracy based on the reaction start time point accurately detected as described above.

More specifically, an antibody is immobilized on the surface of the optical waveguide in advance, and excitation light is introduced into the optical waveguide, which is preferably filled with a buffer solution for favorably performing the antigen-antibody reaction. Here, a signal corresponding to the intensity of scattered light due to this excitation light is detected as an offset signal. Then, in the state in which the same fluorescent dye as the fluorescent dye alone in addition to the fluorescently labeled antibody previously labeled with the fluorescent dye in the target solution to be subjected to immunoassay in this state is injected, the antigen or An antigen-antibody reaction in an amount corresponding to the amount of antibody is performed, and the amount of the fluorescent dye existing near the surface of the optical waveguide rapidly increases, and the presence of the fluorescent dye added alone also causes the optical waveguide to move. Since the amount of fluorescent dye existing near the surface increases more rapidly, a signal that discontinuously and rapidly changes with the offset signal as a reference is obtained.
Therefore, if this discontinuity is detected as the reaction start time, the accuracy of the immunoassay can be improved along with the improvement in the detection accuracy at the reaction start time.

Then, when the detection signal corresponding to the intensity of the emitted fluorescence is a time differential signal based on the fluorescence intensity detection signal, the time that significantly changes corresponding to the discontinuity point of the fluorescence intensity detection signal. Since the differential signal is obtained, the detection of the reaction start time can be made highly accurate.

If the reaction start time detection device of the above configuration, by introducing excitation light into the optical waveguide, to excite the fluorescent dye that is present near the surface of the optical waveguide as a result of the antigen-antibody reaction, radiate from the fluorescent dye In the case of measuring the presence or absence of an antigen or antibody based on the fluorescence, the measurement in which the same fluorescent dye as the fluorescent dye alone in addition to the fluorescently labeled antibody previously labeled with the fluorescent dye is added When the target solution is injected into the surface of the optical waveguide, the fluorescence intensity emitted due to the fluorescence dye added alone and the antigen-antibody reaction changes discontinuously and rapidly. Then, by supplying an electric signal corresponding to the fluorescence intensity to the differentiating means, it is possible to obtain a time-differentiated signal that significantly changes corresponding to the discontinuous portion. Can be detected as a discontinuity point.

In the case of the fourth reaction start time detection method, since the surface of the optical waveguide in the non-injected state of the solution to be measured is kept in a high scattering state, a considerable proportion of the excitation light is scattered on the surface of the optical waveguide. The highly scattered state is eliminated by injecting the solution to be measured, and the scattered light based on the excitation light is significantly reduced. Therefore, the reaction start point can be easily detected based on the large decrease in the scattered excitation light intensity, which is significantly higher than the fluorescence intensity.

However, in this method, since there is a possibility that a large decrease in the scattered excitation light intensity will be considerably reduced compared to the ideal state, it is preferable to obtain a time-differentiated signal, which improves the detection accuracy at the reaction start point. be able to.

In the case of the fifth reaction start time detection device, since a dry layer containing a substance for carrying out the antigen-antibody reaction is formed on the surface of the optical waveguide into which the measurement reagent should be injected, until the solution to be measured is injected. Has a significantly high scattered excitation light intensity,
If the solution to be measured is injected, the scattered excitation light intensity will be significantly reduced, so that the reaction start point can be accurately detected by detecting the point at which the scattered excitation light intensity significantly decreases.

In the case of the sixth reaction start time detection device, since the solid layer having light scattering property and solubility is formed on the surface of the optical waveguide into which the measurement reagent is to be injected, the solution to be measured is injected. The intensity of scattered excitation light is extremely high up to, and if the solution to be measured is injected, the intensity of scattered excitation light becomes extremely low.Therefore, the reaction start time can be accurately detected by detecting the point at which the intensity of scattered excitation light significantly decreases. be able to.

<Example> Hereinafter, an example will be described in detail with reference to the accompanying drawings.

FIG. 3 is a schematic diagram showing an embodiment of the fluorescence measuring apparatus of the present invention, in which the excitation light emitted from the excitation light source (1) is passed through a collimator lens (2) and an optical filter (3) to form an optical waveguide ( Introduced in 4). A cuvette (5) into which the solution to be measured or the like is injected is formed on the surface of the optical waveguide (4), and a large number of antibodies (6) are immobilized on the bottom surface of the cuvette (5). Further, a photodetector (7) is arranged close to the surface of the optical waveguide (4) opposite to the cuvette (5), and excitation is provided between the optical waveguide (4) and the photodetector (7). An optical filter (8) for cutting the light component is arranged. Also, a differentiation circuit (9) that receives the electric signal output from the photodetector (7) and a peak timing generation circuit (10) that generates a timing signal corresponding to a steep peak of the differentiation signal.
And an arithmetic circuit (11) which receives an electric signal output from the photodetector (7) as an input and performs a predetermined arithmetic operation using the timing signal as a reaction start time point instruction signal to generate an immunoassay signal. There is.

FIG. 1 is a schematic diagram for explaining the immunoassay operation. Initially, as shown in FIG. 1A, the excitation light is guided to the optical waveguide (4) only with the antibody (6) fixed on the bottom surface of the cuvette (5). Have been introduced. Then, put the antigen (1
6) Inject the solution to be measured containing only After that, the content of the cuvette (5) is left with the solution to be measured as it is, or preferably, excitation light is introduced into an optical waveguide (4) filled with a buffer solution for favorably performing an antigen-antibody reaction. To do.

Here, an electric signal (see a region R1 in FIG. 2) corresponding to the intensity of scattered light due to the excitation light is output from the photodetector (7).

Thereafter, as shown in FIG. 1B, the fluorescent dye (15) was added alone to the cuvette (5) in addition to the fluorescently labeled antibody (hereinafter simply referred to as labeled antibody) (12). When the solution to be measured (17) is injected, part of the fluorescent dye (15) added alone is located near the interface of the optical waveguide (4) and is contained in the solution to be measured (17). As a result of the antigen-antibody reaction between the antigen (16) and the immobilized antibody (6) and labeled antibody (12), the fluorescent dye (13) labeling the labeled antibody (12) It will be located near the interface of the optical waveguide (4). The fluorescent dye (15) located near the interface of these optical waveguides (4)
Is excited by the evanescent wave component of the excitation light, the intensity of the fluorescent light emitted by the effect of the fluorescent dye (15) added alone increases discontinuously and drastically,
Then, the intensity of fluorescence emitted continuously increases with the progress of the antigen-antibody reaction. Therefore, the photodetector (7)
Outputs an electric signal (see area R2 in FIG. 2) corresponding to the intensity of the fluorescence.

The electric signal is supplied to the differentiating circuit (9) to obtain a time differential signal, and is also supplied to the arithmetic circuit (11) as it is. Then, a timing signal corresponding to the steep peak of the differential signal is generated in the peak timing generation circuit (10) based on the time differential signal,
It is supplied to the arithmetic circuit (11) as a reaction start time point instruction signal. Therefore, for example, after the reaction start time point instruction signal is supplied, the immunological measurement signal is generated by the arithmetic circuit (11) based on the electric signal corresponding to the fluorescence intensity at the time point when a preset predetermined time has elapsed. You can

As is clear from the above description, in this embodiment, the reaction start time can be detected based on the electric signal output from the photodetector (7), so the fluorescent immunoassay apparatus as a whole can be miniaturized. In addition to being able to do so, the cost can be reduced.

In addition, the labeled antibody (12) labeled with the fluorescent dye (13) is used at a concentration that can optimally perform the antigen-antibody reaction, and also in this respect, cost reduction can be achieved and highly accurate immunoassay. Can be done.

In the above example, the electric signal output from the photodetector (7) is time-differentiated before the reaction start point instruction signal is obtained, but the amount of the fluorescent dye added alone is increased. By doing so, a sufficient amount of discontinuity occurs in the electric signal itself, so that it is possible to directly obtain the reaction start point instruction signal based on the electric signal.

<Example 2> Fig. 4 is a schematic view showing another example of the fluorescent immunoassay device. The difference from the above example is that the immobilized antibody (19) is freeze-dried on the bottom surface of the cuvette (5). And the point that the solution to be measured (17) to which the fluorescent dye (15) is not added alone is used instead of the solution to be measured (17), and the configuration of the other parts is the same. .

Therefore, in the case of this example, a considerable amount of excitation light was generated in a state in which neither the solution to be measured (18) nor the measurement reagent (14) was injected.
As a result of being scattered by 9), a high level electric signal (see area R1 in FIG. 5) is output from the photodetector (7).

Then, by injecting the measuring reagent (14) and the solution to be measured (18) into the cuvette (5) in order to carry out the antigen-antibody reaction, the dried state of the immobilized antibody (19) is eliminated, so that the scattered excitation light Rapidly decreases, and thereafter, the fluorescence intensity emitted increases continuously with the progress of the antigen-antibody reaction. As a result, the electric signal from the photodetector (7) is suddenly lowered in level and then continuously increased (fifth level).
Area R2 in the figure) is output.

The electric signal obtained as described above is supplied to the differentiating circuit (9) as in the above-described embodiment to obtain a time differential signal, and is also supplied to the arithmetic circuit (11) as it is. Then, a timing signal corresponding to a steep peak of the differential signal is generated in the peak timing generation circuit (10) based on the time differential signal, and is supplied to the arithmetic circuit (11) as a reaction start time point instruction signal. Therefore, for example, after the reaction start time point instruction signal is supplied, the immunological measurement signal is generated by the arithmetic circuit (11) based on the electric signal corresponding to the fluorescence intensity at the time point when a preset predetermined time has elapsed. You can

As is clear from the above description, also in this embodiment, since the reaction start time can be detected based on the electric signal output from the photodetector (7), the fluorescent immunoassay apparatus as a whole can be miniaturized. In addition to being able to do so, the cost can be reduced.

Further, since the fluorescent dye is not added alone to the solution to be measured (18), further cost reduction can be achieved and highly accurate immunoassay can be performed.

<Embodiment 3> FIG. 6 is a schematic view showing still another embodiment of the fluorescent immunoassay device, and is different from the embodiment of FIG. 4 in that an optical filter is interposed in addition to the photodetector (7). An electric signal output from the photodetector (20) in place of the electric signal output from the point and the photodetector (7) provided with a photodetector (20) for receiving light emitted without Is supplied to the differentiating circuit (10), and the other parts have the same configuration.

Therefore, in this example, the measurement reagent (1
4) and the electric signal that changes stepwise as shown in Fig. 7 before and after injecting the solution to be measured (18) is output from the photodetector (20), so the reaction start time in the differentiation circuit (10) An instruction signal can be obtained. Since the reaction start time point instruction signal is obtained accurately, the arithmetic circuit (11) can generate an accurate immunoassay signal.

Further, since it is not necessary to use a photomultiplier tube as the photodetector (20), cost increase can be significantly suppressed even if the number of photodetectors increases.

In the case of this embodiment, the output signal from the photodetector (7) can be corrected based on the output signal from the photodetector (20). In this case, the optical filter (8) is used. Thus, even if the excitation light is not completely cut, the influence of the excitation light can be completely eliminated and an accurate immunoassay signal can be generated.

Also in the above embodiment, the electric signal output from the photodetector (20) is time-differentiated before the reaction start point instruction signal is obtained. However, since the signal change amount is sufficiently large, the differentiation is performed. It is possible to directly obtain the reaction start point instruction signal by omitting the circuit (10).

<Embodiment 4> FIG. 8 is a schematic view showing still another embodiment of the fluorescent immunoassay device, and is different from the embodiment in FIG. 6 in that it has an optical filter (3) and an optical waveguide (4). Between half mirror (2
1) is provided so that the light emitted from the optical waveguide (4) and reflected by the half mirror (21) is guided to the photodetector (7) through the optical filter (8). The configuration is the same.

Therefore, in the case of this embodiment, the reaction start time point instruction signal can be generated based on the discontinuity point of the electric signal output from the photodetector (20) as in the embodiment of FIG. The half mirror (21) is emitted from the optical waveguide (4).
The immunoassay signal can be generated by the arithmetic circuit (11) based on the intensity of the fluorescence and the reaction start time point instruction signal guided to the photodetector (7) through the optical filter (8) after being reflected by.

Also in the case of this embodiment, the output signal from the photodetector (7) can be corrected based on the output signal from the photodetector (20). In this case, the optical filter (8) is used. Thus, even if the excitation light is not completely cut, the influence of the excitation light can be completely eliminated and an accurate immunoassay signal can be generated. Further, it is preferable to use a dichroic mirror instead of the half mirror (21).

<Effect of the Invention> As described above, the first invention is to inject a solution to be measured in which the same fluorescent dye as the fluorescent dye is added alone in addition to the fluorescently labeled antibody labeled with the fluorescent dye. , By performing the antigen-antibody reaction, the detection signal corresponding to the intensity of the fluorescence emitted from the fluorescent dye can be changed discontinuously and rapidly at the time when the antigen-antibody reaction is started, and automatic transmission is performed. It is possible to accurately detect the reaction start point for fluorescent immunoassay based on this discontinuity with a simple configuration without the need for a liquid mechanism, etc., and it is possible to miniaturize as a whole. Has a unique effect.

The second aspect of the invention has a unique effect that the detection of the reaction start point can be made highly accurate because a time-differential signal that significantly changes corresponding to the discontinuity point of the fluorescence intensity detection signal is obtained. .

A third aspect of the invention is to inject a solution to be measured in which a fluorescent dye is added alone in addition to the fluorescently labeled antibody, and to cause an antigen-antibody reaction to increase the intensity of the fluorescent light emitted from the fluorescent dye. Since the corresponding detection signal can be changed discontinuously and rapidly at the time of starting the antigen-antibody reaction, the discontinuity is detected only based on the above detection signal, and the fluorescence is detected based on this discontinuity. It is possible to accurately detect the reaction start time point for the immunoassay, and to simplify the configuration as a whole and to achieve a unique size.

The fourth aspect of the invention has a unique effect that the reaction start time can be easily detected based on a large decrease in the scattered excitation light intensity, which is significantly higher than the fluorescence intensity.

The fifth invention has a unique effect that the reaction start point can be accurately detected by detecting a marked decrease point of the scattered excitation light intensity, and that the structure can be simplified and miniaturized. Play.

The sixth invention has a unique effect that the reaction start point can be accurately detected by detecting a marked decrease point of the scattered excitation light intensity, and that the structure can be simplified and miniaturized. Play.

[Brief description of drawings]

FIG. 1 is a diagram for explaining a fluorescent immunoassay procedure of the present invention, FIG. 2 is a diagram showing a signal waveform obtained by the fluorescent immunoassay, and FIG. 3 is an embodiment of the fluorescent immunoassay device of the present invention. FIG. 4 is a schematic diagram showing another embodiment of the fluorescent immunoassay device, FIG. 5 is a diagram showing signal waveforms obtained by the embodiment of FIG. 4, FIG. 6 and FIG. FIG. 7 is a schematic view showing still another embodiment of the fluorescent immunoassay device, and FIG. 7 is a view showing signal waveforms obtained by the embodiment of FIG. (1) ... Excitation light source, (4) ... Optical waveguide, (5) ... Cuvette, (6) ... Antibody, (7) (20) ... Photodetector, (9) ... Differentiation circuit , (10) …… Peak timing generation circuit, (11) …… Operation circuit, (12) …… Labeled antibody, (13) (15) …… Fluorescent dye, (14) …… Measurement reagent, (16 ) …… Antigen, (17) (18) …… Measurement target solution, (19) …… Immobilized antibody

Claims (6)

(57) [Claims] 1. A fluorescent dye (13) existing in the vicinity of the surface of the optical waveguide (4) as a result of an antigen-antibody reaction is excited by introducing excitation light into the optical waveguide (4), and the fluorescent dye ( 1
3) In a fluorescent immunoassay method for measuring immunity based on fluorescence emitted from the fluorescent dye, the same fluorescent dye as the fluorescent dye (in addition to the fluorescently labeled antibody labeled with the fluorescent dye (13) ( 1
For fluorescent immunoassay by injecting the solution to be measured (5), which was added alone, to carry out the antigen-antibody reaction, and detecting the discontinuity of the detection signal corresponding to the intensity of fluorescence. The method for detecting the reaction start time in the fluorescent immunoassay method, which comprises detecting the reaction start time. 2. The reaction start time detection method in the fluorescent immunoassay method according to claim 1, wherein the detection signal corresponding to the fluorescence intensity is a time differential signal based on the fluorescence intensity detection signal. 3. A fluorescent dye (13) existing in the vicinity of the surface of the optical waveguide (4) as a result of an antigen-antibody reaction is excited by introducing excitation light into the optical waveguide (4) and the fluorescent dye ( 1
In a fluorescent immunoassay device for measuring immunity based on the fluorescence emitted from 3), in addition to the fluorescently labeled antibody labeled with the fluorescent dye (13), the same fluorescent dye as the fluorescent dye ( 1
The solution to be measured (5) to which 5) has been added independently, the differentiating means (9) for generating a time differential signal based on the electric signal corresponding to the intensity of the fluorescence, and the differentiating means (9) are output. A reaction start time detection device in a fluorescent immunoassay device, which has a discontinuity detection means (10) for detecting a discontinuity with a time differential signal as an input. 4. A fluorescent dye (13) existing in the vicinity of the surface of the optical waveguide (4) as a result of an antigen-antibody reaction is excited by introducing excitation light into the optical waveguide (4), and the fluorescent dye ( 1
3) In the fluorescence immunoassay method in which immunity is measured based on the fluorescence emitted from, the surface of the optical waveguide in the non-injection state of the measurement reagent is kept in a highly scattered state, and the scattered excitation light accompanying the injection of the solution to be measured A method for detecting a reaction start time in a fluorescent immunoassay method, which comprises detecting a reaction start time for a fluorescent immunoassay by detecting a sharp decrease point in the reaction. 5. A fluorescent dye (13) existing in the vicinity of the surface of the optical waveguide (4) as a result of the antigen-antibody reaction is excited by introducing excitation light into the optical waveguide (4), and the fluorescent dye ( 1
3) In a fluorescent immunoassay device for measuring immunity based on the fluorescence emitted from, the surface of the optical waveguide into which the solution to be measured (17) (18) is to be injected contains a substance for carrying out an antigen-antibody reaction. A reaction start point detection device in a fluorescent immunoassay device, characterized in that a dry layer (19) is formed. 6. A fluorescent dye (13) existing near the surface of the optical waveguide (4) as a result of an antigen-antibody reaction is excited by introducing excitation light into the optical waveguide (4), and the fluorescent dye ( 1
3) In a fluorescence immunoassay device that measures immunity based on the fluorescence emitted from, the surface of the optical waveguide into which the solution to be measured (17) (18) is to be injected has a light scattering property and is soluble. A reaction start point detection device in a fluorescent immunoassay device, characterized in that a solid layer is formed.