JPH05203574A - Device for measuring fluorescence immunity - Google Patents

Abstract

PURPOSE:To improve measurement sensitivity and accuracy of the degree of immunity reaction based on fluorescence intensity which is excited by evanescent wave component. CONSTITUTION:An antibody 4 is fixed to a slab-type optical waveguide 4 and an antibody 4d which is labeled by BICY5.18-OSu4e is coupled to an antibody 4c which is connected to the antibody 4b by the antigen antibody reaction. An excitation light with a wavelength exceeding 600nm is introduced to the slab-type optical waveguide 4, thus exciting BICY5.18-OSu4e which is constrained near the surface of the optical waveguide 4 by the evanescent wave component.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a fluorescence immunoassay device, and more specifically, it causes an antigen-antibody reaction to occur on the surface of an optical waveguide that propagates the introduced excitation light while totally reflecting it. The labeled substance is reacted, the fluorescence emitted by the fluorescent substance excited by the evanescent wave component of the excitation light is introduced into the optical waveguide, propagated while being totally reflected, and the immune reaction is caused based on the fluorescence emitted from the optical waveguide. The present invention relates to a fluorescence immunoassay device for measuring the degree of

[0002]

2. Description of the Related Art Conventionally, an evanescent wave component generated by introducing excitation light into an optical waveguide and propagating in the optical waveguide while totally reflecting the excitation light is confined to the vicinity of the surface of the optical waveguide by an antigen-antibody reaction. Fluorescence that indirectly measures the degree of immune reaction by exciting the emitted fluorescent substance, propagating the excited fluorescence through the optical waveguide while totally reflecting it, emitting it from the optical waveguide, and measuring the intensity of the emitted fluorescence. Research on immunoassay devices is being conducted.

Specifically, an optical waveguide molded of synthetic resin is used, fluorein isothiocyanate (hereinafter abbreviated as FITC) is used as a fluorescent substance, and a wavelength region of 450 to 550 nm (excitation light) is used. The visible light region) was used.

[0004]

In the conventional fluorescence immunoassay device described above, not only is the evanescent wave component of the excitation light excited by the fluorescent substance corresponding to the degree of the immune reaction, but also the fluorescence contained in the optical waveguide itself. Since the substance is also excited, the intensity of the fluorescence emitted from the optical waveguide becomes a value obtained by superimposing the fluorescence intensity corresponding to the degree of immune reaction and the fluorescence intensity from the optical waveguide itself. Since the fluorescent substance bound in the vicinity of the surface of the optical waveguide is excited only by the evanescent wave component, the fluorescent substance in the optical waveguide is excited by the excitation light itself, so that it is included in the optical waveguide. Despite the small amount of the fluorescent substance, there is a disadvantage that the optical waveguide itself excites fluorescence that cannot be ignored and the measurement accuracy of the degree of immune reaction is reduced.

In order to solve such inconvenience, it has been proposed to use allophycocyanin as a fluorescent substance, but allophycocyanin has a high molecular weight (30,00).
Since it is a protein (about 0), its mobility in solution is low, and it takes a long time until the required amount of allophycocyanin is bound to the vicinity of the surface of the optical waveguide. When bound to, the stability of the conjugate is poor, and there is no guarantee that the amount of allophycocyanin corresponding to the degree of immune reaction will continue to be bound near the surface of the optical waveguide. Has the disadvantage that it decreases.

Further, if the optical waveguide is made of quartz glass, almost no fluorescence generated from the optical waveguide itself can be eliminated. However, in this case, the optical waveguide becomes expensive and compared with synthetic resin. Since cracks and the like are likely to occur, great care must be taken in storage and handling, and there is the inconvenience that workability is reduced.

[0007]

SUMMARY OF THE INVENTION The present invention has been made in view of the above problems, and when an optical waveguide made of a synthetic resin is used, the intensity of the fluorescence generated from the optical waveguide itself is significantly reduced, and the immunoreaction is further suppressed. It is an object of the present invention to provide a fluorescence immunoassay device capable of significantly improving the measurement sensitivity and the measurement accuracy of the degree.

[0008]

In order to achieve the above object, the fluorescence immunoassay apparatus according to claim 1 is such that the fluorescence excitation wavelength of the optical waveguide and the fluorescence excitation wavelength of the fluorescent substance are different from each other. And the wavelength of the excitation light introduced into the optical waveguide is set to the wavelength included in the fluorescence excitation wavelength of the fluorescent substance.

In the fluorescence immunoassay device of claim 2, the fluorescent substance is a cyanine-based fluorescent dye, and the fluorescent substance is bound to a substance that specifically reacts with the antigen or antibody bound near the surface of the optical waveguide by the antigen-antibody reaction. It was done. Claim 3
The fluorescent immunoassay device described in (1) above has a cyanine-based fluorescent dye bound to an antibody.

[0010]

According to the fluorescence immunoassay device of claim 1, an antigen-antibody reaction is carried out on the surface of the optical waveguide that propagates the introduced excitation light while totally reflecting it, and a substance labeled with a fluorescent substance is further reacted. In the case where the fluorescence emitted by the fluorescent substance excited by the evanescent wave component of the excitation light is introduced into the optical waveguide and propagated while being totally reflected, and the degree of the immune reaction is measured based on the fluorescence emitted from the optical waveguide. Since the fluorescence excitation wavelength of the optical waveguide and the fluorescence excitation wavelength of the fluorescent substance are set to be different from each other, the wavelength of the excitation light introduced into the optical waveguide is included in the fluorescence excitation wavelength of the fluorescent substance. By introducing excitation light of a wavelength into the optical waveguide, it is possible to excite only the fluorescent substance that is bound near the surface of the optical waveguide, and at the same time, it is excited in the optical waveguide itself. The fluorescence can be almost nil in. As a result, even if the optical waveguide is made of synthetic resin, it can receive the excited fluorescent light with a significantly reduced noise component,
The degree of immune reaction can be measured with extremely high sensitivity and accuracy based on the intensity of fluorescence.

In the fluorescent immunoassay device according to claim 2, the fluorescent substance is a cyanine-based fluorescent dye, and the substance reacts specifically with the antigen or antibody bound near the surface of the optical waveguide by the antigen-antibody reaction. Since it is coupled to, it is possible to adopt long-wavelength light of 600 nm or more as excitation light, and it is possible to significantly reduce the intensity of the fluorescence generated from the optical waveguide itself made of synthetic resin, and based on the intensity of the fluorescence emitted from the optical waveguide. The degree of immune reaction can be measured with extremely high sensitivity and accuracy. In addition, since the excitation light is in the long wavelength region of 600 nm or more, it is possible to easily use a light source having excellent monochromaticity as the excitation light source, and also from this aspect, it is possible to achieve high sensitivity and high accuracy of the degree of immune reaction. ..

According to the fluorescence immunoassay device of claim 3, since the cyanine fluorescent dye is bound to the antibody, the cyanine-based fluorescent dye is bound to the antigen in the solution to be measured with the antibody previously fixed on the surface of the optical waveguide. By performing an antigen-antibody reaction between the antigen-antibody reaction and the fluorescent-labeled antibody bound by the antigen bound near the surface of the optical waveguide, the amount of the fluorescent-labeled antibody corresponding to the degree of the immune reaction can be surely obtained. It can be bound near the surface of the optical waveguide, and the degree of immune reaction can be measured with high sensitivity and accuracy based on the excited fluorescence from the bound fluorescent-labeled antibody.

[0013]

Embodiments will now be described in detail with reference to the accompanying drawings showing embodiments. FIG. 1 is a schematic diagram showing an embodiment of the fluorescence immunoassay device of the present invention. Excitation light (long wavelength light of 600 nm or more) emitted from a semiconductor laser 1 is narrowed down by a lens system 2 and reflected by a dichroic mirror 3. Then, it is made incident on the slab type optical waveguide 4 made of synthetic resin, the fluorescence emitted from the incident surface of the slab type optical waveguide 4 is transmitted through the dichroic mirror 3, and the noise component is removed by the lens system 5 including the sharp cut filter. At the same time, the light is sufficiently condensed and guided to the photomultiplier tube 6 as a light receiving element. Reference numeral 7 denotes a light receiving element that receives the excitation light transmitted through the dichroic mirror 3, and detects the change in the intensity of the excitation light output from the semiconductor laser 1 and feedback-controls the semiconductor laser 1 to keep the excitation light intensity constant. Can be held at In addition, the slab type optical waveguide 4 is, for example, as shown in FIG.
As shown in FIG. 5, the solution containing portion 4a is formed so as to surround a predetermined area of the surface, and the antibody 4b is fixed in advance on the surface of the optical waveguide main body included in the solution containing portion 4a.
Instead of arranging the slab type optical waveguide main body so as to face the horizontal direction, the slab type optical waveguide main body may be arranged so as to face the vertical direction to form the solution storage portions 4a on both sides. In this case, the intensity of the excited fluorescence is increased. Can be doubled, which is advantageous for higher sensitivity and higher accuracy. Further, 4c represents an antigen, 4d represents a labeled antibody, and 4e represents a fluorescent substance.

When the test solution is measured by using the fluorescence immunoassay device having the above-described structure, first, the test solution having the same concentration or the test solution diluted to a predetermined magnification is injected into the solution container 4a. As a result, an antigen-antibody reaction is carried out with the antibody immobilized on the surface of the slab type optical waveguide body. Then, the test solution is discharged from the solution storage section 4a, the semiconductor laser 1 is driven, and a solution containing a fluorescent labeled antibody labeled with a fluorescent substance is injected into the solution storage section 4a in place of the test solution, and the antigen antibody The reaction causes the antigen-antibody reaction again with the antigen bound near the surface of the optical waveguide body. Here, as the fluorescent substance, BICY5.18-OSu as a cyanine dye {biochemical
Detection Systems (Biochemical Detectio
n Systems Inc. 4516 Henry Street, Pittsburgh, PA 15
213 USA)}, and mixed with anti-β-2 microglobulin antibodies at a predetermined ratio and bound to each other to obtain BICY.
The solution containing the 5.18-OSu-labeled antibody is replaced with the test solution and injected into the solution container 4a. The semiconductor laser 1 used is one that emits light having a wavelength of 650 nm. BICY5.18-OSu is excellent not only in fluorescence intensity but also in Stokes shift (difference between excitation light wavelength and excited fluorescence wavelength).
In addition, since the molecular weight is significantly smaller than that of allophycocyanin, the stability of BICY5.18-OSu, which is a fluorescent substance bound near the surface of the optical waveguide, is significantly superior to that of allophycocyanin.

Therefore, the intensity of the excited fluorescence increases with the progress of the antigen-antibody reaction based on the BICY5.18-OSu labeled antibody, and finally becomes the fluorescence intensity corresponding to the degree of the immune reaction. This based on the fluorescence intensity curve is shown in Chart was obtained a calibration curve 3 (A), 10 ^-11 M
The measurement sensitivity of As a comparative example, as shown in FIG. 4, the heat ray absorption filter 42a, the condenser lens 42b, and the interference filter 42 are used for the excitation light from the halogen lamp 41.
c, the optical chopper 42d, the slit 42e, the achromat lens 42f, and the dichroic mirror 43 to guide it to the optical waveguide 44 made of synthetic resin, and the fluorescence emitted from the optical waveguide 44 is reflected by the dichroic mirror 43 through the achromat lens 42f, and a sharp cut filter A calibration curve was obtained using an immunoassay device which was designed to lead to a photomultiplier tube 46 through 45. A lock-in amplifier 47b, which converts the current obtained by the photomultiplier tube 46 into a voltage by the I / V converter 47a, and is supplied with the synchronizing signal from the optical chopper 42d as a control signal.
Is amplified by the A / D converter 47c, converted into a digital signal by the A / D converter 47c, and supplied to the computer 47d for signal processing. In addition, FITC is used as a fluorescent substance, and anti-β-2 microglobulin antibody is mixed at a predetermined ratio,
The solution containing the FITC-labeled antibody obtained by binding to each other was injected into the solution container.

The calibration curve obtained by this comparative example is shown in FIG.
As in (B), the measurement sensitivity of 10 ⁻¹⁰ M was achieved. As is clear from the above, BIC is used as the fluorescent dye.
By using Y5.18-OSu and using excitation light having a wavelength of 650 nm, the measurement sensitivity of the degree of immune reaction can be improved by one digit.

The present invention is not limited to the above-mentioned embodiment, and for example, instead of immobilizing the antibody on the surface of the optical waveguide, the antigen or hapten is immobilized, and the cyanine dye is used as the antigen or hapten. Cyanine dyes other than BICY5.18-OSu can be used, and various design changes can be made without departing from the scope of the present invention. is there.

[0018]

As described above, according to the invention of claim 1, the wavelength of the excitation light introduced into the optical waveguide is introduced into the optical waveguide by introducing the excitation light having a wavelength contained in the fluorescence excitation wavelength of the fluorescent substance. It is possible to excite only fluorescent substances that are bound near the surface of the waveguide, and it is possible to virtually eliminate the fluorescence that is excited in the optical waveguide itself. Even if the optical waveguide is made of synthetic resin, it is excited in a state where the noise component is extremely small. The unique effect of being able to receive the emitted fluorescence and being able to measure the degree of the immune reaction based on the intensity of the fluorescence with extremely high sensitivity and high accuracy is exhibited.

According to the second aspect of the invention, the excitation light is 600 nm.
By adopting the above long-wavelength light, the intensity of fluorescence generated from the optical waveguide itself made of synthetic resin can be remarkably reduced, and the degree of the immune reaction can be remarkably highly sensitive based on the intensity of the fluorescence emitted from the optical waveguide. High-accuracy measurement is possible. Furthermore, since the excitation light is in the long wavelength region of 600 nm or more, it is easy to adopt a monochromatic excitation light source.
From this aspect as well, there is a unique effect that it is possible to achieve high sensitivity and high accuracy of the degree of immune reaction.

According to a third aspect of the present invention, an antigen-antibody reaction is carried out with an antigen in a solution to be measured in the state where an antibody is immobilized on the surface of the optical waveguide in advance, and then the antigen bound near the surface of the optical waveguide. By carrying out the antigen-antibody reaction with the fluorescence-labeled antibody and the fluorescence-labeled antibody, it is possible to reliably bind the fluorescence-labeled antibody in an amount corresponding to the degree of the immune reaction to the vicinity of the surface of the optical waveguide, and to excite fluorescence from the bound fluorescence-labeled antibody. Based on this, the unique effect that the degree of immune reaction can be measured with high sensitivity and high accuracy is achieved.

[Brief description of drawings]

FIG. 1 is a schematic diagram showing an embodiment of a fluorescent immunoassay device of the present invention.

FIG. 2 is a vertical sectional view showing a configuration of a slab type optical waveguide.

FIG. 3 is a diagram showing an obtained calibration curve.

FIG. 4 is a schematic diagram showing a fluorescence immunoassay device as a comparative example.

[Explanation of symbols]

4 Slab type optical waveguide 4d Labeled antibody 4e Fluorescent substance

Claims (3)

[Claims] 1. An antigen-antibody reaction is carried out on the surface of an optical waveguide (4) which propagates the introduced excitation light while totally reflecting it, and a substance (4d) labeled with a fluorescent substance (4e) is further reacted. The fluorescence emitted by the fluorescent substance (4e) excited by the evanescent wave component of the excitation light is introduced into the optical waveguide (4), propagated while being totally reflected, and immunized based on the fluorescence emitted from the optical waveguide (4). In the fluorescence immunoassay device for measuring the degree of reaction, the fluorescence excitation wavelength of the optical waveguide (4) and the fluorescence excitation wavelength of the fluorescent substance (4e) are set to be different from each other, and the optical waveguide ( The fluorescence immunoassay device characterized in that the wavelength of the excitation light introduced into 4) is set to a wavelength included in the fluorescence excitation wavelength of the fluorescent substance (4e). 2. The fluorescent substance (4e) is a cyanine-based fluorescent dye, which is bound to a substance which specifically reacts with an antigen or antibody bound near the surface of the optical waveguide (4) by an antigen-antibody reaction. The fluorescent immunoassay device according to claim 1. 3. The fluorescent immunoassay device according to claim 2, wherein a cyanine-based fluorescent dye is bound to the antibody.