JPH05249114A - Method and instrument for immunological measurement - Google Patents

Abstract

PURPOSE:To perform highly sensitive immunological measurement by irradiating a magnetic fluorescent-labeled specimen complex with laser light and, after condensing generated fluorescence, leading the condensed fluorescence to a photodetector through an optical fiber for guiding fluorescence. CONSTITUTION:After a magnetic fluorescent-labeled specimen complex composed of fluorescent micro-beads, an antigen, and fine magnet particles is manufactured, the complex is transferred to a small diameter tube 5 for measuring samples. When an electromagnet 6a is energized, a magnetic field is generated between a lower magnetic core 6 and an upper magnetic core 7 and the complex moves to a position immediately below the magnetic core 7 in the tube 5, resulting in the formation of an aggregate. On the other hand, the fluorescent micro-beads which do not react to the antigen do not form the aggregate. Then the aggregate of the complex is irradiated with laser light by leading the laser light to a laser light irradiating short-focus lens 1 through an optical fiber for leading laser light. The fluorescence radiated from the microbeads which are excited by the laser light is led to an optical fiber 4 for guiding fluorescence and the intensity of the fluorescence is measured with a photodetector 9. A data processing computer 10 analyzes the measured data.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to an immunoassay utilizing an antigen-antibody reaction.

[0002]

2. Description of the Related Art As a conventionally known detection method utilizing an antigen-antibody reaction, a radioion assay (RI
A), enzyme immunoassay (EIA), fluorescent immunoassay (FIA), laser immunoassay (LIA) and the like have already been put to practical use. These methods are methods for detecting the presence or absence of an antigen to which isotopes, enzymes, and fluorescent substances are added as labels, respectively. However, the EIA method,
The FIA method and the LIA method have a sensitivity of 10 ⁻⁶ gram to 10 ⁻¹⁰ gram at most, and are insufficient in sensitivity as an antigen test which is particularly useful in an antigen-antibody reaction, which is a problem in practice. In addition, the RIA method has a sensitivity of 10 ^-12 g and is a measurement method capable of ultra-trace analysis and antigen test, but it requires special equipment because it uses radioactive substances, and it is versatile and costly. I had a problem with.

Therefore, the inventors of the present invention have been able to perform antigen testing 10
^A laser magnetic immunoassay method and a laser magnetic immunoassay apparatus have been proposed as a highly versatile measuring method having a sensitivity of ¹² grams or more (Japanese Patent Application No. 61-224567, 61).
-252427, 61-254164, 62-2206
2, 62-22063, 62-152791, 62-1
84902). These new measurement methods have a feature that after the antigen-antibody reaction, the magnetic fine particles used for the labeling material are aggregated by a magnetic force, and the size of the aggregate is measured using laser light by the intensity of laser interference light. Equivalent to the RIA method,
Alternatively, it is possible to detect an extremely small amount.

The laser magnetic measurement method will be described below with reference to FIG.
This will be described in detail with reference to the detection principle diagram of the laser magnetic immunoassay method (interference type). The laser magnetic immunoassay method aims to improve the detection sensitivity by locally concentrating a magnetically labeled virus in a magnetic field. That is, a virus is subjected to an antigen-antibody reaction between a magnetic labeling reagent in which an antibody is bound to magnetic fine particles and a virus are magnetically labeled to remove unreacted magnetic labeling reagent, and then the amount of the magnetic fine particles is determined by the method shown in FIG. Optically measure with.

For example, magnetite (Fe ₃ O ₄ ) microparticles or magnetite microparticle surfaces are used as the magnetic microparticles, and microparticles of sugar such as dextran and proteins such as protein A are used, and an antibody (or antigen) such as an IgG antibody is added thereto. By binding and binding the magnetic substance-labeled substance to an antigen (or antibody) such as an influenza virus which is a sample and an antigen-antibody reaction, the antigen (or antibody) as a sample is magnetically labeled to prepare a magnetic substance-labeled substance. Next, as shown in FIG. 5, the magnetic label and the dispersed liquid are placed in the inspection container 1.
5 and put this inspection container 15 in the electromagnet 16 and the upper magnetic core 1
It is set in the gradient magnetic field generator configured in 7. When the electromagnet 16 is excited, the magnetic field on the liquid surface immediately below the upper magnetic core 17 is the highest, so that the magnetic marker is concentrated in this portion. When the magnetic field in this portion exceeds several kG, the liquid surface microscopically rises due to the magnetic attraction force in the direction perpendicular to the magnetic marker and the surface tension of the liquid. When laser light 19 is incident on this raised portion 18 from an oblique direction and reflected light 20 from the liquid surface thereof is received by the screen, interference fringes appear. By measuring the light intensity of this interference fringe, the amount of the magnetic substance-labeled substance can be known. Further, not only the measurement of the intensity of the interference light but also the number of the interference fringes and the intensity of the scattered light can be counted to quantify the magnetic label. Furthermore, in order to improve the detection sensitivity, an alternating current can be applied to the electromagnet to measure the interference light, the reflected light, and the scattered light synchronized with the fluctuating magnetic field. Note that this laser magnetic immunoassay method has an extremely high sensitivity (1 × 10
^-13 g / ml) can detect a trace amount of virus.

As described above, the laser magnetic immunoassay method is
This method has a very high detection sensitivity and has a drawback that it is easily affected by dust. That is, if, for example, one dust particle of 0.5 μm floating in the air is mixed in the concentration position of the sample irradiated with the laser beam during the measurement, one virus may cause a misdiagnosis. Of.

Further, in this measuring method, the magnetic substance-labeled complex is guided to the surface of the liquid to be measured, and the projections of the complex gathered on the liquid surface are measured by laser light, so that the liquid surface shape (meniscus) is stabilized. Sex has a great influence on quantification. In particular, regarding the interferometry disclosed in Japanese Patent Application No. 62-184902, it has been clarified that it is inevitable that the water surface is downwardly convex, that is, a meniscus is formed in the mechanism of interference. .. Also, it was revealed that the shape on the water surface has a great influence on the quantitativeness and reproducibility of the measured values even in scattered light and transmitted light.

Therefore, as a method of eliminating the influence of the above-mentioned obstacles such as dust, the concentrated magnetic substance-labeled specimen complex is AC-driven by magnetic force to selectively detect the signal component synchronized with the driving cycle. The previous patent application for the method (Japanese Patent Application No. 62-1
52792, 62-184902). Further, Japanese Patent Application No. 63-6050 discloses a technique for ejecting an air stream to a locally concentrated position of a magnetically labeled sample to eliminate suspended matter such as dust that hinders detection.

Further, in order to obtain a meniscus with good reproducibility, Japanese Patent Application No. 63-102915 of "Inspection container used for laser magnetic immunoassay" by the present inventor discloses the technique. Then, as a result of further research, in order to reliably bring out the performance of the inspection container, after injecting the excess solution into the inspection container, the excess solution was poured until a predetermined liquid level was reached. It was clarified that sucking and removing from the surface ensures the reproducibility of the meniscus and is also a preferable means for removing suspended dust.

[0010]

However, in the above-described laser magnetic immunoassay method, in measuring the intensity of laser interference light, the external light is used as an optical measurement system in order to reduce the background and improve the S / N ratio. It was necessary to optically seal the device to prevent it from entering. Furthermore, since the free surface of the measurement solution is used, it is necessary to use vibration control measures such as an anti-vibration table to prevent fluctuations in the liquid surface due to vibration, and a control mechanism to keep the distance between the water surface and the magnetic pole of the measurement solution constant. It had the drawback of being complicated and bulky. A step of separating the unreacted magnetic substance is required, and there is a drawback that sample preparation takes time.

Further, as an immunoassay method using the fluorescence method so far, Japanese Patent Laid-Open No. 61-419 of Love Systems Co. has been used.
66, JP-A-62-501647, JP-A-62-502
There is 708. In these patents, unreacted phosphors are introduced into a two-liquid structure cell to reduce the background, and there is a problem in terms of operability and sensitivity.

Accordingly, the present invention has been made in view of the above circumstances, and an object thereof is to provide a simple, rapid, and highly sensitive immunoassay method.

[0013]

In order to solve the above-mentioned problems, the immunoassay method according to claim 1 is a first method of capturing an antigen with fluorescent microbeads containing an antibody-bound fluorescent dye and magnetic particles. Step, a second step in which the fluorescent microbeads produced in the first step, a magnetic substance fluorescent-labeled analyte complex that is a complex of an antigen and magnetic fine particles are aggregated by an external magnetic field, and the assembly point is irradiated with laser light. In the immunoassay method consisting of the third step of detecting the fluorescence generated by the above, after the laser light is guided by the optical fiber for laser light irradiation, the converging point is irradiated by the condenser lens for laser light irradiation After condensing with a condenser lens for condensing fluorescence,
It is characterized by having a fluorescence detection system introduced into a photodetector by an optical fiber for fluorescence guiding.

In order to solve the above-mentioned problems, an immunoassay method according to a second aspect of the present invention is the method of the first aspect, wherein the second step is performed in an optically transparent thin tube, and the fluorescent micro wall is attached to the wall surface of the thin tube. The present invention is characterized in that an aggregate of magnetic substance fluorescent-labeled specimen complex comprising a complex of beads, an antigen and magnetic fine particles is produced, and a third step is carried out to quantify the antigen.

In order to solve the above-mentioned problems, the immunoassay method according to claim 3 is the method according to claim 1, wherein after the second step, a biochemically inactive liquid is injected into the thin tube, and The method is characterized in that the third step is carried out after removing the microbeads containing the fluorescent dye in the reaction.

In order to solve the above-mentioned problems, an immunoassay method according to a fourth aspect of the present invention is characterized in that, in the first aspect, a pulsed laser beam is irradiated and the generated fluorescence is measured after a predetermined time has elapsed from the laser irradiation. It is characterized by doing.

In order to solve the above-mentioned problems, an immunoassay device according to a fifth aspect of the present invention comprises an inspection container for containing a solution containing a fluorescent-microbead, a magnetic substance-labeled sample complex composed of a complex of an antigen and magnetic fine particles. An incident optical system that guides laser light to the surface of the inspection container, a magnetic field generator that collects the magnetic substance fluorescent label analyte complex at one point of the laser light irradiation region on the surface of the inspection container, and the magnetic substance fluorescent label In an immunoassay device including at least a light receiving system that receives laser light emitted from an aggregate of specimen complexes, the incident optical system is a laser light introducing light for guiding the laser light from the laser light source. A fiber; and a laser light irradiation condenser lens for irradiating the gathering point of the magnetic substance fluorescent labeled analyte complex with the laser light guided by the laser light irradiation optical fiber. The light receiving system is for collecting a fluorescence condensing lens for condensing fluorescence generated from the magnetic substance fluorescent labeled analyte complex and for guiding the fluorescence condensed by the fluorescence condensing lens. An optical fiber for fluorescence guiding and a photodetector for measuring the amount of fluorescence guided by the optical fiber for guiding fluorescence are provided.

In order to solve the above-mentioned problems, an immunoassay device according to a sixth aspect of the present invention is characterized in that the test container according to the fifth aspect is an optically transparent thin tube. ..

[0019]

According to the present invention, an antigen-antibody reaction is used to specifically capture an antigen on the surface of fluorescent microbeads, and a magnetic label and a fluorescent substance-labeled magnetic substance-labeled analyte complex are aggregated in a magnetic field to emit a laser beam. Is an immunoassay method for detecting by irradiating a laser beam in an incident system with an optical fiber for introducing a laser beam and a condenser lens for irradiating the laser beam to the collecting point, and in a light receiving system, collecting the laser beam to the collecting point. The greatest feature is that the fluorescence generated from the included fluorescent microbeads is collected by the collecting lens for collecting fluorescence and then introduced into the photodetector by the optical fiber for guiding the fluorescence. The laser beam is focused by the focusing lens for irradiating the laser beam, and the fluorescence is focused by the focusing lens for focusing the fluorescence, and then introduced into the optical fiber for guiding the fluorescence, so that the laser is selectively applied to the minute gathering points. Since it is possible to irradiate light and collect only the fluorescence generated from the gathering point, it is not necessary to shield the optical system including the incident system and the light receiving system from external light.
Therefore, it is possible to reduce the size and weight of the device and improve operability.

Further, in the present invention, it is also possible to collect the reaction product on the water surface of the inspection container by external magnetism and measure the fluorescence in the same manner as in the above-described laser magnetic immunoassay method. In place of the above, an optically transparent thin tube is used, and a magnetic fluorescent labeling complex consisting of a fluorescent microbead, a complex of an antigen and magnetic fine particles is placed inside the thin tube, and the magnetic substance is applied by an external magnetic field. An incident system in which fluorescence-labeled analyte complexes are aggregated and a short-focus condenser lens is used at the gathering point, and a light-receiving system in which a short-focus lens is used as a condenser lens that collects fluorescence from the gathering point It is also possible to use an optical measuring system consisting of Therefore, when the thin tube as described above is used, the magnetic pole spacing in the optical measurement system can be reduced, so that the magnetic field strength can be increased with a relatively small electromagnet or permanent magnet, and the size can be reduced.

In the present invention, it is not necessary in principle to separate the unreacted fluorescent beads from the reacted fluorescent beads, but in order to improve the sensitivity in the trace antigen region, a biochemically inactive liquid is placed in the thin tube. It is also possible to remove the microbeads containing unreacted fluorescent dye by injecting into

Furthermore, by measuring the pulsed laser light using an excitation light source after a lapse of a fixed time from the laser excitation, the excitation light can be removed and a high S / N ratio can be measured.
In this case, it is not necessary to remove the excitation light from the spectroscope or the like, so that further miniaturization is possible.

On the other hand, in the case of measuring the conventional antigen-antibody reaction by fluorescence, a method of blocking external light is generally used. In particular, when the sensitivity becomes high, the influence of external light becomes large, and there is a drawback that the device becomes large because the external light is blocked. Further, in the conventional fluorescent bead measuring method, it is necessary to remove the unreacted fluorescent beads by some method, and the above-mentioned JP-A-61-41966 and JP-A-62-501 of Love Systems Co.
In 647, the sample is moved to the lower part of the reaction vessel to separate it,
The dye that absorbs the excitation wavelength is removed by mixing it into the unreacted layer.

In the immunoassay method according to the present invention, the reaction product is concentrated at one point by a magnetic field, the laser light is condensed to several μm by the condensing lens system, and the fluorescence is condensed by the short focus lens. None, and unreacted fluorescent beads are not excited. As a result, a simple and quick measurement system can be provided.

[0025]

The present invention will be described in more detail below with reference to the drawings, but the embodiments disclosed below are merely examples of the present invention and do not limit the scope of the present invention.

1 and 2 are schematic views of an immunomagnetic measuring apparatus for explaining an embodiment of the present invention, in which reference numeral 1 indicates
Condensing lens for irradiating laser light, 2 optical fiber for introducing laser light, 3 converging lens for condensing fluorescence, 4 optical fiber for guiding fluorescence, 5 small diameter tube for sample measurement, 6 lower magnetic core, 6a is an electromagnet, 7 is an upper magnetic core, 8 is a laser light source, 9 is a photodetector, 10 is a data processing computer, 11 is a micropump, 12 is a measurement sample, 13 is a washing liquid, and 14 is a magnetic fluorescent label. It is a sample complex.

In the schematic side view of the immunoassay device shown in FIG. 1, a thin tube 5 for measuring a sample, which acts as a test container containing a magnetic substance fluorescent-labeled sample complex, is provided with a magnetic substance fluorescent-labeled sample complex. A body 14 is contained. As the sample measuring thin tube 5, in addition to a commercially available Crypto tube, a glass-made optically transparent tube having a tube diameter of 10 μm to 10 nm is desirable.

The lower magnetic core 6 and the upper magnetic core 7 are arranged so as to face each other, and the thin tube 5 is inserted in the space between them. The upper magnetic core 7 and the lower magnetic core 6 in this example are preferably made of a high magnetic permeability material having a small residual magnetization, for example, pure iron having high purity is recommended. The diameter of the lower magnetic core 6 is
Sufficiently larger than the diameter of the thin tube 5 and the upper magnetic core 7
It is essential that the diameter of is sufficiently smaller than the diameter of the thin tube. For example, it is preferable that the tip of the upper magnetic core 7 on the side facing the lower magnetic core 6 is sharp such as a needle.

The lower magnetic core 6 has an electromagnet 6
a and 6a are fixed. Further, one end of a substantially C-shaped yoke (not shown in the drawing) is connected to the lower magnetic core 6, and the other end of the yoke is connected to the upper magnetic core 7. And, for example, pure iron is used for this yoke, and a magnetic circuit including an electromagnet 6a, a lower magnetic core 6, an air gap portion, an upper magnetic core 7 and an electromagnet 6a is configured in the magnetic field generating device in this immunoassay device. In this magnetic circuit, when the magnetic flux generated from the electromagnet 6a is guided from the lower magnetic core 6 to the upper magnetic core 7, the magnetic flux that passes through the small-diameter tube 5 and is collected in the upper magnetic core 7 passes through the yoke and the electromagnet. 6a.

Further, since the magnetic circuit as described above effectively collects the magnetic flux from the electromagnet 6a directly below the needle-like end of the upper magnetic core 7 on the lower magnetic core 6, the magnetic substance fluorescent-labeled sample composite. The body is effectively collected.

The laser light emitted from the laser light source 8 is guided to the short-focus laser light irradiation condenser lens 1 by the laser light introducing optical fiber 2, and the thin tube 5 is introduced.
It is installed so as to pass through the central portion of the small-diameter tube 5 in parallel with the axis of, and illuminates the aggregate of the magnetic substance fluorescent labeled analyte complex 14. At this time, by making the spot of the condenser lens 1 for laser light irradiation as small as about 10 to 100 μm, it is possible to suppress the excitation of the fluorescent microbeads that have reacted with the antigen.

Then, the focusing lens 3 for collecting fluorescence with a short focus.
The fluorescence guiding optical fiber 4 and the photodetector 9 form a light receiving system for receiving the fluorescence emitted from the fluorescence microbeads excited by the laser light irradiation. The fluorescence emitted from the fluorescent microbeads excited by the laser light irradiation is collected by the fluorescence collecting condenser lens 3,
It is installed so as to be introduced into the fluorescence introducing optical fiber 4, and further, the axis of the fluorescence introducing optical fiber 4 is set so as to avoid reflected light from the water surface of the laser light irradiation which becomes an obstacle. The axis of the laser light irradiating optical fiber 2 which is the incident axis of the laser light is at a lower angle than the axis of the fluorescence introducing optical fiber 4.

That is, in order to efficiently collect the fluorescence emitted from the aggregate of the magnetic fluorescent labeled analyte complex 14, the fluorescent condensing lens 4 has a large lens diameter,
It is desirable that the working distance be reasonably short.
The lens diameter and working distance are restricted by the spatial arrangement of the upper magnetic core 7, the lower magnetic core 6, and the small diameter tube 5 as shown in FIG. Therefore, the spatial arrangement of the upper magnetic core 7, the lower magnetic core 6, and the small diameter tube 5 efficiently collects the fluorescence emitted from the aggregate of the magnetic substance fluorescent labeled analyte complex 14 aggregated at one point, and The influence of external light can be almost ignored.

Further, the immunoassay apparatus of the present invention having the above-mentioned structure can perform the measurement without the mechanism (so-called B / F) separation for removing the unreacted substance of the magnetic substance fluorescent labeled analyte complex 14. Although possible, it is possible to improve the S / N in the trace antigen region by additionally providing the B / F separation device mechanism as shown in FIG. That is, the measurement sample 12 containing the magnetic fluorescent labeled sample complex 14 and the fluorescent microbeads of the unreacted substance is transferred to the thin tube 5, and after being aggregated by the magnetic field, connected in parallel with the measurement sample 12. A container storing the cleaning liquid 13 is provided so that the cleaning liquid 13 can be delivered into the small-diameter pipe 5 by the micropump.

The immunoassay method of the present embodiment using the immunoassay device will be described while explaining the operation principle of the immunoassay device of the present embodiment configured as described above.

First, the fluorescent microbeads to which the antibody has been bound are reacted with the antigen in advance, and the reaction product is added with the magnetic fine particles to which the antibody has been bound to cause a reaction, whereby the magnetic substance of the complex of the fluorescent microbeads, the antigen and the magnetic fine particles is added. The fluorescent labeled analyte complex 14 is prepared. Here, the fluorescent beads are a material in which the fluorescent dye contained therein is chemically stable, has an absorption band that can be excited at the oscillation wavelength of a commercially available laser, and does not cause nonspecific adsorption of beads and magnetic fine particles. It is desirable to have such characteristics.

Examples of the fluorescent dye include the commercially available products shown in Table 1. Desirable characteristics of fluorescent dyes are that the difference between the maximum absorption wavelength and the maximum fluorescence wavelength is large, and that the existing laser oscillation wavelength has an absorption wavelength.

[0038]

[Table 1]

The magnetic substance fluorescent-labeled analyte complex 14 produced as described above is transferred to the sample measuring thin tube 5. A micro pump 11 is used as the transfer means.
Here, when the electromagnet 6 is energized, a strong magnetic field is generated between the electromagnet 6 and the upper iron core 7, and the magnetic substance fluorescent labeled analyte complex 14
Moves under the upper magnetic core 7 in the small-diameter tube 5 due to the magnetic field,
Form an aggregate. On the other hand, the fluorescent microbeads that have not reacted with the antigen do not combine with the magnetic fine particles and thus do not aggregate by the magnetic field.

Then, the laser light emitted from the laser light source 8 is guided by the laser light introducing optical fiber 2 to the laser light irradiating short-focus lens 1, and the aggregate of the magnetic substance fluorescent labeled analyte complex 14 is irradiated. To do.

The fluorescence emitted from the fluorescent microbeads excited by the laser light irradiation is collected by the fluorescence focusing short focus lens 3 and introduced into the fluorescence guiding optical fiber 4. Then, the introduced fluorescence is input to a photodetector 9 such as a photomultiplier tube, a photodiode or photon counting, and the light intensity is measured. The output of the photodetector 9 is input to the data processing computer 10 for storage and analysis of data. If it is necessary to remove the excitation light, it is possible to put it in front of the photodetector 9 such as a spectroscope or an interference filter to remove the influence of the excitation light. further,
As a method of removing the excitation light, it is possible to completely eliminate the influence of the excitation light by exciting with pulsed laser light and measuring fluorescence after a certain period of time.

In principle, the immunoassay method according to the present invention can be carried out without an operation of removing unreacted substances (so-called B / F separation), but in order to improve the S / N in the trace antigen region, the cleaning liquid 13 is used. B / F separation can also be carried out by flowing the above into the small diameter tube 5. That is, the magnetic substance fluorescent labeled analyte complex 14 composed of fluorescent microbeads, antigens and magnetic fine particles is aggregated by a magnetic field, and then the washing liquid 13 is delivered into the thin tube 5 by the micropump 11. At this time, the reacted fluorescent microbeads remain below the upper iron core 7 (upper magnetic pole) due to the magnetic field, but the unreacted fluorescent microbeads move to the outside of the thin tube 5. As a result, B / F separation is performed and S / N
Is improved. As the washing liquid 13, a buffer such as HEPES or PBS is used so that the reaction complex is not biochemically broken. In addition, a buffer containing a surfactant added in the range of 0.001% to 1% can be used to further improve the cleaning property.

The fluorescence intensity is proportional to the amount of the microbeads of the magnetic substance fluorescent labeled analyte complex 14 composed of the fluorescent microbeads, the antigen and the magnetic fine particles, and when the number-converted amount of the antigen is smaller than the number of the fluorescent microbeads used in the reaction. Since the probability of one or more antigens reacting with the fluorescent microbeads is small, the fluorescence intensity is proportional to the amount of antigen. As a result, the amount of antigen can be quantified from the fluorescence intensity.

(Test Example) Next, a test example in which the immunoassay method of the present invention is applied to the detection of α-fetoprotein will be shown. 20 μl of fluorescent micro beads labeled with α-fetoprotein antibody (bead concentration 0.2 wt% solution), α-
Example of dilution of fetoprotein (protein concentration 10 ^-15 g / ml
10-fold dilution in the range of -10 ^-8 g / ml 8 steps) 20 each
μl was mixed and allowed to react with stirring at 37 ° C. for 1 hour. Here, the fluorescent beads are polymer beads dyed with Oxazine 170 (9-ethylamino-5-ethylamino-10-methyl-5H-benzophenoxazonium perchlorate). As the α-fetoprotein antibody and antigen protein, commercially available products were used.

Then, 10 μl of a magnetic fine particle solution (antibody concentration 10 ⁻² mg / ml) bound with α-fetoprotein antibody was added to the reaction solution having each antigen concentration, and the mixture was reacted at 37 ° C. for 1 hour. About 20 μl of the reaction solution was introduced into a commercially available hematocrit tube (tube diameter 2.0 mm, inner diameter 1.0 mm) and set in a fluorescence measuring device. A He-Ne laser (wavelength 633 nm) 5 mW was used as an excitation light source, and the fluorescence emitted from the sample was separated by a commercially available spectroscope (wavelength resolution 1 nm).
It was detected with a photomultiplier tube. The result is shown in FIG.

FIG. 4 shows the result of fluorescence measurement under room light lighting, in which the alpha fetoprotein antigen concentration was 10 ^{−9 for} A.
g / ml, B is 10 ⁻¹² g / ml, and C is the amount of fluorescence in the case of control (antigen amount 0). The significant difference between B and C shows that the sensitivity of the immunoassay according to the present invention is 10 ⁻¹² mg / ml or more. Also, by obtaining the above sensitivity under room light lighting,
It can be seen that the immunoassay method of the present invention does not require a special optical shield effect.

[0047]

As described above, in the laser magnetic immunoassay method using the fluorescent microbeads and the magnetic fine particles, the fluorescence detection system using the condenser lens enables the fluorescence measurement without being influenced by the external light. , Small and highly sensitive antigen detection is possible.

[Brief description of drawings]

FIG. 1 is a schematic view of an immunoassay device for explaining an embodiment of an immunoassay method according to the present invention, and is a front view seen from the end of a sample measuring thin tube.

FIG. 2 is a schematic view of an immunoassay device for explaining an embodiment of the immunoassay method according to the present invention, and is a side view of a thin tube for measuring a sample as viewed in the longitudinal direction.

FIG. 3 is a schematic diagram of an immunoassay device for explaining an embodiment of the immunoassay method in the present invention, showing an optical system for fluorescence irradiation and fluorescence collection, and a thin tube for measuring a sample. FIG.

FIG. 4 is a fluorescence spectrum when a standard sample of AFP antigen is used in a test example in the immunoassay method of the present invention, where A is a fluorescence spectrum of AFP antigen concentration of 10 ⁻⁹ g / ml, and B is B. Is a fluorescence spectrum with an APF concentration of 10 ⁻¹² g / ml, and C is a fluorescence spectrum with an APF antigen concentration of 0.

FIG. 5 is a detection principle diagram of a laser magnetic immunoassay method (interference type).

[Explanation of symbols]

 1 Condensing lens for laser light irradiation 2 Optical fiber for introducing laser light 3 Condensing lens for fluorescence condensing 4 Optical fiber for fluorescence guiding 5 Small diameter tube for sample measurement 6 Lower magnetic core 6a Electromagnet 7 Upper iron core 8 Laser light source 9 Light Detector 10 Data processing computer 11 Micro pump 12 Measurement sample 13 Washing liquid 14 Magnetic substance fluorescent labeled sample complex

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Claims (6)

[Claims] 1. A first step of supplementing an antigen with fluorescent microbeads containing a fluorescent dye bound to an antibody and magnetic fine particles, and a magnetic substance which is a complex of the fluorescent microbeads produced in the first step with the antigen and magnetic fine particles. In the immunoassay method comprising a second step of assembling the body fluorescence-labeled analyte complex with an external magnetic field and a third step of detecting the fluorescence generated by irradiating the assembly point with the laser light, the laser light is used as the laser light. After the light is guided by the introduction optical fiber, the converging point is irradiated by the condensing lens for laser light irradiation, the generated fluorescence is condensed by the condensing lens for condensing the fluorescence, and the photodetector is formed by the optical fiber for the fluorescence guiding. An immunoassay method characterized by having a fluorescence detection system to be introduced into. 2. The magnetic fluorescent labeled analyte complex according to claim 1, wherein the second step is carried out in an optically transparent thin tube, and the wall surface of the thin tube is composed of a complex of fluorescent microbeads, an antigen and magnetic fine particles. An immunoassay method characterized in that a body assembly is manufactured, and a third step is performed to quantify an antigen. 3. The method according to claim 1, wherein after the second step, a biochemically inert liquid is injected into the small diameter tube to remove the unreacted microbeads containing the fluorescent dye, and then the third step. An immunoassay characterized by carrying out. 4. The immunoassay method according to claim 1, wherein the pulsed laser light is irradiated and the generated fluorescence is measured after a lapse of a predetermined time from the laser irradiation. 5. A test container containing a solution containing a fluorescent microbead, a magnetic substance-labeled sample complex composed of a complex of an antigen and magnetic fine particles, an incident optical system for guiding laser light to the surface of the test container, and A magnetic field generator that collects the magnetic fluorescent-labeled analyte complex at one point on the surface of the inspection container where the laser light is irradiated, and a light-receiving system that receives laser light emitted from the aggregate of the magnetic fluorescent-labeled analyte complex. In the immunoassay device including at least, the incident optical system is a laser light introduction optical fiber for guiding a laser light from the laser light source, and a laser light guided by the laser light irradiation optical fiber. And a condensing lens for irradiating a laser beam for irradiating a collection point of the magnetic substance fluorescent-labeled analyte complex with the light-receiving system generated from the magnetic substance fluorescent-labeled analyte complex. By a fluorescent light collecting condenser lens for collecting light, a fluorescent light guiding optical fiber for guiding the fluorescent light collected by the fluorescent light collecting condenser lens, and the fluorescent light guiding optical fiber An immunoassay device comprising a photodetector for measuring the amount of guided fluorescence. 6. The immunoassay device according to claim 5, wherein the inspection container is an optically transparent thin tube.