JPH0750113B2 - Laser magnetic immunoassay - Google Patents

Description

TECHNICAL FIELD The present invention relates to an immunoassay method using an antigen-antibody reaction. More specifically, the present invention relates to a laser magnetic immunoassay method capable of quantitatively detecting a specific antibody or antigen (hereinafter referred to as a target sample) from an extremely small amount of sample.

2. Description of the Related Art Development of an immunoassay using an antigen-antibody reaction as an early test method for new types of viral diseases such as acquired immunodeficiency syndrome, adult T-cell leukemia, etc., or various cancers is currently on a global scale. It is being promoted.

As a conventionally known microimmunoassay method, a radioimmunoassay method (hereinafter referred to as RIA method), an enzyme immunoassay method (EIA method), a fluorescence immunoassay method (FIA method), and the like have already been put into practical use. These methods are methods in which the presence or absence of an antibody or an antigen that specifically reacts with an antigen or antibody to which an isotope, an enzyme, or a fluorescent substance is added as a label, respectively.

The RIA method quantifies the amount of the specimen that contributed to the antigen-antibody reaction by measuring the radiation dose of the labeled isotope, and is currently the only method capable of measuring an ultratrace amount of picogram. However, since this method uses a radioactive substance, special equipment is required, and attenuation of the labeling effect due to half-life and the like must be taken into consideration, so that there is a large limitation in its implementation. Furthermore, considering the current situation where radioactive waste treatment is a social issue, its implementation is naturally limited.

On the other hand, the EIA method and FIA method using an enzyme or a fluorophore as a label are methods for detecting the amount of a sample that has contributed to an antigen-antibody reaction by observing color development or luminescence, and there are restrictions on implementation such as the RIA method. Absent. However, since the EIA method and FIA method are difficult to completely separate the unreacted label, the background is high and the detection limit is about nanogram.

In addition, as a method of detecting the presence or absence of an antigen-antibody reaction using laser light, for example, there is a method using AFP (alpha-fetoprotein) developed mainly for the purpose of detecting liver cancer. The fundamental drawback of this conventional laser light scattering method is that, in principle, it is unexpected to further increase the detection sensitivity because only one part of the solution in which the sample is dispersed is irradiated with the laser for detection. . In addition, because of such an essential defect, a large amount of sample was required.

As described above, the RIA method, which has a high detection sensitivity among the conventional immunoassays, has many restrictions on its implementation because it uses radioactive substances, and the EIA method and FIA method, which are easy to implement, have low sensitivity. There was a problem that precise quantitative measurement could not be performed.

In order to deal with these problems, the present inventors have conducted research on immunoassay methods having principles different from those of conventional methods, and previously disclosed Japanese Patent Application Nos. 61-224567, 61-252427, 61-254164, 62-22
062, 62-22063, 62-152791, 62-152792, 62-18490
2, 62-264319 and 62-267481 have filed patent applications for inventions relating to the laser magnetic immunoassay method and measuring apparatus. These new immunoassays are characterized by using magnetic fine particles as a labeling material, and can detect picograms in ultratrace amounts without using isotopes. Any of scattered light of laser light, transmitted light, reflected light, interference light, and diffracted light may be used for the detection method. According to the immunoassay method provided by the present inventors by labeling magnetic particles with an antigen or an antibody, it is possible to detect even a very small amount of target. For example,
The present inventors have concluded that the 35th General Meeting of the Virology Society of Japan
As we announced in Mon. No. 4011 "Experiment of virus detection using newly developed immunoassay device"), we conducted virus detection experiments using inactivated influenza viruses A and B as virus models. By the way, 1m
It was possible to detect even if an estimated 1 virus was present in l.

Problems to be Solved by the Invention Generally, currently used labeling methods such as the RIA method, the EIA method, and the FIA method are not reacted by repeated washing during preparation of the sample in order to improve detection sensitivity. There is the trouble of removing the labeled body.

Further, also in the immunoassay method previously proposed by the present inventors, in the preparation of the sample, the step of separating the unreacted magnetic substance-labeled substance after reacting the target sample and the magnetic substance-labeled substance with the antigen-antibody Was needed.

In contrast to these measuring methods, the unlabeled method of fine particle agglutination (PA) has the drawback that the sample preparation is simple but the sensitivity is extremely low.

Therefore, in a labeling method having high detection sensitivity, it has been desired to establish a measurement method capable of selectively detecting only a target sample without separating and removing unreacted labeling material.

An object of the present invention is to provide a novel measurement method which has a detection sensitivity and accuracy higher than those of the RIA method, is easy to prepare a sample, and has no practical limitation.

Means for Solving the Problems In the present invention, a first step of capturing a target analyte on non-magnetic fine particles to which a fluorescent dye is added, and a magnetic substance label on which an antibody or an antigen for the target analyte is immobilized A second step of reacting the body with the non-magnetic fine particles treated in the first step by an antigen-antibody reaction, and locally concentrating the non-magnetic fine particles bound to the magnetic substance-labeled body at a laser irradiation position in a gradient magnetic field by an external magnetic force By a laser magnetic immunoassay method comprising three steps, and a fourth step of irradiating the local concentration point with a laser to excite the fluorescent dye of the non-magnetic fine particles bound to the magnetic label, and detecting the fluorescence. The said subject was solved.

Here, the target sample refers to a specific antibody or antigen to be detected.

In the first step of capturing the target analyte on the non-magnetic fine particles to which the fluorescent dye is added, not only the method of specifically capturing the analyte but also the method of capturing the analyte nonspecifically can be applied. In the first step (specimen trapping step) of capturing the specimen on the non-magnetic fine particles, the present inventors have previously proposed Japanese Patent Application No. 63-10.
2912 The method used in “Sample Preparation Method for Performing Laser Magnetic Immunoassay” can be diverted. That is, a method of non-specifically adsorbing a target analyte by activating the surface of the non-magnetic fine particles, or by previously fixing a known antibody or antigen on the surface of the non-magnetic fine particles, the target analyte is subjected to an antigen-antibody reaction. A method of specifically binding can be used. The former non-specific method is effective for screening tests and for detecting influenza virus in the mouthwash of patients. There are only a few hundred viruses at most in the mouthwash, and in addition, type A,
Since there are a plurality of mutant strains such as type B, it is suitable for the purpose of reliably capturing the virus on the non-magnetic particles without first identifying the virus. In addition, the latter specific method is suitable for a precision test that excludes non-specific reactions as much as possible and surely detects only a specific virus.

As the non-magnetic fine particles used in the method of the present invention, various non-magnetic fine particles can be used as long as they can impart a fluorescent dye. The fluorescent dye can be generally applied by a method of dyeing the surface of the non-magnetic fine particles with the fluorescent dye, but if the non-magnetic fine particles are made of a material transparent to the laser light used for exciting the fluorescent dye, the fluorescent dye It is also possible to apply a method of previously mixing the material with the material. The non-magnetic fine particles are
Unlike the previously proposed Japanese Patent Application No. 63-102912, there is no particular restriction on the weight because it is not centrifuged. Regarding the size of the fine particles, an appropriate value should be selected depending on the sample. Further, from the viewpoint of quantitativeness and reproducibility of measurement, it is preferable that the particle size is uniform. For example, when detecting a virus, microspheres having a diameter of several μm or less and a small particle size distribution width, which are also used in the conventional fine particle agglutination method (PA), are suitable as the nonmagnetic fine particles. 1 if the particle size exceeds several μm
A large number of viruses may be bound to one non-magnetic fine particle, and if the distribution width of the particle diameter is wide, the number of viruses and the number of the non-magnetic fine particles are not proportional to each other, which makes it difficult to quantify the virus.

As the non-magnetic fine particles, for example, particles made of an organic material such as acrylic polymer beads and polystyrene beads, particles made of an inorganic material such as silica fine particles, and the like can be used. It is preferable that these particles have a uniform particle size because they are easily available. Anything can be applied to the fluorescent dye,
In order to improve the detection sensitivity, one having a high fluorescence quantum yield is preferable. For example, coumarin derivatives, fluorescent dyes such as xanthene type, rhodamine type, oxazine type, cyanine type and merocyanine type are suitable.

In the second step of the measuring method of the present invention, it is extremely important that the magnetic substance-labeled substance specifically reacts with the non-magnetic fine particles that have captured the target sample treated in the first step. In order to prevent non-specific adsorption of the magnetic substance to the non-magnetic substance fine particles, the antigen-antibody reaction in the second step is
It is preferably carried out in a solution to which an adsorption inhibitor such as HEPES buffer is added. It is particularly preferable to use a monoclonal antibody as the antibody. The magnetic substance-labeled substance has magnetic fine particles made of various ferromagnetic materials such as various compound magnetic substances such as magnetite and γ-ferrite or metal magnetic substances such as iron and cobalt as cores, and sugars such as dextran around the magnetic fine particles. Alternatively, it can be obtained by coating a protein such as protein A and then binding an antibody or an antigen that specifically reacts with the target sample to these sugars and proteins. Needless to say, the magnetic substance-labeled body used in the present invention requires the utmost consideration for contamination of fluorescent impurities or fluorescence contamination. This is because in the fourth step described below, the unreacted magnetic substance-labeled substance that has not reacted with the non-magnetic substance fine particles is locally concentrated and is also irradiated with laser, so that the magnetic substance-labeled substance is contaminated with fluorescence. And it will interfere with the measurement.

Next, in the third step of the measuring method of the present invention, the laser magnetic immunity apparatus described in Japanese Patent Application Nos. 62-152792 and 62-184902 previously filed by the present inventors is preferably used. This laser magnetic immunity device is composed of a thin magnetic pole piece made of a highly magnetically permeable material, which is arranged on the open water surface of the inspection container, and an electromagnet which is installed at a position facing the magnetic pole piece with the inspection container interposed therebetween. .

According to these devices, it is possible to efficiently locally concentrate the non-magnetic fine particles bound to the magnetic substance-labeled body via the target sample in the laser irradiation position in the gradient magnetic field.

In the fourth step of the measuring method of the present invention, the local concentration point is irradiated with a laser to excite the fluorescent dye of the non-magnetic fine particles bound to the target sample and detect the fluorescence. This 4th
In the process, two methods can be applied. One of them is a method of exciting a fluorescent dye with a pulsed laser.
The laser used in this method should be selected from among high-power semiconductor short-light pulse lasers, dye lasers of synchronous pumping with mode-locked Ar lasers, and solid-state lasers such as Nd: YAG. You can The pulse width of the laser used in this method is preferably several tens of nanoseconds to several picoseconds. If fluorescence is measured after a certain time from the laser pulse, the laser pulse light is attenuated and does not exist, so that the influence of scattered light can be removed. The other one
One method is to continuously excite the fluorescent dye,
Ordinary gas lasers such as He-Ne and He-Cd can also be used. The laser light source should be selected according to the wavelength of the fluorescent dye used. In this case, since scattered light of the laser interferes, it is important to spectrophotometrically measure only the excited fluorescence. Various types of fluorescence such as a photomultiplier tube and a semiconductor diode can be used for detection of fluorescence, but a photocounting method using a photomultiplier tube is preferable for ultrasensitive detection.

As the fluorescence detecting optical system used in the fourth step, it is preferable to use one having a slit arranged so as to detect only the fluorescence from the laser irradiation position. By arranging the slits in this way, the influence of unreacted non-magnetic fine particles can be more reliably avoided.

Action In the laser magnetic immunoassay method of the present invention, after the target sample is captured by the non-magnetic fine particles to which the fluorescent dye is added in advance, the target sample is further reacted with the magnetic substance-labeled body for the antigen-antibody reaction. As a result, the target sample is doubly labeled with the fluorescent dye and the magnetic substance. In the thus prepared specimen suspension, a magnetic substance-labeled substance bound to non-magnetic substance fine particles via a target substance (hereinafter, abbreviated as target substance-conjugated substance) and unreacted non-magnetic substance fine particles , And unreacted magnetic substance-labeled substances are mixed.

When an external magnetic force is applied to this sample suspension, the unreacted magnetic substance-labeled substance and the target sample-bound substance are guided and concentrated at the laser irradiation position.

When the laser is irradiated to this concentration point, the probability of irradiating the laser with the unreacted non-magnetic fine particles is reduced, and only the fluorescent dye of the non-magnetic fine particles bound to the target sample is excited.

In this state, if you measure the fluorescence different from the wavelength of the laser light,
The target sample can be detected with high sensitivity and accuracy because it is not affected by the non-magnetic fine particles not bound to the target sample and the magnetic label, and the influence of dust and scattered light from the container can be eliminated.

EXAMPLES Hereinafter, the present invention will be described in more detail with reference to the drawings, but the following are merely examples of the present invention and do not limit the technical scope of the present invention.

(Embodiment 1) FIG. 1 is a process chart illustrating an embodiment of the present invention.
(A) is a step of dispersing non-magnetic fine particles dyed with a fluorescent dye in a liquid, (b) is a step of capturing a target sample on the non-magnetic fine particles, (c) is a magnetic labeling step, and (d) is This is a local concentration / measurement process.

In this example, the principle of the present invention was confirmed by using inactivated influenza virus, which is highly safe in experiments.

First, the surface of the non-magnetic fine particles 1 made of a monodisperse acrylic polymer having an average particle diameter of 1 μm is coated with Rhodamine B (wavelength 520 nm).
After staining with the fluorescent dye 2 consisting of BSA, it was coated with BSA in order to prevent a decrease in antibody activity. Then, a hyperimmune antiserum against influenza virus was immobilized on this as antibody 3. The non-magnetic fine particles thus prepared were freeze-dried and stored, put into a reaction container at the time of use and dispersed in a HEPES buffer solution (dispersion step in liquid (a)).

Next, influenza virus (A / Ishikawa (H3N2)) as a target sample 4 was added to the non-magnetic fine particle dispersion liquid obtained in the liquid dispersion step, and the virus was incubated at 37 ° C for 2 hours. It was captured by the non-magnetic fine particles (specimen capturing step (b)).

Next, this was further added with a magnetic substance-labeled substance 5 to cause an antigen-antibody reaction with the target specimen 4 (magnetic labeling step (c)). As the magnetic substance-labeled substance 5, a magnetic fine particle 5a made of magnetite coated with dextran and having an average particle diameter of 40 nm was covalently bound to an IgG antibody 5b isolated from an antiserum obtained by immunizing a ferret. . This magnetic labeling was performed under the conditions of incubation at 35 ° C. for 2.5 hours.

Transfer all the solution processed in the above steps to the inspection container 6,
The gradient magnetic field generator proposed by the present inventors (for example,
Japanese Patent Application No. 62-184902). Then, when the electromagnet 7 immediately below the inspection container 6 was excited to concentrate the magnetic field on the magnetic pole piece 8 installed directly above the inspection container 6, the target analyte conjugate A (target analyte) A was detected on the water surface immediately below the magnetic pole piece 8. The non-magnetic fine particles 1 and the magnetic substance labeled body 5 are bound via 4) and the unreacted magnetic substance labeled substance C (the unreacted magnetic substance labeled substance 5) are locally concentrated (local concentration step).

Next, at the local concentration point, He-C with an oscillation wavelength of 441.6 nm and an output of 20 mW
The laser was irradiated with d laser to continuously excite the fluorescent dye of the non-magnetic fine particles 1. Only the fluorescence from the local concentration point was guided to the spectroscope by the slit and lens system, and the fluorescence was detected by the photon counting method. By passing through the spectroscope, the laser scattered light was cut and only the fluorescence could be continuously measured. As a result, it was confirmed that the influenza virus was detected without separating and removing the unreacted non-magnetic fine particles B and the unreacted magnetic label C.

Example 2 The sample preparation process is the same as in Example 1, except that rhodamine B is used as the fluorescent dye instead of the merocyanine dye (wavelength 60).
The measurement process was performed with a pulse laser using a 0 nm range and a fluorescence lifetime of about 20 ns). That is, a dye laser (Rhodamine 6G, output 1W) is excited by a mode-locked Ar laser,
The fluorescent dye was excited at 590 nm with a pulse width of 10 ps, and the fluorescence after a delay time of 1 ns was detected by the photon counting method. FIG. 2 shows changes with time in the laser excitation pulse and fluorescence. By measuring the fluorescence after the laser excitation pulse is extinguished, it is possible to avoid the influence of scattering from the container, dust and the like. Compared to the case of the first embodiment, the output of the pulse laser is large in this embodiment and the detection sensitivity is improved by about one digit because the spectrophotometry is not performed.

(Example 3) The sample preparation process is the same as in Example 1, except that rhodamine B is used as the fluorescent dye instead of the acridine dye (fluorescence lifetime 10
The measurement process was performed with a semiconductor pulse laser. That is, the wavelength is 420 nm and the pulse width is 50 p at the local concentration point.
The fluorescent dye was excited by the laser pulse emitted from the laser diode of s, output of 1 mW, and the fluorescence after the delay time of 1 ns was detected by the avalanche photodiode (APD). High detection sensitivity could be achieved also in this example.

The reason why the fluorescent dye was changed in Examples 2 and 3 is to match the wavelength of the laser light source. Needless to say, it is necessary to use a laser light source having a wavelength shorter than the absorption wavelength of the fluorescent dye.

Further, in Example 1, after the magnetic labeling step (c), a method in which only the magnetically attracted material is collected by a magnet and transferred to an inspection container can be applied. In this case, the influence of the background fluorescence can be eliminated as much as possible.

Further, in the case of efficiently testing a large number of samples such as a screening test, virus capturing, magnetic labeling, local concentration, and measurement can be performed without transferring the samples in the same container. That is, also in the method of the present invention, for example,
It is used in EIA and particle agglutination methods. A 96-well connected container (microplate) can be used for sample preparation and measurement.

EFFECTS OF THE INVENTION As described in detail above, in the laser magnetic immunoassay method of the present invention, a fluorescent dye is added in advance to the non-magnetic fine particles that capture the target sample, and the target sample is magnetically labeled to duplicate the target sample. The target sample is locally concentrated using magnetic force after the labeling of the above, and the fluorescent dye is excited by irradiating the target sample with a laser, so that the sample can be measured without separating the unreacted labeled substance. became.

That is, with the characteristic configuration of the present invention, the conventional RIA
Method, EIA method, FIA method, and the laser magnetic immunoassay method previously proposed by the present inventors, the step of washing and separating the unreacted labeled substance becomes unnecessary, which is unavoidable. Moreover, highly sensitive detection, which is a feature of the labeling method, can be performed.

As described above, the laser magnetic immunoassay method of the present invention has high sensitivity and extremely easy sample adjustment, so that automation can be easily achieved.

In addition, in the laser magnetic immunoassay method of the present invention, since the non-magnetic fine particles that have captured the target analyte are magnetically labeled and locally concentrated, the influence of background is small, and high sensitivity detection with a high S / N ratio is the first time. It became possible.

Further, in the laser magnetic immunoassay method of the present invention, since fluorescently stained non-magnetic fine particles are used, when the target sample is directly fluorescently stained, the sample is damaged and the specificity of the antigen-antibody reaction is impaired during magnetic labeling. While there is a concern that
Such concerns can be avoided. In addition, when detecting a minute target sample such as a virus, the fluorescence intensity can be increased and the detection sensitivity can be improved by using non-magnetic fine particles that are about one digit larger than the virus. In addition, when directly fluorescent staining the target sample,
Care must be taken so that the fluorescent dye does not flow out into the sample suspension, but by using fluorescently dyed non-magnetic fine particles, such an operation becomes unnecessary, and the sample adjustment can be simplified in this respect as well.

The method of the present invention is particularly suitable for mass screening and is effective for screening tests for various infectious diseases. Further, the present invention is not limited to the detection of the viruses mentioned in the examples, early diagnosis of cancer, allergy, various hormones such as peptide hormones to which the RIA method and the conventional RIA method have been applied, or various enzymes or vitamins. In principle, it can be applied to the measurement of drugs, etc. Furthermore, it becomes possible to perform precise measurement widely in a general environment, which could not be performed by the RIA method in limited facilities in the past. As described above, the effect of the present invention in the medical and medical fields is immeasurable.

[Brief description of drawings]

FIG. 1 is a process chart for explaining one embodiment of the sample preparation method of the present invention, wherein (a) in FIG. 1 is a process for dispersing non-magnetic fine particles in a liquid, and (b) is a process for capturing a sample. Step, (c) is a magnetic labeling step, (d) is a local concentration / measurement step, and FIG. 2 is a diagram showing changes over time in laser excitation pulse and fluorescence in Example 2. 1 ... Non-magnetic fine particles, 2 ... Fluorescent dye, 3 ... Antibody,
4 ... Target specimen, 5 ... Magnetic label, 5b ... Antibody.

 ─────────────────────────────────────────────────── --- Continuation of front page (56) Reference JP-A-54-101439 (JP, A) JP-A-61-41966 (JP, A) JP-A-61-96467 (JP, A) JP-A-61- 128168 (JP, A) JP-A-63-290964 (JP, A)

Claims (3)

[Claims] 1. A first step of capturing a target sample on a non-magnetic fine particle to which a fluorescent dye is added, and a magnetic label having an antibody or an antigen for the target sample immobilized on the first step. A second step of reacting the non-magnetic fine particles treated with the antigen-antibody reaction with the non-magnetic fine particles, and a third step of locally concentrating the non-magnetic fine particles bound to the magnetic label to the laser irradiation position in a gradient magnetic field by an external magnetic force, A laser magnetic immunoassay method comprising a fourth step of irradiating a laser to a local concentration point to excite a fluorescent dye of non-magnetic fine particles bound to a magnetic substance label and detecting the fluorescence. 2. The laser magnetic immunoassay method according to claim 1, wherein in the fourth step, the fluorescent dye is excited by a pulse laser and the fluorescence is measured after the pulse laser is extinguished. 3. The laser magnetic immunoassay method according to claim 1, wherein in the fourth step, only the fluorescence is spectrophotometrically measured while continuously exciting the fluorescent dye with a laser.