JPS63315951A - Laser magnetic immunoassay instrument - Google Patents

Abstract

PURPOSE:To shorten the time for measurement and to improve the accuracy of measurement by switching and applying DC excitation and intermittent pulse excitation to electromagnets for local concentration of a magnetic material labeled body and selectively detecting only the signal components. CONSTITUTION:A specimen vessel 1 is mounted to the center of the electromagnet pair 2 consisting of circular conical magnetic pole pieces 4-1, 4-2 and a laser light source 100 is so installed that a laser beam 101 passes the center of the specimen vessel in parallel with the axis of the vessel 1. Further, a photoelectron multiplier 105 is installed right above the specimen vessel. The excitation of the electromagnet pair 2 is executed by a circuit having a DC power supply 107 and an intermittent pulse power supply 108. The electromagnet pair 2 is first subjected to DC excitation to collect the magnetic material labeled body to the inside wall surface in the specimen vessel in the peak parts of the magnetic pole pieces 4-1, 4-2 and is then changed over to the intermittent pulse excitation. Only the scattered light of the laser beam at the time of the non- excitation is selectively detected. The detection signals are calculated and averaged repeatedly by which the magnetic material labeled body is detected.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to an immunoassay device that utilizes antigen-antibody reactions. More specifically, the present invention relates to a laser magnetic immunoassay device capable of quantitatively detecting a specific antibody or antigen from an extremely small amount of specimen.

[Conventional technology]

The development of immunoassay methods that utilize antigen-antibody reactions is currently being promoted on a global scale as an early detection method for new viral diseases such as acquired immunodeficiency syndrome and adult T-cell leukemia, as well as various cancers. There is.

As microimmunoassay methods using conventionally known primary reactions, radioimmunoassay (hereinafter referred to as RIA method), enzyme immunoassay (EIA), fluorescence immunoassay method, etc. have already been put into practical use.

These methods use an antigen or antibody labeled with an isotope, an enzyme, or a fluorescent substance, respectively, and detect the presence or absence of an antibody or antigen that specifically reacts with the antigen or antibody.

The RIA method quantifies the amount of specimen contributing to the antigen-antibody reaction by measuring labeled isotope radioworms, and is currently the only method capable of measuring ultra-trace amounts on the order of picograms. However, since this method uses radioactive substances, it requires special equipment, and the attenuation of the labeling effect due to half-life and other factors must be taken into consideration, so there are significant restrictions on its implementation. Furthermore, considering the current situation where radioactive waste disposal has become a social issue, its implementation is naturally limited.

On the other hand, a method using an enzyme or a fluorophore as a label is a method for detecting the amount of a specimen that has contributed to an antigen-antibody reaction by observing color development or luminescence, and there are no restrictions on implementation as in the RIA method. However, it is difficult to precisely define color development or luminescence, and the detection limit is on the order of nanograms.

Furthermore, as a method of detecting the presence or absence of antigen anti-reaction using laser light, there is a method using AFP (alpha-fetoprotein), which was developed mainly for the purpose of detecting liver cancer.

In this method, an antibody against AFP is added to plastic particles, and the mass change caused by the aggregation of the plastic particles due to the antigen-antibody reaction is investigated.
A detection sensitivity of 1.5 g has been achieved.

This is more than 100 times more sensitive than the conventional method using laser light, but only one hundredth or less compared to the RIA method. Furthermore, since this method utilizes changes in the Brownian motion of antigen-antibody complexes in aqueous solutions, it is extremely susceptible to the effects of the temperature and turbulence of the aqueous solution containing antibodies, as well as the effects of impurity particles mixed in the aqueous solution. In principle, it is undesirable to increase the detection sensitivity beyond this point.

As mentioned above, in the above-mentioned immunoassay methods, the RIA method, which has high detection sensitivity, has many restrictions on its implementation due to the use of radioactive substances.On the other hand, the enzyme immunoassay method, which is easy to implement, Fluorescent immunoassays and the like have low sensitivity and cannot perform precise quantitative measurements.

Therefore, the present inventors provided Japanese Patent Application No. 61-224567 [Laser Magnetic Immunoassay J] as an immunoassay method that eliminates the above drawbacks.

In this method, first, magnetic ultrafine particles are used as a label,
This label is attached to a specific or unknown antigen or antibody to make it a magnetic label. Next, the antibody or antigen as a specimen is subjected to an antigen-antibody reaction with a known immobilized antigen or antibody,
Alternatively, an antibody or antigen as a specimen is directly immobilized on a solid phase, and an antigen-antibody reaction is caused with the magnetic label. Thereafter, after removing the unreacted magnetic label, the specimen is dispersed in the liquid phase. In this case, if the specimen is an antigen or antibody that causes a specific antigen-antibody reaction with the magnetic label, the magnetic label remains in the liquid phase containing the specimen; , no magnetic label exists in the liquid phase.

Therefore, by knowing the presence or absence and amount of the magnetic label in the liquid phase, it is possible to identify the specimen and determine its constant fluorescence. The presence or absence of the magnetic label and the amount present can be determined by measuring the scattering of laser light by the specimen dispersed in the liquid phase and the intensity change of the transmitted light.

This method has the advantage of having detection sensitivity and accuracy comparable to the RIA method, but with no practical limitations.

Furthermore, the present inventors have proposed a laser magnetic immunoassay device and device that further embodies the above-mentioned measurement method in Japanese Patent Application No. 61-25.
It was provided in No. 2427. The laser magnetic immunoassay device according to this application performs the concentration of the magnetic label and the control of the scattered light of the sample mechanically using a permanent magnet or by using an array of a plurality of electromagnets, etc. This is how it was done.

[Problem that the invention seeks to solve]

The present invention deals with the following problems that the above-mentioned laser magnetic immunoassay device has.

In other words, in the above device, the mechanical method using a permanent magnet requires a complicated mechanism, and the method using an electromagnet has a structure that is not suitable for generating a strong magnetic field. This method has the disadvantage that it takes a long time to locally concentrate the labeled substance.

SUMMARY OF THE INVENTION An object of the present invention is to provide a laser magnetic immunoassay device that has a simple configuration and can locally concentrate a magnetic label in a short time.

[Means for solving problems]

The present invention is an immunoassay device to which a laser magnetic immunoassay method (Japanese Patent Application No. 61-224567) using magnetic ultrafine particles as a label is applied. A pair of electromagnets that sandwich a sample container, a power source that generates two types of direct current and intermittent pulses and supplies them to the electromagnets, a laser beam irradiation optical system that guides laser light to the sample container, and a magnetically labeled sample. A light receiving system is installed to receive the scattered light of the laser beam, and an electronic system that selectively detects only the scattered light synchronized with intermittent pulses from this light receiving system and repeatedly adds and averages the scattered light signals. the electromagnet pair has a magnetic core and a magnetic pole piece made of a material with small residual magnetization so that the magnetic field is maximum at the center of the magnetic core and increases toward the center of the magnetic core. It is characterized by being configured.

According to a preferred embodiment of the present invention, the sample container is a capillary, and a mechanism for holding the capillary horizontally between the pair of electromagnets, and a mechanism for directing the irradiation laser beam along the axis of the capillary to the center of the capillary. and an optical system that extracts scattered light from the specimen at the center of the electromagnet pair from above or below the specimen and guides it to a photomultiplier tube.

According to another preferred aspect of the present invention, after the power source continuously outputs a large direct current for a predetermined period of time, the power source outputs an intermittent pulse having a period within a range of 0.05Hz to 10Hz, the peak value of which is an intermittent pulse. It is controlled to output a pulse that is smaller than the continuous DC value and has no DC offset.

Furthermore, according to another preferred aspect of the present invention, the electromagnet pair can be selectively excited between independent excitation and summation excitation.
A current circuit and a magnetic circuit are provided.

[Effect]

In the present invention, by using a pair of special electromagnets and switching between DC excitation and intermittent pulse excitation, the concentration of the specimen and the control of the scattered light of the specimen were effectively performed. For this reason, it has become extremely advantageous in shortening measurement time and improving measurement precision.

Furthermore, when measuring laser scattered light, in addition to the above-mentioned method of detecting scattered light synchronized with the variation period of the magnetic field, measurement sensitivity can be improved by repeatedly adding and averaging the scattered light signals from the specimen. We were able to significantly improve the reproducibility of measurements.

〔Example〕

The present invention will be described in more detail below with reference to the drawings, but what is shown below is only one embodiment of the present invention, and does not similarly limit the technical scope of the present invention.

FIG. 1 is a schematic diagram of a laser magnetic immunoassay device illustrating an embodiment of the present invention, in which l is a sample container, 2 is an electromagnet,
2-1.2-2 is an electromagnet coil, 3-1, 3-2 is a magnetic core of the electromagnet, 4-1, 4-2 is a magnetic pole piece of the electromagnet, 5-
1, 5-2 is a yoke for the electromagnet, 6 is a magnetic flux control piece for controlling the magnetic flux generated by the electromagnet, 7 is a stand, 100 is a laser light source (laser light irradiation optical system), 101 is an irradiation laser beam, 102 is a laser scattered light beam from the specimen, +03 is a slit, 104 is a lens for condensing the laser scattered light beam, 105 is a photomultiplier tube for receiving the laser scattered light, and 106 is a photomultiplier tube. This is an electronic circuit section that processes output. The sample container 1 is a thin tube with an inner diameter of 2.5 mm, and the thin tube is horizontally attached to the center of the electromagnet pair. The laser light source 100 emits a laser beam 101
is placed parallel to the axis of the thin tube and passing through the center of the thin tube. The slit 103, the lens 104, and the photomultiplier tube 105 (these members constitute a light receiving system that receives the laser scattered light beam) are connected to the sample container at the center of the pair of electromagnets. It is installed directly above to receive the scattered light from the magnetic label inside. The electromagnet pair is composed of a coil, a magnetic core made of pure iron, a magnetic pole piece, and a yoke.
It has been wound 0 times. The yokes 5-1 and 5-2 are mounted on a stand 7 made of non-magnetic material, and are slidable on the stand 7 so that the length of the magnetic gap between the electromagnet pairs can be adjusted. The magnetic flux control piece 6 has a wedge shape,
It can be attached and detached between the yoke 5-1 and 5-2. The magnetic flux generated by the electromagnet 2 is controlled by a magnetic flux control piece 6, and when the magnetic flux control piece 6 is inserted between the yoke 5-1, 5-2, the magnetic flux density in the magnetic gap where the specimen is inserted increases. do.

On the other hand, when the magnetic flux control piece 6 is removed from between the yoke 5-1, 5-2, the electromagnet pair acts as two independent electromagnets. Since the magnetic flux control piece 6 is wedge-shaped as described above, by moving the magnetic flux control piece 6 in and out between the city yoke, the magnetic flux density in the magnetic gap between the electromagnet pair is kept within a certain range. It can be adjusted arbitrarily. Said magnetic pole piece 4-1.4
-2 preferably has a conical shape, and the magnetic pole piece 4-1.4-
2 is bolted onto the magnetic core 3-1, 3-2.

FIG. 2 shows a power supply and a power supply circuit for exciting the electromagnet pair, and this power supply is used for concentrating the magnetic label and driving the magnetic label after concentration. The electromagnetic coils 2-1, 2-2 can be connected or disconnected by a switch SWI, which is simultaneously connected to a turn-on switch of the DC power source I07. On the other hand, the electromagnetic coil 2-2 passes through the switch SW2,
It is connected to an intermittent pulse power source 108. The switch S
The opening/closing direction of W2 is opposite to that of the switch SWI, and when the switch SW+ is closed, the switch SW2 is opened.

3 and 4 are plan views of the magnetic pole pieces 4-1 and 4-2 and the sample container 1 when the electromagnet pair is viewed from above in FIG. 1, and 8 is a plan view of the sample container 1. Vaseline 9 is a magnetic label. FIG. 3 illustrates the above-mentioned concentration process of the magnetic label, and FIG. 4 illustrates the driving process of the magnetic label after concentration.

Next, the step of concentrating the magnetic label will be explained. First, the second
Close the switch SWl in the figure and close the coils 2-1 and 2-2.
A DC power supply 107 is connected to. At this time, the magnetic flux control piece 6 shown in FIG. 1 is inserted between the yokes 5-1 and 5-2. Since the magnetic pole piece of the electromagnet pair has a conical shape, when the electromagnet is excited with direct current, the magnetic flux density in the magnetic gap at the center of the electromagnet pair becomes maximum. In this example, the magnetic gap length 5
mm, and when IA was energized, a maximum of 12,000 G was obtained at the center of the pair of electromagnets.

FIG. 3(a) schematically shows the distribution of the magnetic label inside the sample container before concentration, and FIG. 3(b) schematically shows the distribution of the magnetic label inside the sample container after concentration. (
(a) In the figure, before the electromagnet pair is excited, the magnetic label inside the sample container is uniformly distributed in the solution, but after the electromagnet pair is DC excited, (b) As shown in the figure, the magnetic label is collected on the inner surface of the sample container wall at the apex of the magnetic pole piece.

Next, the process of driving the magnetic label after concentration will be explained.

The switch SWI in FIG. 2 is opened, the switch SW2 is closed, and the intermittent pulse power source 108 is connected to the electromagnet 2-2. At this time, the magnetic flux control pieces 6 in FIG.
Then, the electromagnetic coils 2-1 and 2-2 are cut off.

FIG. 4(a) shows that the electromagnetic coil 2-1 is operated at a pulse height of 0.2 in the step of driving the magnetic label after concentration.
A schematically shows the distribution of magnetic labels while being excited by intermittent pulses with a period of 0.2 Hz, and (b) schematically shows the distribution of magnetic labels while the electromagnetic coil is not excited. That is, in (a), the magnetic label is concentrated and distributed on the inner surface of the sample container wall at the apex of the magnetic pole piece, but in (b), the magnetic label is concentrated on the inner surface of the sample container wall at the apex of the magnetic pole piece. Since the residual magnetization of the magnetic pole pieces is small, there is almost no magnetic field between the electromagnet pairs at the center, and the magnetic label diffuses from the wall surface of the sample container to the surrounding area by Brownian motion in the solution.
Over time, the particles will be uniformly distributed inside the sample container centering on the apex of the magnetic pole piece.

Therefore, the laser beam guided to the center of the narrow tube along the axis of the curved narrow tube is scattered by the electromagnet and the specimen at the center, and the intensity of the scattered light changes in accordance with the period of the pulsed power source. do. Scattered light from the specimen can be taken out from above or below the specimen container.

The sample container 1 is preferably a thin tube with a diameter larger than the diameter of the laser beam, and after sucking the sample after antigen-antibody reaction into the inside of the thin tube attached to a microsyringe, the sample container 1 is a thin tube having one opening thereof. If the tube is sealed with Vaseline or the like, it will be convenient for subsequent handling of the specimen. Preferably, the capillary is mounted horizontally at the center of the electromagnet pair. The reason for this is that when the thin tube is held vertically and the electromagnetic coil is de-energized, the magnetic label concentrated in the center of the magnetic pole piece falls from the center of the magnetic pole piece under its own gravity. This is because there is a drawback that the scattering center moves up and down with each pulse excitation.

The period of the pulse excitation is preferably in the range of 0.05 Hz to 1OH2. This is because at 0.05 Hz or less, a long time is required for measurement, and at 10 Hz or more, the magnetic label cannot follow changes in the magnetic field. Further, it is preferable that the pulse has a peak value smaller than the DC excitation current value and has no DC offset. This is because if a DC offset exists, the magnetic label always remains trapped on the inner wall of the thin tube at the center of the commercial magnetic pole piece, and no variation in scattered light intensity occurs.

In addition, in order for the electromagnet pair to efficiently perform concentration and magnetic field drive, the two electromagnetic coils are equipped with, for example, a current circuit and a magnetic circuit as in this embodiment so that independent excitation and summative excitation can be selected. It is preferable that the

Scattered light from the specimen is received by a photomultiplier tube 105. The output of the photomultiplier tube 105 is transmitted to the electronic circuit section 10
6, the electronic circuit unit 106 selectively detects only the scattered light synchronized with the intermittent pulse, and repeatedly adds and averages the scattered light signals. This makes it possible to detect extremely small amounts of magnetic labels on the order of picograms.

〔Effect of the invention〕

As detailed above, the laser magnetic immunoassay device according to the present invention uses a pair of special electromagnets, switches between DC excitation and intermittent pulse excitation, and selectively detects only signal components. It is easy to use and can shorten measurement time and improve measurement variation.

The present invention provides an ultra-sensitive immunoassay device that is particularly suitable for test automation, so it can be used to detect various viruses,
If applied to detailed examinations of cancer, etc., it becomes possible to diagnose cancer or viral diseases at an early stage, and it becomes possible to accurately implement effective early treatment. Furthermore, in addition to antigen-antibody reactions, it can also be applied to the measurement of various hormones such as peptide hormones, various enzymes, vitamins, drugs, etc., to which the RIA method has been conventionally applied.

As described above, the effects of the present invention in the medical and medical fields are immeasurable.

[Brief explanation of drawings]

The drawings are for explaining one embodiment of the present invention, and FIG. 1 is a schematic configuration diagram of a laser magnetic immunoassay device according to the present invention, and FIG. 2 is a circuit diagram of a power source that excites a pair of electromagnets. 3(a), (b), and 4(a), (b) are plan views of the magnetic pole piece and sample container when the electromagnet pair is viewed from above in FIG. (a). 4(b) is an explanatory diagram showing the step of concentrating the magnetically labeled material, and FIGS. 4(a) and 4(b) are explanatory diagrams showing the driving step of the magnetically labeled material after concentration. ■...Sample container, 2...Electromagnet, 3-
1.3-2...Magnetic core, 4-1.4-2-...
・Magnetic pole piece, 9...Magnetic material, 100...
...Laser light source (laser light irradiation optical system), 103...
...Slit (light receiving system), IO2... Lens (light receiving system), 105... Photomultiplier tube (light receiving system), 106... Electronic circuit section, 107,108
······power supply. Applicant Nippon Telegraph and Telephone Corporation Figure 2 Coil 2-1 Coil 2-210
7.108: Power supply

Claims (4)

[Claims] (1) A sample container containing a liquid containing a magnetic label;
A pair of electromagnets that sandwich the sample container, a power source that generates two types of direct current and intermittent pulses and supplies them to the electromagnets, a laser beam irradiation optical system that guides laser light to the sample container, and a magnetic label. A light receiving system is installed to receive the scattered light of the laser light by the specimen, and from this light receiving system, only the scattered light synchronized with the intermittent pulse is selectively detected, and the scattered light signals are repeatedly added and averaged. The electromagnet pair is made of a material with small residual magnetization so that the magnetic field at the center of the magnetic core is maximum and increases toward the center of the magnetic core. and a magnetic pole piece. (2) The sample container is a thin tube, and includes a mechanism for holding the thin tube horizontally between the pair of electromagnets, and an optical system for guiding the irradiated laser beam to the center of the thin tube along the axis of the thin tube. and an optical system that extracts scattered light from the specimen at the center of the pair of electromagnets from above or below the specimen and guides it to a photomultiplier tube. The laser magnetic immunoassay device described. (3) After the power supply continuously outputs a large direct current for a predetermined period of time, the power supply outputs an intermittent pulse with a period in the range of 0.05Hz to 10Hz, the peak value of which is smaller than the continuous DC value, and The laser magnetic immunoassay device according to claim 1, wherein the device is controlled to output a pulse without DC offset. (4) The laser magnetism immunoassay device according to claim 1, wherein the electromagnet pair is provided with a current circuit and a magnetic circuit so that independent excitation or summation excitation can be selected.