JPH01272973A - Method and apparatus for laser magnetic immunoassay - Google Patents

Abstract

PURPOSE:To improve measurement accuracy by subjecting both of an inclined magnetic field generator and the water surface of an inspection vessel to irradiation of laser light and detecting the position of the image of the inclined magnetic field generator formed of the light reflected from the water surface. CONSTITUTION:A magnetic material-labeled specimen complex after the antigen- antibody reaction effected with the specimen and a labeling body of magnetic material is housed in the inspection vessel 1. A magnetic circuit in which the magnetic fluxes emitted from an electromagnet 10a penetrate through the vessel 1 and return again to the electromagnet 10a through a magnetic pole piece 10b, a height adjuster 10c, and yokes 10d-10f is formed. Since the front end of the magnetic pole piece 10b is formed to a sharp pointed shape, the magnetic field on the surface of the vessel 1 right under the front end part of the magnetic pole piece 10b is highest. The magnetic material-labeled specimen complex is, therefore, condensed to this position. The laser beam emitted from a laser light source 50 is made into an incident luminous flux 52 by a laser expander 51. The specimen housing part 1a and the front end of the magnetic pole piece 10b are simultaneously irradiated by this luminous flux. The luminous flux is reflected from the water surface and is formed as an image on a screen 54.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to an immunoassay method and apparatus that utilize antigen-antibody reactions. More specifically, the present invention relates to a laser magnetic immunoassay method and apparatus that can consistently detect a specific antibody or antigen from an extremely small amount of specimen.

[Conventional technology]

Acquired immunodeficiency syndrome, adult engineered cell leukemia, etc.1
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 and various cancers.

As conventionally known microW immunoassay methods using primary reactions, radioimmunoassay (hereinafter referred to as RIA method), oxygen immunoarray (ElΔ), fluorescence immunoarray method, etc. have already been put into practical use. These methods are universal methods that use antigens or antibodies labeled with isotopes, enzymes, or fluorescent substances, respectively, and detect the presence or absence of antibodies or antigens that specifically react with the antigens or antibodies. 1 RIA France has 1 labeled iso-1 to 1 radioisotope each.
This method is used to quantify the amount of a sample that exhibits an antigen-antibody reaction, and is capable of measuring ultrafine H1 in melons. however,
Since this method uses radioactive substances, it requires special equipment, and it is also necessary to take into account the attenuation of the labeling effect due to shelf life, etc., so there are major restrictions on its implementation.

Furthermore, given the current situation in which radioactive waste disposal has become a social issue, its implementation will naturally be limited.

On the other hand, enzymes and fluorophores are e! The method used for detection is a method of detecting the amount of a specimen that has contributed to an antigen-antibody reaction by observing color development or luminescence, and there are no practical restrictions like the RIA method. However, it is difficult to accurately quantify color development or luminescence, and the detection limit is on the order of nanograms.

Furthermore, as a method for detecting the presence or absence of an antigen-antibody reaction using Rede light, there is a method using AFP (alpha-fue-1-protein), which was developed mainly for the purpose of detecting liver cancer.

In this method, antibodies against AFP are added to plasticine particles, and the mass change caused by the aggregation of plasticine particles due to the antigen-antibody reaction is investigated.
A detection sensitivity of 1.5 g has been achieved. This is more sensitive than the conventional method using laser light, but l'<
Compared to the IA method, it is less than one hundredth.3. Furthermore, because this method utilizes changes in the Brownian motion of antigen-antibody complexes in aqueous solutions, it is extremely susceptible to the effects of the humidity of the aqueous solution containing antibodies, the influence of Fukue, or impurity particles mixed in the aqueous solution. , it is fundamentally undesirable to increase the detection sensitivity beyond this.

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

Therefore, prior to the present invention, the present inventors provided a novel immunoassay method and device that has a detection sensitivity and seminal fluid comparable to R■△, but has no practical limitations. That is, the present inventors conducted research on an immunoassay method that differs in principle from conventional methods, and previously filed a patent application No. 61-224.
No. 567, Patent Application No. 1983-252427, Patent Application No. 1983
= No. 254.164, Japanese Patent Application No. 62-22062, Japanese Patent Application No. 1983-16-22063, Japanese Patent Application No. 152791-1983, Japanese Patent Application No. 1983
No. 2-152792, Japanese Patent Application No. 62-18/'1902, Japanese Patent Application No. 62-264319, Japanese Patent Application No. C52-267
/I No. 81 has filed a patent application for the invention of a laser magnetic immunoassay method and a measuring device.8 These new immunoassay methods are characterized by the use of magnetic particles as labeling materials, and are It is possible to detect ultra-trace amounts of picograms without using a probe. Based on the above-mentioned patent, the present inventors labeled magnetic fine particles with antigens or antibodies and detected viruses for the first time. It is being confirmed that this new laser magnetic immunoassay method has higher detection sensitivity than the R1 method, which has traditionally been considered to have the highest detection sensitivity. For example, the present inventors published the 35th general meeting of the Japanese Society of Virology (November 1988, No. 4011r).
Virus detection experiments using a newly developed immunoassay device (tC) reported that virus detection experiments were carried out using inactivated Innoluune 1A virus types A and B as virus -[dels]. i1. Etsuda place,
1- It was possible to detect even if there was a virus of about 1 degree in the sample. 3. The method of irradiating a concentrated sample with a laser beam and detecting the reflected light from the sample is a method with high detection sensitivity. be.

[Problems that the invention attempts to solve]

The present invention aims to improve quantitative performance and facilitate adjustment of the apparatus in the laser magnetic immunoassay method based on the new principle described above. That is, the problem to be solved unique to the present invention is to solve the problem in a solution of a magnetically labeled complex formed by subjecting a sample to an antigen-antibody reaction with a magnetically labeled body in which an antigen or an antibody is labeled with magnetic fine particles. This is due to the fact that the magnetic field at the concentration location is directly related to the intensity of the reflected light from the composite. Therefore, it is necessary to maintain the magnetic field constant with good reproducibility. The magnetic field at the concentration position depends on the distance between the gradient magnetic field generator and the sample container. In particular, when the gradient magnetic field generator is composed of two magnetic fields and a magnetic field generator installed so as to sandwich a sample container with the sample container above, the gradient magnetic field generator is placed directly above the water surface of the sample container. On the other hand! The distance between 18i and the water surface must be kept constant.

[Means to solve the problem]

The present invention has been made to solve the above problems,
First, a magnetic substance 42 labeled with magnetic particles added to a predetermined antigen or antibody as a label, and a sample A magnetic field is applied to a solution containing a magnetically labeled specimen complex, which is a complex of a magnetically labeled substance and a specimen after the first stage, to avoid an antigen-antibody reaction with the antibody or antigen. A laser magnetic immunoassay method that includes at least a second step of inducing and concentrating the magnetically labeled specimen complex in the laser beam irradiation region by directing the laser beam to both the gradient magnetic field generator and the water surface of the specimen container. and detecting the position of the image of the gradient magnetic field generator formed by the reflected light from the water surface at each time of A, thereby adjusting the magnetic field at the concentration position of the magnetically labeled specimen complex. A laser magnetic immunoassay method characterized by:

In the above structure, the present invention provides, as an image of the gradient magnetic field generator, a solid shadow image formed by the laser beam directly irradiated to the gradient magnetic field generator being reflected from the water surface, and a solid shadow image formed by reflecting the laser beam from the water surface. Two images can be used: a projected reflected image formed by the reflection of the Rayleigh light irradiated onto the projected image of the gradient magnetic field generator from the water surface.

Roughly speaking, in the above configuration, in the present invention, a light receiver is provided at a central position that is symmetrical with the solid shadow image and projected reflected image of the tilted 14i5'J projecting device α in the reflected light from the water surface. It is characterized by the installation of

Further, according to the present invention, there is provided a measuring device that can suitably carry out the above measuring method. That is, a test container containing a solution containing a magnetically labeled specimen complex labeled with magnetic fine particles, an incident optical system that guides a laser beam to the surface of the test container, and a laser beam irradiation area on the surface of the test container. A Rede magnetic immunoassay comprising at least a gradient magnetic field generating device that guides and concentrates the magnetically labeled specimen complex at one point, and a light receiving system that receives reflected laser light from the magnetically labeled specimen complex. The apparatus comprises a light receiver that receives reflected light when the laser light is applied to both the gradient magnetic field generator and the water surface of the sample container simultaneously or in chronological order (an image of the gradient magnetic field generator). and a magnetic field adjustment mechanism section that adjusts the magnetic field at the concentration position of the magnetically labeled sample complex by the gradient magnetic field generator based on the detection of the image position by the detection section. This is a laser magnetic immunoassay device characterized by:

In the above configuration, in the present invention, for example, a screen, a light receiving element, or an image pickup tube can be used as the light receiver of the detection section.

(Function) As is well known, in extreme measurement technology for detecting extremely small amounts of objects, the important issue is how to ensure not only detection but also quantitative performance and reproducibility of measurement.

The laser magnetic immunoassay method according to the present invention contributes to improving the quantitativeness and reproducibility of measurements, and reproduces the magnetic field by monitoring the distance between the sample container and the gradient magnetic field generator using a simple method. It is possible to keep the temperature constant.

In particular, when the gradient magnetic field generator is composed of two magnetic fields and a magnetic field generator installed to sandwich a sample container with an upward opening, the gradient magnetic field generator is arranged directly above the water surface of the sample container. The distance between one magnetic pole and the water surface must be constant. A common solution to this problem is to measure the water level in the sample container. You will need something. The present invention does not use a water level gauge, but instead measures the distance between the solid shadow image of the magnetic pole and the projected reflected image obtained by irradiating a laser beam onto both the magnetic pole and the water surface of the sample container, and then receiving the reflected light. Then, since this distance is proportional to the distance between the magnetic pole and the water surface of the sample container, the distance between the magnetic pole and the water surface of the sample container can be known. Normally, it is not necessary to find the absolute value of the distance, and the distance v[+] between the solid shadow image and the projected reflection image is determined in advance, and this distance is maintained for each sample.J: Adjust the position of the sea urchin sample container. If you cover it, J, yes.

For example, two light-receiving elements KM separated by a certain distance can be used to detect the distance.

In this way, the magnetic field at the concentration position is maintained at a constant value each time the measurement is performed, so that quantitative properties and reproducibility of the measured values can be ensured.

Further, as one of the features of the present invention, the central symmetrical point of the solid shadow image and the projected reflection image is the water surface directly below the magnetic pole, and this water surface is nothing but the concentration position. Therefore, since the reflected light from the magnetically labeled specimen complex concentrated at this concentration position is emitted with the central symmetry point as the center, if the light receiver is installed at this central symmetry point, the magnetic The signal of the body-labeled analyte complex can be detected. Utilizing this feature, you can effectively adjust the position of the receiver.
I can do it. As mentioned above, as a light receiver,
A screen, light receiving element, or image pickup tube can be selected. In particular, when quantifying a sample using the interferometry method previously invented by the present inventors, the center symmetry point becomes the center of the interference fringes reflected on the screen. The center always appears in the same position, which is very useful when measuring interference fringes.

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 limit the technical scope of the present invention in any way.

〔Example〕

FIG. 1 is a schematic diagram of a laser magnetic immunoassay device illustrating a first embodiment of the present invention, in which reference numeral 1 denotes a sample container made of acrylic resin, and 1a denotes a sample storage part in this sample container. , 2 is a vertically moving table on which the sample container 1 made of non-magnetic material is mounted, 3 is a moving screw, 4 is a moving guide, 5 is a left and right moving table,
6 is a moving motor, 7 is a guide surface, 8 is a pedestal, 10 is a gradient magnetic field generator, 50 is a He-'Ne laser light source, 5
1 is a beam expander, 52 is an incident light beam, 53 is a reflected light beam, and 54 is a screen (light receiver).

The sample container 1 contains a magnetically labeled sample complex after an antigen-antibody reaction between the sample and the magnetically labeled body. The method for preparing the sample is described in Japanese Patent Application No. 111-2245678, Patent Application No. 111 If (61
-25'1164 No. V, published in Me, can be applied.

The 1B1 oblique magnetic field generating device 10 is an electromagnet 10a, etc.! 1
Pole piece 10b,! :The vAJ4J-ilo EN7)Qg
- Consisting of a regulator 10C1 and joints n10d, 10e, 10f, the magnetic flux emitted from the electromagnet 10a passes through the sample container 1, and then passes through the magnetic pole piece 10b, the height adjuster 10c of the magnetic pole piece 10b, and the yoke. 10d, 10e, and 10f, a magnetic circuit is formed so as to return to the electromagnet 10a again. 9. The electromagnet 10a is fixed on the right moving table 5 made of non-magnetic stainless steel. It is attached on top of the pure iron yoke 10f. The magnetic pole piece 1
0b has a sharp tip on the side of the sample container 1, so the magnetic field on the surface of the sample container 1 directly below the tip of the magnetic pole piece 10b is highest. At this point, the magnetically labeled analyte complex is concentrated at this position. The height of the sample container 1 is determined by the moving table 2 rotating the Tanabata 6 attached to the left and right moving table 5 at a low speed, the moving screw 3 and the moving guide 4.
"J" means "J" for moving up and down, and "J" means "J" for moving up and down. In addition, since the left-right movable table 5 can move left and right on the guide surface 7, it can be moved from side to side on the guide surface 7.
E1 can be set and covered just below the magnetic pole piece 10b. The height of the magnetic pole piece 10b can be arbitrarily adjusted by the height adjuster 10c. With this configuration, the magnetic field at the concentration position is applied to the sample container 1.
And it can be arbitrarily adjusted by moving the magnetic pole piece 10b up and down. Of course, the magnetic field at the concentration position can be adjusted by changing the excitation current of the electromagnet.
The laser beam emitted from the C1A1-spander 51 becomes an incident light beam 52 whose beam diameter is expanded, and simultaneously irradiates the specimen accommodating section 1a and the tip of the magnetic pole piece 10b. Although the screen 5/I is not shown in the figure, it is fixed to the pedestal 8 so as to receive the reflected light beam 53 at its central position.

2(a) and 2(b) are enlarged views of the screen 5/1 in FIG. 1, and FIG. 2(E) is an enlarged view of the screen 5/1 in FIG. −
1 and a projected reflection image 10b-2 are schematically drawn. The solid shadow image 10b-1 is the magnetic pole piece 10b.
The projected reflected image 10b-2 is an image formed on the soil of the screen 54 by the laser beam directly irradiated on the water surface, and the projected reflected image 10b-2 is the image irradiated onto the projected image of the magnetic pole piece 10b reflected on the water surface. This is an image formed on the screen 54 by the reflected light from the water surface. These images 10b
-1 and 10b-2 are observed as Silera 1 to reflected light. Two pinballs 55as and 55b each having a diameter of 0.5# are provided on the screen 54 at positions 80all apart from each other. FIG. 2(b) is a schematic diagram of the back side of the screen, with the pinholes 55a, 5E5
Light receiving elements (light receivers) 57a and 57b of the SL photodiode are fixedly placed at the position b (=j'G).

Since the screen 5/l has such a structure, when the reflected light beam 553 illuminates the screen 54, a part of the reflected light is detected by the light receiving elements 57a, 57b through the pinholes 55a, 55b. Ru. The sample container 1
If the distance between the magnetic pole piece 10b and the magnetic pole piece 10b is widened, the solid shadow image 101) -1 and the projected reflection image 10 b -2 of the magnetic pole piece 10b are obtained.
The distance between them will correspondingly widen, and if you move them closer together, the images will also get closer together. FIG. 2(a) shows a state in which the solid shadow image 10b-1 and the projected reflection image 10b=2 in the shape of the silera 1 just fit into the pinholes 55a and 55b.

This position of the light receiving elements 57 El and 57b has an accuracy of J,
Since the sample container 1 and the magnetic pole 10b can be easily detected,
You can always keep the interval constant. Therefore, by repeatedly using the method of the present invention, it is easy to reproduce the magnetic field and keep it constant. Note that the height of only one of the magnetic pole piece 10b and the back of the sample container 1 is variable. In this case, once the position of the screen 54 has been adjusted, the magnetic field can be adjusted as described above with just one bottle ball on the screen 54, but in this embodiment, the height of both can be adjusted independently. In this case, it is preferable to have two pinholes, because if the heights of both pinholes are adjusted independently, the interference fringes are often deviated from the center position of the screen 54.

In this case, if there are two pinholes, the magnetic pole piece 10b
Since it is possible to detect in which direction the image has deviated, there is an advantage that adjustment can be made easily.

In the application of this example, the magnetic field at the concentration position can always be kept at a constant value, so there is no variation in data due to the measurement method, and quantitative performance is greatly improved.

As a modification of this example, the interference fringes on the screen were photographed with an ITV camera (image tube), and the diameter, number, brightness, etc. of the interference fringes were measured by image processing. In this method, the interference fringe image from the specimen is recorded on the VTR.
-, it is useful for saving data on row seedlings. The records of 120 samples can be stored in 11 volumes of VTR/C0. Figure 3 shows another modification of this embodiment, in which a light shielding plate 54-1 is installed at the position of the screen of the measuring device shown in Figure 1. did.

The light-shielding plate 5/l-1 having an opening in the center has two pinholes 55a and 55b on the right as in the above embodiment, and has light-receiving elements 57a and 57b on the back side and one for receiving interference fringes. Dimensional image sensor type light receiving element (light receiver)
58 is fixed. As mentioned above, by applying the method of the present invention to the actual IM, not only the magnetic field is adjusted to a constant value, but also the center position of the interference fringes appears at a specific position on the light receiving element 58 with good reproducibility, so that the number of interference fringes can be reduced. it = always easier. This is because when the center position of interference fringes fluctuates, in order to count the diameter or number of interference fringes, the center position must first be determined using some method, such as photographing the interference fringes with an ITV camera and using image processing. Pre-processing is required.

This preprocessing generally requires a complex algorithm, which reduces the processing cost! There is a drawback caused by ~.

As another modification, a deflection element is used instead of the beam expander 51 to manipulate the incident laser beam up and down to irradiate the sample container 1 and the magnetic pole piece 10b in a time-series manner. The distance between the sample container 1 and the magnetic pole piece 10b was adjusted. This modification detects the difference between the scattered light from the concentrated position and the non-concentrated position by scanning the incident beam, which is disclosed in Japanese Patent Application No. 62-264319, which was previously invented by the Ministry of the Invention and others. The effect is great if used in conjunction with the method. In the case of a method that detects scattered light, it is also advantageous to use a beam expander, since measurements with a high S/N ratio can be made with a narrower beam diameter.

Of course, a modification is also possible in which the beam expander is used only when adjusting the magnetic field, and the beam expander is retracted from the input optical system when measuring the specimen.

In addition, in the said structure, the screen 54, pinhole 55a, 55b, the light receiving element 57a, 57b, the light shielding plate 54-1, and the pinball 55a.

The 55b1 light receiving elements 57a, 57b, and 58 constitute a detection section (light receiving system) 60. In addition, the sea 1F light source 50,
The beam expander 51 constitutes an input optical system 61.

Although not shown, a magnetic field adjustment mechanism is provided between the detection unit 60 and the gradient magnetic field generation device 10, and this magnetic field adjustment mechanism allows the detection unit 60 to detect the position of the image. Based on this, the gradient magnetic field generator 10 adjusts the magnetic field at the concentration position of the magnetically labeled specimen complex.

〔Effect of the invention〕

As described in detail above, the laser magnetic immunoassay method and measuring device according to the present invention improve the quantitative performance of the laser magnetic immunoassay method based on a new principle using magnetic fine particles as a labeling substance, improve the reproducibility of measured values, and improve the device. In addition to making adjustment easier, when measuring by interferometry, the center of interference fringes always appears at the same position, making it very easy to measure interference fringes and improving measurement accuracy.

The Rede magnetic immunoassay method and measuring device according to the present invention are applicable not only to antigen-antibody reactions, but also to the measurement of various hordes such as peptide hormones, various enzymes, vitamins, drugs, etc., to which the RIA method has traditionally been applied. It is possible to apply it. Therefore, in the past, RI was performed at limited facilities.
Precise measurements that could not be carried out without Method A can now be widely carried out in a general environment. In general situations such as mass medical examinations, accurate measurements such as screening tests for various viruses, cancer, etc. can be carried out widely.If implemented, early diagnosis of cancer or viral diseases etc. will be possible, and effective early treatment will be possible. Thus, the effects of the present invention in the medical and medical fields are immeasurable.

[Brief explanation of the drawing]

FIG. 1 is a schematic diagram of a laser magnetic immunoassay device explaining the first embodiment of the present invention, and FIG. 12 is a screen 5 of FIG. 1.
4, FIG. 2(a) schematically depicts the interference fringes 56 of the screen operator, the solid shadow image 10b-L and the projected reflection image 10b-2 of the VItlKi1'7, FIG. 2 (b> is a schematic diagram of the back side of the screen, and FIG. 3 is another modification of the present embodiment in which a light shielding plate 54-1 is installed at the screen position of the measuring device flf'/ of FIG. 1. 1... Sample container, 1a... Sample storage part in the sample container, 2... 10F moving table, 3... Moving screw, 4... Moving guide, 5... Left and right Moving table, 6... Moving motor, 7... Guide surface, 8... Frame, 10... Gradient magnetic field generator/I device, 50... Rake light source, 51... Beam expander , 52... Incident light flux, 53... Reflected light flux, 54... Screen (receiver>, 55a, 55
b... Pin small, 56... Interference fringes, 57a, 57
b-... Light receiving element (light receiver), 58... Light receiving element (light receiver), 60... Detecting section (light receiving system), 61... Incoming optical system.

Claims (7)

[Claims] (1) A first step of causing an antigen-antibody reaction between a predetermined antigen or antibody labeled with magnetic fine particles as a label and an antibody or antigen as a specimen; and a magnetic label after the first step. A second step of applying a magnetic field to a solution containing a magnetically labeled specimen complex that is a complex with a specimen to induce and concentrate the magnetically labeled specimen complex in a laser beam irradiation area. In the measurement method, a laser beam is irradiated onto both the gradient magnetic field generator and the water surface of the sample container, and the position of the image of the gradient magnetic field generator formed by the reflected light from each of the water surfaces at that time is detected. . A laser magnetic immunoassay method, characterized in that the magnetic field at the concentration position of the magnetically labeled specimen complex is adjusted. (2) An image of the gradient magnetic field generator is a solid shadow image formed by reflection of a laser beam from the water surface after direct irradiation of the gradient magnetic field generator, and a solid shadow image of the gradient magnetic field generator reflected on the water surface. 2. The laser magnetism immunoassay method according to claim 1, wherein the projected image is a projected reflected image formed by the laser beam irradiated on the water surface and the projected reflected image is formed. (3) The laser magnetism according to claim 1, characterized in that a light receiver is installed at a central position where a solid shadow image and a projected reflected image of the gradient magnetic field generating device in the light reflected from the water surface are symmetrical. Immunoassay method. (4) A test container containing a solution containing a magnetically labeled specimen complex labeled with magnetic fine particles, an incident optical system that guides a laser beam to the surface of the test container, and a laser beam on the surface of the test container. A laser magnetic immunocomputer comprising at least a gradient magnetic field generating device that guides and concentrates the magnetically labeled specimen complex at one point in a light irradiation region, and a light receiving system that receives reflected laser light from the magnetically labeled specimen complex. The measuring device is a measuring device that detects an image of the gradient magnetic field generator by receiving reflected light on a receiver when the laser beam is irradiated to both the gradient magnetic field generator and the water surface of the sample container simultaneously or in chronological order. and a magnetic field adjustment mechanism section that adjusts the magnetic field at the concentration position of the magnetically labeled specimen complex by the gradient magnetic field generator based on the position detection of the image by the detection section. Characteristic laser magnetic immunoassay device. (5) Claim 4, wherein the light receiver of the detection section is a screen.
The laser magnetic immunoassay device described. (6) The laser magnetism immunoassay device according to claim 4, wherein the light receiver of the detection section is a light receiving element. (7) The laser magnetic immunoassay device according to claim 4, wherein the light receiver of the detection section is an image pickup tube.