JPH01272970A - Laser magnetic immunoassay method and measuring instrument and labeling superparamagnetic material used for laser magnetic immunoassay and its production - Google Patents

Abstract

PURPOSE:To enable the inspection of an antigen-antibody reaction with good efficiency and high sensitivity by identifying an unreacted labeling superparamagnetic material and the labeling superparamagnetic material-specimen complex after the antigen-antibody reaction by a difference in the arrival time of laser light at a concn. position. CONSTITUTION:The labeling superparamagnetic material 7 is obtd. by immobilizing an antibody 2 consisting of an immune globulin antibody to protein A coated on the surface of the superfine superparamagnetic material particles 1. On the other hand, the surface of nonmagnetic microspheres 5 consisting of an acrylic polymer having 1mum grain size is activated and thereafter, the antibody 4 consisting of the immune globulin antibody similar to the immune globulin antibody of the antibody 2 is adsorbed on the surface thereof to obtain a nonmagnetic antibody complex 8. Influenza virus 6 existing in a patient's mouth wash is used as a specimen and the antigen-antibody reaction is effected in the liquid contg. this virus 6, the labeling material 7 and the complex 8. A ferromagnetic field is applied to the liquid during this reaction to uniformize the directions of easy magnetization of the complex 9 of the labeling material 7, the complex 8 and the virus 6. The concn. position is then irradiated by the laser light and the labeling material 7 and the complex 9 are identified from the difference in the arrival time thereof.

Description

Detailed Description of the Invention [Industrial Application Field] The present invention relates to a laser magnetic immunoassay method and a method for quantitatively detecting a specific antibody or antigen from an extremely small amount of a sample by utilizing an antigen-antibody reaction. Measurement'IA Fl i
The present invention relates to a superparamagnetic label used in ID and laser magnetic immunoassays and a method for producing the same.

(Prior art and its problems) ((The development of an immunoassay method using antigen-antibody reactions as a preliminary test for new viral bamboo diseases such as congenital immunodeficiency syndrome, adult cell leukemia, etc., and various cancers. Currently, I11 is being promoted on an international scale C'.

Conventionally known fine f7+ immunoassay methods include radioimmunoarrray (hereinafter referred to as RIA method), oxygen immunoarray ([IΔ), and fluorescent immunoarray ([IΔ]).
△) Laws, etc. have already been put into practical use. These methods use antigens or antibodies labeled with iso-1, enzymes, or fluorescent substances, respectively, and are specifically reacted with the antigen or antibody.
This method detects the presence or absence of antibodies or antigens.

However, although RIA detection has high detection sensitivity, there are many restrictions on its implementation because radioactive substances are used as labels. J, T, EIA method and F7Aθ
However, there are fewer restrictions on implementation than the RrA method; it is easy to implement, but test 1J1 sensitivity was low, and precise and early measurements were required.

In order to overcome the drawbacks of the above-mentioned immunoassay methods, the present inventors conducted research on a laser magnetic immunoassay method that differs in principle from the above-mentioned methods, and the results were first published in Japanese Patent Application No. 61-224.
567.61-252/127.61-25416=1
．． 62-22062.62-22063.62'-15
2791,62-152792,62-18/l 90
A patent application has been filed as No. 2.62-26/1319.62-267481. These new immunoassay methods are characterized by the use of laser light to detect the presence or absence of antigen-antibody reactions, and the use of I6 magnetic particles as the labeling material.
It is possible to detect ultra-trace amounts of picograms without using a ~-p.

By the way, in such a RayIf magnetic immunoassay fjFA, a magnetic label in which one antigen or antibody is added to magnetic fine particles as a labeling material, and this magnetic label and an antibody or antigen as a specimen are used. The antigen-antibody reaction is performed on the magnetically labeled sample complex, which is distinguished by magnetic force, and the unreacted magnetically labeled substance is separated and removed. This separation/removal operation is relatively troublesome;
If only the magnetically labeled specimen complex could be identified and detected without performing this operation, the advantages of step (1) would be immeasurable.

Furthermore, the method d1 of using magnetic fine particles as described in (a) above for separating cells and transporting drugs has been known for a long time.

Example Eva, j′S<ha, c: aev of JWi released in 1975
American g'+ in the invention of er! '! 397051
8” Magnetic separation of
"Biological particles" include antibodies to colloidal particles with large 10 μm serrations, F-JD magnetic, F-1, F-1, etc. A method for coating and isolating 101 cells and the like is disclosed.

However, until now, cell separation and drug delivery (
, 1 is at the research stage and has hardly been put into practical use.

Now, if we list in chronological order the summaries of the inventions and A's that have been made so far as this type of magnetic labeling material used for cell separation, 1') drug transport, etc., it will be as shown in I'. be.

(1) Davies et al. US Pat. No. 4,177,253"
Magnetic particle for imm
UI (OaSSay) has a particle size ranging from 1 μm to 1 μm, coated with a metal magnetic material such as NL and biologically active substances such as anti-1jrj and antibodies on the surface of low-density = 17/1'81. cm composite magnetic particles are described.

(2) Ho1daV US Patent No. 4452773”M
agnetic 1ron-dextran m1cr
osphcres" describes magnetite 1 fine particles of FJO magnetic material coated with dex 1 to run, etc., with a preferred particle size of 30 to 4 QIllll.

(3) Czerlinski U.S. Patent No. 44542
3'1“Co11led magnetizable
m1croparticles.

reversible 5uspensions th
ereof, and processes relat
ing tberOto''' has a temperature of 5 to 6
The surface of fine particles of ferromagnetic material such as ferrite 1 ~, iso 1 helium iron garnet 1 ~, etc. in the range of 5℃ is coated with a polymeric polymer etc. based on acrylamide, and the magnetic domain is 0.1 μm F ? Magnetic particles of up to 100% have been described.

(4) Ikeda et al. U.S. Patent No. 4582622“M
agnetic particulate for i
mmmobilizationofbiologica
l protein and process, of
"Producing Same" describes particles having a grain size of 3 μ7n and a culm degree containing Nijilabun as a main component and a FxD magnetic material consisting of 0.00001 to 2% fly l-.

(5) Hargel's U.S. Patent No. 11324923
Metal coated polyaldehyde
"m1crospheres" describes boreal atom microspheres coated with transition metals, and strong iron, nickel, and cobalt as +1fil materials.

Each of the magnetic phases (1) to (5) above is at least 3Qn
They are ferromagnetic or ferromagnetic with a particle size of m or more, and are classified as ferromagnetic. This ferromagnetic yield varies depending on the type of material, but the grain size is usually several tons or more, and residual magnetization is observed even after the external magnetic field is removed. On the other hand, magnetic materials have the property that no magnetization remains when the external magnetic field is removed.
There is a private magnetic moon r1 which has a smaller particle size than the above ferromagnetic material. These super-magnetic grains and ferromagnetic materials are highly sensitive to hysteresis curves, magnetic susceptibility measurements, and the Mössbauer effect, and are not suitable for conventional cell separation or drug delivery. Since the magnetic particles used in the labeling need to be effectively guided by weak magnetic forces, ferromagnetic particles are most suitable for this purpose, but the new magnetic particles according to the present invention, which will be described in detail below, are suitable for this purpose. In the magnetic immunoassay method, it is necessary that the magnetic label alone is difficult to be induced by magnetic force, so privately-produced porcelain bamboo materials are most suitable for this purpose.

The present invention was made in view of the above circumstances, and its purpose is to provide a novel laser magnetic immunotherapy that has detection sensitivity higher than that of the RIA method, has simple specimen preparation, and does not have the limitations of Embodiment 1. 3. [Means for Solving the Problems] The first aspect of the present invention is to provide a superparamagnetic label that can be suitably used for a measurement method, a measurement neck j19, and a laser magnetic immunoassay, and a method for producing the same. According to the invention, superparamagnetic ultrafine particles have 1
A superparamagnetic labeled substance to which one antigen or antibody has been added, and a 1-1 plate culm in which an antigen-antibody reaction occurs between the antibody or antigen as a specimen, and the superparamagnetic substance after the culm = 9 - A magnetic field is applied to a solution containing a superparamagnetic label (complex A), which is a complex of a paramagnetic label and an analyte (the superparamagnetic label analyte complex is guided to a predetermined position and concentrated). Distinguish the second culm from the unreacted superparamagnetic substance-labeled substance and the superparamagnetic substance-labeled sample complex after the antigen-antibody reaction by concentrating (l/reaching to the neck) 1 hour difference. '' A laser magnetic domain immunoassay method is provided, which comprises at least two culms.

Further, according to the second invention, the first culm-C1 antigen-antibody reaction of the first invention is carried out in a magnetic field, and the magnetization direction of the super II single-labeled specimen complex is aligned after the antigen-antibody reaction. A magnetic immunoassay method is provided which is characterized in that J and M are combined.

In these first or second inventions, the time difference between the superparamagnetic label and the superparamagnetic labeled sample complex to reach the concentration position is determined by directing the laser beam to the concentration position and scattering from the concentration position. This is obtained by detecting the output light of light, transmitted light, reflected light, interference light, diffracted light. It is not possible to use a method that selectively detects signal components. Also, d3 is placed in the second culm of IC1, and the same ray light is applied to the concentration position and non-concentration position.
1. Detect the emitted light of ``1'' or chronologically, detect the emitted light of
Furthermore, according to the third invention, a superparamagnetic label in which 1' antigen or antibody is added to superparamagnetic ultrafine particles, and the superparamagnetic The antibody labeled body and the sample antibody or [31 antigen] are
(A test container containing a solution containing a superparamagnetic target labeled specimen complex for antibody reaction; a gradient magnetic field generator that guides and concentrates the superparamagnetic labeled specimen complex to one point in the test container; An entrance optical system that guides the SE1A light to the WA neck of the superparamagnetically labeled specimen complex in the test container, the exit light from the superparamagnetically labeled specimen complex, and a solution that does not contain the superparamagnetically labeled specimen complex. A laser magnetic immunoassay device comprising at least an optical system for receiving the emitted light from each of A laser magnetic immunoassay device having the following characteristics is provided.

The antigen-antibody reaction can be carried out by a direct method in which the sample or superparamagnetic label is reacted directly with the sample, or by immobilizing a known antigen or antibody on the surface of a non-magnetic microsphere and then combining the J1 magnetic microspheres with the sample. After the reaction, a superparamagnetic label is added and the sample is reacted with the superparamagnetic label (a non-German method or the like can be used).

In addition, in a3 of the third invention, the identification mechanism [well, the time course of the emitted light from the superparamagnetic material labeled sample complex after the reaction is based on the time course of the emitted light from the unreacted superparamagnetic material labeled material. It is preferable to include an electronic circuit section that changes the change. In addition, the gradient magnetic field generator N LJ,' is composed of an electromagnet and a magnetic pole J4 which is arranged to face the electric field 1f ('''i and sandwich the inspection container between them, and either of the magnetic pole pieces of the electromagnet or the inspection Preferably, the container is configured to be movable in a horizontal plane.

Further, according to a fourth invention, the surface of ultrafine particles made of a superparamagnetic substance is coated with a biologically active substance, in the label used in the laser magnetic immunoassay method according to the first or second invention, There is provided a superparamagnetic bamboo label for Ray-11 magnetic immunoassay, characterized in that an antigen or antibody is immobilized on the coating layer.

Roughly speaking, according to the fifth invention, among the magnetic solid ultrafine particles on which the coating layer has been formed, there is a king culm that forms a coating layer of t15 material on the surface of the superparamagnetic ultrafine particles, and a privately produced magnetic solid ultrafine particle on which the coating layer is formed. A superparamagnetic material for magnetic immunoassays characterized by comprising at least a step of fractionating and collecting only the magnetic substance, and a step of immobilizing an antigen or antibody on a coating layer of superparamagnetic ultrafine particles. A method for producing a labeled body is provided. .

Here, it goes without saying that the superparamagnetic ultrafine particles used in the above-mentioned superparamagnetic label (1) do not have any problems such as radiation or aI properties, and there are no particular restrictions on the use of these particles. > Yes. Also, these superparamagnetic ultrafine particles are
Any ferromagnetic material can be obtained by converting it into ultrafine particles (1). For example, strong! The i-type material can be appropriately selected from various compound magnetic materials such as magnetite 1- and γ-ferrite 1-, or metal magnetic materials such as iron, niracle, and =13-=I-pal 1-.

As a method for making these ferromagnetic materials into ultrafine particles, various conventionally known gas phase methods and liquid phase methods can be used, excluding mechanical pulverization methods. For example, heat of evaporation in gas, evaporation method, co-precipitation method, etc. can be applied.
, ultrafine particles created by these gas phase methods and liquid phase methods [31,
Superparamagnetic material and ferromagnetic bamboo forest particles coexist, and it is necessary to separate and collect only the superparamagnetic material. Various mechanical, chemical, and physical methods can be applied to separation and recovery. For example, there are methods such as centrifugation, liquid chromatography, and magnetic filters. The particle size of the superparamagnetic ultrafine particles differs depending on the ferromagnetic abrasive, but it must be less than the critical size of single-domain particles and must be 4 particles. For example, magnetite 1~, γ-
In the case of Ferrite 1- (. tionm or less, in the case of pure iron, it is preferably 3 nm or less.

Such superparamagnetic ultrafine particles are coated with a biologically active substance in order to immobilize antigens or antibodies on their surfaces. Examples of biologically active substances that can be used include sugars such as dex1-ran, proteins such as protein 1/l-A49, and polymeric coatings such as medyl methacrylate. Antibodies are immobilized on the surface of biologically active substances.

Then, the superparamagnetic substance labeled with C1 can be lyophilized and stored at a C length. When used as a labeled test sample, the labeled substance may be dispersed in an aqueous solution containing a surfactant iodine.1. It is also possible to add a surfactant to suppress aggregation and store the solution in a cool place. Examples of surfactants include Twcen 20, which is also used as a cleaning solution for ISA;
arboxy methyl cellulose)
(A dry well, etc. used for draining and drying photographic film is effective.) As is known, ferromagnetic particles can easily change their magnetization direction when their particle size becomes very small. Due to thermal motion, it becomes a renderer 11 and becomes a superparamagnetic material.For example, in the case of magnetite 1~,
It is known that when the particle size becomes 10 nm or less, the material changes to a ferromagnetic material or a superparamagnetic material. Ferromagnetic materials and private magnetic f1 materials can be easily distinguished from hysteresis curves, magnetic susceptibility measurements, or the Mössbauer effect. In other words, the retention horn of a superparamagnetic material is 0, and the magnetic susceptibility becomes a band (the effect of the grain size is anti-Φλ on the t1 ratio, with the critical grain size changing from a ferromagnetic material to a superparamagnetic material as a boundary). It decreases as the particle size becomes smaller.Also, in the case of ferromagnetism, the female bow width of iron is 1.
-C is divided into six lines, and when it becomes superparamagnetic, two absorption lines appear in the center, so superparamagnetism can be quantified. In the case of ultrafine iron particles, the thermomagnetic relaxation time at which magnetization is reversed due to thermal disturbance is 1 second at room temperature for a particle size of 2.9 nm, and 3.6 nm for a particle size of 3.6 nm at room temperature.
In m, it is calculated to be about 30 sweats per h1. The difference in particle size is only 111 m (magnetic substances vary greatly).

The superparamagnetic label of the present invention has magnetic fine particles made of the above-mentioned superparamagnetic material as a core, and the surface thereof is coated with an antigen or an antibody. These analytes can be selectively reacted to be captured and magnetically labeled. This superparamagnetic labeled material is smaller than target cells such as cancer and lymphocytes by more than two orders of magnitude, and has the same or smaller serrations than viruses. Generally, the antigen-antibody binding sites of these specimens (there are many of them), so if the privately-produced 11-mer labeled compound of the present invention is added in excess of the specimen,
For example, multiple superparamagnetic labels will bind to one virus. Because such binding exhibits magnetic amplification effects and volumetric amplification effects, the superparamagnetic label (J) is superparamagnetic before the reaction, and the complex bound to the sample after the antigen-antibody reaction is ferromagnetic. Therefore, in the laser magnetic immunoassay method according to the present invention,
Due to the superparamagnetic properties of superparamagnetic labels used as magnetic labels, even if the labels are concentrated in a gradient field, the direction of easy magnetization of the labels is random and Brownian motion is active. Therefore, it takes a long time for the marker to reach the m-line position.

On the other hand, the superparamagnetic labeled analyte complex, which is a composite of the superparamagnetic label and the analyte, has a larger particle size than the superparamagnetic label, so it becomes ferromagnetic and exhibits inactive Brownian motion. Therefore, when placed in a gradient magnetic field, the superparamagnetic label can reach the concentration position more quickly than the superparamagnetic label.

Therefore, the unreacted superparamagnetic label and the reacted superparamagnetic label analyte complex can be distinguished based on the time difference in arrival at the concentration level.

Therefore, according to the laser magnetic immunoassay method according to the present invention, since the above superparamagnetic label is used as a label, due to the difference in arrival time to the concentration position, the unreacted superparamagnetic label and the superparamagnetic label after the reaction are Since the magnetic labeled substance (complex A) can be easily identified in a liquid containing both, there is no need to separate the two, and measurements with higher detection sensitivity than the IIA method can be carried out extremely easily. A monkey. Therefore,
According to the present invention, it becomes possible to automatically quantify antigens or antibodies as specimens. .

Moreover, according to the immunoassay method 1 according to the present invention,
During the antigen-antibody reaction in the culm, the directions of easy magnetization of the superparamagnetic material-labeled specimen complex were aligned, so the complex shown in - after the antigen-antibody reaction was made into a ferromagnetic material, and the complex was placed at the concentration position. It can be efficiently guided to the backside for a short time and concentrated there.

Furthermore, the immunoassay method neck device according to the present invention includes J.
The immunoassay method described in 1- can be suitably carried out.

. These characteristic configurations of the present invention eliminate unreacted labeled substances that were unavoidable in the conventional R1Δ method, [IΔv1, "■Δ method, and laser magnetic immunoassay method that the present inventors previously applied for. The T culm to be washed and separated becomes unnecessary. Due to this feature, the detection sensitivity is reduced, making it possible to automate the measurement extremely easily.

In the following, the present invention will be described in more detail with reference to the drawings.
However, what is shown below is only one example of the present invention, and does not limit the technical scope of the present invention in any way.

First, an embodiment of the laser magnetic immunoassay method according to the present invention will be described.

[Example 1] Figures 1 to 4 are for schematically explaining an example of the laser magnetic immunoassay method according to the present invention.
The figure shows a superparamagnetic label dispersed in a dispersion medium, Figure 2 shows a known antibody immobilized on the surface of a non-magnetic microsphere, and Figure 3 shows a superparamagnetic label dispersed in a dispersion medium. Figure 4 shows the superparamagnetic substance-labeled specimen complex with its easy magnetization direction aligned by a 5 KG magnetic field. Figure 4 shows the complex shown in Figure 3 concentrated in a gradient magnetic field and the complex detected quantitatively. This shows the process.

In the figure, 1 is a superparamagnetic ultrafine particle, 2 is an antibody, 3 is an arrow indicating the direction of easy magnetization of the superparamagnetic ultrafine particle, 4 is an antibody,
5 is a non-magnetic microsphere, 6 is a virus (antigen), 71. L
superparamagnetic label, 8 (non-magnetic antibody complex, 9 superparamagnetic label sample complex, 10 laser incident beam, 11
is an emitted light beam, 12 is a magnetic pole piece, 13 is a test container, and 1/I is an electromagnet.

Superparamagnetic ultrafine particles 1 are ultrafine particles made of iron with an average particle size of 2 nm, and their surfaces are coated with protein Δ. In order to collect only the body, we used a magnetic field filter to separate the ferromagnetic body and the privately produced magnetic body 4I!ζ.

As shown in FIG. 1, T
A superparamagnetic material label 7 was obtained by immobilizing the antibody 2 consisting of an antibody. For immobilization of Antibody 2, a method was used in which rabbit hyperimmune serum, which is anti-influenza 1F virus (B/% Shiro/2/85), was allowed to act on Prodein A.

On the other hand, as shown in FIG. 2, after activating the surface of nonmagnetic microspheres 5 made of acrylic polymer with a particle size of 1 μm, antibody/I made of an IgG antibody similar to antibody 2 was adsorbed onto the surface. Non-magnetic antibody complex 8 is toga.

Next, as shown in FIG. 3, the influenza virus 6 present in the patient's gargle fluid was used as a sample, and this virus 6, the privately produced 1i bamboo label 7 and the non-vJ1+1 antibody complex 8 were combined. 3. Carry out antigen-antibody reaction = 21 - reaction in the solution containing /i. During this reaction, a strong magnetic field is applied, and the direction of easy magnetization of the complex 9 of the labeled body 7, the non-magnetic antibody complex 8, and the virus 6 is shifted forward, and the complex 9 is moved away from the ferromagnetic material.

Next, a solution containing the complex 9 was placed in the test container 13. As shown in FIG. 4, this test container 13 is sandwiched between a magnetic pole piece 12 and an electromagnet 1/1 that face each other.
On the surface of the test container 13, a magnetic pole piece 12 and an electromagnet 1 are arranged so that the magnetic field directly below the pole piece 12, which is made of pure iron and has a rod shape with a diameter of 8 InIn and has a sharp tip, is highest.
The configuration is such that a gradient magnetic field is applied by a gradient magnetic field generator consisting of . Using this gradient magnetic field, the magnetic pole piece 1
Complex 9 was induced and concentrated on the liquid surface directly below 2. This concentrated position was irradiated with incident He-Ne laser light 10 with a wavelength of 632.8 nm, and the scattered light (A) was detected as the output light 11. Since the change over time of the emitted light 11 was detected, it was possible to reliably distinguish between the complex 9, which took a short time to reach the concentration position, and the unreacted superparamagnetic label 7, which took a long time to reach the concentration position. Incidentally, by selecting and detecting reflected light, interference light, and diffracted light in addition to scattered light from the concentration position and transmitted light as emitted light, labeled body 7 and complex 9 could be similarly identified.

Next, an example of a measuring device that can suitably carry out the immunoassay according to the present invention will be explained. .

[Example 2] FIG. 5 shows an example of the immunoassay device according to the present invention.
1 is a schematic configuration diagram, where the reference mark >210 in the figure represents an incident light flux, and 11
a and 11b are scattered light fluxes, 12a and 12b are magnetic pole pieces,
13a and 13b μ test container 13, 14 is an electromagnet, 15 is a laser light source, 16 is a deflector, 17a and 17
b is a condenser lens, 18a and 18b are slits, 19a
and 19 b 1. ; 20 is a horizontal movement device for the test container 13; 21 is an electronic circuit section forming an identification mechanism R4;

In the Rayleigh light source 15, the laser incident light beam 10 is caused to travel within a plane by a deflector 16, and is directed to both wells 13a and 13b.
The sea urchin is taken at a angle of 30 degrees from the horizontal plane. Light q1 of both wells 13a and 13b
Directly above the position are both vA pole pieces 12a and 12b. Further, the scattered light beams 11a and 11b pass through condensing lenses 17a and 17b, slits 1 to 18a and 18b, and are respectively detected by photomultiplier tubes 19a and 19b. 13a and 1
3b respectively contain a solution containing the complex 9, and the scattered light beams 11a and 11[) from both the tubes 13a and 13b are detected by photomultiplier tubes 19a and 19b, and the multiplex circuit section 21 is analyzed. This made it possible to efficiently measure two samples. Then, the horizontal movement device 20 has J:
By sequentially moving the test containers 13, it was possible to measure a larger number of specimens. In addition, the wells 13a and 13b can be used for comparative measurements by storing a sample in one side and a sample control in the other. Even if I try to irradiate it, it still doesn't work. In this case, needless to say, the divided beams need to be irradiated onto J, which includes the concentration position.

In addition, in order to eliminate the influence of background light,
The above-mentioned deflector 16 is inserted just before dividing into T-13, and each
It is desirable to simultaneously run between the condensation positions a and 13b and the 11m contraction position. The S/N ratio can be further improved by lock-in amplification of the signal from the sample synchronized with the scanning frequency.

Figures 6 and 7 are graphs showing examples of measurement results using the laser magnetic immunoassay device according to the present invention. ,
FIG. 7 shows the change over time in the scattered light area for a specimen control, that is, when the specimen was subjected to the same magnetic field treatment as the specimen but no virus was present. Measurement of the scattered light part (well, a method was adopted in which the outputs of the photoelectron amplifier tubes 19, 1, and 19b were amplified by a lock-in amplifier, and a signal synchronized with the scanning frequency of the incident light beam 10 was recorded and read. In this case, when the electromagnet is excited, the scattered light part from the specimen increases steadily and shows a constant value.On the other hand, the scattered light part from the specimen control increases only slightly over time. In this way, by detecting the change over time in the scattered light area and subtracting the scattered light area of the sample control, it is possible to determine only the sample ff1-J. It is not always necessary to measure the sample at once, and it is of course possible to measure it once before measuring the sample and store the results in a memory.

According to the present invention, virus culture is required before conventional detection.
It was possible to directly detect influenza viruses without culturing them, and it was possible to measure about 10 different influenza viruses present in the gargle fluid.

By the way, in the case of the conventional RIA method, several thousand viruses/
There are no more than d (couldn't be detected if pulled).

Next, another example of the immunoassay method according to the present invention will be explained.

[Example 3] An attempt was made to detect a virus by conducting an antigen-anti-Iho reaction with a labeled virus by a direct method.

The ultra-fine magnetic particles used for the label have an average particle size of 9 nm.
These are ultrafine particles of Magnetile 1, the surface of which is coated with Dex 1 to Ran. These Ngunnetai 1 to ultrafine particles were prepared using the method of Holday et al. (J, Imm, Heth
, Vol. 52, pp. 353-367, 1982), and in order to remove the ferromagnetic particles, they were centrifuged (the supernatant remaining in the supernatant was used). An IQG antibody obtained from a rabbit hyperimmune serum against influenza 1 virus was covalently bound to Dex 1 Heran to obtain a privately available 1! bamboo body labeled substance.

Note that the method for separating the superparamagnetic material and the superparamagnetic material may be a method using known liquid chromatography or a magnetic filter alone or in combination.

Next, 1 x 10'g of the superparamagnetic substance labeled substance is added to the specimen 1d collected from the patient's gargling fluid, and a superparamagnetic substance labeled specimen complex is obtained by directly causing an antigen-antibody reaction between the specimen and the superparamagnetic substance labeled substance, This complex was measured by the interferometry method described in Japanese Patent Application No. 18/1983, ``Laser Magnetic Immunoassay Method and Apparatus'' previously filed by the present inventors.
. In other words, this measurement method projects the light emitted from the composite onto a screen and observes interference fringes.

Clear interference fringes were observed from the interference light from the specimen, and only a few interference fringes were observed from the interference light from the specimen control in which the specimen (·ν virus) was not present.

On the 3fi IJ of these interference fringes, the size of the virus is 1
2 Qnm, whereas the size of the superparamagnetic label is about 90 nm, so the antigen-antibody complex between the virus and the superparamagnetic label is about one order of magnitude larger than the superparamagnetic label. , resulting in different degrees of enrichment. ′? l'/
:I As mentioned above, superparamagnetic labels take time to concentrate due to active Brownian motion, but antigen-antibody complexes have Brownian motion and magnetic attraction by gradient magnetic fields is effective. It's for a reason. .

In addition, as a modification, the antigen-antibody reaction between the specimen and the superparamagnetic label was performed in a magnetic field of 10 kG, and then measured by the same interference method as above. As a result, the interference fringes of the specimen control were the same as when the antigen-antibody reaction was performed without applying a magnetic field, but the interference fringes of the specimen appeared faster than when the antigen-antibody reaction was performed without applying a magnetic field. Measurement time was reduced.

For comparison, the average particle size of the magnetic label was 5Q.
The same experiment as above was carried out by binding virus antibodies to nanomagnetite ferromagnetic particles using the method described above, but there was a difference in concentration time between the antigen-antibody complex and the unreacted magnetite 11 to ferromagnetic label. The specimen could not be identified because no

The present invention is not limited to the above-mentioned interference method, and the measurement strip fI (
Of course, it is also possible to select a method (magnetic field, time, etc.) and measure the scattered light, transmitted light W, reflected light m, etc. at a certain time after electromagnet excitation IIjl.

[Example 4] In order to investigate the possibility of application to leukemia and cancer, a model experiment for target cell detection was conducted. Acrylic particles with a particle size of 3 μm (manufactured by Toshi, trade name: Tresfuure) were used as target cells, and after the surface of the acrylic particles was activated, they were sensitized with an ether-treated antigen of influenza T virus as an antigen.

As the superparamagnetic material W and the magnetic material, the magnetite superwoven magnetic bamboo material of Example 3 was used. Although the antigen-sensitized particles and the superparamagnetic label were directly subjected to an antigen-antibody reaction, the unreacted superparamagnetic label was separated, and the unreacted superparamagnetic label was separated and measured using the interference method described above, even one antigen-sensitized particle was detected. I was able to detect it. Therefore, the laser magnetic immunoassay method of the present invention can be applied to early diagnosis of abnormal cells such as cancer cells infected with AIDS.

Next, an example of a method for producing a superparamagnetic label suitably used in carrying out the immunoassay method according to the present invention will be described.

[Example 5] FIG. 8 shows the manufacturing process of a superparamagnetic label according to the present invention.

Daiichichloride 69 (FeCf2.4 +-120)2. o
Dissolve y and 5.4 g of ferric chloride (FeCf3 .6H20) in distilled water, add 50 mQ of 50% dex1-ran aqueous solution to a colander, keep the liquid temperature at 85°C, and while stirring well, add 59150 ae of caustic soda. The solution was added dropwise at a constant rate of 25 d/min. When the precipitate had settled to Ef 1, the supernatant liquid was discarded again, and the washing operation was repeated 8 times. Next, as a dispersion stabilizer, 0°5% 1ween
After adding 20 m of aqueous solution to bring the total volume to 100 m, the samples were collected in centrifugal tubes at intervals of 10 m. The solution collected in a centrifuge tube was centrifuged at 20,000 revolutions per minute for 30 minutes (), the precipitate containing the ferromagnetic material was discarded, and the superparamagnetic ultrafine particles were collected from the clear liquid (9). The privately produced ultrafine ferromagnetic particles produced in this way had an average particle size of 90, and the surface m1 was coated with dextran.

A superparamagnetic label was obtained by adding influenza 1 virus IQG antibody isolated from rabbit serum to a solution in which privately produced ultrafine ferromagnetic particles were dispersed, and covalently bonding Dex 1-Ran with the IgG antibody. .

The liquid (0,1d) in which this superparamagnetic label was suspended was 4
I was able to catch the 01-1△ influenza virus.

[Example 6] FIG. 9 shows an example of a manufacturing apparatus suitably used for manufacturing the superparamagnetic label according to the present invention.

In the figure, 1': January 31 is an atmosphere tank, 32 is a superparamagnetic ultrafine particle raw material, 33 is a laser light source, 34 is a laser beam, 35 is a mirror, 36 is a vacuum pump, 37 is a collection tube for evaporated particles, 38 is a recovery tank for superparamagnetic ultrafine particles, 39 is a vacuum pump, 40 is a dispersion solution, 41 is a magnetic filter,
42 is an atmospheric gas, 43 is a leak valve, 4.4 is a liquid nitrogen container, 45 is liquid nitrogen, and 46 and 47 are valves.

First, 50% of a 10% dextran aqueous solution (manufactured by Pharmacia Rei, FF1LJ, average molecular weight 7700) was poured into the recovered W438, and then 45% of liquid nitrogen was poured into the liquid nitrogen container 44 to freeze the dextran aqueous solution. . Next, turbo seven and Kueller Bonp 36 J:
Then, after evacuating the inside of the atmosphere tank 31 to 2×1Q-7 Torr, the valve 46 was closed, and the argon gas was
2 was introduced by opening the leak valve 43. Next, the valve 47 was opened and the oil rotary pump 39 was used to exhaust the argon gas. At this point, the pressure in the atmosphere tank 31 was maintained at 5 x 10' Torr by adjusting the leak valve 43. As a result, argon gas/12 is constantly guided from the atmosphere tank 31 to the collection tank 38 through the collection tank 37,
It was exhausted to the outside by an oil rotary pump 39.

Next, the magnetic filter 41 consisting of an electromagnet was activated.

When the magnetic filter 41 was in operation, a magnetic field of 1000 Gauss was applied inside the collection tube 37 in a direction perpendicular to the collection tube 37. After the above operations, the laser beam 34 of the YAG laser 33 with an output of 10 W was guided to the atmosphere tank 31 by the mirror 35, and the evaporation raw material 32 made of pure iron with a purity of 99.9% was heated and evaporated. The evaporated iron particles together with argon gas passed through the magnetic filter 41 and reached the collection tank 38.

At this time, since the ferromagnetic particles are captured by the magnetic filter 41, only the superparamagnetic ultrafine particles reach the collection tank 38. Since the dextran to run solutions are frozen in the interior of the recovery tank 38 by the above-described operation, the superparamagnetic ultrafine particles are deposited on the surface of the frozen dextran solution. After evaporating a predetermined amount, the liquid nitrogen container 44
is lowered and the recovery tank 38 is returned to room temperature. The iron superparamagnetic ultrafine particles deposited on the surface of the frozen solution are dispersed in the solution as the Dex 1-Ran solution is melted. At this time,
Dex 1-Ran was adsorbed on the surface of iron, and superparamagnetic ultrafine particles coated with dextran and having an average particle diameter of 10 nm were obtained.

Next, the laser output, argon gas pressure, magnetic filter 4
By adjusting the magnetic field etc. of 1, the average particle size is 5 nm ~
Using 20 nm superparamagnetic ultrafine particles, the above Example 5
In the same manner as above, IgG antibodies against influenza virus types A and B were covalently bonded to obtain superparamagnetic labeled substances.

Using the interference method previously applied by the present inventors, the above superparamagnetic label was added to one sample collected from the patient's gargle fluid.
X10'S? As a result of trying to detect the virus using a direct method in which the blood cells are added, it was possible to identify virus types A and B with a detection sensitivity 100,000 times higher than that of the conventional hemagglutination method.

〔Effect of the invention〕

As described in detail above, the laser magnetic immunoassay method and measuring device according to the present invention can move the magnetically labeled sample complex after antigen-antibody reaction and the unreacted superparamagnetic label to the concentration position. Since discrimination is made based on the arrival time difference, there is no need to separate the complex and the label when detecting the complex, and the antigen-antibody reaction test can be carried out with extremely high efficiency and a detection sensitivity higher than that of the RIA method.

In addition, the superparamagnetic labeled substance according to the present invention has a size comparable to or smaller than a virus, and also has a size similar to that of a virus,
It is 3 mm smaller than a cyst. Therefore, for example, when a t-clonal antibody that specifically captures lymphocytes infected with a virus or the like is used in the superparamagnetic label of the present invention, lymphocytes 1
A magnetic amplification effect occurs because a large number of superparamagnetic labels bind around the individual. For example, scattered light, interference light,
When optically detected using reflected light or diffracted light, the volume of the antigen-antibody complex of lymphocytes and superparamagnetic label increases significantly, resulting in a volumetric amplification effect. Due to these amplification effects, the detection limits of superparamagnetic labels are generally
Although it is 1X10-12g, it is improved by more than one order of magnitude, so even one virus or lymphocyte can be detected.

Therefore, the present invention can be applied to the measurement of various hormones such as peptide hormone, various enzymes, vitamins, and drugs, to which the rllA method has conventionally been applied. Therefore, in the past, RI was performed with limited capacity.
Precise measurements that could not be carried out otherwise can now be carried out in a general environment. If precise measurements such as screening tests for various viruses and cancers can be carried out widely in common situations such as mass medical examinations, early diagnosis of cancer or viral diseases, etc. will be possible and effective. It becomes possible to carry out period treatment accurately. In this way, the present invention is medical. The effects it will have in the medical field are immeasurable.

[Brief explanation of the drawing]

Figure 1 and Figure 4 are for explaining an example of the C1F magnetic immunoassay method according to the present invention, and Figure 1 is a schematic diagram showing a superparamagnetic label dispersed in a dispersion solvent. ,
Figure 2 is a schematic diagram showing an antibody immobilized on the surface of a non-magnetic microsphere, and Figure 3 is a schematic diagram showing a superparamagnetic material-labeled specimen complex whose magnetization direction is easily aligned by a magnetic field during antigen-antibody reaction. , FIG. 4 is a schematic diagram showing the process of concentrating and detecting the complex shown in FIG. 3. FIG. 5 is a schematic configuration diagram without showing an example of the laser magnetic immunoassay device according to the present invention, and FIG. 6 and FIG. 7 show measurement results obtained using the measuring device shown in FIG. 6
The figure is a graph showing the time dependence of the amount of scattered light in the presence of a specimen, Figure 7 is a graph showing the time dependence of scattered light 1 in the absence of a specimen, and Figure 8 is a superparamagnetic label according to the present invention. FIG. 9 is a process diagram showing an example of the method for manufacturing the superparamagnetic material W! according to the present invention. FIG. 1 is a schematic cross-sectional view showing an example of a manufacturing apparatus suitably used in carrying out a method for manufacturing a medical body. 1... Superparamagnetic ultrafine particles, 2... Antibodies, 3...
Arrow indicating the direction of easy magnetization of superparamagnetic ultrafine particles, 4...
・Antibody, 5... Nonmagnetic microspheres, 6... Virus (antigen), 7... Superparamagnetic label, 8... Nonmagnetic antibody complex, 9... Superparamagnetic label Specimen complex, 10.
... Laser incident beam, 11... Output beam, 11a, 1
1b...Scattered light flux, 12.12a, 12b...Magnetic pole piece (gradient magnetic field generator), 13...Test container, 13a
, 13b...well in test container 13, 14...electromagnet (gradient magnetic field generator), 15...laser light source (incident optical system), 16...deflector (incident optical system), 17a
, 17b... converging lens (light receiving optical system), 18a, 1
8b...Slit (light receiving optical system), 19a, 19b.
... Photomultiplier tube (light receiving optical system), 20 ... Horizontal movement device for inspection container, 21 ... Electronic circuit section (identification mechanism),
31... Atmosphere tank, 32... Superparamagnetic ultrafine particle raw material, 33... Laser, 34... Laser beam, 35
... Mirror, 36 ... Vacuum pump, 37 ... Collection tube for evaporated particles, 38 ... Collection tank for superparamagnetic ultrafine particles, 39 ... Vacuum pump, 40 ... Dispersion solution, 41・
...Magnetic filter, 42...Atmosphere gas, 43...
・Leak valve, 44...Liquid nitrogen container, 45...
Liquid nitrogen, 46... valve, 7'I7... bulge. 5C

Claims (5)

[Claims] (1) A first step of causing an antigen-antibody reaction between a superparamagnetic label in which one antigen or antibody is added to superparamagnetic ultrafine particles and an antibody or antigen as a specimen, and a superparamagnetic label after the first step. a second step in which a magnetic field is applied to a solution containing a superparamagnetic-labeled specimen complex, which is a complex of a body and a specimen, to guide and concentrate the superparamagnetic-labeled specimen complex to a predetermined position; A laser magnetic immunoassay method comprising at least the step of identifying the superparamagnetic labeled substance and the superparamagnetic labeled specimen complex after antigen-antibody reaction based on the difference in arrival time to a concentration position. (2) In the first step according to claim 1, the antigen-antibody reaction is performed in a magnetic field, and a process is performed to align the magnetization direction of the superparamagnetic material-labeled specimen complex after the antigen-antibody reaction. Immunoassay method. (3) A superparamagnetic labeled substance in which one antigen or antibody is added to superparamagnetic ultrafine particles, and a superparamagnetic labeled specimen complex in which the superparamagnetic labeled substance is subjected to an antigen-antibody reaction with the antibody or antigen serving as the specimen. a gradient magnetic field generator that guides and concentrates the superparamagnetically labeled specimen complex to one point in the test container; A laser magnetic immunoassay that includes at least an input optical system that guides to a concentration position and an optical system that receives emitted light from a superparamagnetically labeled analyte complex and emitted light from a solution that does not contain the superparamagnetically labeled analyte complex, respectively. 1. A laser magnetic immunoassay device, comprising a mechanism for distinguishing between the superparamagnetic label sample complex and unreacted superparamagnetic label. (4) A superparamagnetic label used in the laser magnetic immunoassay method according to claim 1 or 2, wherein the surface of the ultrafine particles made of a superparamagnetic substance is coated with a biologically active substance;
A superparamagnetic label for laser magnetic immunoassay, characterized in that an antigen or antibody is immobilized on the coating layer. (5) A step of forming a coating layer made of an active substance on the surface of the superparamagnetic ultrafine particles, a step of separating and collecting only the superparamagnetic substance from the magnetic ultrafine particles on which the coating layer has been formed, and 1. A method for producing a superparamagnetic label for laser magnetic immunoassay, the method comprising at least the step of immobilizing an antigen or antibody on a coating layer of ultrafine particles.