JPH05322894A - Method and instrument for measuring biomass - Google Patents

Abstract

PURPOSE:To measure the biomass concentration of a sample liquid with high sensitivity in a short time by causing a reaction between fine magnetic particles in which a labeled linkable phygiologically active substance is immobilized and bio-substance by impressing alternating magnetic fields. CONSTITUTION:Electrophoretic vibrators 6 which are used for accelerating the dispersion of fine magnetic particles in which a labeled linkable phygiologically active substance is immobilized are arranged around an installing section 5 in which a reactive container 4 of a sample liquid 3 containing a bio-substance to be measured and the fine magnetic particles in a dispersed state is installed. In addition, at least four solenoids 7 for impressing alternating magnetic fields are arranged on the outside of the vibrators 6 so as to facilitate the reaction between the bio-substance and magnetic particles. The reaction between the bio-substance and magnetic particles is caused by impressing alternating magnetic fields from the solenoids 7. The quantity of emitted light from the sample solution 3 from which the resulted aggregate of the reaction is magnetically separated is measured and, when the light quantity is larger than a prescribed value, the quantity of the bio-substance is calculated based on the quantity of emitted light from the separated aggregate.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for measuring the amount of a biological substance using luminescent substance-labeled magnetic fine particles as a carrier, and an apparatus therefor.

[0002]

2. Description of the Related Art As a method for measuring biological substances in a liquid sample using magnetic fine particles, an example of using latex particles (trade name: Dynabeads) containing magnetic fine particles as an immunoassay for cell measurement (Journ
al of Immunological Methods, 137 (1991) 1-8), and “Method for measuring antigen or antibody” by the present inventor (Japanese Patent Laid-Open No. 2-281142, which is a method for detecting an antigen-antibody and has a particle size of 50 μm). To immobilize the antibody on the magnetic particles of ~ 1000A and react it, and optically measure the agglutination, and apply the alternating magnetic field of 0.05-10Hz at the time of reaction), and to solve the above two problems. In addition, the “invention method for detecting antigen and antibody” (Japanese Patent Application No. 3-74003).
No.) and "Methods for measuring bacteria, cells and viruses" (Japanese Patent Application No. 3-
No. 292546) These are methods for detecting an antigen antibody and the like, and there is a method of immobilizing a fluorescently labeled antibody on magnetic bacterial particles and utilizing it for measurement.

[0003]

However, each method has the following problems.
There are the following problems. The operation including separation and washing operation is complicated and has multiple steps, the measurement time is long (2 to 3 hours), and the measurement sensitivity is low.

[0004] reaction is completed in a relatively short time (less than 10 minutes in the IgG model system), but the measurement itself is
It is a measure of scattering degree, etc., and the sensitivity is extremely low.

[0005] In the case of (1), since the measurement sensitivity was improved by utilizing fluorescence, the reaction time was long, and therefore the whole measurement time was long. At the time of proposing this invention, a stable dispersion system of magnetic fine particles on which an antibody labeled with a luminescent substance was immobilized was not found in the reaction solution, and the effect of accelerating the reaction by the alternating magnetic field could not be confirmed.

Therefore, an object of the present invention is to provide a method for measuring the concentration of a biological substance in a sample liquid in a short time with high sensitivity, and a measuring device therefor.

[0007]

The above objects can be achieved by the present inventions (1) to (10) below. (1) A method of measuring the concentration of a biological substance in a sample solution, which comprises labeling a bioluminescent substance on a magnetic fine particle on which a physiologically active substance that specifically binds to the biological substance is immobilized, with a labeled binding physiology The active substance-immobilized magnetic fine particles are prepared, and a sufficient amount of labeled binding bioactive substance-immobilized magnetic fine particles are dispersed in the sample liquid as compared with the biological substance in the sample liquid to obtain the labeled binding bioactive substance. After the immobilized magnetic fine particles are reacted with the biological substance to generate an aggregated substance, the aggregated substance is magnetically separated from the sample liquid, and the amount of luminescence is measured to obtain the biological substance existing in the sample liquid. A method for measuring the amount of a substance, which comprises reacting the labeled binding bioactive substance-immobilized magnetic fine particles with a biological substance while applying an alternating magnetic field.

(2) The amount of luminescence emitted from the solution from which the agglomerate has been removed is measured, and when the amount of luminescence is equal to or more than a predetermined value, the living body is detected on the basis of the amount of luminescence from the magnetically separated agglomerate. The method for measuring the amount of a biological substance according to (1) above, which calculates the amount of the substance.

(3) The method for measuring the amount of biological substance according to the above (1) or (2), wherein the luminescent substance is a chemiluminescent substance or a fluorescent substance.

(4) The physiologically active substance is an immune-related substance,
The method for measuring the amount of a biological substance according to (1), (2) or (3) above, which is a nucleic acid-related substance.

(5) The particle size of the magnetic fine particles is 5 nm to 1 μm
In the above (1), the particle concentration during the reaction is 1 mg / ml or less.
(4) The method for measuring the amount of a biological substance according to any one of (4) to (4).

(6) The method for measuring the amount of a biological substance according to any one of (1) to (5) above, wherein the magnetic fine particles are magnetic bacterial particles.

(7) In an apparatus for reacting a labeled magnetic fine particle having a binding physiologically active substance immobilized thereon and a biological substance which is a substance to be bound which complementarily binds to the binding physiologically active substance, in a liquid, An alternating magnetic field applying means for applying an alternating magnetic field to the labeled magnetic fine particles in order to accelerate the reaction, and a one-way magnetic field applying means for applying a magnetic field in one direction to separate the aggregated substance generated by the reaction from the liquid. An apparatus for measuring the amount of a biological substance, comprising:

(8) The apparatus for measuring the amount of biological material according to (7), further comprising means for keeping the liquid temperature constant.

(9) The apparatus for measuring the amount of biological material according to the above (7) or (8), further comprising a dispersing means for dispersing the fine particles in a liquid.

(10) The apparatus for measuring the amount of biological material according to (9), wherein the dispersing means is an ultrasonic wave generating means of 0.3 W / mm ² or less.

[0017]

Specific Structure of the Invention The specific structure of the present invention will be described in detail below. Magnetic Fine Particles In the present invention, the material of the magnetic fine particles used for immobilization of the physiologically active substance is not particularly limited as long as it is insoluble in an aqueous solution and exhibits magnetism. For example, Fe 2 O, γ-Fe 2 O 3, Co-γ-Fe 2 O, (NiCuZn) O
Zn) O ・ Fe O, (MnZn) O ・ Fe O, (NiZn) O ・ Fe O, SrO ・ 6Fe O, B
FeO coated with aO ・ 6Fe 2 O 3 and SiO 2 (particle size about 200 A) [Enzy
Me Microb.Tecnol., vol2, p.2-10 (1980)], and composite fine particles of various polymer materials (nylon, polyacrylamide, polystyrene, etc.) and ferrite.

The particle size of these magnetic particles is 5 nm.
The range is ˜1 μm, and the range of 10 nm to 0.1 μm is particularly preferable. Regarding the particle size of the magnetic fine particles, if the particle size is large and exceeds 0.1 μm, it becomes difficult to maintain stable dispersion for a long time, depending on the concentration of the magnetic fine particles, and reproducibility deteriorates. On the other hand, if the particle size is small, the secondary particle size after the rain in the industry is small, which makes magnetic separation operation difficult. In the present invention, the above particle size is preferable for the above reasons.

Among the materials having the above-mentioned materials and particle diameters, as particularly preferable ones, iron as a main component, which was previously devised by the present inventors, produced by a thermal plasma method in a liquid phase in which carbon coexists, Ni , Co-containing fine particles (particle size approx.
300 A), and these particles are described in, for example, JP-A-04-04
It can be easily produced by the method disclosed in No. 59903.

Substances to be Measured and Physiologically Active Substances The substances to be measured in the present invention are not particularly limited as long as they specifically react with and bind to the physiologically active substance immobilized on the magnetic fine particles, and how they are exemplified. And the antigen-
An antigen or antibody utilizing binding by an antibody reaction is a typical one. Further, for a specific substance to be measured, a specific physiologically active substance is selected and provided for measurement.

Substances to be measured: IgG, IgA, IgM, IgE,
Albumin, HCG, AFP, cardiolipin antigen, blood group substance, concanavalin, DNT, prostaglandin, CRP, HBs, human growth hormone, steroid hormone, CEA, IgD, etc. Bioactive substance: anti-albumin antibody,
Anti-HCG antibody, anti-IgG antibody, anti-IgA antibody, anti-IgM antibody, anti-
IgE antibody, anti-IgD antibody, anti-AFP antibody, anti-DNT antibody, anti-prostaglandin antibody, anti-human coagulation factor antibody, anti-antibody
Biological substances such as CRP antibody, anti-HBs antibody, anti-human growth, hormone antibody, anti-steroid hormone antibody, serum containing them, and monoclonal antibody.

In the present invention, a physiologically active substance capable of reacting with the substance to be measured is immobilized on such magnetic fine particles.

The immobilization method may be either physical adsorption or formation of a chemical covalent bond, but physical adsorption is preferable for immobilizing a protein having a high physical adsorption ability such as an antibody or a high molecular weight protein. The formation of chemical covalent bonds is preferably used for immobilization of hormones and haptens having low physical adsorption ability. Many immobilization methods have already been proposed, and it is advisable to select an immobilization method from known methods according to the characteristics of the physiologically active substance to be immobilized. Generally, magnetic fine particles may be mixed with an antibody or an antigen in a dispersion medium in the presence of a buffer solution or a cross-linking agent, if necessary.

The dispersion medium of the magnetic fine particles on which the physiologically active substance is immobilized is not particularly limited, but from the viewpoints of stability of the magnetic fine particles during storage and reproducibility of reaction during agglutination reaction,
A buffer solution such as glycine-sodium hydroxide buffer solution Tris-hydrochloric acid buffer solution, ammonium chloride-ammonia buffer solution, phosphate buffer solution is preferably used.

The concentration of the magnetic fine particles on which the physiologically active substance is immobilized is not particularly limited, but in general, the concentration is preferably 1 mg / ml or less at the time of the binding reaction.

Luminescent substance As the luminescent substance used as the luminescent substance label, any of fluorescent substances and chemiluminescent substances known to be used for this kind of luminescence analysis can be used.

Fluorescent substances: estrone, aureomycin, quinine, chlorophyll, benzopyrene, fluorescein, prednisolone, reserpine, folic acid, and derivatives thereof.

Chemiluminescent substances: luminol, dioxetane, lophine, lucigenin, acridinium salt, acridinium ester, tetrakisethylene, indole, and their derivatives.

Among these luminescent substances, particularly preferable examples of the fluorescent substance include fluorescein isothiocyanate (FITC) which is a derivative of fluorescein, and chemiluminescent substances include luminol derivative, pyrene and luciferin derivative.

The luminescent substance may be labeled on the magnetic fine particles, may be labeled on the immobilized binding bioactive substance, or may be labeled on both of them.

Reaction To carry out the method of the present invention, a certain amount of the above-mentioned binding bioactive substance-immobilized magnetic fine particles labeled with a luminescent substance is added to a liquid sample that may contain a substance to be measured. ..
When the substance to be measured is present in this liquid sample, a specific binding reaction occurs, and aggregation of magnetic fine particles is formed. At this time, in the present invention, an alternating magnetic field is applied to the reaction system to accelerate the reaction. The aggregate is magnetically separated by suction to separate the aggregate from the solution, and the amount of the luminescent substance contained in each is measured to determine the concentration of the measurement object.

The physiologically active substance-immobilized magnetic fine particles labeled above are usually added to a liquid sample in a state of being suspended in an aqueous medium. The composition and addition method of the aqueous medium are not particularly limited, but an isotonic salt aqueous solution containing a surface active agent is preferable as the aqueous medium. As the isotonic salt aqueous solution, for example, 0.9% NaCl aqueous solution, 0.025M sucrose aqueous solution can be used, and as the surfactant added thereto, a nonionic surfactant such as Tween 80 is used. Be done.

In this method, ultrasonic waves can be used to disperse the magnetic fine particles in the liquid sample, and it is preferable to use a dedicated reaction apparatus as in the present invention. Due to the dispersion treatment of the sample in the suspension, the measurement target existing in the liquid sample and the physiologically active substance are bound to each other, and aggregates of magnetic fine particles are generated.

In the present invention, an alternating magnetic field is applied to the reaction system when causing a reaction. As a result, the physiologically active substance-immobilized magnetic fine particles perform a reversal motion by themselves, the contact frequency with the measurement target existing in the liquid is increased, and the bond formation reaction is promoted. As a result, aggregates of the combination of the substance to be measured-physiologically active substance-magnetic fine particles are generated in a short time. Since the opportunity of contact between the immobilized physiologically active substance and the measurement object in the test liquid is greatly enhanced by the reversal movement of the magnetic fine particles, stirring by other stirring means is not particularly necessary.

The waveform of the alternating magnetic field applied is not particularly limited, and examples thereof include a rectangular wave, a triangular wave, a pulse wave such as an impulse, and a sine wave. The strength of the alternating magnetic field is about 100
Gauss or more, especially about 100 to 350 gauss is preferable. If the magnetic field strength is too low, the reaction time will be long, but if it is too strong, nonspecific aggregation will occur. The frequency of the alternating magnetic field is
0.05 to 10 Hz is preferable.

The method of applying an alternating magnetic field to the reaction system is not particularly limited, and any method may be applied as long as a required alternating magnetic field is applied so that the magnetic field lines of the alternating magnetic field effectively pass through the reaction system containing dispersed magnetic fine particles. The method may be, for example, a method of installing an electromagnet outside the reaction vessel, or a method of arranging the permanent magnets such that the N pole and the S pole of the permanent magnet face each other so as to sandwich the reaction vessel, and rotating the two permanent magnets in the same cycle. Method or Z
There is a method in which four magnets are opposed to each other in the axial and Y-axis directions to further improve the motion controllability of the magnetic fine particles to accelerate the reaction. In any case, from the viewpoint of the stability of the dispersed state, it is desirable that the force that moves the dispersed magnetic fine particles in one biased direction does not act. In this case, disposing a plurality of electromagnets around the reaction vessel as in the apparatus of the present invention is a preferable method because the dispersed state is easily maintained.

By accelerating the reaction as described above, the reaction can be completed in about one-third of the time required in the conventional case.

Immediately after the separation reaction, magnetic attraction separation is performed. The method of magnetic attraction is not particularly limited. It is performed by bringing electromagnets and permanent magnets close to each other.

Preferably, it is desirable to attract with an electromagnet in the same direction as gravity.

As in the apparatus of the present invention, the method of attracting to the bottom surface of the reaction vessel using an electromagnet is simple.

Measurement As in the conventional method (Japanese Patent Laid-Open No. 3-74003), the separated magnetic fine particles are dispersed again in another solution to measure fluorescence intensity or chemiluminescence intensity.

For the separated solution, fluorescence intensity measurement or chemiluminescence intensity measurement is performed as it is. As an example of measurement, when the fluorescent labeling substance is FITC, fluorescence is emitted with excitation light of 490 nm.
Observed at 0 nm, when the chemiluminescent labeling substance is luminol,
Add equal amount of pH8.5NaOH solution with hydrogen peroxide concentration of 0.02% to the measurement solution and add the amount of luminescence while stirring.

Although only the conventional method was used, the ratio of the amount of the physiologically active substance fixed to the measurement object and the magnetic fine particles was 1:
Carry out for verification in case of 10 or more or 1: 1 or less.

An example of the reaction device used for the device measurement will be described below. The reaction device 1 has a machine casing 2. At the center of the upper part of the machine frame 2, there is an installation unit 5 for installing a reaction container 4 containing a sample liquid 3 in which a biological substance to be measured, labeled binding bioactive substance-immobilized magnetic fine particles and the like are dispersed. It is provided.
An electrostrictive vibrator 6 for promoting the dispersion of the magnetic fine particles is arranged around the installation portion 5. On the further outside thereof, at least four alternating magnetic field applying solenoids 7 are arranged for promoting the reaction between the biological substance and the labeled binding bioactive substance-immobilized magnetic fine particles.
It is desirable to place the four alternating magnetic field applying solenoids 7 in positions of orthogonal relationship as much as possible. A magnetic attraction solenoid 8 for attracting the aggregate generated by the reaction to the bottom of the container 4 is provided below the installation unit 5. At the bottom of the machine casing 2, a controller 9 composed of a microcomputer or the like is arranged in order to control the various elements described above, and executes the steps of dispersion, reaction and separation.

[0045]

EXAMPLES Examples of the present invention will be described below together with comparative examples. 2 ml of γ-aminopropyltriethoxysilane (hereinafter simply referred to as “γ-APTES”) was added to 2 mg of magnetic fine particles having a particle size of 500 A (hereinafter simply referred to as “magnetic fine particles”), and ultrasonic waves were applied at 30 W for 20 seconds. After dispersion, the mixture was reacted at room temperature for 1 hour. Then, it was centrifuged at 15,000 × g for 20 minutes to obtain a precipitate. To the obtained precipitate, 4 ml of distilled water was added and the operation of centrifuging under the same conditions as above was repeated to remove excess γ-APTES.

Next, 4 ml of physiological saline containing glutaraldehyde at a concentration of 25% was added to the precipitate of the obtained magnetic fine particles, and ultrasonic dispersion was carried out at 30 W for 20 seconds while cooling with ice, followed by reaction at room temperature for 1 hour. Let After reaction, 2 at 15,000 × g
The operation of centrifuging for 0 seconds was repeated to remove the excess glutaraldehyde.

Next, 4 ml of physiological saline was added to the precipitate of the magnetic fine particles subjected to the above operation, and ultrasonically dispersed at 30 W for 20 minutes to obtain a suspension. To this suspension, 2 mg of an anti-human IgG antibody having fluorescein isocyanate (FITC) immobilized as a fluorescent substance-labeled antibody was added, and the mixture was reacted overnight at 4 ° C. to immobilize the anti-human IgG on the magnetic fine particles. Then, the suspension was centrifuged at 15,000 × g for 20 minutes to obtain a precipitate. Saline containing Tween 80 at 0.6% in this precipitate 4
After adding ml and stirring, the operation of centrifuging at 15,000 × g for 30 minutes was repeated to remove excess anti-human IgG antibody. In this way, FITC-labeled anti-human IgG antibody-immobilized magnetic fine particles (hereinafter, simply referred to as "FITC-labeled antibody magnetic fine particles") were obtained. Using a luminol-labeled anti-human IgG antibody obtained by reacting an anti-human IgG antibody with diazotized luminol, a luminol-labeled anti-human IgG antibody-immobilized magnetic fine particle (hereinafter simply referred to as “luminol-labeled antibody Magnetic particles ").

Example 1 The concentrations of anti-human IgG were 0, 2.5, 10, and 1, respectively.
The FITC-labeled antibody magnetic fine particle antibody suspension prepared above was added to a physiological saline solution of 00, 1000 and 10000 ng / ml so as to have a concentration of 0.075 mg / ml, and 1 ml of the suspension was injected into a reaction cuvette. This was mounted in the reaction apparatus of the present invention, reacted to cause magnetic attraction, and the reaction supernatant and the agglomerated magnetic particles were separated. 2 minutes after ultrasonic dispersion, 29
The reaction was performed for 20 minutes while applying an alternating magnetic field of 0 Gauss and a pulse width of 0.2 seconds. Subsequently, a magnetic field of 500 gauss was applied from the lower part for 2 minutes to perform magnetic attraction.

The obtained agglomerated magnetic fine particles were 1% gelatin.
GVB including Thermomixer (Thermonics MODEL TM-
105) to disperse, glory spectrophotometer (Hitachi F-1200)
The glory intensity was measured at. Similarly, the supernatant was measured. Excitation light is 490nm, fluorescence is 520nm, 10mm × 1
The measurement was carried out using a 0 mm quartz cell.

As shown in the graph of FIG.
A calibration curve with good linearity was obtained up to 0 ng.

Comparative Example 1 A sample was prepared under the same conditions as in Example 1, the reaction was performed under the same conditions except that no alternating magnetic field was applied during the reaction, and the measurement was also performed under the same conditions.

As shown in the graph of FIG. 2, the relative fluorescence intensity was extremely small and the measurable region was narrow as compared with Example 1.

Comparative Example 2 The same operation as in Comparative Example 1 was performed except that the reaction time was changed to 2 hours.

The reaction time was set to 2 hours, but as shown in the graph of FIG. 2, the relative fluorescence intensity was smaller and the measurable region was narrower than in Example 1.

Example 2 The concentrations of anti-human IgG were 0, 2.5, 10, and 1, respectively.
The luminol-labeled antibody magnetic fine particle antibody suspension prepared above was added to a physiological saline solution of 00, 1000 and 10000 ng / ml in the same manner as in Example 1, and the reaction was carried out under the same conditions.

The agglomerated magnetic fine particles obtained were gelatin 1%.
GVB including Thermomixer (Thermonics MODEL TM-
Disperse with 105) and sample 1 ml as the measurement solution. This is a weak light measurement device (Tohoku Electronics Industry Co., Ltd.)
The luminescence was measured using CLD-100. P for measurement sample
A NaOH solution was added to H12.6 and left for 15 minutes, 100 μl of hydrogen peroxide concentration of 0.02% was further added, and the luminescence amount was integrated for 15 minutes while stirring. As shown in the graph of FIG. 3, as in the case of Example 1, the emission amount was large and a calibration curve with good linearity was obtained over a wide range.

[0057]

As described above, according to the present invention,
The biological substance concentration in the sample liquid can be measured with high sensitivity in a short time.

[Brief description of drawings]

FIG. 1 is a schematic diagram showing an example of a reaction apparatus used for measuring the amount of a biological substance of the present invention.

FIG. 2 Anti-human IgG in Example 1 and Comparative Examples 1 and 2.
It is a graph which shows the relationship between concentration and relative fluorescence intensity.

FIG. 3 is a graph showing the relationship between anti-human IgG concentration and luminescence integrated value in Example 2.

[Explanation of symbols]

 1 Reaction Device 2 Machine Frame 3 Sample Liquid 4 Reaction Vessel 5 Reaction Vessel Installation Section 6 Electrostrictive Oscillator 7 Solenoid for Applying Alternating Magnetic Field 8 Solenoid for Magnetic Suction 9 Controller

Front Page Continuation (72) Akira Shibue Inventor Akira Nihonbashi 1-13-1, Chuo-ku, Tokyo TDC Corporation (72) Inventor Shun Tanaka 1-13-1 Nihonbashi, Chuo-ku, Tokyo TDC Corporation (72) Inventor Shinji Kamiya 1-13-1 Nihonbashi, Chuo-ku, Tokyo Inside TDC Corporation

Claims (10)

[Claims] 1. A method for measuring the concentration of a biological substance in a sample solution, which comprises labeling a luminescent substance on a magnetic fine particle on which a physiologically active substance that specifically binds to the biological substance is immobilized, and labeling the bound substance. Magnetic microparticles immobilized with a physiologically active substance are prepared, and a sufficient amount of labeled binding bioactive substance-immobilized magnetic microparticles than the biological substance in the sample solution is dispersed in the sample solution to obtain the labeled binding physiology. After the active substance-immobilized magnetic fine particles are reacted with the biological substance to generate an aggregated substance, the aggregated substance is magnetically separated from the sample liquid, and the amount of luminescence is measured to determine whether the aggregated substance is present in the sample liquid. In the method for measuring the amount of the biological substance, the reaction between the labeled binding bioactive substance-immobilized magnetic fine particles and the biological substance is performed while applying an alternating magnetic field. 2. The amount of luminescence emitted from the solution from which the aggregating substance has been removed is measured, and when the luminescence amount is equal to or more than a predetermined value, the biological substance is based on the amount of luminescence from the magnetically separated aggregating substance. The method for measuring the amount of a biological substance according to claim 1, wherein the amount of the biological substance is calculated. 3. The method for measuring the amount of a biological substance according to claim 1, wherein the luminescent substance is a chemiluminescent substance or a fluorescent substance. 4. The method for measuring the amount of a biological substance according to claim 1, 2 or 3, wherein the physiologically active substance is an immune-related substance or a nucleic acid-related substance. 5. A magnetic fine particle having a particle size of 5 nm to 1 μm and a particle concentration during the reaction of 1 mg / ml or less.
A method for measuring the amount of biological material according to any one of 1. 6. The method for measuring the amount of a biological substance according to claim 1, wherein the magnetic fine particles are magnetic bacterial particles. 7. An apparatus for reacting, in a liquid, a labeled magnetic fine particle having a binding physiologically active substance immobilized thereon, and a biological substance which is a substance to be bound that complementarily binds to the binding physiologically active substance, in the liquid. An alternating magnetic field applying means for applying an alternating magnetic field to the labeled magnetic fine particles, and a one-way magnetic field applying means for applying a magnetic field in one direction to separate the aggregated substance generated by the reaction from the liquid. An apparatus for measuring the amount of a biological substance, which is provided. 8. The apparatus for measuring the amount of biological material according to claim 7, further comprising means for keeping the liquid temperature constant. 9. The apparatus for measuring the amount of biological material according to claim 7, further comprising a dispersing means for dispersing the fine particles in a liquid. 10. The apparatus for measuring the amount of biological material according to claim 9, wherein the dispersing means is an ultrasonic wave generating means of 0.3 W / mm ² or less.