JPS6390766A - Suspension of fine magnetic particles antigen-antibody with immobilized and method for measuring concentration of antigen or antibody by using said suspension - Google Patents

Abstract

PURPOSE:To permit preservation of a liquid suspension for a relatively long period of time before use by dispersing fine magnetic particles of 50-500Angstrom grain size immobilized with an antigen or antibody into an aq. salt soln. contg. a surface active agent to prepare said liquid suspension. CONSTITUTION:This liquid suspension is prepd. by dispersing fine magnetic particles of 50-500Angstrom grain size with immobilized antigen or antibody into >=5ml (per mg of fine magnetic particle) of isotonic aq. salt soln. with concn. of surface active agent >=0.5wt%. The grain size of fine magnetic particles is preferably in a 100-300Angstrom range. The flocculation operation by an external magnetic field is not possible or an extremely strong magnetic field is required and such is not practicable if the grain size of fine magnetic particles is below 50Angstrom . Long-time maintenance of the stable dispersion state is difficult if the grain size exceeds 500Angstrom . The concn. of the surface active agent in the aq. isotonic salt soln. is preferably 1.0-5.0wt%.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to a stable suspension of antigen/antibody-immobilized magnetic particles useful in clinical tests, etc., and a method for measuring antigen/antibody concentration using the same. .

[Conventional technology]

Latex agglutination method is conventionally known as one of the methods for measuring the concentration of antigen or antibody in a liquid. In this method, latex particles such as polyethylene on which an antibody or antigen that specifically reacts with the antigen or antibody to be measured is immobilized are dispersed in a sample solution, and the latex particles generated due to the antigen-antibody reaction are The amount of aggregates is optically measured using light scattering, thereby measuring the concentration of antigen or antibody in the sample solution. However, this latex aggregation injection has the disadvantage that it takes a long time, 24 hours or more, for the aggregation of latex particles accompanying the antigen-antibody reaction.

Therefore, the present inventors previously proposed a method for measuring antigen/antibody concentration using magnetic microparticles on which antigens or antibodies are immobilized (Japanese Patent Application No. 130506/1982). In this measurement method, magnetic microparticles with immobilized antigens or antibodies are dispersed in a liquid containing the antibody or antigen to be measured, and a magnetic field is applied to promote the antigen-antibody reaction. This is a method of measuring the concentration of antigen or antibody in a sample solution by aggregating the conjugate and measuring the concentration of the generated aggregate. This method has advantages such as high sensitivity and high reliability, and because it uses a magnetic field, magnetic fine particles can be rapidly agglomerated, and rapid measurement is possible. “By the way, if the antigen- or antibody-immobilized magnetic particles used in the above method could be supplied or stored as a stable suspension, the antigen- or antibody-immobilized magnetic particles could be There is no need to prepare a suspension of magnetic microparticles, and the concentration of antigen or antibody can be determined by simply adding the sample to a pre-prepared or commercially available suspension of antigen/antibody-immobilized magnetic particles and performing the necessary operations. It is extremely convenient because it allows the measurement of 20% of the human body, and greatly contributes to speeding up and streamlining clinical testing.However, such a stable antigen/antibody-immobilized magnetic particle suspension has not been known to date.

[Problem that the invention seeks to solve]

According to the research of the present inventors, if even some aggregation or precipitation occurs between the preparation of a suspension of magnetic particles immobilized with antigens or antibodies and the time of use, even if dispersion treatment is performed again, However, due to van der Waals forces, etc., it is impossible to completely disaggregate the particles and return them to their original uniformly dispersed state. Therefore, once such aggregation occurs, it is difficult to accurately determine the concentration of antigen or antibody. It turned out that it could not be measured.

Therefore, an object of the present invention is to provide a highly stable suspension of antigen/antibody-immobilized magnetic particles and a method for measuring antigen/antibody concentration using the same.

[Means for solving problems]

The present invention solves the above-mentioned problems by providing magnetic particles having a particle size of 50 to 500 on which antigens or antibodies are immobilized, with a surfactant concentration of 0.5 weight or more per 1 mg of the magnetic particles. % or more of an isotonic salt aqueous solution.

In the present invention, the material of the magnetic particles used for immobilizing the antigen or antibody is not particularly limited, and examples include metal iron, Fe
at pe3o4. T FezO3, Cor-r-
FezO5+ (NiCuZn)O1ezoz+ (Cu
Zn)O4ez03. (Mn-Zn)0・FezO5
+ (NiZn)O・FezO:++ SrO・6Fe
Fe30g (particle size: 200) coated with BaO・6Fez03.5i02 (Enzyme M
iclob, Technol, vol, 2+ p
, 2 10 (1980)], and composite fine particles of various polymeric materials (nylon, polyacrylamide, etc.) and ferrite. Further, the particle size of these magnetic fine particles is in the range of 50 to 500 particles, preferably 10 to 500 particles.
A range of 0 to 300 people is preferred. The particle size of magnetic fine particles is 5
If the number is less than 0, the aggregation operation using an external magnetic field becomes impossible or requires a very strong magnetic field, which is not practical. Moreover, when the particle size exceeds 500 particles, it is difficult to maintain a stable dispersion state for a long time.

Among those having the above-mentioned materials and particle sizes, a particularly preferred one is F with a particle size of about 200 coated with Sin.
e, 0. particles, and γFezO with a particle size of 200-300 people
One particle can be mentioned. Further, as such preferable magnetic fine particles, fine particles (particle size of about 500 particles) made of magnetite (Fe30.) obtained from magnetotactic bacteria can be mentioned. The above-mentioned magnetotactic bacteria are, for example,
It can be easily collected from freshwater or seawater by the method and collector disclosed in No. 3129.

The antigen or antibody-immobilized magnetic microparticles used in the present invention can be produced by immobilizing the required antigen or antibody onto the above-mentioned magnetic microparticles. The immobilization of the antigen or antibody onto the magnetic microparticles can be carried out by a method known as an antigen or antibody immobilization technique. , and a method of binding antibodies.

The type of antibody or antigen immobilized on the magnetic fine particles has an antibody or antigen relationship with the specific antigen or antibody to be measured, and is selected depending on the antigen or antibody in the sample solution. . Examples of such antigens or antibodies include the following:

Antigens: IgG, IgA, IgM, IgE
, albumin, 11cG 5AFP, cardiolipin antigen, blood group substance, concanavalin A, DNT
, prostaglandin, CRP, HBs human growth hormone, steroid hormone, CEA, Ig
D, etc.; Antibodies: anti-albumin antibody, anti-CR antibody, anti-IgG antibody, anti-1. ^Antibody, anti-IgG antibody, anti-IgG antibody, anti-Ig
G antibodies, anti-CRP antibodies, anti-DNT antibodies, anti-prostaglandin antibodies, anti-human coagulation factor antibodies, anti-CRP antibodies, anti-HBs antibodies, anti-human growth hormone antibodies, anti-steroid hormone antibodies, and serum containing these, and monoclonal antibody.

The dispersion medium used in the suspension of the present invention is an isotonic salt aqueous solution containing a surfactant at a concentration of 0.5% by weight or more. Examples of the isotonic salt aqueous solution include 0.9% NaCl aqueous solution,
A 0.025 ML saccharide aqueous solution can be used, and as a surfactant added to this, Tween
Examples include surfactants having groups such as 80 and -COOHSCOO-. The surfactant concentration in the isotonic salt aqueous solution needs to be 0.5% by weight or more, and preferably,
It is 1.0 to 5.0% by weight.

In the suspension of the present invention, the isotonic salt aqueous solution containing the above-mentioned surfactant as a dispersion medium is 5 ml or more, preferably 8 to 10 ml per 1 mg of magnetic fine particles on which an antigen or antibody is immobilized as a dispersoid. used within the range. When the ratio of the isotonic salt aqueous solution is less than 5 ml per 1 mg of magnetic fine particles,
Suspensions that are stable over long periods of time cannot be obtained.

To prepare the suspension of the present invention, magnetic microparticles with immobilized antigens or antibodies may be dispersed in a predetermined amount of an isotonic salt aqueous solution using, for example, ultrasound.

The suspension of the present invention thus obtained can maintain a stable dispersion state for 10 days or more.

The present invention also provides a method for measuring antigen/antibody concentration using a suspension of magnetic microparticles on which the above-mentioned antigen or antibody is immobilized.

The antigen/antibody concentration measurement method is a method for measuring the concentration of an antibody or antigen in a sample solution, and the sample solution is added to a suspension of magnetic particles on which the antigen or antibody is immobilized. , disperse, and then apply a magnetic field to the suspension to aggregate the antigen-antibody-magnetic particle conjugate to form an aggregate, and then stop applying the magnetic field to remove unreacted antibodies or This is an antigen/antibody concentration measurement method that consists of dispersing magnetic fine particles on which antigens have been immobilized into a suspension, and then measuring the concentration of the aggregates suspended in the liquid.

According to the method of the present invention, if an antibody or antigen that specifically reacts with the antigen or antibody immobilized on the magnetic particles is present in the sample solution, the sample solution is dispersed into the sample suspension. The antibody or antigen present on the magnetic particle binds to the antigen or antibody immobilized on the magnetic microparticle to produce a ternary complex consisting of antigen-antibody-magnetic microparticle. These conjugates tend to aggregate with each other as the antigen-antibody reaction progresses, but this rate is extremely slow if left alone.

In the method of the present invention, a magnetic field is applied to the sample liquid at this stage.

Then, the antigen-antibody reaction and the aggregation of the conjugates are promoted, and the agglutination is completed in a very short time, that is, 1 to 10 minutes, and an aggregate is formed. The amount of aggregates produced (aggregate size x number) is proportional to the concentration of antigen or antibody present in the sample. Note that the method of applying a magnetic field to the sample liquid is not particularly limited, and examples include a method of arranging a magnet facing the outside of the sample liquid container, a method of placing the sample container inside the coil of an electromagnet, etc. .

When the above-mentioned aggregates are formed, unreacted antigen- or antibody-immobilized magnetic particles may also physically aggregate due to the action of the magnetic field, Coulomb force, etc., or may adhere to the aggregates, but in the next step, When the application of the magnetic field is stopped and a dispersion process is performed by stirring, vibration, etc., such aggregates and deposits of magnetic fine particles containing unreacted antigens or antibodies are again finely dispersed in the liquid.

On the other hand, the aggregates generated by the antigen-antibody reaction will remain in the liquid in that state.

Next, the amount of produced aggregates (size x loss) present in the liquid is measured. Although the measurement method is not particularly limited, a method using an image sensor is a preferred method since it is suitable for automatic measurement. In this method, a sample containing aggregates is subjected to an optical microscope, and an image of the microscope field is captured using a COD camera.

Agglomerates are shown as black pixels while the background is shown as white pixels.

The size and number of aggregates are converted into a histogram using computer processing based on the number of black pixels, and the amount of aggregates produced is determined. Alternatively, a method of measuring the degree of scattering of infrared light depending on the size and number of aggregates can also be used.

Examples of antigens or antibodies in a sample solution that can be measured by the above method include the antigens and antibodies exemplified above.

The use of the suspension of magnetic particles immobilized with antigens or antibodies of the present invention is not limited to the above-mentioned antigen/antibody concentration measurement method. It can also be used for separation, recovery, etc. The suspension is suitable for being stored in a suitable container and sold as a measurement kit.

〔Example〕

Hereinafter, the present invention will be specifically explained with reference to Examples.

Example 1 (1) Production of particle size 100-3
00 FeO fine particles (hereinafter simply referred to as "magnetic fine particles") 2 γ-aminopropyltriethoxysilane 2
ml was added and subjected to ultrasonic dispersion at 30 W for 20 seconds, followed by reaction at room temperature for 1 hour. Then 3000 x g
A precipitate was obtained by centrifugation for a minute.

To the obtained precipitate, 4 ml of physiological saline (0.9% NaCl aqueous solution) containing Tween 80 at a concentration of 1.0% was added, and the operation of centrifugation under the same conditions as above was repeated three times to remove excess γ. - Removed aluminum pro and lutriethoxysilane.

Next, 4 ml of physiological saline containing glutaraldehyde at a concentration of 2.5% was added to the resulting precipitate of magnetic fine particles, and after cooling with ice, ultrasonic dispersion was performed at 30 W for 20 seconds, and then at room temperature for 1 hour. Made it react. After the reaction, the mixture was centrifuged at 3000×g for 30 minutes to obtain a precipitate. Twee is applied to the precipitation of these magnetic particles.
Add 4 ml of physiological saline containing 1.0% n80, stir, and centrifuge at 3000 x g for 30 minutes.
Excess glutaraldehyde was removed by repeating the process several times.

Next, Tw
20 ml of physiological saline containing i, o% of een 80
In addition, ultrasonic dispersion was performed at 30 W for 20 seconds to obtain a suspension.

Add 0.5 mg of anti-human IgG to this suspension and incubate at 4°C.
The anti-human 1gG was immobilized on the magnetic particles by reacting overnight. The suspension was then centrifuged at 3000×g for 30 minutes to obtain a precipitate. Add 1 portion of Tween 80 to this precipitate.
．． Add physiological saline 4a+1 containing 0%, stir and add 300%
Centrifugation at 0×g for 30 minutes was repeated three times to remove excess anti-Human 1gG antibody. In this way, anti-human IgG-immobilized FeO microparticles were obtained.

(2) Preparation of Suspension of Hypoxia' U
20 m of physiological saline containing 1.0% Tween 80
1 (equivalent to 10 ml per 1 mg of magnetic fine particles),
Ultrasonic dispersion treatment was performed at 30 W for 20 seconds to obtain a suspension in which the target anti-IgG immobilized magnetic particles were uniformly dispersed.

Comparative Example 1 PeO microparticles immobilized with anti-arthropod gG were produced in the same manner as in (1) of Example 1, except that FeQ having a particle size of about 600 was used as the magnetic particles to immobilize anti-aromaine gG. . A suspension of antibody-immobilized magnetic particles was prepared in the same manner as in Example 1 (2) except that the anti-Human IgG-immobilized magnetic particles were used.

Comparative Example 2 When dispersing anti-human IgG-immobilized magnetic fine particles obtained in the same manner as in Example 1 (1) in physiological saline, a physiological saline containing 0.1% by weight of Tween 80 was used. A suspension was prepared in the same manner as in Example 1 (2) except that the following ingredients were used.

Comparative Example 3 When anti-human IgG-immobilized magnetic fine particles obtained in the same manner as in Example 1 (1) were dispersed in physiological saline containing 1.0% Tween 80, the antibody-immobilized magnetic fine particles 2 ■ to 4 ml of the physiological saline (2 ml per 1 mg of magnetic particles)
A suspension of antibody-immobilized magnetic particles was prepared in the same manner as in (2) of Example 1, except that the antibody-immobilized magnetic particles were dispersed in ml (equivalent to ml).

Example 2 (1) Percentage of arsenic grains 11% grain size 100
~300 human FeQ microparticles 6-■, 20 mg of human serum albumin and 0 Staphylococcus-derived protein A5■
．． In addition to 5 ml of distilled water, 10 ml of cottonseed oil was added, and the mixture was ultrasonically dispersed at 30 W for 1 minute. Then, this suspension was gradually added to 40 ml of cottonseed oil incubated at 50°C, and after reacting for 10 minutes with stirring,
The precipitate was obtained by centrifugation at X g for 20 minutes. Add 2 ml of anhydrous ether per 1 mg of this precipitate and incubate at 3000×
The cottonseed oil was removed by repeating the centrifugation at 100 g for 20 minutes twice. Per 1 mg of the obtained precipitate, Tween
Physiological saline 21111 containing 1.0% of 80 was added,
Repeat the operation of stirring and centrifuging at 3000 x g for 20 minutes three times, and add Tween 8 per 1 mg of precipitate.
Add 10ml of physiological saline containing 1.0% of
Ultrasonic dispersion was carried out for 20 seconds.

To the FeO fine particles treated in this manner, 6 µm of anti-Les GG goat serum was added, and the mixture was allowed to react at 4°C overnight.

It was then centrifuged at 3000×g for 20 minutes. Add 4 ml of physiological saline containing 1% Tween 80 per 1 mg of precipitate, stir, and centrifuge at 3000 x g for 20 minutes. Repeat this procedure three times to remove excess anti-human I.
gG goat serum was removed. In this way, anti-human IgG-immobilized FeO microparticles were obtained.

(2) Preparation of ancient magnetic particle turbidity (1)
Twe
Add 20 ml of physiological saline containing 1.0% en 80 (equivalent to 10 ml per 1 mg of antibody-immobilized magnetic particles),
Ultrasonic dispersion was carried out at 0 W for 20 seconds to obtain a suspension in which the objective anti-human IgG immobilized magnetic particles were uniformly dispersed.

Foot 1 Wiping The suspensions of Examples 1 and 2 and Comparative Examples 1 to 3 were left at 25°C, and the stability of the dispersion state over time was observed with the naked eye.
The following results were obtained.

Example 1: Even after 10 days, the antibody-immobilized magnetic particles remained uniformly dispersed as at the beginning.

Comparative Example 1 After 10 days, agglomeration occurred to form a precipitate.

Slight magnetic particles were observed in the supernatant. It was done.

Comparative Example 2: Almost 100% of the magnetic particles were aggregated and precipitated after 10 days.

Comparative Example 3: Formation of precipitate was observed after 24 hours, and 1
Most of the precipitation occurred after 0 days.

Example 2: As in Example 1, a highly stable dispersion state was exhibited even after 10 days.

Example 3 t″″-Voice measurement (ヰ'EWの乍)Human I
GG? JA degree is Oμg/ml and 50μg/m respectively
l.

200μg/ml and 1,000. crg/ml physiological saline (containing 801.0% Tween) was prepared in advance as a sample solution.

0.8 ml of the suspension of anti-arborvitae gG-immobilized magnetic particles obtained in Example 1 was placed in a test tube, and 0.2 ml of each of the above sample solutions was added to each sample to form a 1 ml test sample. After reacting these at room temperature (25° C.) for 1 hour, each was dropped onto a slide glass to prepare a preparation for microscopic observation. COD with these attached to a microscope
When the image was processed using an image sensor (magnification: 600 times) and the number of black pixels was determined, the results shown in the table below were obtained.

From the above results, the calibration curve shown in FIG. 1 was obtained, and it was found that with the suspension of Example 1, it was possible to measure up to about 50 μg/ml by an antigen-antibody reaction at room temperature for 1 hour.

〔Effect of the invention〕

Since the suspension of antigen/antibody-immobilized magnetic particles of the present invention has high stability, it can be made into a kit, and the suspension can be stored for a relatively long period of time until use. Therefore, there is no need to prepare it each time it is used, which is convenient for measuring antigen/antibody concentrations, and greatly contributes to streamlining and speeding up clinical tests. In addition, the method for measuring antigen/antibody concentration of the present invention can be carried out by using such a stable suspension that can be made into a kit.
Accurate measurements can be made quickly.

[Brief explanation of the drawing]

FIG. 1 shows an example of a calibration curve determined in Example 3 and used in the antigen/antibody concentration measurement method according to the method of the present invention.

Claims (2)

[Claims] (1) Particle size 50-50 on which antigen or antibody is immobilized
A suspension of antigen/antibody-immobilized magnetic particles, in which 0 Å magnetic particles are dispersed in an isotonic salt aqueous solution containing 5 ml or more per 1 mg of the magnetic particles and having a surfactant concentration of 0.5% by weight or more. (2) A method for measuring the concentration of an antibody or an antigen in a sample solution, which comprises converting the sample solution into particles with a size of 50 to 500 Å on which an antigen or antibody that can specifically bind to the antibody or antigen is immobilized. Magnetic fine particles are added to a suspension formed by dispersing them in an isotonic salt aqueous solution with a surfactant concentration of 0.5% by weight or more and 5 ml or more per 1 mg of the magnetic fine particles, and then dispersed. Apply a magnetic field to aggregate the antigen-antibody-magnetic microparticle complex to form an aggregate, then stop applying the magnetic field and suspend the unreacted antibody or magnetic microparticles on which the antigen is immobilized. A method for measuring antigen/antibody concentration comprising dispersing in a liquid and then measuring the concentration of the aggregate suspended in the liquid.