JPS5814057A - Measurement of biologically active substances and labelling agent used therefor - Google Patents

Abstract

PURPOSE:To determine antigen or the like in a sample by measuring the intensity of scattered light of active fine particles by employing active fine particles with a fine particle connected thereto as labeling agent for a matter (antibodies or the like) peculiarly combined with a biologically active matter (antigen, hormon, or the like). CONSTITUTION:A dispersion liquid of active fine particles is prepared for a matter (antibodies agent antigens) peculiarly combined with biologically active matters (antigens, hormons or the like) to be measured by connecting fine particles 0.03mu-3mu in the particle size such as synthetic resin thereto as a labelling agent. On the other hand, a partner (an antibody, an immunoglobulin, or the like) peculiarly combined with the matter being measured is solidified on a hole wall of a microplate and synthetic resin particles (with the particle size of 4mu or more) to prepare a solid phase. After reacting the matter being measured in the sample, the solid phase is separated and reacts a fixed amount of the dispertion liquid the active fine particles. Then, the intensity of scattered light of the active fine particles. Then, the intensity of scattered light of the active fine particles. Then, the intensity of scattered light of the active fine particles left from the reaction with the solid phase is measured. The results are compared to a curve of the intensity of scattered light and the concentration of the matter to be measured previously obtained for a known amount thereof thereby assuring a highly accurate determination thereof.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for measuring biologically active substances using microparticles and a labeling agent used therein.

In recent years, methods for quantitatively measuring trace components using antigen-antibody reactions have been used in clinical testing and research fields such as medicine, veterinary medicine, pharmacy, and microbiology.

In addition to classical methods such as hemagglutination and immunodiffusion, recent methods include nephelometry, which uses light scattering to measure immune complexes generated by antigen-antibody reactions, and nephelometry, which uses fluorescence, radioactive isotopes, or enzymes to measure immune complexes generated by antigen-antibody reactions. Alternatively, there is a labeled immunoassay method in which the antigen is labeled and the antigen or antibody is measured.In addition, the antigen or antibody is immobilized on synthetic polymer particles, and the degree of aggregation of the particles that occurs in the presence of the antibody or antigen is measured on a glass plate or microplate. Methods are also being developed to measure antibodies and antigens by observing them with a spectrophotometer or by measuring changes in light transmittance due to aggregation using a spectrophotometer.

In particular, radioisotope-based immunoassays (RIA)
) is widely used in practical use as the most sensitive analysis method, but it has major drawbacks such as the risk of radiation exposure and the need for special facilities because it handles radioactive isotopes. Therefore, there is a need for a highly sensitive measurement method to replace RIA. One of these is enzyme immunoassay (EIA), which has been developed as a highly sensitive and easy-to-handle method by using enzymes instead of radioactive isotopes while retaining the advantages of labeled immunoassay. Although EIA does have a measurement sensitivity comparable to RIA for some substances to be measured, it does not necessarily have the same sensitivity as RIA for all substances, and the reagents are relatively expensive. It has not yet surpassed the RIA method.

In general, immunoassay methods typified by the RIA method can be broadly classified into two methods in principle. One is the competition method and the other is the so-called sandwich method. The competitive method is a measurement sample solution containing a substance to be measured, which is an antigen or an antibody,
An antigen-antibody complex is formed by mixing a known concentration of a substance to be measured that has been previously labeled with a labeling agent such as a radioactive isotope, and then mixing it with an antibody or antigen and allowing it to react.
The complex or free substance that did not form a complex contains labeled analyte and unlabeled analyte, and by measuring the ratio, the analyte in the sample can be measured. This is the way to do it. Another method, the sandwich method, is a two-step measurement method. In other words, a solid phase on which a binding partner that specifically binds to the analyte is prepared is prepared, and after a step of reacting with the analyte, the solid phase is separated from the liquid phase, and then A labeled binding substance obtained by labeling a substance that specifically reacts with the analyte in the process with a radioactive isotope, etc. is reacted with the analyte on the solid phase, and the labeled substance in the solid or liquid phase is removed. This is a method of measuring the substance to be measured by measuring .

As mentioned above, the sandwich method is a two-step measurement method in which the analyte in the sample is specifically reacted with a binding partner for the analyte immobilized on a solid phase. Since only the analyte is bound to the solid phase and other substances and ions are washed away, there is no need to introduce substances that interfere with the label other than the analyte in the sample into the second step. The substance to be measured is also concentrated in the measurement liquid, which is advantageous in terms of measurement sensitivity and accuracy.

As a result of our study to develop a safe, inexpensive, and highly sensitive measurement method to replace the RIA method, the present inventors discovered a novel method that can measure biologically active substances with high sensitivity by using fine particles in the sandwich method. We discovered a measurement method and arrived at the present invention.

That is, the present invention involves reacting a biologically active analyte in a sample solution with a solid phase on which a partner that specifically binds to the analyte is immobilized, and then separating the solid phase from the reaction mixture. After that, reacting the solid phase with a substance that has the property of specifically binding to the analyte and is labeled with a labeling agent,
Next, in the method of measuring the substance to be measured by measuring the labeling substance remaining in the liquid phase, a particle size of 0 is used as the labeling agent.
The present invention relates to a method for measuring biologically active substances characterized by using fine particles of 506 to 6 μm, and a labeling agent used therein.

The feature of the present invention is that instead of using a labeling substance in which a substance that specifically reacts with the analyte (hereinafter referred to as a binding substance) is labeled with a radioactive isotope or an enzyme in the second step of the side-witch method, Binding substance-immobilized fine particles (
(hereinafter referred to as active fine particles). The active fine particles bind to the solid phase via the substance to be measured, but if an excess of active fine particles is used, some of the active fine particles do not bind to the solid phase and remain dispersed on the liquid phase side. By counting the active particles remaining on the liquid phase side, the substance to be measured can be quantitatively measured. Counting methods include a method in which the intensity of scattered light generated by shining light on the active fine particle dispersion is measured, a method in which transmitted light is similarly measured, and a method in which the number of active particles is counted using a particle counter. .

The solid phase used in the present invention preferably has a shape or material that can be separated from the active fine particles.

In other words, the solid phase can be extracted from the mixed system with the active particles as a spherical or plate-shaped substance that is much larger than the active particles, or even if the solid phase is made into fine particles, the particle size is larger than the active particles, so that the solid phase can be formed into a membrane, By making it possible to separate the particles using a filter or centrifuge, or by making the specific gravity of the solid phase particles larger than that of the active particles without changing the particle size,
It is necessary to make it easy to settle so that it can be separated by centrifugation, or to make it possible to separate it by enclosing a magnetically sensitive substance inside the solid phase fine particles and attracting it with a magnet.

The above specific examples have been described to facilitate understanding of the separation between the solid phase and active fine particles, and are not intended to particularly limit the solid phase. The material for the solid phase may be biological components, metals, etc., but organic polymer compounds whose shape can be freely adjusted and are stable are preferred. For example, hydrophobic polymers such as polystyrene, polyacrylonitrile, polyacrylonitrile, polymethyl methacrylate, poly-ε-capramide, polyethylene terephthalate, or polyacrylamide, polymethacrylate, poly-N-vinylpyrrolidone, polyvinyl alcohol, Hydrophilic crosslinked poly(2-oxyethyl acrylate), poly(2-oxyethyl methacrylate), poly(2,3-dioxypropyl acrylate), poly(2,3-dioxypropyl methacrylate, polyethylene glycol methacrylate, etc.) There is a group of polymers or a group of coelectrics that have both properties.

Further, the shape of the solid phase may be a plate, a test tube, a microplate, etc., but if fine particles are used as the solid phase, the surface area can be easily increased.

Methods for immobilizing a bonding partner to a solid phase include physical adsorption and chemical bonding. For example, proteins can be immobilized on a hydrophobic solid phase by physical adsorption, and substances with carboxyl or amino groups can be covalently bonded to a solid phase that has amino or carboxyl groups as functional groups. A substance with an amine group can be immobilized on a solid phase with an aba) group by covalent bonding with glutaraldehyde, and a substance with an abano group can be immobilized on a solid phase with a hydroxyl group with cyanogen bromide. Substances can be immobilized by covalent bonds. A cleaning solution containing a surfactant may be used to wash the solid phase, and a covalent bonding method is preferable to a physical adsorption method in which there is a risk of detachment of the immobilized substance.

The binding partner used in the present invention is a substance that specifically binds to the analyte. For example, when the substance to be measured is an antigen, an antibody, a hormone, an antigen-antibody complex, a sugar chain, an immunoglobulin, a lymphokine, a complement, etc., the following order is applied: the antibody, the antigen, the hormone receptor, the rheumatoid factor, the lectin, the protein. A1 The combination of the lymphokine receptor, the complement receptor, etc. is a binding partner for the analyte, respectively.
It is a combination of vines.

After separating the solid phase and liquid phase, the solid phase is washed.

The washing method may be appropriately selected depending on the shape of the solid phase. For example, if the solid phase is a test tube or microplate,
It is easy to remove the liquid by decantation or suction, and if the particles are fine, they can be sedimented in a centrifuge and the supernatant can be removed by suction, or a magnetically sensitive substance can be encapsulated inside the solid phase. For example, the solid phase may be attracted with a magnet and the liquid may be removed by suction.

Next, the active fine particles are caused to react with the solid phase to which only the analyte obtained by washing is bound via a binding partner. Active microparticles are microparticles on which a substance (binding substance) that specifically binds to a substance to be measured is immobilized. It is preferable that the fine particles have a uniform particle size and shape in order to obtain measurement accuracy. From the point of view of reaction efficiency, the smaller the particle size, the better.
The particle size is at least large enough to cause Brownian motion, i.e. 3μ.
It is preferable that it is below m.

However, even if the particle size is too small, it is not suitable for operational and measurement purposes, so the particle size is suitably in the range of 0.03 μm to 3 μm, and particularly preferably in the range of 0.1 μm to 0.8 μm.

The material for the active fine particles is preferably an organic polymer in order to obtain fine particles that are uniform and have an appropriate particle size.

For example, there are hydrophobic polymers similar to the solid phase described above, hydrophilic polymers, and copolymers of both. The fine particles of the present invention can be prepared by emulsion polymerization or precipitation polymerization.

These polymerization methods are suitable for obtaining fine particles with uniform particle size and shape. In particular, in emulsion polymerization, the particle size can be adjusted from 0.5μm to 0.03μm by adjusting the emulsifier and monomer concentration.
A polymerization method that can prepare fine particles with a uniform particle size in the range of m.
This polymerization method is suitable for obtaining the desired fine particles. The binding substance can be bound to the fine particles by physical adsorption or chemical bonding. The present fine particles must have good dispersibility and must not be bound to the solid phase without the analyte being involved. It is a substance that specifically binds to
It may be the same substance as the binding partner immobilized in the same phase, or it may be a different substance.

For example, when the substance to be measured is immunoglobulin, protein A is used as the binding partner to bind to the solid phase.
An anti-immunoglobulin antibody may be used as the binding substance for the active microparticles, or an anti-immunoglobulin antibody may be used for both.

In the present invention, the substance to be measured is a substance that has a biologically specific affinity to the substance, and specifically, for example, streptococcus, staphylococcus,
Antibodies against bacteria such as Diphtheria, Salmonella, and Shigella and their constituents; Antibodies against spirochetes such as Treponema pallidum and their constituents; Antibodies against mycoplasma and their constituents; Antibodies against protozoa such as malaria parasites and their constituents; Antibodies against richchaa and its constituents; influenza, adenovirus, polyoma, measles, rubella, hepatitis,
Viruses such as mumps and their components, and antibodies against them; Heterologous antigens and enzymes such as polysaccharides, human albumin, and ovalbumin; Enzymes such as ribonuclease, creatine phosphokinase, and asparaginase; Kidney, liver, α-fetoprotein, and CEA
Organ-specific antigens or receptors such as; collagen,
Connective tissue components such as amyloid; blood cell antigens such as red blood cells and red platelets, or receptors; plasma proteins such as fibrin and plasinodan; pathological globulins such as rheumatoid factor and C-reactive protein; immune complexes; Autoantibodies against cell membranes, etc.

After the reaction between the solid phase and the active particles, the remaining active particles that are not bound to the solid phase are measured. The measurement method may be any method that allows quantitative measurement of fine particles. Among these methods, the method of measuring the intensity of scattered light by fine particles is to irradiate the fine particles with light of a wavelength similar to the particle size of the fine particles, and measure the scattered light with the same wavelength as the wavelength of the irradiated light, which is called Mie scattering. This is a method that allows high sensitivity measurement. Below, some examples are shown to facilitate understanding of the present invention.

Example 1゜((Measurement of anti-serum Alban antibody)) (Preparation of fine particles for solid phase) The fine particles used as the same phase were glycidyl methacrylate, 2-oxyethyl It was prepared by mixing and polymerizing fine particles of methacrylate and triethylene glycol methacrylate in a molar ratio of 85.7:9.5:4.8, followed by amination and hydrolysis. These are hydrophilic fine particles with an average particle size of 4.6 μm.

(Immobilization of bovine serum albumin (abbreviated as BSA) to fine particles for solid phase) Patent application No. 55-437S18 for aminated and hydrolyzed fine particles
Microparticles activated with glutaraldehyde according to the described method were mixed with BSA (Mj les ) I Dml/
Disperse to a concentration of 1% in 0.15 molar physiological phosphate buffer (PBS) at pH 7.2 containing 60 ml of
After reacting at ℃ for 6 hours, the particles were washed by sedimentation by centrifugation at 3 DO Orpm.
BSA-immobilized microparticles were prepared by dispersing them in 1% human serum albumin (abbreviated as ISA).

(Preparation of fine particles for immobilizing binding substances) Glycidyl methacrylate, methacrylic acid, and ethylene glycol dimethacrylate were mixed at a molar ratio of 85:10:5, and 0.1% of sodium dodecyl sulfate and 0.01 M of ammonium persulfate were added. In addition to the aqueous solution containing the monomer, the aqueous solution with a monomer concentration of 10% (W/V) was reacted at 60°C for 22 hours in an argon gas atmosphere, and the resulting fine particles were aminated and hydrolyzed in the same manner as the solid phase fine particles. (Immobilization of anti-rabbit immunoglobulin G'' (abbreviated as anti-rabbit IgG) antibody to microparticles for immobilizing binding substances) For solid phase use. According to the method for immobilizing BSA on microparticles, the microparticles were activated with glutaraldehyde, and anti-heron IgG antibody (produced in goat) was dissolved at a concentration of 1%/g to give a particle concentration of 1%. dispersed in PBS containing 30
React at 50'C for 2 hours, add ISA to 1%, continue reaction at 50'C for 1 hour, and repeat at 110,000 rpm.
After washing the microparticles by sedimenting them with a centrifugal operation, they were dispersed in PBS containing 0.1% H5A to a particle concentration of 1% to prepare anti-heron IgG antibody-immobilized microparticles. did.

(Quantitative measurement of anti-BSA antibody) Anti-BSA antibody prepared by immunizing a rabbit with BSA was
μg/mis 1 μg/wl, 100ng/m7?
, PBS solution 2 containing 10ng/mlsIng/mA'
Add 50μ of 1% BSA-immobilized solid phase solution to 00μ2 in a glass test tube and heat to 1.5μ at 37°C with shaking.
Allowed time to react. After washing the particles with PBS, the particles were sedimented by centrifugation at 3000 rpm, and then
Disperse the solid phase in 112 PBS and then add IgG
25μ2 of antibody-immobilized fine particles (0.01% dispersion) were added, shaken, and reacted at 60°C for 2 hours. After standing overnight at 4°C, 2.5 WLe of PBS was added and the solid-phase microparticles and anti-heron IgG antibody-immobilized microparticles were filtered through a membrane with 1.2 μm diameter holes (ξ Lipore Filter RA). The light scattering intensity of the anti-heron IgG antibody-immobilized fine particle dispersion that had been separated and passed through the membrane was measured using a fluorophotometer manufactured by Aminco Pomann.
It was measured by irradiating with 00 nm light. Since the light scattering intensity is proportional to the number of particles, the number of particles was determined from the light scattering intensity using a calibration curve prepared in advance. As shown in FIG. 1, by this method, the anti-BSA antibody was
It can be seen that quantitative measurement is possible in the range of al to 10 μg/ml.

Example 2 (Measurement of Human Ciliary Gonadotropin (abbreviated as HCG)) (Immobilization of anti-HCG antibody onto solid phase fine particles) A glutaraldehyde-treated solid prepared in the same manner as in Example 1 was used. Phase fine particle 1% dispersion and anti-HCG antiserum 16000 I
U/rnl CMiles] were mixed in equal amounts and reacted at 30°C for 6 hours, and then 1% BSA was added to the particle dispersion.
Add so that
After washing by centrifugation at pm, the particles were dispersed in PBS containing 0.1% BSA to prepare an anti-)ICG antibody-immobilized microparticle solid phase.

(Immobilization of anti-HCG antibody onto fine particles for immobilizing a binding substance) The fine particles for immobilizing a binding substance were 0.27 mm as in Example 1.
Fine particles with a particle size of μm were used. The immobilization was carried out in accordance with the method of immobilizing anti-HCG antiserum on solid phase microparticles. Washing is 110
This was done with PBS by sedimentation of the microparticles at 000 rpm.

(Measurement of HCG) Prepare a 10-fold dilution series (each 90μ!) from HCGCDI O'U/43 to IU/-g, and dilute 10 times in a glass test tube.
% of anti-HCG-immobilized solid-phase microparticle dispersion m1ooμ2 without shaking, reacted at 30'C for 2 hours, and washed three times with PBS by centrifuging at 3000 rpm.
Obtained a solid phase bound via CG Anti-HCG antibody immobilized fine particles (0.27ttm) 0.02% dispersion 1
0μ! This and 100 μm of this 1% dispersion of solid phase fine particles were mixed in a glass test tube, and reacted at 60° C. for 2 hours. After the reaction, PB52.5m6 was added and centrifuged at 300 rpm for 10 minutes to precipitate the solid phase. A fine particle dispersion of the supernatant was taken. The scattered light intensity was measured in the same manner as in Example 1, and the number of fine particles was determined from the intensity.

FIG. 2 shows the relationship between HCG concentration and scattered light intensity in the range from HCG 104 U/4 to 1 U/43.

Example.

((Measurement of HCG)) (Adsorption of anti-HCG antibody to microplate) Example 3
The anti-HCG antiserum used in
Add 200μ of a solution containing an equal amount of IC-6 (pH 8,0) to each well of a polystyrene microplate.
After standing at 25°C for 6 hours, wash with PBS and remove anti-HCG.
Antibody adsorption microplates were prepared.

(Reaction between HCG and solid phase) HCG 10'% 103% 102% 1.0UZ
PBS solution containing 1% rabbit serum each containing 2.
Stones were added to each well of a microplate coated with anti-HCG antiserum and allowed to react at 25°C for 3 hours. 0.1 after reaction
The cells were washed once with l''BS containing % Triton X-100 and twice with PBS.

(Measurement of HCG) The anti-HCG antibody-immobilized fine particles were the fine particles of Example 2 ([1,
27 μm) was used. Add 10 μ2 of 0.02% fine particle dispersion to a solid-phase microplate containing 140 μ of PBS, react at 25°C for 6 hours, leave to stand at 4°C overnight, and pipette into each hole to remove the fine particle dispersion. was aspirated and added to s2.5WLll of PBS, and the scattered light intensity of the particles was measured in the same manner as in Example 1, and the number of particles was determined from the intensity. A calibration curve in the range of HCG104 U to 10'' U/, I3 prepared as described above is shown in FIG.

(17) Example 4 ((Measurement of HCG)) Anti-HCG antibody-bound microparticles (4μ) were used as a solid phase. HCG (7) 105 U/,, & ~I CJU/
A 10-fold dilution series (90μ2) in the range of 43 was prepared and reacted with 100μ of a 1% anti-HCG immobilized solid phase microparticle dispersion in a glass test tube at 25°C for 6 hours with shaking.
The particles were washed three times with PBS by centrifugation at 000 rpm to obtain particles in which HCG was bound via anti-HCG.

0.02% polystyrene latex (Gestate Slide Eiken) with a particle size of approximately 0.2 μm sensitized with anti-HCG antiserum
10 μ2 of the solution and 100 μ2 of this solid phase fine particle 1% dispersion
μ! were mixed in a glass test tube and allowed to react at 25°C overnight. After the reaction, add 32.5 m-g of PB and heat at 3000 r.
P'm for 10 minutes, the solid phase was sedimented, the supernatant microparticle dispersion was taken, and the intensity of scattered light was measured in the same manner as in Example 1, and the number of microparticles was determined from the intensity.

FIG. 4 shows the relationship between the (20) HCG concentration and the scattered light intensity in the range of HCG 105 U/, 8 to 10 U/β.

[Brief explanation of the drawing]

Figures 1 to 4 show the measurement results of Examples 1 to 4, respectively. Figure 1 shows the measurement of anti-BSA antibodies,
The figure shows HCG measurements. Patent applicant Azuma Shi Co., Ltd. Tomi 1 time 1 BsA pile skin (ng/ml) ¥52 Figure HCG crush degree (u/I) Figure 3 HCG concentration (u/I) Tomi 41!1 HCG concentration ( u/I)

Claims (2)

[Claims] (1) A biologically active analyte in a solution was reacted with a solid phase on which a binding partner that specifically binds to the analyte was immobilized, and the solid phase was separated from the reaction mixture. After that, the solid phase is reacted with a substance that has the property of specifically binding to the analyte and is labeled with a labeling agent, and then the analyte is measured by measuring the labeling substance remaining in the liquid phase. In the method of
- A method for measuring biologically active substances, characterized by using microparticles of 5 μm. (2) One meter consisting of fine particles with a particle size of 0.06 μm to 3 μm
Labeling agent for immunoassay.