JPS5860256A - Measurement of biologically active substance and label agent used therefor - Google Patents

Abstract

PURPOSE:To enable a safe and highly sensitive measurement in place of the RIA method by employing fine particles of an organic high polymer as label agent in a method of measuring biologically active substance using fine particles. CONSTITUTION:A label substance (active fine particles) labeled by fine particles 0.03-3mum, especially 0.1-0.8mum in the particle size is used to measure substance in place of that labeled by a radioactive isotope or enzyme in the competitive methods. When a fixed amount of active fine particles are caused to react to a solid phase where a bond partner reacted with a test object is solidified, the active fine particles bonded with the solid phase decrease as the quantity of substance in a solution of the object solution increases, while those left in the liquid phase increase. The substance to be measured can be measured quantitatively by counting the active fine particles left in the liquid phase. The solid phase herein used is preferably separable from the active fine particles in the shape or quality, for example, organic high polymer compound such as polystylene. The fine particles serving as carrier of the active fine particles should be uniform in the particle size and shape and made of preferably a hydrophobic or hydrophilic organic high polymer and that of both natures.

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, the method described above, which utilizes antigen-antibody reactions to quantitatively measure trace components, has 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, a labeled immunoassay method in which the antigen is labeled and the antigen or antibody is measured, and in addition, the antigen or antibody is immobilized on synthetic polymer particles.
Antibodies and antigens can be measured by observing the degree of aggregation of fine particles on a glass plate or microplate due to the presence of antibodies and antigens, and by measuring changes in light transmittance due to aggregation with a spectrophotometer. Methods to do this are also being developed.

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 related to 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 gIA does have a measurement sensitivity comparable to RIA for some analyte substances, it does not necessarily have the same sensitivity as RIA for all substances, and the reagents are relatively expensive. For these reasons, it has not yet surpassed the RIA method.

In general, immunoassay methods represented by the R and IA methods basically have two
It is broadly divided into methods. One is the competition method and the other is the so-called sandwich method. What is competition law?
When a measurement sample solution containing a substance to be measured, which is an antigen or an antibody, is mixed with a known concentration of the substance to be measured, which has been previously labeled with a labeling agent such as a radioactive isotope, and an antibody or antigen is mixed therein and reacted. An antigen-antibody complex is formed, but the free substances that have not yet formed a complex include labeled and unlabeled analyte substances, and the ratio can be measured. This method measures the substance to be measured in a sample. Another method, the sandwich method, is a two-step measurement method. In other words, a binding partner that specifically binds to the analyte
After a step of preparing a solid phase with immobilized substances and reacting it with the analyte, the solid phase is separated from the liquid phase, and in the next step, a substance that can specifically react with the analyte is extracted. The analyte is measured by reacting the labeled binding substance obtained by labeling with a radioactive isotope with the analyte on the solid phase and measuring the labeled substance in the solid or liquid phase. It's a method.

Compared to the sandwich method, the competition method is easier to operate and moreover, it is possible to measure even if there is only one antigenic site of the substance to be measured, whereas the sandwich method is easy to use when there is only one antigenic site. is impossible to measure.

Therefore, competition law is more commonly used in current RIA law.

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 microparticles in a competitive method. They discovered a measurement method and arrived at the present invention.

That is, the present invention combines a biologically active analyte in a sample solution and an analyte labeled with a known amount of a labeling agent by immobilizing a binding partner that specifically binds to the analyte. In a method of measuring a substance to be measured by competitively reacting with a solid phase that has been converted into a solid phase, and then measuring the labeling substance remaining in the liquid phase, a particle size of 0.03 μm or more is used as a labeling agent.
In a method for measuring biologically active substances characterized by using fine particles of 6 μm, particles with a particle size of 0.03 μm are used as a labeling agent.
The present invention relates to a method for measuring biologically active substances characterized by using fine particles of ~3 μm, and a labeling agent used therein.

The feature of the present invention is that instead of using a labeling substance in which the substance to be measured is labeled with a radioactive isotope or an enzyme in the competitive method, particle size is reduced to 0.
．． The reason is that a labeling substance labeled with fine particles of 06 μm to 3 μm (hereinafter abbreviated as active fine particles) was used. When a certain amount of active microparticles is reacted with a solid phase on which a binding partner is immobilized, either simultaneously with the analyte or with the analyte reacted first, the analyte in the analyte solution is measured. As the amount of active particles increases, the amount of active particles that bind to the solid phase decreases, and conversely, the amount of active particles that remain in the liquid phase increases. 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 of measuring the intensity of scattered light generated by irradiating the active fine particle dispersion with light, a method of measuring transmitted light in the same way,
There is a method of counting the number of active particles 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 thicker than the active particles, or even if the solid phase is made into fine particles, the particle size is larger than the active particles and the membrane is formed. , 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, polymethacrylamide, poly-N-vinylpyrrolidone, polyvinyl alcohol, poly( Crosslinked hydrophilic polymers such as poly(2-oxyethyl acrylate), poly(2-oxyethyl methacrylate), poly(2,3-dioxypropyl acrylate), poly(2,3-dioxypropyl methacrylate), polyethylene glycol methacrylate, etc. The solid phase can be in the form of a plate, test tube, microplate, etc., but the surface area can be easily increased by using fine particles or filaments as the solid phase. I can do it.

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 immobilized with carbodiimide on a solid phase that has amino or carboxyl groups as functional groups through covalent bonding. A substance with an amine group can be covalently immobilized with glutaraldehyde on a solid phase with an amino group, and a substance with an amino group can be covalently immobilized with cyanogen bromide on a solid phase with a hydroxyl group. can be converted into 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 can specifically bind to the analyte. For example, when the substance to be measured is an antigen, an antibody, a hormone, an antigen-antibody complex, a saccharide, an immunoglobulin, a lymphoid complex, a complement, etc., the order is as follows: antibody, antigen, hormone receptor, rheumatoid factor, lectin, protein A1. The combination with the lymphokine receptor, the complement receptor, etc. can be a combination of the analyte and the binding partner, respectively.

In order to obtain measurement accuracy, it is preferable that the fine particles serving as carriers for the active fine particles that cause the solid phase to react competitively with the substance to be measured in the specimen have uniform particle size and shape. From the viewpoint of reaction efficiency, the smaller the particle size, the better, and it is preferably at least a particle size that causes Brownian motion, that is, 3 μm or less. However, even if the particle size is too small, it is not suitable for operational and measurement purposes, so if the particle size is 0.06 μm to 6 μm.
It is appropriate that the range is 0.1 μrrt-
o, a particle size of 8 μm is preferred.

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 methods for obtaining microparticles of uniform size and shape. In particular, emulsion polymerization is a polymerization method that can prepare fine particles with a uniform particle size in the range of 1 μm to 0.05 μm by adjusting the emulsifier and monomer concentration, and is a polymerization method suitable for obtaining the desired fine particles. be. The substance to be measured can be bound to the fine particles by physical adsorption or chemical bonding. The microparticles must have good dispersibility;
Furthermore, since there must be no bonding to the solid phase that does not involve the analyte, fine particles prepared by covalently bonding a binding substance to a hydrophilic organic polymer compound are particularly preferred, similar to the solid phase.

In the present invention, substances that can be measured are substances that have a biologically specific affinity as a countersubstance, and specifically include streptococcus, staphylococcus, diphtheriae, and salmonella. , antibodies against bacteria such as 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 components; viruses such as influenza, adenovirus, polyoma, measles, rubella, hepatitis, mumps, etc. and their constituent components and antibodies against them; xenoantigens such as polysaccharides, human albumin, ovalbumin and antibodies against them; insulin, thyroid Hormones such as hormones and chorionic gonadotropin; enzymes such as ribonuclease, taleatin phosphokinase, and asparaginase; antigens or receptors specific to fat ducts such as kidney cell membranes, liver cell membranes, α-fetoprotein, and OEA; binding of collagen and amyloid, etc. Tissue components; blood cell antigens such as red blood cells and platelets, or receptors; blood proteins such as fibrin and plasminogen; pathological globulins such as rheumatoid factor and 0-reactive protein; immune complexes; autoantibodies against cell membranes, etc. be.

After the solid phase reacts with the substance to be measured and the active particles, the number of remaining active particles that are not bound to the solid phase is measured. The measurement method may be any method that allows quantitative measurement of fine particles.

Among them, the method of measuring the intensity of light scattered by fine particles is
This is a method in which microparticles are irradiated with light of a wavelength similar to the diameter of the microparticles, and the scattered light with the same wavelength as the irradiated light is measured, which is called Mie scattering, and is a method that allows for highly sensitive measurements.

Some examples are shown below to facilitate understanding of the present invention.

Example 1 (Measurement of insulin) (Preparation of fine particles for solid phase) The fine particles used as the solid phase were glycidyl methacrylate, 2-oxy7, as described in Japanese Patent Application No. 55-43618.
Ethyl methacrylate and triethylene glycol dimethacrylate, 85.7: 9.5:
The fine particles mixed and polymerized at a molar ratio of 4.8 are aminated,
Prepared by hydrolysis, average particle size 4.3 μm
It is a hydrophilic fine particle.

(Preparation of anti-pig insulin antiserum solid phase) Aminated and hydrolyzed fine particles were activated with glutaraldehyde according to the method described in Japanese Patent Application No. 55-43618, and the pH was 7.
, 2 in 0.15M physiological phosphate buffer (PBs) to a concentration of 1%. Mix equal amounts of this dispersion and swine insulin antiserum (Miles), and!
After reacting at 10°C for 5 hours, 1% bovine serum albumin (hereinafter abbreviated as BSA) was added to the particle dispersion.
Added so that
After washing by centrifugation at 00 rpm, a B of 0.196
A microparticle solid phase immobilized with anti-porcine insulin antiserum was prepared by dispersing it in PBs containing SA.

(Preparation of microparticles for active microparticles) Glycidyl methacrylate, methacrylic acid, and ethylene glycol dimethacrylate were mixed in a molar ratio of 85:10:5, containing 0.1% sodium dodecyl sulfate and o, oi mol/l of ammonium persulfate. In addition to the aqueous solution, the aqueous solution with a 7-mer concentration of 10% (W/V) was reacted at 60°C for 22 hours in an argon gas atmosphere, and the resulting fine particles were subjected to amination and hydrolysis in the same manner as solid phase fine particles. Uniform fine particles having a particle size of 0.27 μm were prepared.

(Preparation of insulin-immobilized active microparticles) Insulin was immobilized according to the method for immobilizing anti-pig insulin IJ 7 antiserum onto solid phase microparticles. That is,
Equal amounts of 1% glutaraldehyde-treated fine particle dispersion and 4D units/- of butane insulin (N0VO) were mixed, reacted at 30°C for 2 hours, and then 1% BSA was added.
%, and continued the reaction at 60°C for 1 hour.
After washing the microparticles by centrifuging at 0,000 rpm for 30 minutes, they were dispersed in PBS containing 0.1% BSA to a particle concentration of 1% to prepare active microparticles immobilized with porcine insulin.

(Measurement of insulin) Porcine insulin 25μU/7.12.5μU/d,
Add 100 μt of anti-porcine insulin antiserum-immobilized solid phase fine particles to 100 μt of PBS solution containing 6.25 μU/g.
After adding /2 and reacting at 25°C for 2 hours, 1 of insulin-immobilized active fine particles (o, oichi dispersion) was added.
0 μt was added, and the reaction was further allowed to proceed overnight at 25°C. 2.5-PBS was added to the reaction solution, the solid phase and the active particles that had reacted with the solid phase were precipitated by centrifugation at 3000 rpm, and the fine particle dispersion that did not react with the solid phase was used as the supernatant. U
/degree. , the light scattering intensity of the active fine particle dispersion was measured by Aminco・
It was measured by irradiating 400 In of light using a Porman fluorophotometer. 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, it can be seen that constant t[K can be measured for insulin in the range of 25 μU/ml to 6.25 pU/ml.

Example 2 Measurement of 1B8A) (Preparation of anti-BSA antiserum-immobilized solid phase) A dispersion of glutaraldehyde-treated fine particles for solid phase prepared in the same manner as in Example 2 and anti-BSA antiserum (Mi
After mixing equal amounts of les) and reacting at 30°C for 6 hours, human serum albumin (hereinafter abbreviated as BSA) was added to the particle dispersion so that it was 1 g, and the reaction was further continued for 1 hour. After washing by centrifugation at 5000 rpm, the particles were dispersed in PBS containing 0.1% BSA to prepare anti-BSA antiserum-immobilized microparticles.

(Preparation of BSA-immobilized active fine particles) BSA was added to a 1% dispersion of active fine particles with a particle size of 0.27 μm that had been treated with glutaraldehyde and prepared in the same manner as in Example 1.
Add 10 μt of A to 1 g/ml and incubate at 30°C for 3
After reacting for an hour and washing by centrifugation at 110,000 rpm, the B
SA-immobilized active microparticles were prepared.

(Measurement of BSA) 10μg/mj of BSA! , 111g/ml, 10
0℃g/m/.

Add 100μ of a 1% dispersion of anti-BSA antiserum-immobilized solid-phase microparticles to 90μt of PB8 solution containing 10μ/−, and stir for 1 hour.
After reacting at 0°C, BSA-immobilized active microparticles were added, and the reaction was further carried out at 30°C overnight. 2.5-PBS was added to the reaction solution, and the active particles that were not bound to the surface phase were bound to the solid phase using a membrane with 1.2 μm diameter holes (Millipore filter-RA). It was separated from the occupied fine particles and dispersed in the same manner as in Example 1.

Ta.

Example 6 (Measurement of human chorionic gonadotropin (abbreviated as HOG))
) (Preparation of anti-HOG antiserum immobilized same phase) A glutaraldehyde-treated solid phase microparticle 1T dispersion prepared in the same manner as in Example 1 and anti-HOO antiserum 160
00 I U/d (Mi 1es) were mixed in equal amounts, and 3
After reacting at 0°C for 6 hours, BSA was further added to the particle dispersion so that the amount was 1 inch, and the reaction was further continued for 1 hour.
After washing by centrifugation at 000 rpm, 0.1% B
An anti-HOG antiserum-immobilized microparticle solid phase was prepared by dispersing it in pBs containing SA.

(Preparation of microparticles for active microparticles) Glycidyl methacrylate, ethylene glycol dimethacrylate sulfopropyl me! Acrylate sodium salts were mixed at a molar ratio of 88:IG+20 and added to an aqueous solution containing 0.125 mol/l of sodium dodecyl sulfate and 0.01 mol/l of ammonium persulfate to give a monomer concentration of 10% (W/L).
After reacting as an aqueous solution of V) at 60°C in an argon gas atmosphere for 6 hours, the generated fine particles were heated to 11,000 or
The particles were washed with a centrifugal operation of P, and were aminated and hydrolyzed in the same manner as the solid phase particles to prepare uniform particles with a particle size of 0.1 μm.

(Preparation of HOG-immobilized active fine particles) According to the method for immobilizing BSA on solid phase fine particles, HC
G was immobilized. That is, HOG (S
igma) at 6200IU/1nI! , and reacted at 30° C. for 3 hours, 88 particles were added to the particle dispersion so as to make 1 g, and the reaction was further continued for 1 hour. After washing by centrifuging at 15,000 rpm for 60 minutes, the particles were dispersed in PBS containing 0.1% BSA to prepare HCG-immobilized active microparticles.

(Measurement of HOG) 10-fold dilution series of HOG from 104 to 102/l (
90 μt each) and 1% anti-HO in a glass test tube.
G-immobilized solid phase fine particle dispersion 5011t and 0.01%
10μ of HOG fine particle dispersion was added and reacted overnight at 25°C. Adding 2.5-PB8 to the reaction solution, Example 2
In the same manner as above, active particles that did not bind to the solid phase were separated using a membrane, the intensity of the scattered light was measured, and the number of particles was determined from the intensity.

υ υ H in the range of HOG1o'/l to 102mu/l
FIG. 3 shows the relationship between CG concentration and scattered light intensity.

[Brief explanation of the drawing]

Figures 1 to 3 show the measurement results of Examples 1 to 3, respectively. Figure 1 shows the measurement of insulin, Figure 2 the measurement of BSA, and Figure 3 the measurement of the sword. shows. Patent applicant Higashi Shi Co., Ltd. Figure 1 Insulin immersion and 7U/mrll) Ko 2 ward (xlO7 ko/,) BSA concentration (J hawk) Tori 3 (xlo' duck ward) Hccr concentration 7! (v/1.)

Claims (2)

[Claims] (1) Biologically active analyte in the sample solution;
A known amount of the analyte labeled with a labeling agent is reacted with a solid phase on which a binding partner that specifically binds to the analyte is immobilized, and then the labeling substance remaining in the liquid phase is measured. A method for measuring a biologically active substance, characterized in that fine particles having a particle size of 0.03 μm to 3 μm are used as a labeling agent. (2) A labeling agent for competitive immunoassay consisting of fine particles with a particle size of 0.03 μm to 3 μm.