JPH0862214A - Method for measuring substance in vivo - Google Patents

Abstract

PURPOSE: To obtain a highly sensitive and highly reliable measurement result in a short time without being affected by a measurement-interfering substance in a humor even when a humor specimen is measured without pretreatment. CONSTITUTION: Reagents herein used are a magnetic particle supporting a substance to be coupled to a substance to be measured, a non-magnetic coloring particle and a magnetic particle to be coupled to a measurement interfering substance. A humor is mixed with the reagents to remove the magnetic particle and the substance coupled thereto by magnetization. Then, the amount of the non-magnetic coloring particle left is measured.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention is a method for measuring an in-vivo substance in an immune reaction using magnetic particles and non-magnetic particles, which can be measured using a body fluid sample. The present invention relates to a measurement method capable of obtaining highly sensitive and highly reliable measurement results in an extremely short time without being affected by the measurement interfering substances.

[0002]

The use of magnetic particles in immunoassays involving the step of reacting an antigen with an antibody is disclosed in US Pat. No. 4,177,253. In addition, an insoluble carrier particle is loaded with an antibody or an antigen, and the loaded antibody or antigen is reacted with the antigen or the antibody or a mixture thereof in a liquid medium to measure the degree of change of the reaction product. In the method of measuring an antibody, a reaction is carried out using magnetic particles and non-magnetic particles as carrier particles, and then a magnetic field is applied to the reaction mixture to separate it from a reaction mixture containing unreacted magnetic particles and the like. A measurement method for qualitatively or quantitatively determining the antigen or antibody in a liquid medium by detecting the remaining non-magnetic particles is disclosed in, for example, Japanese Patent Application Laid-Open No. 1-193647.

[0003]

However, the accuracy and reliability of the measurement could not be expected unless the sample used in the measuring method in the prior art as described above is an in-vivo substance from which the measuring interfering substance is removed. .

That is, the sample used for measuring the substance in the living body must be measured with high sensitivity, high accuracy and promptly, without undergoing a pretreatment process such as serum separation.
For example, red blood cells in whole blood without going through processes such as serum separation
White blood cells, platelets, hemoglobin, bilirubin, lipoprotein, etc. became measurement interfering substances, and highly sensitive measurements were impossible. Proceeding with a pretreatment process such as serum separation to remove interfering substances makes rapid measurement difficult. Therefore, an object of the present invention is to solve the above-mentioned problems in the measurement of the prior art and to develop a method for rapid qualitative or quantitative measurement of an in-vivo substance having high sensitivity and high reliability.

[0005]

Means for Solving the Problems The present invention includes the following (1) to
Provided is a method for measuring an in-vivo substance, which is characterized by sequentially performing the step (4). (1) A step of mixing particles (a) to (c) below with a body fluid (a) Magnetic particles carrying a substance that binds to a measurement-interfering substance in the body fluid by an immune reaction (b) In the body fluid Magnetic particles carrying a substance that binds to the substance to be measured by an immunological reaction (c) Colored particles that do not have magnetic substances that carry a substance that binds to the substance to be measured in a body fluid by an immunological reaction (2) In the mixture Step of applying a magnetic field (3) Step of removing the complex bound to magnetic particles by immunoreaction and particles of unreacted magnetism by magnetism (4) The amount of colored particles not magnetized is 350 nm or more The step of qualitatively or quantitatively measuring the substance to be measured in the body fluid without being affected by the measurement-interfering substance in the body fluid by measuring the absorbance at.

Here, the magnetic particles are one or more polymers of polystyrene or (meth) acrylic acid ester having an iron oxide-based ferrite coating layer on the surface thereof, and have a diameter of 0.1 to 5 or even 0.2. It is preferably ˜3 μm.

Also, the body fluid is whole blood, the measurement-interfering substance is red blood cells, and the colored particles are one or more of white, black, red, blue and yellow of a polymer having a diameter of 0.1 to 5 μm, and these. It is preferable that the colored particles are one or more kinds selected from the mixed color, and the absorbance measurement wavelength is 350 to 1000 nm.

The object to be measured by the measuring method of the present invention is an in-vivo substance such as human or animal, and a body fluid such as blood or urine having this substance is measured. However, in body fluids, there may be in-vivo substances such as erythrocytes, hemoglobin, and bilirubin, or drugs such as phenobarbital, phenytoin, digoxin, imipramine, theophylline, penicillin, etc. ,
It adversely affects the measurement accuracy and becomes a measurement interfering substance.

The substance to be measured in the present invention is a specific antigen or antibody as an in-vivo substance contained in the body fluid to be measured, and the following can be exemplified.

Antigens; IgG, IgA, IgM, Ig
E, albumin, HCG, AFP, cardiolipin antigen, blood group substance, concanavalin A, DNT, prostaglandin, CRP, HBs, human growth hormone, steroid hormone, CEA, IgD, Lp (a), Apo
-AI, Apo-AII, Apo-CII, Apo-CII
I, Apo-B, Apo-E, hemoglobin and the like. Antibodies; albumin antibodies such as anti-albumin antibody, anti-AFP antibody; anti-HCG antibody, anti-IgG antibody, anti-IgA antibody, anti-IgM antibody, anti-IgE antibody, anti-IgD antibody, anti-C
Globulin antibodies such as RP antibody; hormone antibodies such as anti-human growth hormone antibody, anti-steroid hormone antibody; anti-DNT antibody, anti-prostaglandin antibody, anti-human coagulation factor antibody, anti-HBs antibody.

In the measuring method of the present invention, the following (a) to (c)
Of particles are used. (A) Magnetic particles carrying a substance that binds to a measurement-interfering substance in a body fluid by an immune reaction (b) Magnetic particles carrying a substance that binds to a substance to be measured in a body fluid by an immune reaction (c) ) Colored particles that do not have magnetism and carry a substance that binds to the substance to be measured in body fluid by an immune reaction

The non-magnetic particles used in the present invention are
Organic polymeric substances and inorganic substances can be used. As the organic polymer substance, there are latexes of organic polymer substances obtained by emulsion polymerization such as gelatin particles, polystyrene, and styrene-based polymer particles such as styrene-butadiene copolymer, and polystyrene latex and polyvinyltoluene latex are useful. is there. Also, one or more polymers of polystyrene or (meth) acrylic acid esters can be used.

As the (meth) acrylic acid esters,
For example, 2-hydroxyethyl (meth) acrylate, 2-hydroxypropyl (meth) acrylate, 1-methyl-2-hydroxyethyl (meth) acrylate, glycerol monomethacrylate, 2-acrylamido-2-methylpropanesulfonic acid , 2-sulfoethyl methacrylate,
Acid phosphoroxyethyl methacrylate, 3-chloro-2-acid phosphoroxypropyl methacrylate, acid phosphoroxypropyl methacrylate, ethyl (meth) acrylate, n-butyl (meth) acrylate, i-methacrylate acrylate Butyl, 2-ethylhexyl (meth) acrylate, lauryl methacrylate, cyclohexyl methacrylate, (meth) acrylic acid amide, N-methylolacrylamide, N-butoxymethylacrylamide,
Glycidyl (meth) acrylate, methylglycidyl (meth) acrylate and the like can be used.

The particle size is not particularly limited, but 0.01 to 1
00 μm and more preferably 0.1 to 30 μm. The reason for this is that particles with good floating properties that do not settle in a short time in an aqueous solution are preferable for immune reaction. Particles that have no magnetism
A colorant is added to obtain colored particles. Preferably 350-
A colorant whose absorbance can be measured at a wavelength of 1000 nm is used.

The magnetic particles to be used in the present invention are obtained by incorporating iron or magnetic iron oxide into the above-mentioned organic polymer substance or inorganic substance particles, or by coating the organic polymer substance or inorganic substance particles as a core with a ferrite coating. Then, ferrite-coated particles are formed.

In the present invention, the magnetic particles obtained as described above may be further treated with a polymer before use. As the polymer compound used for the polymer treatment, for example, silane, nylon, polystyrene or the like can be used.

Each of these particles carries a substance that immunologically binds to the substance to be measured in the body fluid or the substance that interferes with the measurement. The substance that binds immunologically is an antibody against the substance to be measured or the substance that interferes with the measurement when the substance is a specific antigen, etc. It is an antigen or the like for this.

The above-mentioned method of supporting a substance that binds immunologically to the particles is common to both particles having or not having magnetism, and the antibody or antigen is physically adsorbed or chemically Carry it. The physical adsorption method is carried out by reacting the particles with an antigen or an antibody in an appropriate buffer solution. The buffer solution used in this reaction includes a phosphate buffer solution, a Tris-hydrochloric acid buffer solution,
For example, carbonate buffer solution. The reaction can easily proceed by mixing both at room temperature to obtain the desired product. Further, as the chemical bonding method, a carbodiimide method or a glutaraldehyde method in the so-called peptide bonding method can be adopted.

As the antibody to be carried, a polyclonal antibody or a monoclonal antibody is used, and its form is
γ-globulin, IgG, IgM, F (ab ′) 2, F
It may be any of ab 'and the like. When an antigen is used as a carrier, cell debris, hapten, antigen protein, immune complex, natural or synthetic high molecular antigen, etc. are used. Generally, the amount of the antigen or antibody to be carried varies greatly depending on the type of colored particles, but in the general range, it is 0.001 mg / ml to 20 mg / ml, preferably 0.005 mg / ml to 5 mg / ml. Selected from the range.
The particles supporting the substance to be bound by the immune reaction are dispersed in a water solvent such as a buffer solution so as to have a concentration of 0.01 to 10% by weight and used as latex particles as a suspension.

In the measuring method of the present invention, the particles (a) to (c) described above are mixed with the body fluid as the first step (1).
Magnetic particles carrying a substance that immunologically binds to the substance to be measured when the amount of the non-magnetic colored particles (c) carrying a substance that immunologically binds to the substance to be measured is 1 volume (B) is used in the range of 1/4 to 4 times the capacity. Further, the magnetic particles (a) carrying a substance that immunologically binds to a substance that interferes with the measurement are 1/10 to 10
It is preferably used in a range of 10 times the capacity.

The mixing order of the particles (a) to (c) and the body fluid is not particularly limited, but the particles (a) having magnetism,
The mixture of (b) and the non-magnetic particles (c) may be mixed with the body fluid, or the non-magnetic particles (c) and the body fluid may be mixed with each other and then the magnetic particles ( You may mix with a) and (b). Particularly preferred is a method in which particles (a) and (b) having magnetism are mixed with particles (c) not having magnetism, and then the mixture is mixed with a body fluid. The sample may be diluted with a buffer or the like, if necessary.

The mixing is usually carried out at room temperature, but it may be carried out by heating at 30 to 40 ° C. to improve the immune reaction or cooling at 4 to 20 ° C. to maintain the stability of the reagent.

The step (2) of the measuring method of the present invention is a step of applying a magnetic field to the mixture obtained in the step (1). The strength of the magnetic field is not particularly limited, but the total amount of the mixture is 2
In the case of 00 μl, it is 1,000 to 10,000 gauss. Commercially available magnetic flux collector, such as GATHER manufactured by Nippon Paint Co., Ltd.
IN or the like may be used.

Next, in the third step (3), a complex of unreacted simple particles (a) and (b) having magnetism and particles (c) or substances bound to these by immunoreaction is collected by magnetizing. Remove. The step (3) is not particularly limited, but is preferably performed for 10 seconds to 5 minutes.

In the step (4), the absorbance of the uncollected colored particles (c) is measured at a wavelength of 350 nm or more, preferably at a wavelength of 500 to 700 nm. The measurement is preferably carried out at a wavelength in the range where the dye of the colored particles exhibits absorption. The amount of the remaining colored particles (c) that have not been magnetized depends negatively on the amount of the measurement object in the sample.

Generally, a calibration curve is prepared by measuring the absorbance of a sample having a known concentration of a target object to be measured, and a calibration curve is prepared in advance. The concentration of the target substance is quantified.

An example of measuring the amount of CRP (C-reactive protein) in whole blood using the method of the present invention will be described with reference to the drawings. CRP is β-globulin in serum, which is increased in the case of inflammatory diseases involving tissue destruction.

When whole blood is measured as a body fluid as shown in FIG. 1, CRP as a substance to be measured and erythrocytes, hemoglobin, etc., which act as a measurement interfering substance in this case, exist in the whole blood. The reagent used for the measurement is anti-human red blood cell rabbit-derived antibody-bearing magnetic particles (a), which are magnetic particles (a) carrying a substance that immunologically binds to the measurement-interfering substance in the body fluid, As magnetic particles (b) carrying a substance that immunologically binds to the substance to be measured,
Anti-human CRP goat-derived antibody-supported magnetic particles (b) and non-magnetic colored particles (c) carrying a substance that immunologically binds to a substance to be measured in a body fluid are used as anti-human CRP.
The goat-derived antibody-carrying colored particles (c) are mixed and used.

When whole blood is mixed with a reagent and a magnetic field is applied, it is magnetized as shown in FIG. The particles collected at this time are
(A) Particles and immunocomplexes of (a) and measurement-interfering substances, (b) particles and CRP bound to them, (b) particles and (c) particles bound to CRP. The particles remaining without being magnetized are anti-human CRP goat-derived antibody-bearing colored particles that did not bind to CRP.

FIG. 2 shows a schematic diagram of a calibration curve obtained by measuring the absorbance at a wavelength of 570 nm for blood having a known CRP concentration. It is possible to measure the components in blood directly without sample pretreatment such as serum separation, rapid measurement is possible, and it is useful for bedside and emergency measurement. Especially in the case of bedside measurement, whole blood can be directly used as a sample, which is useful.

The method of the present invention is an immunoassay method using magnetic particles, but it can also be applied as an assay method such as the EIA method and the luminescence method.

[0032]

The present invention will be described below with reference to examples, but the present invention is not limited thereto. (Example 1) [Preparation method of reagent] Preparation method of anti-CRP sensitized colored particles (c) 0.2 μm colored particles (styrene polymer colored particles prepared by suspension polymerization method) have a solid content of 1%. So 1 ml
1% solids in 20 mM phosphate buffer (pH 7.4)
It was dispersed so that Further, 0.25 mg of anti-human CRP goat-derived antibody was added, and the mixture was stirred and then sensitized in the refrigerator for 5 days. Then, centrifuge (12000 rpm × 20
min) to remove the supernatant and remove the separated colored particles by 1
This operation was repeated 3 times by redispersing in 20 ml of a 20 mM phosphate buffer solution (pH 7.4) containing 1% bovine serum albumin.

Preparation Method of Anti-CRP Sensitized Magnetic Particles (b) Magnetic particles of 2 μm (prepared by a ferrite coating method with a styrene polymer prepared by a suspension polymerization method) have a solid content of 1%.
1 ml of 20 mM phosphate buffer (pH 7.
4) dispersed in. Further, 0.25 mg of an anti-human CRP goat-derived antibody was added, and the mixture was stirred and then sensitized in a refrigerator for 3 days. Then, the supernatant was removed using a magnetic separation device, and the separated magnetic particles were redispersed in 1 ml of a 20 mM phosphate buffer solution (pH 7.4) containing 1% bovine serum albumin, and this operation was repeated 3 times.

Preparation Method of Anti-Human Erythrocyte Sensitized Magnetic Particles (a) Magnetic particles of 2 μm (same as above) were added to 1 ml of 20 mM phosphate buffer (pH 7.
4) dispersed in. Further, 0.25 mg of anti-human red blood cell rabbit-derived antibody was added, and the mixture was stirred and then sensitized in a refrigerator for 3 days. Then, remove the supernatant using a magnetic separator,
The separated magnetic particles were redispersed in 1 ml of a 20 mM phosphate buffer solution (pH 7.4) containing 1% bovine serum albumin, and this operation was repeated 3 times.

[Selection of measurement wavelength] The absorption wavelength of the colored particles is detected by scanning the wavelength. The absorption wavelength of the dye of the colored particles was used as the measurement wavelength.

[Operation Method] 1. The anti-CRP sensitized colored particles (c), the anti-CRP sensitized magnetic particles (b) and the anti-human red blood cell sensitized magnetic particles (a) were 1:
The particles were mixed at a ratio of 2: 1 and used as a particle preparation reagent. 2. CRP standard diluted to various concentrations (25.3 mg /
dl) was mixed with blood to obtain a sample. 1 μl of the sample and 200 μl of the particle preparation reagent were added to a 3.96-well microplate, mixed, and reacted at room temperature for 5 minutes. 4. Next, a magnetism collecting device GATHERIN (manufactured by Nippon Paint Co., Ltd.) was inserted into a microplate, and an immune complex of magnetic particles (b) and colored particles (c) bound via CRP antigen, magnetic particles (a bound via erythrocytes). ), The immune complex and the unreacted magnetic particles (a) and (b) are magnetized. 5. The concentration of the remaining colored particles (c) was measured with a microplate reader at a wavelength of 570 nm to measure the absorbance,
A calibration curve was created.

Comparative Example 1 [Preparation Method of Reagent] Preparation Method of Anti-CRP Sensitized Colored Particles (c) 0.2 μm colored particles are added to 1 ml so that the solid content becomes 1%.
1% solids in 20 mM phosphate buffer (pH 7.4)
It was dispersed so that Further, 0.25 mg of an anti-human CRP goat-derived antibody was added, and the mixture was stirred and then sensitized in a refrigerator for 3 days. Then, centrifuge (12000 rpm × 20
min)) and the separated latex particles were redispersed in 1 ml of a 20 mM phosphate buffer (pH 7.4) containing 1% bovine serum albumin, and this operation was repeated 3 times.

Preparation Method of Anti-CRP Sensitized Magnetic Particles (b) 2 μm of magnetic particles were added to 1 ml of 2 so that the solid content was 1%.
It was dispersed in 0 mM phosphate buffer (pH 7.4). Further, 0.25 mg of an anti-human CRP goat-derived antibody was added, and the mixture was stirred and then sensitized in a refrigerator for 3 days. After that, the supernatant is removed using a magnetic separation device, and the separated magnetic particles are separated by 1 m.
This operation was repeated 3 times by redispersing in 1 mM 20 mM phosphate buffer (pH 7.4) containing 1% bovine serum albumin.

[Selection of Measurement Wavelength] The absorption wavelength of the colored particles is detected by scanning the wavelength. The absorption wavelength of the dye of the colored particles was used as the measurement wavelength.

[Operation Method] 1. The anti-CRP sensitized colored particles (c) and the anti-CRP sensitized magnetic particles (b) were mixed at a ratio of 1: 2, and this was used as a particle preparation reagent. 2. CRP standard diluted to various concentrations (25.3 mg /
dl) was mixed with blood to obtain a sample. To a 3.96-well microplate, 1 μl of the sample and 200 μl of the particle preparation reagent were added and mixed, and the mixture was reacted at room temperature for 5 minutes. 4. Next, a magnetizer GATHERIN (manufactured by Nippon Paint Co., Ltd.) was inserted into a microplate, and an immune complex of magnetic particles (b) and colored particles (C) bound through a CRP antigen, and magnetic particles (b) that did not react Collected magnetism. 5. The concentration of the remaining colored particles (c) was measured with a microplate reader at a wavelength of 570 nm to measure the absorbance,
A calibration curve was created.

In Example 1, since the anti-human erythrocyte antibody-sensitized magnetic particles (a) were contained in the particle preparation reagent, the absorbance decreased as the antigen concentration increased even when blood was used as the sample, The antigen concentration could be measured accurately. In Comparative Example 1, no decrease in absorbance was observed with an increase in antigen concentration.

[0042]

According to the method of the present invention, in an immunoassay method using magnetic particles and non-magnetic particles, a magnetic substance carrying a substance that immunologically binds to a measurement interfering substance in a liquid is contained. By adding the particles described above, even if a body fluid is used as a sample, the measurement can be performed quickly and with high sensitivity without being affected by the measurement-interfering substance in the body fluid.

[Brief description of drawings]

FIG. 1 is a schematic diagram illustrating a method of the present invention.

FIG. 2 is a schematic diagram showing an example of a calibration curve used in the present invention.

Claims (4)

[Claims] 1. A method for measuring an in-vivo substance, which comprises sequentially performing the following steps (1) to (4). (1) A step of mixing particles (a) to (c) below with a body fluid (a) Magnetic particles carrying a substance that binds to a measurement-interfering substance in the body fluid by an immune reaction (b) In the body fluid Magnetic particles carrying a substance that binds to the substance to be measured by an immunological reaction (c) Colored particles that do not have magnetic substances that carry a substance that binds to the substance to be measured in a body fluid by an immunological reaction (2) In the mixture Step of applying a magnetic field (3) Step of removing the complex bound to magnetic particles by immunoreaction and particles of unreacted magnetism by magnetism (4) The amount of colored particles not magnetized is 350 nm or more The step of qualitatively or quantitatively measuring the substance to be measured in the body fluid without being affected by the measurement-interfering substance in the body fluid by measuring the absorbance at. 2. The magnetic particles are one or more polymers of polystyrene or (meth) acrylic acid ester having an iron oxide-based ferrite coating layer on the surface thereof, and have a diameter of 0.2 to 3 μm. The method for measuring an in-vivo substance described. 3. The method for measuring an in-vivo substance according to claim 1, wherein the body fluid is whole blood, and the measurement-interfering substance is red blood cell, white blood cell, platelet, hemoglobin, bilirubin, or lipoprotein. 4. The colored particles have a polymer diameter of 0.1 to 5.
It is one or more kinds of colored particles selected from white, black, red, blue, yellow of 1 μm or more and a mixed color thereof, and the absorbance measurement wavelength is 350 to 1000 nm. The method for measuring an in-vivo substance according to 1.