JPH07306204A - Immunoassay - Google Patents

Abstract

PURPOSE:To realize a high sensitivity assay by dripping microparticles coupled with ligands dispersed into a liquid, a specimen containing an object to be assayed, and a ligand having to form an immune complex and then measuring the quantity of compositional substance therein. CONSTITUTION:Ligands dispersed into a liquid and ligand having a bonded labeled substance exhibit bonding properties specifically to an objective substance being assayed. The ligand includes antibodies, antigens, etc., and a monoclonal antibody is preferably employed. The microparticle includes red blood cell, kaolin, etc. The object being assayed includes any antigens and antibodies for normal immunoassays. The specimen containing the objective substance includes a humor, e.g. blood. The porous matrix includes a glass fiber filter having pore smaller than the microparticle of average particle size. The quantity of labeled substance in an immune complex is measured by means of a colorimeter or a fluorescent measuring apparatus.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to an immunoassay method.
More specifically, the present invention relates to a heterogeneous immunoassay method, which is a simple immunoassay method suitable for automating the measurement.

[0002]

2. Description of the Related Art Conventionally, in heterogeneous immunoassays, beads and test tubes have been used as solid phase carriers. Further, a measuring method using a porous matrix such as a glass fiber filter or a nitrocellulose filter as a solid phase (JP-A-3-233359, JP-A-2-108971 and JP-A-1-244368, JP-A-1-244368). 63-18659, JP 62-9.
8258, JP-A-62-98260, JP-A-61-173160, etc.) are known. Furthermore, a method of using fine particles as a solid phase, reacting the fine particles with a sample, and then capturing the mixture on a porous matrix to quantify the amount of immune complex (JP-A-63-238559 and JP-A-3-72261). Japanese Patent Laid-Open No. 63-250567
JP-A-62-98257, etc.) are known. Further, a measurement method in which fine particles bound to a ligand are bound to a porous matrix in advance as a solid phase (Japanese Patent Laid-Open No. 3-120467 and Japanese Patent Laid-Open No. 6-120647).
4-10176, JP-A-63-42471, JP-A-61-292059, and JP-A-62-1.
No. 94458, etc.) are known. The porous matrix used for these measurements is usually a cartridge integrated with a reaction tube or the like.

[0003]

In recent years, there has been a demand for a more simple and highly sensitive assay method in which the reaction operation can be automated in the immunoassay method. The conventional measurement method using beads or test tubes as the solid phase requires complicated operations such as moving and washing the solid phase, and is not suitable for automation.

When the porous matrix is directly used as a solid phase, a special technique is required for binding the ligand, and it is difficult to store the porous matrix to which the ligand is bound.
Further, there is a drawback that the measurement sensitivity is low. Furthermore, in automation, when measuring a plurality of items, there is the difficult problem of mechanically selectively moving different types of porous matrices.

In a measuring method in which fine particles are used as a solid phase, the fine particles are reacted with a specimen, and then the complex is supplemented on a porous matrix to quantify the amount of immune complex, there is an advantage that a common porous matrix can be used. Since it is necessary to prepare a reaction tube for reacting the fine particles with the sample and a step of moving the reaction liquid to the matrix, a complicated movement of the device is required for automation. Further, when the porous matrix is formed into a cartridge shape, it is necessary to additionally provide a reaction tube portion inside the cartridge.

In the case of a measuring method in which fine particles bound to a ligand are bound to a porous matrix in advance as a solid phase, it is difficult to store the porous matrix, and moreover, it is automated to measure a plurality of items. In this case, there is a difficult problem of mechanically selectively moving a porous matrix having different fine particles bound thereto.

[0007]

Generally, it is considered to be advantageous in terms of immunoreactivity to react the fine particles with the sample in a separate reaction tube in advance, but the present inventor is keen to solve the above problems. As a result of the investigation, they found that high immunoreactivity was exhibited by sequentially performing a specific series of steps, and arrived at the present invention. That is, the present invention is an immunoassay method for forming an immune complex on a porous matrix and quantifying the amount of the immune complex, wherein at least the following steps are sequentially performed. Step 1) A step of dropping the fine particles (b), to which the ligand (a) is bound, dispersed in a liquid onto the porous matrix (c) and capturing them on the porous matrix (c). Step 2) A step of dropping the specimen (e) containing the substance (d) to be measured on the porous matrix to form the immune complex (f). Step 3) A step of forming the immune complex (i) by dropping the ligand (h) to which the labeling substance (g) is bound onto the porous matrix. Step 4) A step of measuring the amount of the labeled substance (g) in the immune complex (i) on the porous matrix.

In the measuring method of the present invention, as described above, the measurement can be carried out simply by dropping the reagent on the porous matrix, and after reacting the fine particles with the sample in a separate reaction tube in advance, the porous matrix From the measurement method of dripping
A measurement method suitable for extremely simple automation can be provided. Further, there is an advantage that it is not necessary to separately prepare a reaction tube section.

In the present invention, the substance (d) to be measured is not particularly limited as long as it is an antigen or antibody that can be usually measured by immunoassay. For example, AFP, CEA, CA19-9,
Tumor markers such as CA125, POA, ferritin;
TSH, LH, FSH, hCG, prolactin, hG
H, gastrin, somatostatin, glucagon, hormones such as insulin; IgG, IgM, IgA, Ig
Proteins such as E, IgD, TBG, CRP, β2-microglobulin; enzymes such as elastase, alkaline phosphatase, amylase, protease, lipase, ribonuclease, enolase; viruses such as hepatitis virus, AIDS virus and antibodies against viruses, etc. To be

Examples of the sample (e) containing the substance to be measured (d) include body fluids such as blood (including serum and plasma), lymph, saliva, urine, and extracts of feces and tissues derived from living bodies.

In the present invention, the ligands (a) and (h) have specific binding properties to the substance to be measured (d), and examples thereof include antibodies, antigens, lectins, protein A and the like. However, it can be freely selected within a range corresponding to the measurement target substance (d). A preferable example of the ligand is a monoclonal antibody against the substance to be measured (d). In this case, the ligand (a) and the ligand (h) are more preferably monoclonal antibodies that recognize different epitopes of the substance to be measured (d).

As the fine particles (b) used in the present invention, conventionally known fine particles can be used. For example, erythrocytes; inorganic fine particles such as kaolin and carbon; rubber particles in natural rubber latex; polymer particles in latex of organic polymer compounds such as polystyrene. Of these, preferred are the copolymers of polystyrene, styrene, and other monomers with monomers having functional groups such as amino groups, thiol groups, carboxyl groups, active ester groups, and aldehyde groups, and polyacrolein. Is mentioned. Particularly preferably, a copolymer of styrene and phenylmethyl sulfonium sulphate methacrylate having an active ester group, a copolymer of diethylene glycol dimethacrylate and N-acryloyloxysuccinimide, a copolymer of diethylene glycol dimethacrylate and 1-methacryloyloxybenzotriazole. It is a polyacrolein having a polymer and an aldehyde group.

The fine particles (b) may be single particles or secondary particles formed by combining a plurality of smaller fine particles (b1). The average particle diameter of the fine particles (b) is preferably within the range of 0.5 to 200 μm. Further, it is preferable that the distribution of particle diameters is narrow. When the average particle size of the fine particles (b) is large, the dispersibility in an aqueous solution is low, and the surface area per particle weight is low, so that the measurement sensitivity is low. On the other hand, if the average particle size is too small, the capturing efficiency with the filter is lowered and the final measurement sensitivity is lowered.

When the fine particles (b) are secondary particles composed of the fine particles (b1), the average particle diameter of the fine particles (b1) is 0.
It is preferably in the range of 1 to 5 μm. Various methods can be used to form the fine particles (b) depending on the properties of the fine particles (b1). Examples of the method of binding a plurality of fine particles (b1) include a method of changing the surface charge of the fine particles (b1) to agglomerate, a method of aggregating (b1) by adding a surfactant or a change in pH, and a fine particle ( b1) There may be mentioned a method of crosslinking the two. As a preferred example, as the fine particles (b1), those having a functional group such as an amino group, a thiol group, a carboxyl group, and an active ester group on the surface are used, and (b1) a method of binding each other with a crosslinking reagent, or a similar functional group. There is a method in which (b1) having a group is linked via a protein that is not involved in an immune reaction. Fine particles (b) Complexing fine particles (b1) per one
The number of can be various values depending on the conditions,
In order to make the particle size of the formed fine particles (b) uniform, the number of particles is remarkably small by centrifugation or the like.
Large items can be separated and removed.

For binding the fine particles (b) and the ligand (a), a conventionally known method can be used. For example, using a method by physical adsorption or using (b) having a functional group on the surface,
There is a method of binding the functional group of the ligand (a) with a cross-linking reagent. It is also possible to bind via a protein that does not participate in an immune reaction such as BSA, or indirectly bind using an antibody or avidin / biotin binding to the ligand (a).

In order to prevent non-specific adsorption, the thus obtained (a) -bound (b) is treated on the surface thereof with an appropriate protein, surfactant, etc. in the same manner as a usual carrier for immunoreaction. It is preferable to block with and use.

As the porous matrix (c) used in the measuring method of the present invention, any material can be used as long as it has a pore diameter smaller than the average particle diameter of the fine particles (b) used. Examples thereof include a glass fiber filter, a quartz fiber filter, a silica fiber filter, a nitrocellulose filter, a polyacetate filter, a filter paper, a porous Teflon membrane and a porous ceramic. B
/ F separation efficiency and time reduction,
A porous matrix having a high filtration rate is preferable, and a non-specific adsorption of proteins and the like is preferable. Preferred examples include filters made of glass fibers and porous Teflon membranes. To prevent non-specific adsorption, the porous matrix should be
It may be coated with sugar or polymer.

The porous matrix (c) is preferably used in the form of being incorporated in a suitable cartridge. More preferably, the filter is incorporated in the cartridge-shaped formation, and the cartridge-shaped formation has a liquid absorbing portion (j) which is in contact with the lower portion of the filter and absorbs excess reaction liquid and washing liquid. It is something that exists. Efficient B / F without using power by having the liquid absorbing part (j)
Separation is possible. Any material can be used as the material of the liquid absorbing part (j) as long as it absorbs and retains the reaction liquid and the cleaning liquid in a short time. For example, pulp fiber, filter paper, sponge, water-absorbent polymer and the like can be used alone or in combination. From the viewpoint of water absorption rate and retention capacity, the super absorbent polymer is preferably used alone or in combination with other materials.

In the present invention, the bond between the labeled substance (g) and the ligand (h) is prepared by a conventionally known method using the conventionally known labeled substance (g) and the ligand (h). You can Examples of the labeled substance (g) include isotopes (
¹²⁵ I), enzymes (peroxidase, alkaline phosphatase, β-galactosidase, luciferase, etc.), fluorophores (fluorescein, europium derivatives, etc.), luminescent substances (acridinium ester, N-aminobutyl-N-ethylisoluminol, etc.) Can be given.

In the present invention, as a method for measuring the amount of the labeled substance (g) in the immune complex (i) on the porous matrix (c), there is a conventionally known method depending on the labeled substance (g). Can be used. For example, when the label is an enzyme, a color-developing substrate is dropped on the porous matrix, and the color development on the matrix surface is measured by a colorimeter using an integrating sphere.
Examples include a method in which a fluorescent substrate is dropped and the fluorescence intensity on the matrix surface is measured by a reflection type fluorescence measuring device, and a method in which a luminescent substrate is dropped on the matrix surface and the emission intensity from the matrix surface is measured. When the labeling substance is a fluorescent substance, the fluorescence generated by irradiating the matrix with appropriate excitation light can be measured. When the labeling substance is a luminescent substance, the generated luminescence can be measured by adding an appropriate trigger.

The present invention is characterized in that steps 1) to 4) are sequentially carried out, but a washing step and a standing time for the reaction can be provided between the steps, if necessary. is there.

[0022]

EXAMPLES The present invention will be further described below with reference to examples, but the present invention is not limited thereto.

1) Preparation of anti-AFP antibody-bound single dispersion microparticles 2.5% polyacrolein microparticles aqueous dispersion (average particle size 0.8 μm, Matsumoto Microspheres-A-0.8,
1 ml of Matsumoto Yushi-Seiyaku Co., Ltd.) was washed twice with distilled water and centrifuged, and the washed microparticles were diluted with 1 mg / ml of phosphate buffer to 1 mg / ml of anti-AFP antibody (rabbit antibody, Dako) solution.
ml was added and the reaction was carried out at room temperature for 8 hours. 50 μl of 1 mg / ml dimethylamine-borane aqueous solution was added, and the mixture was reacted at room temperature for 2 hours. The microparticles were centrifuged, 1 ml of a phosphate buffer containing 0.1% bovine serum albumin was added, and after reacting for 2 hours, 0.5 mg of dimethylamine-borane was added and reacted for 1 hour. The particles were centrifuged, washed with 0.1% bovine serum albumin-containing phosphate buffer 3 times, and then washed with 0.1%.
The microparticles were suspended in 10 ml of bovine serum albumin-containing phosphate buffer to obtain an anti-AFP antibody-bound single dispersion microparticle suspension. Stored at 4 ° C until use. It was confirmed by observation with an electron microscope that the particles were in a monodispersed state.

2) Preparation of anti-AFP antibody-bound secondary particles 2.5% polyacrolein fine particle aqueous dispersion (average particle size 0.8 μm, Matsumoto Microspheres-A-0.8,
1 ml of Matsumoto Yushi-Seiyaku Co., Ltd.) was washed twice with distilled water and centrifuged, and the washed microparticles were diluted with 1 mg / ml of phosphate buffer to 1 mg / ml of anti-AFP antibody (rabbit antibody, Dako) solution.
ml was added and the reaction was carried out at room temperature for 8 hours. 50 μl of 1 mg / ml sodium borohydride aqueous solution was added, and the mixture was reacted at room temperature for 2 hours. The microparticles were centrifuged, 1 ml of a phosphate buffer containing 0.1% bovine serum albumin was added, and after reacting for 2 hours, 0.5 mg of sodium borohydride was added and reacted for 1 hour. The particles were centrifuged, washed with 0.1% bovine serum albumin-containing phosphate buffer 3 times, and then washed with 0.1%.
The fine particles were suspended in 10 ml of a bovine serum albumin-containing phosphate buffer solution to obtain an anti-AFP antibody-bound secondary particle suspension. 4 ° C
Saved until use. Observation by an electron microscope revealed that the fine particles had an average particle size of 5 μm.

3) Alkaline phosphatase labeled anti-AF
Preparation of P antibody To 1 ml of a phosphate buffer containing 1 mg of anti-AFP antibody (rabbit antibody, manufactured by Dako), 100 μl of 1 mg / ml 2-iminothiolane hydrochloride (2-IM) aqueous solution was added, and reacted at room temperature for 1 hour. did. The reaction solution was passed through a gel filtration column to remove unreacted 2-IM to obtain a thiol group-introduced anti-AFP antibody. 1 mg of a thiol group-introduced anti-AFP antibody and alkaline phosphatase (manufactured by Toyobo Co., Ltd.) in 1 ml of a phosphate buffer solution were added to 0.1 mg of the dicrosslinking reagent GMBS [N-
(Γ-maleimidobutyryloxy) succinimide: manufactured by Dojindo], 20 μl of dimethylformamide
Was added and reacted at room temperature for 8 hours. The reaction solution was passed through a gel filtration column, and a fraction having enzyme activity and antibody activity was used as an alkaline phosphatase-labeled anti-AFP antibody. It was diluted with a phosphate buffer containing magnesium chloride before use.

4) Preparation of glass filter for measurement A glass filter (GA200 manufactured by Toyo Roshi Kaisha, Ltd., retention particle size 0.8 μm) was immersed in a phosphate buffer containing 0.05% skim milk for 1 hour and then sufficiently air-dried. . Cut the filter into a circle with a diameter of 8 mm, diameter 10 mm, length 2
The glass filter for measurement was adhered to the upper part of a cylindrical water absorption filter of 5 mm to obtain a glass filter for measurement.

5) Standard AFP solution As the standard AFP solution used for the measurement, the standard AFP solution of the commercially available AFP measurement kit "Glazyme AFP-EIA TEST" (released by Wako Pure Chemical Industries, Ltd.) was used.

6) Method for measuring alkaline phosphatase activity The glass filter part of the measuring filter was transferred to a 96-well microplate, 10 μl of 1.2 mM 4-methylumbelliferyl phosphate solution was added dropwise, and a fluorometer (microplate) was used. The fluorescence intensity on the filter surface was measured with a reader MTP-32: manufactured by Corona Electric Co., Ltd., with excitation light 365 nm and measurement light 450 nm.

Example 1 50 μl of a suspension of anti-AFP antibody-bound single-dispersed fine particles or a suspension of anti-AFP antibody-bound secondary particles was dropped onto a glass filter for measurement, and then 50 μl of a standard AFP solution was dropped to 37
It was left at ℃ for 10 minutes. Further, 200 μl of a phosphate buffer containing 0.1% bovine serum albumin was added dropwise to wash the fine particles and the filter. Next, 50 μl of an alkaline phosphatase-labeled anti-AFP antibody solution was added dropwise and left at room temperature for 5 minutes. Further, 400 μl of a phosphate buffer containing 0.1% bovine serum albumin was added dropwise to wash the fine particles and the filter, and then the alkaline phosphatase activity on the filter was measured. The calibration curve for AFP measurement is shown in FIG.

[0030]

[Figure 1]

COMPARATIVE EXAMPLE 1 Monodisperse fine particles and standard AFP were first placed in another test tube 37
The alkaline phosphatase activity on the filter was measured in the same manner as in Example 1 except that the reaction was carried out at 10 ° C for 10 minutes. The calibration curve for AFP measurement is shown in FIG.

[0032]

[Fig. 2]

Comparative Example 2 This comparative example shows the measurement of AFP using a glass filter directly as a solid phase.

1) Preparation of a glass filter for measurement A glass filter (GA200 manufactured by Toyo Roshi Kaisha, Ltd., retention particle size 0.8 μm) was immersed in an acetone solution containing 0.5% γ-aminopropyltriethoxysilane for 1 hour and then removed. After washing with ionized water, it was immersed in a 5% glutaraldehyde aqueous solution for 1 hour. After washing with deionized water, 1 mg with phosphate buffer
The plate was immersed in an anti-AFP antibody (rabbit antibody, Dako Co.) solution diluted to 1 ml / ml for 1 hour. After washing with a phosphate buffer containing 0.85% sodium chloride, it was immersed in a phosphate buffer containing 0.1% bovine serum albumin and freeze-dried. This filter was cut out into a circular shape having a diameter of 8 mm, and was adhered to the upper portion of a cylindrical water-absorbing filter having a diameter of 10 mm and a length of 25 mm to obtain a measuring glass filter.

2) Measurement procedure Using the same labeled antibody and standard solution as in Example 1, measurement was carried out by the following procedure. 50 μl of the standard AFP solution was dropped on the glass filter for measurement and left at 37 ° C. for 10 minutes. Furthermore, 0.
200 μl of a phosphate buffer containing 1% bovine serum albumin was added dropwise to wash the filter. Next, 50 μl of an alkaline phosphatase-labeled anti-AFP antibody solution was added dropwise and left at room temperature for 5 minutes. Further, 400 μl of a phosphate buffer containing 0.1% bovine serum albumin was added dropwise to wash the filter, and then the alkaline phosphatase activity on the filter was measured.

3) Measurement Results A calibration curve for AFP measurement is shown in FIG.

[0037]

[Figure 3]

As can be seen from FIGS. 1 to 3, the measurement method according to the present invention has the same measurement sensitivity as the measurement method (Comparative Example 1) in which the fine particles and the sample are reacted beforehand in the measurement using the monodispersed fine particles. The sensitivity is higher than that of the measurement method (Comparative Example 2) using a filter in which a ligand is directly bound. Furthermore,
Higher sensitivity measurement was possible by using secondary particles. Further, the filter prepared in Comparative Example 2 has a temperature of 1 at room temperature.
When left for a week, the measurement count decreased to 50% immediately after preparation. When the fine particles used in Example 1 were previously dropped on a filter and dried and stored, the measurement count was lowered to 20% immediately after preparation when left for 1 week at room temperature. On the other hand, when the filter and the fine particles in Example 1 were stored as they were, no deterioration in performance was observed.

[0039]

INDUSTRIAL APPLICABILITY According to the present invention, there are the following effects in the immunoassay method for forming an immune complex on a porous matrix and quantifying the amount of the immune complex. Since the porous matrix is not directly used as the solid phase or the fine particles having the ligand bound thereto are not bound to the porous matrix in advance, it is possible to use a common porous matrix for measurement. No selection mechanism is needed. Moreover, since the ligand is not bound, the porous matrix is stable and easy to store. Since it is not necessary to react the ligand-bonded fine particles with the sample in advance, a reaction tube for that purpose is not necessary, and a reaction liquid transfer mechanism is also unnecessary. Further, when the porous matrix is formed into a cartridge shape, the attached reaction tube portion is not required, and therefore the cartridge can be downsized. By using the secondary fine particles, it is possible to measure with higher sensitivity. As described above, the present invention can provide a simple and highly sensitive immunoassay method suitable for automation.

[Brief description of drawings]

1 is a calibration curve for AFP measurement according to Example 1. FIG.

FIG. 2 is a calibration curve for AFP measurement according to Comparative Example 1.

FIG. 3 is a calibration curve for AFP measurement according to Comparative Example 2.

Claims (6)

[Claims] 1. An immunoassay in which an immune complex is formed on a porous matrix and the amount of the immune complex is quantified,
An immunoassay method characterized by carrying out at least the following steps in sequence. Step 1) A step of dropping the fine particles (b), to which the ligand (a) is bound, dispersed in a liquid onto the porous matrix (c) and capturing them on the porous matrix (c). Step 2) A step of dropping the specimen (e) containing the substance (d) to be measured on the porous matrix to form the immune complex (f). Step 3) A step of forming the immune complex (i) by dropping the ligand (h) to which the labeling substance (g) is bound onto the porous matrix. Step 4) A step of measuring the amount of the labeled substance (g) in the immune complex (i) on the porous matrix. 2. The immunoassay method according to claim 1, wherein the average pore diameter of the porous matrix (c) is smaller than the average particle diameter of the fine particles (b). 3. The fine particles (b) are smaller than the fine particles (b).
The immunoassay method according to claim 1 or 2, wherein 1) is a secondary particle formed by combining a plurality of particles. 4. The immunoassay method according to claim 1, wherein the porous matrix (c) is formed of glass fiber or a porous Teflon membrane. 5. The immunoassay method according to claim 1, wherein the ligand (a) and the ligand (h) are antibodies or antigens. 6. The porous matrix (c) is a porous matrix having a liquid absorbing portion (j) in contact with a portion for capturing the fine particles (b), and the liquid absorbing portion (j).
Is a polymer having at least a superabsorbent polymer as a constituent, The immunoassay method according to claim 1.