JPH08146001A - Immunoassay and apparatus - Google Patents

Abstract

PURPOSE: To qualitatively and quantitatively measure an antigen or antibody in a specimen by adding an enzyme-labelled antigen or antibody to aggregated magnetic particles judged to be positive to react with each other and measuring the activity of enzyme bonded to the magnetic particles. CONSTITUTION: In an indirect aggregation measuring method, magnetic particles to which an antigen or antibody is bonded and a specimen are mixed in a reaction container to be reacted and the magnetic particles in the container are sedimented by a magnet. The inner surface of the reaction container is inclined by 20-40 deg. and the length of the particles flowing out after 1-5min is measured. The specimen wherein the measured length of the particles does not reach definite length by the agglutination due to immunoreaction is judged to be positive. The magnetic particles obtained by removing a supernatant liquid from a particle soln. judged to be positive are washed to be mixed with an enzyme-labelled antigen (antibody) to be reacted therewith. Further, the magnetic particles are collected by a magnet and, after the unreacted enzyme- labelled antigen (antibody) is removed, the magnetic particles are washed to be subjected to B/F separation and, thereafter, the activity of enzyme bonded to the magnetic particles is measured using a chemoluminescent substrate. By this constitution, the object to be measured in a specimen can be measured qualitatively and quantitatively.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention mixes magnetic particles bound to an antigen or antibody with a specimen in a reaction vessel and causes magnetic particles to settle on the inner surface of the vessel by an external magnetic force, and the vessel is tilted at a predetermined angle. Indirect agglutination immunoassay for measuring the flow-out state of magnetic particles, and after reacting by adding an enzyme-labeled antigen or enzyme-labeled antibody to the agglutinated magnetic particles determined to be positive by the indirect agglutination immunoassay, the magnetic particles are An immunoassay for measuring an antigen or an antibody in a sample by continuously performing a chemiluminescent enzyme immunoassay in which the activity of a bound enzyme is measured using a chemiluminescent substrate, and immunity for performing this immunoassay. Regarding measuring device.

[0002]

2. Description of the Related Art Conventionally, the indirect agglutination immunoassay method has been widely put to practical use as a simple assay method for a large number of specimens, which uses particles bound with an antigen or an antibody as a reagent and does not use a special device for the measurement. In addition, the method of using magnetic particles for this particle is to measure the particle from the state in which the particle is forcibly settled by reacting the particle and the specimen and then the particle is forcibly settled, and then the container containing the particle is tilted at a predetermined angle to flow out the settling particle. In order to obtain the result, it is known as a simple measuring method that can obtain the result of determination of the presence or absence of immune reaction in a short time (Japanese Patent Laid-Open No. 3-14436)
(See No. 7). On the other hand, the enzyme immunoassay (EIA) is a quantitative method for obtaining a result by comparing a result obtained by measuring the activity of an enzyme bound to a solid phase by an immunoreaction with a calibration curve previously obtained from a sample having a known concentration. It is a measurement method that enables measurement (precision test).

[0003]

When performing quantitative measurement of a sample by the above-mentioned indirect agglutination immunoassay, a dilution series of the sample is formed and the measurement is performed based on the visual judgment result of each agglutination image. Only the measurement result of was obtained. Therefore, from such characteristics, the indirect agglutination immunoassay method is
It has been mainly used as a qualitative measurement (screening method) of a sample. In addition, the chemiluminescent enzyme immunoassay method that uses a chemiluminescent substrate for EIA is a method that can obtain the measurement result in a short time, but it requires the use of an expensive chemiluminescent substrate, and bind / free (B / F) separation. In addition, it is not a practically satisfactory method as a method for qualitatively measuring a large number of samples, such as using an automatic measuring device because complicated operations such as washing of the solid phase are repeated. Therefore, in order to perform qualitative measurement and quantitative measurement by the conventional method and obtain each result, first perform the qualitative measurement of the sample and obtain the result, and then repeat the complicated sample selection operation and dispensing operation from this result. Quantitative measurement was performed. Therefore, it takes a lot of labor and time to obtain quantitative results from many samples.

[0004]

Means for Solving the Problems As a result of intensive studies, the inventors of the present invention, in measuring an antibody or an antigen in a specimen, first mix the antigen or the magnetic particles to which the antibody is bound with the specimen,
After the magnetic particles are settled on the inner surface of the container by the external magnetic force, the container is tilted at a predetermined angle, and the indirect agglutination immunoassay is performed to measure from the flow-out state of the settled magnetic particles.
Then, a chemiluminescent enzyme for measuring the activity of the enzyme bound to the magnetic particles by using a chemiluminescent substrate by reacting by adding an enzyme-labeled antibody or an enzyme-labeled antigen to the aggregated magnetic particles determined to be positive in this indirect agglutination immunoassay. The present invention has been completed by finding an immunoassay method for carrying out the immunoassay method and an immunoassay apparatus for performing the immunoassay method.

In the indirect agglutination immunoassay method of the present invention, first, magnetic particles to which an antigen or an antibody is bound and a sample are placed in a reaction container and mixed to carry out a reaction, and a precipitation promoting device in which a magnet is arranged in the reaction container is used. This is a method in which the magnetic particles in the container are settled on the top by magnetic force, then the magnetic force is removed and the container is tilted at a predetermined angle, and then the flow-out state of the magnetic particles settled on the inner surface of the container is measured. In this measuring method, the reaction between the magnetic particles and the sample can be performed by stirring for 2 minutes to 15 minutes. In order to settle the magnetic particles by magnetic force after the reaction, they can be placed on a settling promoting table for 30 seconds to 5 minutes, but the treatment time depends on the particle size of the magnetic particles used or the strength of the magnetic force. I can decide. Further, the flow-out state of the magnetic particles is measured by measuring the length of the flow-out particles 1 to 5 minutes after the reaction container is tilted. The measurement can be performed by a method of visually measuring with a scale or a measuring device using an optical sensor such as a transmissive optical sensor or a reflective optical sensor. To determine negative or positive with this method, after inclining the inner surface of the container for forming an image that normally flows out to 20 ° to 40 ° with respect to the horizontal plane, a sample in which the flowout of a certain value or more is recognized is negative. The specimen is judged to be positive if it is aggregated by an immune reaction and does not reach the length.

The magnetic particles used in the present invention are, for example, 5
Gelatin magnetic particles containing ferromagnetic particles of 0 to 500 Å (Japanese Patent Publication No. 3-17103), particles coated with a protein such as serum albumin which is a magnetic material, particles similarly coated with a synthetic polymer, and a synthetic polymer as a core. Examples thereof include particles coated with a ferrite layer and further coated with a silane polymer. Among these magnetic particles, it is preferable to use gelatin magnetic particles containing a ferromagnetic material that exhibits good dispersibility because it is less likely to cause a non-specific reaction for measurement and has a small residual magnetism.

The magnetic particles of the present invention have a particle size of 0.5 μm to 5.0 μm.
It is preferable to use particles having a particle size of μm. Generally, chemiluminescent enzyme immunoassays have good reaction efficiency and give good results when particles with a small particle size are used, whereas indirect agglutination immunoassays have the disadvantage that agglutination may not occur when particles with a small particle size are used. There are cases. In view of these circumstances, the lower limit of the particle size of the magnetic particles used is preferably particles larger than 0.5 μm. As described above, when the particles are made larger, the chemiluminescent enzyme immunoassay cannot be efficiently performed, but the indirect agglutination immunoassay is likely to give good results. In view of these circumstances, the upper limit of the particle size of the magnetic particles used is preferably particles smaller than 5 μm. Furthermore, in order to perform both measurements efficiently, the particle size of the magnetic particles should be 1.0μ.
m to 3.0 μm is preferable.

Further, various kinds of antigens or antibodies can be bound to the above-mentioned magnetic particles according to the object to be measured in the sample and used for immunoassay. Antibodies bound to particles include:
For example, drugs such as theophylline, phenytoin, valproic acid, low molecular hormones such as siloxy, estrogen, estradiol, carcinoembryonic antigen (CEA), cancer markers such as α-fetoprotein (AFP), thyroid stimulating hormone (TSH), insulin, etc. Polymeric hormone of I
L-1, IL-2, IL-6 and other cytokines, EG
Growth factors such as F and PDGF, DNA, RNA
And the like, antibodies against hemoglobin and the like, antibodies against immunoglobulins (antibodies), and the like. These antibodies may be monoclonal antibodies or polyclonal antibodies, and may be fragments of F (ab ') ₂ , Fab', Fab, etc., which are degradation products of the antibodies. On the other hand, as an antigen, for example, H
Examples thereof include virus-related antigens such as IV, ATLV and HBV, the above-mentioned viral DNA, high molecular hormones such as insulin and TSH. Examples of the sample include body fluid containing the measurement target, such as blood, serum, urine and the like. As a method of binding these antigens or antibodies to the magnetic particles, a physical adsorption method or a chemical binding method can be adopted, but a chemical binding method in which the antigens or antibodies are firmly bound is preferably used. This chemical bonding method is a known method, using a binding reagent such as a carbodiimide reagent as a binding reagent, glutaraldehyde, cyanuric chloride, benzoquinone, tannic acid, tolylene diisocyanate, etc., magnetic particles in a buffer solution and an antigen or It can be performed by reacting with an antibody.

As a container for receiving a sample and a reagent for carrying out the main measurement, a U-shaped, V-shaped, or flat container made of plastic or glass having no magnetic response, such as polystyrene resin or ABS resin, is used. It is a container with a bottom. The size of these measuring containers is not limited, but a polystyrene V-shaped microplate is suitable for processing a large number of specimens, easy handling, and obtaining a clear image. This microplate has a total of 96 wells with 12 rows in the vertical direction and 8 rows in the horizontal direction, 12 rows, 10 rows.
A large number of samples can be measured by using a column having a total of 120 wells. Further, an electromagnet, a permanent magnet, or the like can be used as a means for causing the magnetic particles to settle on the inner surface of the container, but it is usually preferable to use a permanent magnet that is easy to handle.

When the magnetic particles are settled in the above-mentioned container having a flat bottom, the magnetic particles can be forced to settle at a predetermined position on the bottom of the well by causing the magnetic force of the magnet to act through an adapter. it can. As this adapter, a tip portion in contact with the bottom surface of the well has a sharp shape and is formed using a magnetized material such as magnet or iron.

Next, the supernatant is removed from the particle solution which has been agglutinated by the indirect agglutination immunoassay and determined to be positive, and the magnetic particles are washed, an enzyme-labeled antigen or an enzyme-labeled antibody is added to the particles and mixed to carry out a reaction. The unreacted enzyme-labeled antigen or enzyme-labeled antibody is removed by collecting the magnetic particles with the aid of a supernatant solution, washing the magnetic particles with physiological saline or distilled water, and performing B / F separation. The amount of the target substance in the sample can be measured by measuring the activity of the enzyme bound to the enzyme using a chemiluminescent substrate. The reaction between the aggregated magnetic particles and the enzyme-labeled antigen or enzyme-labeled antibody is preferably performed by mixing at room temperature for 2 minutes to 10 minutes.

The enzyme-labeled antibody used in the present measurement can be produced by labeling the above-mentioned polyclonal antibody or monoclonal antibody or a fragment of these antibodies with an enzyme. Further, as the antibody used in this measurement, it is advantageous to use an antibody that recognizes an epitope different from that of the antibody bound to the magnetic particles for the sample, in order to perform the measurement with high sensitivity.
Examples of the enzyme bound to this antibody include alkaline phosphatase, β-galactosidase, glucose oxidase and the like. In order to bind these enzymes to the antibody, a known method for producing an EIA reagent may be used (see "Enzyme-linked immunosorbent assay", Medical Shoin, (1987)).

The activity of these labeled enzymes can be measured using a substrate. It goes without saying that the chemiluminescent substrate used for the measurement should be one suitable for the labeling enzyme, for example, methylumbelliferyl phosphate, 3
-(2'-Spiro adamantane) -4-methoxy-4-
(3 ″ -phosphoryloxy) phenyl-1,2-dioxetane disodium salt (AMPPD) (for alkaline phosphatase), methylumbelliferyl-β-D-galactoside (for β-D-galactosidase), etc. are used. The amount of luminescence can be measured at room temperature to 40 ° C. for 1 minute to
The reaction is carried out for 30 minutes, and the amount of luminescence is measured by a measuring device such as a photon counter. Other measurement is 4 ℃ -40 ℃
It is also possible to employ a so-called rate method in which heating is performed within the range. By comparing the luminescence amount obtained by this measurement with the calibration curve, the target measurement object can be quantified.

For the measurement of chemiluminescence, it is preferable that the reaction solution to which the chemiluminescence substrate is added is transferred to a chemiluminescence measurement test tube, a container such as a cell, and the amount of luminescence is measured.

Further, the chemiluminescent enzyme immunoassay is usually carried out by measuring the activity of the enzyme bound to the magnetic particles, but the enzyme-labeled antibody or enzyme-labeled antigen not bound to the magnetic particles is separated,
The amount of the measurement target in the sample can also be measured by measuring the activity of the unbound enzyme.

On the other hand, the measuring device of the present invention comprises a reaction container for receiving a magnetic particle having an antigen or an antibody bound thereto and a sample, and a dispensing device for dispensing the magnetic particle, sample or reagent into the reaction container. A stirrer for mixing the reagents in the reaction vessel, a settling promoting apparatus for settling the magnetic particles by magnetic force, an inclination processing apparatus for inclining the reaction vessel, and a determination apparatus for measuring the flow-out state of the magnetic particles from the reaction vessel And a cleaning device for performing B / F separation, a chemiluminescence measuring device for measuring the enzyme activity bound to the magnetic particles using a chemiluminescent substrate, and the like. More specifically, as shown in the block diagram of FIG. 2 of the present invention, the immunoassay device includes a reaction container supply device 1, a reagent and sample dispensing device 2, a stirring device 3, a magnetic settling promoting device 4, and an inclination treatment. Device 5, Judgment device 6 for measuring the outflow length of magnetic particles, Washing device 7, Dispensing device 8 for dispensing enzyme labeled antigen or enzyme labeled antibody, Stirrer 9, Unreacted enzyme labeled antigen from magnetic particles or Cleaning device 10 for removing the enzyme-labeled antibody and B / F separation, chemiluminescent substrate dispensing device 11, chemiluminescent measuring device 12, reaction container collecting device 13
Etc. can be provided.

Further, in these devices, the dispensing device, the stirring device, and the cleaning device can be miniaturized by using them a plurality of times in the measurement process. The configuration example shown in FIG. 2 is an example of the measuring apparatus of the present invention and does not limit the present invention.

[0018]

The present invention will be described in more detail with reference to the following examples. Example 1 Production of anti-AFP antibody-sensitized magnetic particles Gelatin magnetic particles containing a ferromagnetic substance having each particle size and produced according to the method described in Japanese Patent Publication No. 3-17103, were used to prepare anti-human AFP antibody (rabbit). , DACO) using the method of Barnard et al. (Clin. Chem.,
27 (6) 832 (1981)), anti-AFP antibody-sensitized magnetic particles were prepared.

Example 2 Qualitative and Quantitative Measurement of AFP In order to perform qualitative measurement, first, a V-shaped microplate having 96 wells was sensitized with the anti-AFP antibody prepared in Example 1 to prepare 25 μl of magnetic particles having different particle sizes ( Magnetic particle 0.0
(9% dispersion) and 25 μl of a 40-fold diluted sample of each concentration were added and stirred for 5 minutes. After stirring, V
The mold microplate was set on the sedimentation promoting device in which the magnet was placed for 1 minute, and the magnetic particles were collected at the center of the bottom of the well. Place the plate at 60 ° (about 12 at the bottom of each well).
It is V-shaped at 0 °, and is substantially inclined to the ground by about 30 °. ) Tilt and hold for 2 minutes. Particles did not flow out in the strongly positive sample (FIG. 1, (a)), and particle outflow was observed in the negative sample (FIG. 1, (b)). The length of the magnetic particles that flowed out was measured and adjusted so that the cutoff value was in the middle, and positive and negative were determined. Table 1 shows the results.
In this measurement, 25 ng / ml was used as the cutoff value.

Quantitative measurement was carried out on a sample judged to be positive by qualitative measurement. In this measurement, magnetic particles that became positive in the plate as a result of qualitative measurement were selected, and the particles were washed 1, and 20 μl of the anti-AFP antibody labeled with alkaline phosphatase (the labeled antibody concentration was 0.5 μg)
/ Ml; 0.1 M Tris-hydrochloric acid, 2% BSA, 1 mMM
gCl ₂ , 0.1 mM ZnCl ₂ , pH 7.5) was added and stirred for 5 minutes. Furthermore, after washing with physiological saline three times to remove unbound labeled anti-AFP antibody, 200 μl of AMPPD as a chemiluminescent substrate (AMPPD concentration: 100 μm)
g / ml; 0.1 M Tris-hydrochloric acid, 1 μM MgC
_{l 2, 0.1mM ZnCl 2, pH9.8} ) to obtain the solution measured AFP concentration in a luminometer and the light emission amount in a container for chemiluminescence measurement of that added to the magnetic particle solution.
The results are shown in Table 2. If the result is negative, it is below the cutoff, so quantitative measurement is not performed.

[0021]

[0022]

[0023]

INDUSTRIAL APPLICABILITY In the present invention, since the indirect agglutination immunoassay method using magnetic particles and the chemiluminescent enzyme immunoassay method are continuously carried out, a qualitative measurement result and a quantitative measurement result can be simultaneously obtained by a simple operation. be able to. This indirect agglutination immunoassay requires about 10 minutes, and the chemiluminescent enzyme immunoassay requires 10 to 3 minutes.
It takes 0 minutes. Therefore, the measurement results cannot be obtained at the same time,
Compared with the conventional method that requires a measurement time of at least about 1 hour, in the main measurement in which continuous measurement is performed, 20 to 20
A qualitative measurement result and a quantitative measurement result can be obtained in 40 minutes. Further, when microplates are used, 96 tests can be processed simultaneously for each microplate, and the results can be obtained, so that 576 to 1152 samples can be measured in one hour. In addition, for samples that were determined to be positive by non-specific agglutination by the indirect agglutination immunoassay, re-measurement is performed by the chemiluminescent enzyme immunoassay, so it is possible to easily exclude false-positive measurement results due to non-specific reactions. it can.

[Brief description of drawings]

FIG. 1 is an example of measurement by an indirect agglutination immunoassay using a V-shaped microplate. (A) is a positive sample,
(B) is an example of measurement of a negative sample.

FIG. 2 is a block diagram showing a configuration example of an immunoassay device according to the present invention.

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 1 Reaction container supply device 2 Dispensing device 3 Stirring device 4 Settling promotion device 5 Gradient treatment device 6 Judgment device 7 Cleaning device 8 Dispensing device 9 Stirring device 10 Cleaning device 11 Substrate dispensing device 12 Chemiluminescence measuring device 13 Reaction container recovery apparatus

Claims (8)

[Claims] 1. A magnetic particle bound with an antigen or an antibody and a sample are mixed in a reaction vessel, the magnetic particle is allowed to settle on the inner surface of the vessel by an external magnetic force, and the vessel is inclined at a predetermined angle to flow out the magnetic particle. After the indirect agglutination immunoassay for measuring the condition and the agglutinated magnetic particles determined to be positive by the indirect agglutination immunoassay are reacted with the enzyme-labeled antigen or enzyme-labeled antibody, the activity of the enzyme bound to the magnetic particles is measured. An immunoassay characterized by continuously performing a chemiluminescent enzyme immunoassay in which a chemiluminescent substrate is used for measurement. 2. The immunoassay method according to claim 1, wherein the magnetic particles are particles of 0.5 μm to 5.0 μm. 3. The immunoassay method according to claim 2, wherein the magnetic particles are gelatin magnetic particles. 4. The immunoassay method according to claim 1, wherein the labeling enzyme is alkaline phosphatase. 5. A reaction container that receives a magnetic particle having an antigen or an antibody bound thereto and a sample, a dispensing device that dispenses the magnetic particle, sample or reagent into the reaction container, and a reagent in the reaction container are mixed. Stirrer, a settling promoting device that settles the magnetic particles by magnetic force, a tilting device that tilts the reaction container, a determination device that measures the flow state of the magnetic particles from the reaction container, and a B / F separation. An immunoassay device comprising a washing device for performing and a chemiluminescence measuring device for measuring an enzyme activity bound to magnetic particles using a chemiluminescent substrate. 6. The apparatus according to claim 5, wherein the reaction container is a V-shaped microplate. 7. The inner surface of the reaction vessel is 20 ° to 4 with respect to the horizontal.
The device according to claim 5, which is a tilting device that tilts at 0 °. 8. The device according to claim 5, which is a determination device for measuring the flow-out state of magnetic particles by laser light.