JP3216452B2 - Immunoassay method and device - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method of mixing a magnetic particle with an antigen or an antibody and a specimen in a reaction vessel, sedimenting the magnetic particles on the inner surface of the vessel by an external magnetic force, and tilting the vessel at a predetermined angle. Indirect agglutination immunoassay to measure the flow-out state of the magnetic particles, and after reacting by adding an enzyme-labeled antigen or an enzyme-labeled antibody to the aggregated magnetic particles determined to be positive by the indirect agglutination immunoassay, A chemiluminescent enzyme immunoassay for measuring the activity of a bound enzyme using a chemiluminescent substrate is performed continuously, and an immunoassay for measuring an antigen or an antibody in a sample, and an immunoassay for performing the immunoassay. It relates to a measuring device.

[0002]

2. Description of the Related Art Conventionally, an indirect agglutination immunoassay has been widely put into practical use as a simple assay for a large number of samples using a particle to which an antigen or an antibody is bound as a reagent and using no special device for the assay. In the method using magnetic particles for the particles, the particles and the sample are allowed to react, then the particles are forcibly settled by a magnetic force, and then the container containing the particles is tilted at a predetermined angle and measured from a state in which the settled particles flow out. It is known as a simple measurement method that can obtain the result of the determination of the presence or absence of an immune reaction in a short time in order to obtain the result (JP-A-3-14436).
No. 7). On the other hand, enzyme immunoassay (EIA) is a quantitative method for comparing the result obtained by measuring the activity of an enzyme bound to a solid phase by an immune reaction with a calibration curve previously obtained from a sample of known concentration. This is a measurement method capable of measurement (precision test).

[0003]

When a quantitative measurement of a sample is carried out by the indirect agglutination immunoassay method, a semi-quantitative measurement is performed because a dilution sequence of the sample is prepared and the measurement is performed based on the visual judgment result of each agglutination image. Was obtained only. Therefore, indirect agglutination immunoassay is based on these characteristics.
It has been mainly used as a qualitative measurement (screening method) of specimens. The chemiluminescent enzyme immunoassay using a chemiluminescent substrate for EIA is a method capable of obtaining a measurement result in a short time, but requires the use of an expensive chemiluminescent substrate, and bind / free (B / F) separation. As a method for qualitatively measuring a large number of samples, for example, using an automatic measuring device to repeat complicated operations such as washing of a solid phase and the like, it was not a practically satisfactory method. Therefore, in order to perform qualitative measurement and quantitative measurement by the conventional method and obtain the respective results, first perform the qualitative measurement of the sample to obtain the result, and then repeatedly perform complicated sample selection and dispensing operations from the result. Quantitative measurement was performed. Therefore, it takes a great deal of labor and time to obtain a quantitative result from a large number of samples.

[0004]

Means for Solving the Problems As a result of intensive studies, the present inventors have found that, when measuring an antibody or an antigen in a sample, first, a magnetic particle to which the antigen or the antibody is bound is mixed with the sample.
The magnetic particles settle on the inner surface of the container by an external magnetic force, and after performing an indirect agglutination immunoassay in which the container is tilted at a predetermined angle and measurement is performed from the flow-out state of the settled magnetic particles,
Next, an enzyme-labeled antibody or an enzyme-labeled antigen is added to the agglomerated magnetic particles determined to be positive in the indirect agglutination immunoassay to react, and the activity of the enzyme bound to the magnetic particles is measured using a chemiluminescent substrate. The present inventors have found an immunoassay method for continuously performing an immunoassay method and an immunoassay apparatus for performing the immunoassay, and completed the present invention.

In the indirect agglutination immunoassay of the present invention, first, magnetic particles to which an antigen or an antibody is bound and a sample are put into a reaction vessel and mixed to perform a reaction, and the reaction vessel is settled by a sedimentation promoting device provided with a magnet. This is a method in which the magnetic particles in the container are settled by a magnetic force on the container, and the magnetic force is removed, the container is inclined at a predetermined angle, and the flow-out state of the magnetic particles settled on the inner surface of the container is measured. In this measurement method, the reaction between the magnetic particles and the sample can be performed by stirring for 2 minutes to 15 minutes. Further, in order to settle the magnetic particles by magnetic force after the reaction, the magnetic particles can be set on a settling accelerator for 30 seconds to 5 minutes. You can decide. Further, the measurement of the flow-out state of the magnetic particles is performed by measuring the length of the flowed-out particles 1 minute to 5 minutes after the reaction vessel 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 transmission optical sensor or a reflection optical sensor. In order to determine negative or positive by this method, usually, after inclining the inner surface of the container for forming an image to flow out to 20 ° to 40 ° with respect to the horizontal plane, a sample in which the flow out of a certain value or more is recognized as negative. The sample was aggregated by the immune reaction and did not reach its length, and was judged to be positive.

The magnetic particles used in the present invention are, for example, 5 particles.
Gelatin magnetic particles containing ferromagnetic particles of 0 to 500 ° (Japanese Patent Publication No. 3-17103), magnetic particles coated with proteins such as serum albumin, particles coated with synthetic polymer, and synthetic polymer as 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 hardly causes a nonspecific reaction in measurement and has good remanence because of low 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. In general, chemiluminescent enzyme immunoassay has good reaction efficiency when small particles are used, and gives good results.On the other hand, indirect agglutination immunoassay has disadvantages such that aggregation may not occur when small particles are used. There are cases. In view of these circumstances, the lower limit of the particle size of the magnetic particles used is preferably larger than 0.5 μm. Also, as described above, when the size of the particles is gradually increased, the chemiluminescent enzyme immunoassay cannot be carried out efficiently, but good results are easily obtained in the indirect agglutination immunoassay. In view of these circumstances, the upper limit of the particle size of the magnetic particles used is preferably smaller than 5 μm. In order to perform both measurements efficiently, the particle size of the magnetic particles should be 1.0 μm.
m to 3.0 μm is preferred.

Further, various antigens or antibodies can be bound to the above-mentioned magnetic particles in accordance with an object to be measured in a sample and used for immunoassay. Antibodies bound to the particles include:
For example, drugs such as theophylline, phenytoin, valproic acid, low molecular hormones such as siloxy, estrogen and estradiol, carcinoembryonic antigen (CEA), cancer markers such as α-fetoprotein (AFP), thyroid stimulating hormone (TSH), insulin and the like High molecular hormone, I
Cytokines such as L-1, IL-2 and IL-6, EG
Growth factors such as F, PDGF, DNA, RNA
And antibodies against hemoglobin and the like, and antibodies against immunoglobulin (antibody). These antibodies may be monoclonal antibodies or polyclonal antibodies, and may also be fragments of F (ab ') ₂ , Fab', Fab, etc., which are degradation products of the antibodies. On the other hand, as the antigen, for example, H
Examples include virus-related antigens such as IV, ATLV, and HBV, and the above-mentioned viral DNA, insulin, and macromolecules such as TSH. Examples of the sample include a body fluid containing the measurement target, such as blood, serum, and urine. As a method for binding these antigens or antibodies to the magnetic particles, a physical adsorption method or a chemical binding method can be employed, but it is preferable to carry out the chemical binding method in which the antigen or antibody is firmly bound. 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. The reaction can be performed by reacting with an antibody.

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

When the magnetic particles are settled in the container having the flat bottom, the magnetic particles are forcibly settled at a predetermined position on the bottom of the well by applying the magnetic force of the magnet 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 an adapter formed using a magnetic material such as a magnet or iron can be used.

Next, the supernatant is removed from the particle liquid which has been agglomerated 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, mixed and reacted. The unreacted enzyme-labeled antigen or enzyme-labeled antibody is removed by removing the supernatant solution by collecting the magnetic particles, and the magnetic particles are washed with physiological saline or distilled water to perform B / F separation. The amount of the analyte in the sample can be determined by measuring the activity of the enzyme bound to the sample 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 of 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. In addition, it is advantageous to use an antibody that recognizes an epitope different from that of the antibody bound to the magnetic particles for the specimen in order to perform the measurement with high sensitivity.
Examples of the enzyme to be bound to this antibody include alkaline phosphatase, β-galactosidase, glucose oxidase and the like. The binding of these enzymes to the antibody may be performed according to a known method for producing an EIA reagent (see “Enzyme Immunoassay”, Medical Shoin, (1987)).

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

For the measurement of chemiluminescence, it is preferable to transfer the reaction solution to which the chemiluminescent substrate has been added to a test tube or a cell such as a cell for measuring chemiluminescence and measure the amount of luminescence.

[0015] Further, in the chemiluminescent enzyme immunoassay, the activity of an enzyme bound to magnetic particles is usually measured, and an enzyme-labeled antibody or an enzyme-labeled antigen not bound to magnetic particles is separated.
By measuring the activity of the unbound enzyme, the amount of the measurement target in the sample can also be measured.

On the other hand, the measuring device of the present invention comprises a reaction container for receiving a magnetic particle to which an antigen or an antibody is bound and a sample, a dispensing device for dispensing the magnetic particle, the sample or the reagent to the reaction container, A stirrer for mixing the reagents in the reaction vessel, a sedimentation accelerating device for sedimenting the magnetic particles by magnetic force, an inclining device for inclining the reaction vessel, and a determination device for measuring the state of the magnetic particles flowing out of the reaction vessel And a washing device for performing B / F separation, a chemiluminescence measuring device for measuring the enzymatic 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 vessel supply device 1, a reagent and sample dispensing device 2, a stirring device 3, a magnetic sedimentation promoting device 4, a tilting process, and the like. Apparatus 5, determination apparatus 6 for measuring the length of magnetic particles flowing out, washing apparatus 7, dispensing apparatus 8 for dispensing enzyme-labeled antigen or enzyme-labeled antibody, stirring apparatus 9, unreacted enzyme-labeled antigen from magnetic particles or Washing device 10, which removes enzyme-labeled antibody and performs B / F separation, chemiluminescent substrate dispensing device 11, chemiluminescent measuring device 12, reaction vessel collecting device 13
Etc. can be provided.

Further, in these apparatuses, the dispensing apparatus, the stirring apparatus and the washing apparatus are used a plurality of times in the measuring step, so that the apparatus can be downsized. The configuration example shown in FIG. 2 is an embodiment 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 A gelatin magnetic particle containing a ferromagnetic substance having each particle diameter produced according to the method described in Example of Japanese Patent Publication No. 3-17103 and an anti-human AFP antibody (rabbit) , DACO) and 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 Measurements of AFP To perform qualitative measurement, first, 25 μl of magnetic particles having different particle diameters sensitized with the anti-AFP antibody prepared in Example 1 above were placed on a V-shaped microplate having 96 wells ( Magnetic particles 0.0
(A 9% -containing dispersion) and 25 μl of each concentration of a sample diluted 40-fold were added and stirred for 5 minutes. After stirring, V
The mold microplate was set on a settling accelerator equipped with a magnet for 1 minute, and the magnetic particles were collected in the center of the bottom of the well. Place the plate at 60 ° (the bottom of each well is approximately 12
It is a 0 ° V-shape, which is substantially inclined to the ground by about 30 °. ) Tilt and hold for 2 minutes. Particles did not flow out of the strongly positive sample (FIG. 1, (a)), and particles flowed out (FIG. 1, (b)) of the negative sample. The length of the outflowing magnetic particles was measured and adjusted so that the cut-off value was intermediate, 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 performed on a sample which was determined to be positive by qualitative measurement. In this measurement, magnetic particles which became positive in the plate as a result of the qualitative measurement were selected, the particles were washed 1, and the magnetic particles were treated with alkaline phosphatase and labeled with an anti-AFP antibody 20 μl (labeled antibody concentration 0.5 μg).
/ Ml; 0.1 M Tris-HCl, 2% BSA, 1 mM
gCl ₂ , 0.1 mM ZnCl ₂ , pH 7.5) and stirred for 5 minutes. Further, after washing with physiological saline three times to remove unbound labeled anti-AFP antibody, 200 μl of a chemiluminescent substrate, AMPPD (AMPPD concentration of 100 μl)
g / ml; 0.1 M Tris-HCl, 1 μM MgC
l ₂ , 0.1 mM ZnCl ₂ , pH 9.8) was added to the magnetic particle solution, placed in a container for measuring chemiluminescence, and the luminescence was measured with a luminometer to determine the AFP concentration.
Table 2 shows the results. If the result is negative, the quantitative value is not measured because it is below the cutoff.

[0021]

[0022]

[0023]

According to the present invention, since the indirect agglutination immunoassay using magnetic particles and the chemiluminescence enzyme immunoassay are performed continuously, the qualitative measurement results and the quantitative measurement results can be obtained simultaneously with a simple operation. be able to. The indirect agglutination immunoassay requires about 10 minutes, and the chemiluminescence enzyme immunoassay requires 10 to 3 minutes.
It takes 0 minutes. Therefore, no measurement results can be obtained at the same time,
Compared to the conventional method that required at least about one hour of measurement time, in the main measurement in which the measurement is continuously performed, 20 to 20 hours are required.
A qualitative measurement result and a quantitative measurement result can be obtained in 40 minutes. When a microplate is used, 96 tests per microplate are simultaneously processed and the result is obtained, so that 576 to 1152 samples can be measured in one hour. In addition, for samples that were determined to be positive due to non-specific agglutination by indirect agglutination immunoassay, re-measurement by chemiluminescence enzyme immunoassay can be used to easily remove false-positive measurement results due to nonspecific agglutination. it can.

[Brief description of the 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 a measurement example of a negative sample.

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

[Explanation of symbols]

 REFERENCE SIGNS LIST 1 reaction vessel supply device 2 dispensing device 3 stirring device 4 sedimentation promotion device 5 tilt treatment device 6 judging device 7 washing device 8 dispensing device 9 stirring device 10 washing device 11 substrate dispensing device 12 chemiluminescence measuring device 13 reaction vessel collection apparatus

Continuation of front page (56) References JP-A-8-114595 (JP, A) JP-A-4-285857 (JP, A) JP-A-5-281230 (JP, A) JP-A-2-210262 (JP) JP-A-3-14467 (JP, A) JP-A-4-48266 (JP, A) JP-A-5-240859 (JP, A) JP-A-59-195161 (JP, A) 6-324042 (JP, A) (58) Field surveyed (Int. Cl. ⁷ , DB name) G01N 33/543 581 G01N 33/553

Claims (8)

(57) [Claims] 1. A method in which magnetic particles to which an antigen or an antibody is bound and a specimen are mixed in a reaction container, and the magnetic particles are settled on the inner surface of the container by an external magnetic force. An indirect agglutination immunoassay for measuring the state, and after reacting by adding an enzyme-labeled antigen or an enzyme-labeled antibody to the aggregated magnetic particles determined to be positive by the indirect agglutination immunoassay, the activity of the enzyme bound to the magnetic particles is determined. An immunoassay, comprising continuously performing a chemiluminescent enzyme immunoassay using a chemiluminescent substrate. 2. The immunoassay according to claim 1, wherein the magnetic particles are particles of 0.5 μm to 5.0 μm. 3. The immunoassay according to claim 2, wherein the magnetic particles are gelatin magnetic particles. 4. The immunoassay according to claim 1, wherein the labeling enzyme is alkaline phosphatase. 5. A reaction container for receiving a magnetic particle to which an antigen or an antibody is bound and a sample, a dispensing device for dispensing the magnetic particle, the sample or the reagent into the reaction container, and a reagent in the reaction container. A stirrer, a sedimentation accelerating device for sedimenting the magnetic particles by magnetic force, a tilting device for tilting the reaction vessel, a determination device for measuring a state of the magnetic particles flowing out of the reaction vessel, and a B / F separation. An immunoassay device comprising a washing device for performing the measurement, and a chemiluminescence measurement device for measuring the enzyme activity bound to the magnetic particles using a chemiluminescence substrate. 6. The apparatus according to claim 5, wherein the reaction vessel is a V-shaped microplate. 7. The inner surface of the reaction vessel is 20 ° to 4 ° with respect to the horizontal.
6. The apparatus according to claim 5, wherein the apparatus is a tilt processing device for tilting to 0 °. 8. The apparatus according to claim 5, wherein the apparatus is a determination apparatus that measures the flow state of the magnetic particles by using a laser beam.