JPH0472568A - Method and device for measuring immunologically active material - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of the Invention] The present invention relates to a method and apparatus for measuring immunologically active substances such as antigens and antibodies in a specimen using microparticles. More specifically, the present invention relates to a method and apparatus for optically measuring the degree of agglutination caused by an antigen-antibody reaction using solid microparticles carrying an immunologically active substance.

[Description of prior art]

A dispersion (latex reagent) in which microparticles of polystyrene carrying an immunologically active substance, such as an antibody, are dispersed in a liquid medium such as water is selectively reactive with the above-mentioned immunologically active substance. The latex agglutination immunoassay (LAIA), which performs measurements by observing the agglutination caused by the action of a substance (such as an antigen), has been developed by J.M. Singer et al.
et al) and found that 0A, m, J, Mec
i, 21 888 (1956)], and various studies have been made since then. Among these methods, the method of visually determining the degree of aggregation is widely used in practice because it has the advantage of being simple and that results can be obtained in a short time, although quantitative measurement is difficult.

In recent years, attempts have been made to optically measure the degree of aggregation, and Ni Future et al.
al) proposed a method for optically measuring changes in turbidity associated with aggregation reactions and performing quantitative analysis from kinetic analysis [A, Future et al,; Proti
desBiol Fluids, Proc, Co11
oq,, 20 589 (1972)).

However, as will be described later, the measurement values fluctuate greatly due to the instability of the latex reagent itself, and the measurement sensitivity is also not sufficient. In other words, latex reagents consist of fine solid particles dispersed in a liquid dispersion medium, which is an inherently unstable system, so long-term storage may cause aggregation or decrease sensitivity. Easy, and
Freezing destroys the dispersed state, which poses problems such as requiring special consideration for preservation.

In response to this, a method has been proposed to improve the stability of latex reagents by removing and drying the liquid that is the dispersion medium (Japanese Unexamined Patent Publications No. 52-11.7420 and No. 62-46262). .

However, although the storage stability of latex reagents can be improved by drying them, the aggregation reactivity of latex reagents obtained by redispersion varies greatly, and as a result, measurement data often fluctuates. There's a problem.

Therefore, although the conventional techniques can be used for qualitative measurements such as negative or positive determination by visual inspection on a slide, they are not suitable for highly accurate quantification.

In addition, an agglutinating immunoreagent such as a latex reagent is injected into a capillary tube and mixed with the specimen in the freeze-dried capillary tube to cause a reaction, and immunologically active substances in the specimen are detected by observing the state of agglutination. A method has been proposed (Japanese Unexamined Patent Publication No. 73866/1986). This method has the same good storage stability of the reagents as the above proposal, and is attractive as a simple testing method, but the problem is that the reproducibility of measured values is poor and accurate quantification is not possible. be.

[Purpose of the invention]

The present invention solves the problems in the prior art described above, and
An object of the present invention is to provide an immunoassay method and a measuring device that have excellent reproducibility of measured values and enable accurate quantification.

[Structure and effects of the invention]

The present inventors investigated the problems with conventional measurement methods using dry reagents and found that the cause of fluctuations in measured values in the conventional method was due to irregularities in the redispersion state, as well as due to redispersion. It has been revealed that measurement sensitivity decreases because the bond between the solid particles and the immunologically active substance is broken when stirring is continued for a long time or when the stirring intensity is too strong. Furthermore, as a result of intensive studies based on these phenomena, the present inventors found that when redispersing the dry reagent, the dispersion state was optically measured while stirring, and once a suitable dispersion state was reached, the following It was revealed that proceeding to the reaction step has an extremely large effect on performing highly sensitive and stable measurements.

The present invention was completed as a result of further studies based on the above-described facts, and includes the measurement method described below and an apparatus suitable for carrying out the measurement method.

The measuring method of the present invention is as described below.

That is, by chemically bonding a substance that is immunologically active to the analyte in the sample to the surface of solid particles, and making the sample react with the bound immunologically active substance in a liquid medium. In the method of optically measuring the degree of aggregation of the resulting reaction mixture, (I) dry solid fine particles (hereinafter referred to as " (ii) stirring the dispersion medium in the reaction cell, the dry reagent particles, and the sample, and adding the dispersion medium and the sample into the reaction cell containing the dry reagent particles; (III) the step of optically measuring the dispersion state in the step [
measuring the dispersion state of the dry reagent fine particles in the dispersion medium from the optical measurement data obtained in (II), and stopping the stirring in step (II) when a preset dispersion state is reached; (IV) A step of reacting the dispersion containing the specimen that has reached the dispersion state set in the above step (III) to produce an agglomerated state; A method for measuring an immunologically active substance, comprising the steps of: (VT) optically measuring the degree of aggregation of the reaction mixture poured into the measurement cell; The apparatus of the present invention suitable for implementing the measuring method is as described below. That is, by chemically bonding a substance that is immunologically active to the analyte in the sample to the surface of solid particles, and making the sample react with the bound immunologically active substance in a liquid medium. An apparatus for optically measuring the degree of aggregation of a reaction mixture formed, the apparatus comprising:
I) means for fixing the reaction cell; (II) means for injecting a dispersion medium into the reaction cell; (I)
I [) means for injecting the specimen into the reaction cell, (rV)
means for stirring the contents in the reaction cell; (V) controlling the continuation or stop of stirring from optical measurement data obtained from the dispersion state of dry reagent fine particles in the dispersion medium in the stirred reaction cell; (VI) means for fixing a measuring cell for measuring the degree of aggregation of the reaction mixture; (VII) means for flowing the reaction mixture through the measuring cell;
III) A device for measuring an immunologically active substance, characterized in that it has means for optically measuring the degree of aggregation of the reaction mixture flowed into the measurement cell.

The present invention will be explained in detail below.

The solid fine particles used in the present invention include biologically derived fine particles, inorganic fine particles, and organic fine particles. Examples of the microparticles derived from the living organisms include bacteria such as Staphylococcus and Streptococcus treated with red blood cell dispersion. Examples of the inorganic fine particles include silica, alumina, bentonite, and the like. Examples of the organic fine particles include homopolymers and/or copolymers of vinyl monomers such as styrene, vinyl chloride, acrylonitrile, vinyl acetate, acrylic esters, and methacrylic esters, and styrene-butadiene copolymers. Examples include fine particles of butane-based copolymers such as methyl methacrylate-butadiene copolymers. The particle diameter of the above-mentioned fine particles is that of biologically derived fine particles,
The diameter is preferably 0.05 μm to 5 μm for both inorganic and organic fine particles, and particularly preferably 0.5 μm to 5 μm for application to this measurement method. When the particle size exceeds 0.05 μm, it becomes difficult to disperse the dry reagent, and when the particle size exceeds 5 μm, the stability of the dispersed reagent becomes poor.

Examples of immunologically active substances to be bound to the surface of solid microparticles include immunoglobulins such as IgG, IgM, and IgE;
Plasma proteins such as complement, CRP, ferritin, α,-microglobulin, β2-microglobulin and their antibodies α-fetoprotein, carcinoembryonic antigen (CEA)
), prostatic acid phosphatase (PAP), CA-1
Tumor markers such as 9-9, CA-125 and their antibodies Luteinizing hormone (LH), Follicle stimulating hormone (
Hormones such as FSH), human ciliated gonadotropin (hCG), Nistroken, and insulin, and their antibodies HBV-related antigens (HBs, HBe, HBc)
, HTV, ATL, and other viral infection-related substances and their antibodies; Bacteria such as Nottilia, Clostridium botulinum, Mycoplasma pallidum, and Treponema pallidum; and their antibodies; Antibodies and drugs such as anti-epileptic drugs such as phenytoin and phenobarbital, heart and blood medicines such as quinidine and shigoquinin, anti-asthmatic drugs such as theophylline, antibiotics such as chloramphenicol and gentamicin, and their antibodies Other enzymes , bacterial exotoxins (such as Strelisin 0), and their antibodies, and substances that cause an antigen-antibody reaction with the substance to be measured in the specimen are appropriately selected and used depending on the type of specimen.

Among these immunologically active substances, hCG antibody, CRP antibody, β2-microglobulin antibody or α-fetoprotein are particularly preferred.

Methods for immobilizing immunologically active substances onto solid fine particles include physical adsorption and chemical bonding, and chemical bonding is preferred in the present invention. That is, in the present invention, in order to simultaneously disperse dry solid fine particles carrying an immunologically active substance in a dispersion medium and the specimen as an aggregation factor by strong stirring, the immobilization method using physical adsorption of binding force is used to disperse the solid fine particles from the solid fine particles. This is because immunoactive substances may be released. For immobilization methods by chemical bonding, immunologically active substances can be
Since it contains a protein part as a component,
This can be carried out by a known method of chemically bonding the protein to a carrier (see Immobilized Enzyme, Kodansha (1975), edited by Chibata). In addition, for solid particles containing amino groups or carboxyl groups as functional groups, it is possible to immobilize immunologically active substances using carbodiimide as a condensing agent (see Japanese Patent Publication No. 12966/1986 or (Refer to Japanese Patent Application Laid-Open No. 5352620).

Furthermore, for solid microparticles having carbamoyl groups or amino groups, polyaldehydes such as glutaraldehyde can be used to immobilize immunologically active substances thereon via covalent bonds. Furthermore, for solid microparticles containing hydroxyl groups, cyanogen bromide can be used to immobilize immunologically active substances thereto via covalent bonds. Furthermore, for solid fine particles having an epoxy group or an aldehyde group, an immunologically active substance can be directly reacted with the solid particles and immobilized thereon via a covalent bond.

In any case, the above-mentioned binding reactions for immobilizing immunologically active substances on microparticles are performed using water or water and organic solvents that are compatible with water such as alcohols and ketones. Preferably, the reaction is carried out in a mixed solvent. In addition, a phosphate buffer-physiological saline solution was added to the reaction system for the purpose of stabilizing the particles and preventing non-specific aggregation.
It is preferable to add a buffer such as TrisHC1 buffer, an inert protein such as bovine serum albumin, a surfactant, and the like. The pH of the reaction solution used in the above-mentioned binding reaction is usually 6 to 10, preferably 7 to 9. Further, the concentration of fine particles in the reaction solution is usually 0.01 to 20% (by weight).

The dried immunoreagent is obtained by removing the dispersion medium used in the dispersion of microparticles bound to the immunologically active substance.

The dispersion medium is removed under 60'CE, preferably 30'CJ.
It is advantageous to carry out the reaction under J in order to maintain the activity of the immunologically active substance. A particularly preferred embodiment for the removal of the dispersion medium is removal by lyophilization, in which case the sensitivity of the reagent is maintained constantly high.For introducing the dry immunoreagent into the reaction cell, A predetermined amount of a dispersion of microparticles bound to an immunologically active substance may be added and the dispersion medium may be removed by drying as described above, or a predetermined amount of a dried immunoreagent from which the dispersion medium has been removed may be added. may be placed in the reaction cell.

The reaction cell used is one made of transparent glass or plastic (eg, polystyrene, polymethyl methacrylate, polyvinyl chloride, polycarbonate, polysulfone, etc.).

The dispersion medium for dispersing the dried immunoreagent in the reaction cell and the diluent for diluting the reaction mixture are water or a mixed solvent of water and an organic solvent compatible with water such as alcohols or ketones, respectively. .

Further, a pH buffer, a protein, a surfactant, a water-soluble polymer compound, etc. are appropriately added to the dispersion medium and diluent.

Since the antigen-antibody reaction is generally susceptible to the pH of the solvent, a pH buffer is added to adjust the pH to an optimum level, and for example, a phosphate or Tris HCI buffer is used. Proteins are added for the purpose of preventing non-specific reactions, and for example, bovine serum albumin, gelatin, etc. are used.

Surfactants and water-soluble polymer compounds are effective as dispersion aids for dry immunoreagents, such as nonionic surfactants such as Tween 20, anionic surfactants, polyvinyl alcohol, polyacrylamide, and polyacrylic acid. Salt, hydroxyethylcellulose, etc. are used. However, these additives are used to the extent that they do not inhibit the antigen-antibody agglutination reaction.

Further, the dried immunoreagent is diluted with the above-mentioned dispersion medium as appropriate depending on the object to be measured. Although the solid content concentration varies depending on the type and size of the measurement cell used, -
Preferably 0.01-5%, more preferably 0.05-5%
Adjusted within a range of 2%.

To agitate the specimen, dispersion medium, and dried immunoreagents,
A method of injecting a predetermined amount of dispersion medium into a reaction cell containing a specimen and a dried immunoreagent, and inserting and stirring a stirring tool, a method of shaking the reaction cell, etc. can be selected as appropriate.

Among these, stirring treatment using ultrasonic liquid stirring is preferred as it is the most effective for dispersing fine particles. The ultrasonic wave used for ultrasonic stirring varies depending on the type and size of the reaction cell, but - for boat fishing, the vibration frequency is 15 KHz to 50 KH.
z ultrasound is used.

In the above-mentioned dispersion step, the degree of dispersion of the immunoreagent into the dispersion medium is measured using an optical measuring means, and the optical measuring means includes, for example, a method of measuring transmitted light intensity;
A method of measuring the intensity of scattered light, a method of measuring the intensity of transmitted light and scattered light in combination, etc. are used as appropriate.

For example, when measuring the degree of dispersion using transmitted light intensity, the second
As shown in the schematic diagram in the figure, as the dispersion progresses, the amount of light passing through the reaction cell decreases and becomes almost constant in a uniformly dispersed state.

In addition, a preferred method for measuring the dispersion state was derived from the results of the experimental examples described below, and the method for determining the dispersion state is as follows: When monochromatic light passes through, the intensity of the incident light is I0, and the intensity of the transmitted light and 7' or scattered light is I
and fogIo/I=A.

The index A shown in In contrast, when the monochromatic light passes through the reaction cell containing the dry reagent fine particle dispersion obtained in step W (II) above, the intensity of the incident light is I0, and the intensity of the transmitted light and/or scattered light is Sawo ■ and l ogI O/r=A
This method is performed by confirming that the index A shown by is within the following range. A/A.

≦1.1 The above dispersion determination method will be explained in detail based on the following experimental example.

Experimental example”.

A portion of the CRP-sensitized latex obtained by the same method as in Example 1 described below was mixed with phosphate buffer physiological saline (pH 7.5) to which 1% bovine serum albumin and 5% sucrose were added.
After adjusting the reagent solid content concentration to 0.2% using PBS (hereinafter referred to as PBS), it was placed in a glass optical cell (optical path length 2 mm), and the index A was obtained using the above method. was found to be 2.75 (measurement wavelength 633 nm). In addition, P was added to the dry reagent particles for CRP detection obtained by the same method as in Example 2, which will also be described later.
After adding BS and adjusting the reagent solid content concentration to 0.2%, C
RP standard serum (2 mg/df) was added, ultrasonic stirring was performed, and index A was determined by the above method. Next, 60 seconds after each stirring is stopped, the reaction mixture is diluted 500 times with PBS in a dilution cell, the diluted liquid is introduced into a flow cell, and the diluted liquid is diluted by irradiating Ar laser light and detecting backscattered light from particles. The state of aggregation of the reagent particles inside was measured. Compare this result with a pre-prepared calibration curve to determine the CR in the sample.
The results of calculating the P concentration are shown in Table 3 and FIG.

1 example -? An experiment similar to Experimental Example 1 was conducted using fine particles of other compositions such as carboxylated styrene-methyl methacrylate copolymer and polymethyl methacrylate instead of carboxylated polystyrene, and with different particle sizes. The results are plotted on the horizontal axis as the stirring treatment time and on the vertical axis as A/A o.
It is shown in FIG. Note that portions with good measurement sensitivity M degree are connected with solid lines, and other portions are connected with broken lines.

As is clear from the results of Experimental Example 1.2, A/A.

If it exceeds 1.1, the measurement sensitivity is poor. Furthermore, when the same experiment was repeated, it became clear that when A/A o exceeds 1.1, the fluctuations in the measured values are large. In other words, A/A.

It has been found that 1.1 is a critical value from the two aspects of measurement sensitivity and fluctuation of measurement values. Furthermore, as can be seen from Table 3, it was found that the measurement sensitivity tended to decrease as the stirring treatment was performed for a long time.

That is, when stirring dry reagent particles into a dispersion medium containing a specimen, the stirring process is terminated when A/Ao satisfies the above range while performing optical measurement, and the process proceeds to the next step. This makes it possible to perform stable measurements with little variation in measured values even in the presence of trace components, regardless of the type of substance to be measured. Next, the index A of a complete dispersion of dry reagent microparticles. Usually, when chemically bonding an immunologically active substance to solid fine particles, it is carried out in water or a mixed medium mainly composed of water. It is preferable to match the composition of the dispersion medium of the previous sensitizing reagent latex suspension with the composition of the dispersion medium used for redispersing the dried reagent fine particles, and then perform optical measurement to determine the composition.

Furthermore, in the present invention, in the stirring process involving dry reagents and specimens, the dispersion state is checked by optical measurement as described above, and the obtained measurement results of the dispersion state are compared with optical data indicating the dispersion state set in advance. Continuing or stopping the stirring process or controlling the stirring intensity.

After stirring, if the sample contains a substance (analyte) that is reactive with the immunologically active substance bound to the surface of the fine particles in the reagent, the analyte and the immunologically active substance are mixed. An antigen-antibody reaction occurs, and agglutination progresses depending on the concentration of the substance to be measured in the specimen.

Note that stirring can also be carried out by inserting a stirring tool into the reaction cell or shaking the reaction cell within a range where the formed aggregates do not dissociate.

Further, this stirring is preferably performed with a weaker stirring force than the above-mentioned stirring treatment of the dispersion medium containing the dry reagent and the sample.

The reaction mixture in the reaction cell in which the aggregation reaction has progressed is diluted with the aforementioned diluent in the dilution cell. The dilution concentration is subsequently made when directing the reaction mixture dilution to the flow cell.
The concentration is appropriately adjusted so that the aggregates are sent one by one.

The agglomeration state of the diluted reaction mixture is determined by feeding the aggregates one by one into a flow cell and sequentially measuring the optical reaction. , a flow cytometer with the same optical axis, etc. are preferably used.

The concentration of the analyte in the sample can be calculated from the obtained measurement data by, for example, creating a calibration curve in advance that shows the relationship between the concentration of the analyte and the aggregation state of the diluted reaction mixture after the reaction.
This is done by comparing the state of agglutination of a diluted reaction mixture of specimen and reagent. A flowchart showing the basic principle of the measuring method according to the present invention is shown in FIG.

As described above, the measuring device according to the present invention includes a means for fixing the reaction cell, a means for injecting a dispersion medium into the reaction cell, a means for injecting a specimen into the reaction cell, and a means for injecting a sample into the reaction cell. A means for stirring, a means for controlling the continuation and termination of stirring, and a means for determining the degree of aggregation of the reaction mixture from optical measurement data obtained from the dispersion state of dry reagent fine particles in the dispersion medium in the stirred reaction cell. It has means for fixing a measurement cell to be measured, means for flowing the reaction mixture into the measurement cell, and means for optically measuring the degree of aggregation of individual aggregated particles of the reaction mixture flowed into the measurement cell. Also,
It may also include means for diluting the reaction mixture into a diluent before flowing the reaction mixture into the measurement cell. Furthermore, a means for diluting the specimen and a means for checking antigen excess (Brosin phenomenon) may be added.

Although any suitable apparatus may be used to carry out the present invention, an example of a preferable apparatus suitable for carrying out the method of the present invention is shown in FIG.

In the apparatus shown in Figure 1, a reaction cell 2 made of acrylic resin or (quartz) glass containing a dry latex reagent is used.
A bar code 12 is pasted on the top of the bar code 12 displaying a code for calling from memory the calibration curve data determined from the optical data during dispersion of the latex reagent and the degree of aggregation of the reaction mixture. Reaction cell 2 is a cell holder and constant temperature bath 10
The constant temperature bath 10 set in is attached with a stirring device 11 consisting of an ultrasonic vibrator for providing a stirring function. The barcode pasted on the reaction cell 2 is read by the barcode reading device 13 and then sent to the data processing device 1.
Sent to 4. A fixed amount of the dispersion medium is injected into the reaction cell from the dispersion medium container 8 in the constant temperature bath 7 through the liquid feeding valve 17. Further, a fixed amount of the sample is injected from the sample container 9 into the reaction cell 2 through the liquid supply valve 18 .

The reaction cell is immediately subjected to ultrasonic stirring in a constant temperature bath 10. In the stirring process, the light beam emitted from the light source I is introduced into the optical cell 2. When emitting coherent light, the light source 1 is a He-Ne gas laser (wavelength: 632.
8 nm), semiconductor laser (wavelength 78 Q nm, 830
nm) etc. are used. When emitting incoherent light as a light source, a tungsten lamp or a halogen lamp can be used, and an appropriate wavelength is selected using a monochromator or filter.

The light flux introduced into the reaction cell 2 is dispersed or absorbed,
The transmitted light is detected by a photodetector 3 made of a photodiode, and the amount of scattered light is detected by a photodetector 4.

Further, variations in the amount of light from the light source 1 are detected by the photodetector 5 and sent to the data processing device 14.

The detection signal of the photodetector 3.4 is also sent to the data processing device 14, enters the comparison calculation circuit from the A/D conversion circuit, and is compared with the optical data at the time of dispersion of the latex reagent in the memory circuit.

Based on the result, a signal is sent to the ultrasonic agitation controller 15 to control the stoppage, continuation, or dispersion intensity of the ultrasonic agitation.

When the stirring process is completed, aggregation reaction begins in the reaction cell 2. The reaction mixture is appropriately sent to the dilution cell 20 through the liquid sending valve 19 in accordance with the reaction conditions at the time of creating the test line. or,
At the same time, the liquid feed valve 2 is connected to the diluent container 22 in the constant temperature bath 21.
A predetermined amount of diluent is sent through 3 to obtain a diluted reaction mixture (at this time, the diluted solution may be stirred by the stirrer 24). The reaction mixture dilution is sent to the flow cell 26 through the liquid sending valve 25. The light beam emitted from the laser light source 27 is introduced into the flow cell 26, and a photodetector 28 detects the amount of light scattered when the aggregated particles in the diluted reaction mixture pass through. These signals are sent to the data processing device 14,
From the A/D conversion circuit, a measurement calculation circuit performs calculation processing on concentration data based on previously inputted calibration curve data, and the results are displayed on the display device 16.

The present invention will be explained in detail in the following examples and comparative examples.

Preparation of antibody sensitization suspension: Anti-human CRP goat serum (manufactured by Bio Makor) was added to Pr
The otein-A 5epharose (manufactured by Pharmacia) was purified into an IgG fraction by column chromatography, and 10 m
It was diluted to a concentration of g/mf.

Carboxylated polystyrene with a particle size of 0.71 nm (GO70 manufactured by Nippon Synthetic Rubber) 10% water suspension 10rn
j! 1-cyclohexyl-3-[2-morpholinyl-(4)-ethyl]carbonimide meth-
p-Toluenesulfonate (hereinafter referred to as luposiimide Ts)
1% aqueous solution of 25rr+jl! and the above I
Add 20 ml of gG fractionated antibody, stir at room temperature for 3 hours,
A sensitized latex was obtained.

After washing the above sensitized latex by centrifugation, phosphate buffer-physiological saline (hereinafter referred to as PBS) of pH 7.2 prepared to contain 1% bovine serum albumin and 3% sucrose was added to suspend the CRP antibody sensitized latex. It was made into a liquid.

Drying of Reagent The CRP-sensitized latex suspension prepared above was freeze-dried in liquid nitrogen under reduced pressure to obtain dry reagent particles for CRP detection.

Measurement - Reproducibility evaluation: Standard CRP serum (Kyowa Yuka type) was diluted with Tris HCR buffer, 5 μg/m! ! The CRP sample was determined to have a concentration of .

The reagent solid content concentration was 0 in a glass optical cell (optical path length 2 mm) containing 1 or 2 mg of the above-mentioned dry reagent fine particles.
．． Add PBS to 2%.

Furthermore, CRP antibody (5 μg/mf) was added to the above cell at 0.5 μg/mf.
Added 3mI. Immediately, the contents of the cell were subjected to ultrasonic stirring, and during the stirring process, the intensity of the incident light was increased to 10, and the intensity of the transmitted light was increased to 1.
Then, find the index A expressed by βoglo/I-A (
Measurement wavelength λ = 633 nm).

On the other hand, the CRP antibody-sensitized latex obtained in the above [Preparation of antibody-sensitized latex suspension] was adjusted to a concentration of 0.2%, and the transmitted light was measured in the same manner as above. The result was 2.75 (-A.)
Met. In the stirring step, A determined above is A/Ao≦1.
1 (A≦3.03) was satisfied, and 300 seconds after stopping, the reaction mixture was diluted 500 times with PBS and mixed in a dilution cell (20 ml made of polyethylene terephthalate). The obtained diluted reaction mixture was sent to a flow cell and irradiated with a laser beam having a wavelength of 488 nm to measure the state of aggregation of particles in the diluted reaction mixture. Compare this data with pre-measured calibration curve data, and
The concentration of CRP in the sample was measured, and this operation was performed 10 times to evaluate reproducibility.

In the stirring step, reproducibility was investigated in exactly the same manner as in Example 1, except that the ultrasonic dispersion time was kept constant.

Results〉 The results of Example I and Comparative Example 1 are shown in Table 1.

From this result, if you check the dispersion state of the dry reagent and stop stirring when a certain dispersion state is reached, the CRP
The measured value of the concentration is closest to the true value (5.0 μg/ml), and the fluctuation of the measured value is small. Stirring processing time 15
When the time is fixed at seconds, the reagent fine particles are not monodispersed due to insufficient dispersion of the dry reagent, and particles in an aggregated state are present before the reaction with CRP, which seems to increase the apparent measured value.

When the stirring treatment time was fixed at 30 seconds, the stirring treatment time was generally the same as in Example 1, but since the dispersion state varied individually, the measured value of the CRP concentration fluctuated greatly. Furthermore, when the stirring treatment time was set to 300 seconds, the dry reagent was sufficiently dispersed, but the sensitivity was lowered and the CRP measurement value was lower than the true value, probably due to a decrease in antibody activity due to stirring.

2-Preparation of hCG antibody-sensitized latex suspension Anti-hCG antibody (rabbit) (manufactured by Bio Makor) was purified into an IgG fraction by column chromatography using Protein A-3 epharose (manufactured by Pharmacia), and 10 mg of the IgG fraction was purified. /mA was diluted in 0,1 hi myphosphate buffer, pH 7.2.

Add 20 mR of a 1% aqueous solution of carbonimide Ts to 4 ml of a 10% aqueous suspension of carboxylated polystyrene (GO701 manufactured by Nippon Gosei Rubber Co., Ltd.) with a particle size of 0.71 nm, and add 20 m and f' of IgG fractionated antibodies at room temperature. The mixture was stirred for 2 hours to obtain immobilized antibody-sensitized latex.

After the above sensitized latex was centrifugally washed, 1% bovine serum albumin, 3% sucrose, and 2% carboxymethyl cellulose sodium salt were added, and PBS was added to redisperse the hC.
A G antibody sensitized latex suspension was prepared.

Drying of reagent The hCG sensitized latex liquid suspension prepared above was freeze-dried in liquid nitrogen under reduced pressure to obtain dry reagent fine particles for hCG detection.

Measurement - Evaluation of reproducibility: PBS was added to a glass optical cell (optical path length 2 mm) containing 1 or 2 mg of hCG dry reagent fine particles so that the reagent solid content was 0.2%.

Furthermore, in the above cell, hCG standard solution (manufactured by Nippon Chemical Research) was prepared at a concentration of 10 IU/mf (hereinafter h
100 μl of CG sample) was added. Immediately, the inside of the cell was subjected to ultrasonic agitation treatment, and the agitation was at a rate of 0, 2, which was measured in advance.
% bcG sensitized latex suspension at a wavelength of 633 nm A o (2,81), and if the absorbance index A during stirring is A/A o ≦1.1 (i.e. As2.09
) is satisfied. The stirring time in that step was 40 seconds. 300 seconds after stopping the dilution cell (20 m
! ! The reaction mixture was diluted 500 times with PBS and mixed in a polyethylene terephthalate solution. The obtained diluted reaction mixture was sent to a flow cell and irradiated with a laser beam having a wavelength of 488 nm to measure the state of aggregation of particles in the diluted reaction mixture. This data was compared with previously measured calibration curve data to measure the concentration of hCG in the hCG sample, and this operation was repeated 10 times to evaluate reproducibility.

In the stirring process, the ultrasonic stirring time is constant (45 seconds, 3 seconds).
The coefficient of variation was calculated in the same manner as in Example 2, except that the measurement time was changed to 00 seconds) in order to examine reproducibility (measurement was repeated 10 times).

Preparation of antibody-sensitized latex suspension of 3-AFP Anti-human α-fetoprotein (horse) (AFP) serum (Green Cross V) was added to Protein-A 5epha
The IgG fraction was purified by column chromatography using rose (manufactured by Pharmacia), and further purified with 0.5% IgG fraction at pH 7.2.
It was diluted with 1M phosphate buffer to a concentration of 10 mg/mj' to obtain an IgG fraction antibody.

Add 20 ml of a 1% aqueous solution of carbodiimide Ts to 5 mA of a 10% water suspension of carboxylated polystyrene with a particle size of 0.71 nm (GO701 manufactured by Nippon Gosei Rubber), add 20 ml of an IgG fractionated antibody, and incubate for 2 hours at room temperature. The mixture was stirred for a period of time to obtain antibody-sensitized latex.

After washing the above sensitized latex by centrifugation, bovine serum albumin at a concentration of 1% and sucrose were added to a concentration of 3%, pH 7.
, 2 of PBS was added to prepare an AFP antibody sensitized latex suspension.

Drying of reagents.

The AFP antibody-sensitized latex suspension prepared above was frozen in liquid nitrogen and dried under reduced pressure to obtain dry reagent particles for AFP detection.

Measurement - Reproducibility evaluation: A reagent solid concentration of 0.2 was placed in a glass optical cell (optical path length 2 mm) containing 1.2 mg of dry reagent fine particles for AFP detection.
% of PBS.

Furthermore, 200 μl of 50 μg/mf AFP standard solution (hereinafter referred to as AFP sample) is added to the above cell (AFP sample is standard AFP serum (manufactured by Kyowa Yuka) diluted with Tris HC1 buffer to a predetermined concentration). Immediately, the inside of the cell was subjected to ultrasonic agitation treatment, and the agitation was measured in advance at 0,
Extinction index A of 2% AFP sensitized latex suspension at wavelength 633 nm. (2,71), if the extinction index A during stirring is A/Ao≦1.1 (i.e. As2.98)
Stop when satisfied.

The stirring time in that step was 30 seconds.

300 seconds after stopping, the reaction mixture was diluted with PBS in a dilution cell (20 ml polyethylene terephthalate).
00 times diluted and mixed. The obtained diluted reaction mixture was sent to a flow cell and irradiated with a laser beam having a wavelength of 488 nm to measure the state of aggregation of particles in the diluted reaction mixture. Compare this data with pre-measured calibration curve data,
The concentration of AFP in the AFP sample was measured, and this operation was repeated 10 times to evaluate the reproducibility.

In the stirring process, the ultrasonic stirring time is constant (30 seconds, 3 seconds).
The coefficient of variation was calculated in the same manner as in Example 3 except that the measurement time was changed to 00 seconds) in order to examine the reproducibility (measurement was repeated 10 times).

4- Micro Roblin's Preparation of antibody sensitized latex suspension.

Anti-β2-microglobulin (rabbit) (Bi.

Protein-A 5e (manufactured by Makor)
It was purified into an IgG fraction by column chromatography using Pharose (manufactured by Pharmacia), and further purified at pH 7,
2 with 0.1M phosphate buffer] to a concentration of Omg/ml, and used as an IgG fraction antibody.

Add 20 ml of a 1% aqueous solution of carbodiimide Ts to 4 ml of a 10% water suspension of O carboxylated polystyrene (GO701 manufactured by Nippon Gosei Rubber) with a particle size of 0.71 nm, and then add 20 ml of an IgG fractionated antibody, and incubate at room temperature for 3 hours. The mixture was stirred to obtain antibody-sensitized latex.

After the above sensitized latex was centrifugally washed, 1% concentration of raw serum albumin and sucrose were added to a 3% concentration, and the pH was adjusted to 1%.
7,2 PBS was added to prepare a β2-microglobulin antibody sensitized latex suspension.

Drying of reagent: The β2-microglobulin antibody-sensitized latex suspension prepared above was frozen in liquid nitrogen and dried under reduced pressure to obtain dry reagent particles for β2-microglobulin detection.

Measurement - Reproducibility evaluation: Dry reagent fine particles 1 for β2-microglobulin detection,
Glass optical cell containing 2 mg (2 mm optical path length)
PBS is added to the solution so that the reagent solid content concentration is 0.2%.

Furthermore, 100 μl of 5 μg/ml β2-microglobulin standard solution (hereinafter referred to as MG sample) is added to the above cell (M
Sample G is standard β2-microglobulin serum (manufactured by Kyowa Yuka Co., Ltd.) diluted with Tris HCl buffer to a predetermined concentration. Immediately, the inside of the cell was subjected to ultrasonic agitation treatment, and the agitation was performed using the previously measured extinction index A of the 0.2% β2-microglobulin antibody-sensitized latex suspension at a wavelength of 633 nm.

(2,80), if the extinction index A during stirring is A/
The process is stopped when Ao≦1.1 (ie, As3.06) is satisfied. The stirring time in that step was 40 seconds.

300 seconds after stopping, the reaction mixture was diluted with PBS in a dilution cell (made of 20 mf polyethylene terephthalate).
00 times diluted and mixed. The obtained diluted reaction mixture was sent to a flow cell and irradiated with a laser beam having a wavelength of 488 nm to measure the state of aggregation of particles in the diluted reaction mixture. Compare this data with pre-measured calibration curve data,
Measuring the concentration of β2-microglobulin in the MG sample,
This operation was repeated 10 times to evaluate reproducibility.

Further, the coefficients of variation C, V, (%) were calculated in the same manner as in Example 1.

In the stirring process, the ultrasonic stirring time was kept constant (35 seconds, 3
The coefficient of variation was calculated in the same manner as in Example 4 except that the measurement was repeated (00 seconds) to examine the reproducibility (10 measurement repetitions).

Table 2 shows the measurement results of Examples 2 to 4 and Comparative Examples 2 to 4.

From the results in Table 2, even if the substance to be measured is changed to hCG, β2 microglobulin, or AFP, the measurement in the example in which the stirring treatment time is variably controlled is closer to the true value than in the comparative example in which the stirring time is fixed. Data was obtained, and the coefficient of variation was reduced and reproducibility improved.

Table 1 D * CV = -X100 Table O: Good measurement sensitivity, △: Slightly poor, ×: Poor *The apparent measured value is high, but the difference from the true value is large [Summary of the effects of the invention] Explanation above As described above, the present invention can quantify immunologically active substances such as antigens and antibodies in a specimen by utilizing antigen-antibody reactions.

Furthermore, since the present invention uses a dried immunoreagent, it has the following advantages in terms of reagent storage compared to conventional reagents dispersed in water.

1. Since the reagent is in a dry state, natural aggregation does not occur over time unlike reagents dispersed in water.

2. Temperature control during storage of reagents is relaxed.

(Conventional reagents cannot be frozen and must be stored with care.) 3. Dried reagents are stable and can be stored for a longer period of time.

Using a dry immunoreagent with the above advantages, the dispersion is optically measured during the stirring treatment process of the dispersion medium containing the fine particles and the specimen, and the subsequent agglutination reaction is smoothed by controlling the stirring force. This makes it possible to significantly improve the reproducibility and reliability of the data obtained.

Furthermore, since the stirring time can be controlled to the minimum in the reagent stirring process, it is possible to prevent a decrease in sensitivity and at the same time to shorten the measurement time.

[Brief explanation of the drawing]

FIG. 1 is a diagram schematically showing a typical example of an apparatus suitable for carrying out the method of the present invention, FIG. 2 is a diagram showing transmittance changes in each step, and FIG. It is a flowchart of the measurement method of this invention. FIG. 4 and FIG. 5 are diagrams showing the relationship between stirring time and A/A o in Experimental Examples 1 and 2. In FIG. 1, l...Light source 2...Reaction cell 3-5.28...Photodetector 6...Half mirror 7...Dispersion medium thermostat 8...Dispersion medium container 9 ...Sample containers 10, 21...Cell holder and constant temperature bath 11...Stirrer f (ultrasonic vibrator and shaker) 12...Barcode 13...Barcode reader 14... Data processing device 15... Stirring device control device 16... Display device 17.18.19.23.25... Liquid feeding valve 20.
... Dilution cell 22 ... Diluent container 29 ... Stirring device 26 ... Flow cell 27 ... Laser light source 29 ... Liquid feeding tube 1 Itching treatment squeezing tube Sugatsubaki push rotation Moekaku

Claims (5)

[Claims] (1) Chemically bonding a substance that is immunologically active to the substance to be measured in the sample to the surface of solid particles, and causing the sample to react with the bound immunologically active substance in a liquid medium. A method for optically measuring the degree of aggregation of a reaction mixture produced by (I) drying solid microparticles with chemically bonded substances immunologically active to the analyte in the sample on the surface; (
In a reaction cell containing dry reagent particles (hereinafter referred to as “dry reagent particles”),
a step of adding a dispersion medium and a specimen; (II) a step of stirring the dispersion medium, dry reagent fine particles, and specimen in the reaction cell, and optically measuring the dispersion state of the dry reagent fine particles in the dispersion medium; ( III) Measure the dispersion state of the dry reagent fine particles in the dispersion medium from the optical measurement data obtained in step (II) above,
When the preset dispersion state is reached, the stirring in step (II) is stopped; (IV) the dispersion containing the sample that has reached the dispersion state set in step (III) above is reacted to form an agglomerated state. (V) A step of flowing the reaction mixture in the reaction cell generated in the above step (IV) into a measurement cell; (VI) Optically measuring the degree of aggregation of the reaction mixture flowed into the measurement cell. A method for measuring an immunologically active substance, comprising the steps of: (2) To determine the dispersion state in the above step (III), optical measurement data of the dispersion state of the dry reagent fine particles in the dispersion medium is prepared in advance, and the dry reagent fine particle dispersion obtained in the above step (II) is prepared in advance. Compare with the optical measurement data of the body,
The measuring method according to claim 1, wherein the measuring method is carried out by checking dispersion. (3) To determine the dispersion state in step (III) above, the intensity of the incident light when monochromatic light passes through the reaction cell containing the complete dispersion of dry reagent fine particles in the dispersion medium measured in advance is I_0. , where the intensity of transmitted light and/or scattered light is I, and the index A_0 is expressed as logI_0/I=A_0. When monochromatic light passes through, the intensity of the incident light is I_0, and the intensity of the transmitted light and/or scattered light is I
2. The measuring method according to claim 1, wherein the measuring method is carried out by confirming that an index A expressed by logI_0/I=A is within the following range. A/A_0≦1.1 (4) The optical measurement of the aggregation state in the above step (VI) is carried out by diluting the reaction mixture obtained in the above step (V) with a diluent,
The measuring method according to claim 1, wherein the measuring method is carried out while the diluent is flowing. (5) Chemically bonding a substance that is immunologically active with respect to the analyte in the specimen to the surface of the solid fine particles, and causing the specimen to react with the bound immunologically active substance in a liquid medium. An apparatus for optically measuring the degree of aggregation of a reaction mixture caused by (I) means for fixing the reaction cell, (II) means for injecting a dispersion medium into the reaction cell, (III)
) a means for injecting the sample into the reaction cell, (IV) a means for stirring the contents in the reaction cell, (V) a state of dispersion of dry reagent fine particles in the dispersion medium in the stirred reaction cell. means for controlling the continuation or stop of stirring from the obtained optical measurement data; (VI) means for fixing a measurement cell for measuring the degree of aggregation of the reaction mixture; (VII) means for flowing the reaction mixture into the measurement cell. , (VIII
) A device for measuring an immunologically active substance, comprising: means for optically measuring the degree of aggregation of the reaction mixture flowed into the measurement cell.