JPH0472569A - Measuring device for 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 [Industrial Application Field] 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 C.M. Singer et al.
et al) [Am, J, Med,
, 21888 (1956)], and thereafter various studies have been made regarding this method. Although quantitative measurement is difficult, the method of visually determining the degree of aggregation has been widely used in practice because it has the advantage of being simple and allowing results to be obtained in a short time.

In recent years, attempts have been made to optically measure the degree of aggregation, and Nie Fature 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
desBi, ol Fluids, Proc, Co1
1oq, 2589 (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, the latex reagent is a state in which solid particles are dispersed in a liquid dispersion medium, which is an inherently unstable system. (,or,
It has various problems, including the fact that freezing destroys the dispersed state, requiring special consideration for preservation.

On the other hand, a method has been proposed to improve the stability of latex reagents by removing the dispersion medium liquid and drying them (Japanese Patent Laid-Open Nos. 52-117420, 1986).
2-46262).

However, although the storage stability of latex reagents can be improved by drying them, latex reagents obtained by redispersion have large fluctuations in aggregation reactivity, and as a result, measurement data often fluctuates. There is a problem.

Therefore, in the prior art, it can be used for qualitative measurements such as negative or positive determination by visual inspection on a slide, but it is not suitable for highly accurate quantitative measurements.

Alternatively, an agglutinating immunoreagent such as a latex reagent is injected into a capillary tube, lyophilized, and mixed with the specimen in the capillary tube to react, 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. 513-73866). This method has the same good storage stability of the reagents as the above proposal, and is attractive as a simple testing method, but it has the problem of poor reproducibility of measured values and the inability to perform accurate quantification. .

[Purpose of the invention]

The present invention solves the problems in the prior art described above, and
The object of the present invention is to provide an immunoassay device that has excellent reproducibility of measured values and enables 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 causes of fluctuations in measured values in the conventional method were due to variations in the state of redispersion, as well as stirring for redispersion. It has been found that the measurement sensitivity is reduced due to the destruction of the bond between the solid particles and the immunologically active substance due to prolonged stirring or excessively strong stirring. Furthermore, as a result of intensive studies based on these findings, the present inventors found that when redispersing the dry reagent, the dispersion state was optically measured while stirring, and when a suitable dispersion state was reached, It has been found that proceeding to the next reaction 1 culm 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 is an apparatus suitable for carrying out the measurement method described below.

”-The apparatus of the present invention suitable for carrying out the measurement method described above is
The content is as described below.

A substance that is immunologically active to the substance to be measured in the specimen is physically and/or chemically bound to the surface of the solid fine particles, and the specimen is attached to the bound immunologically active substance in a liquid medium. An apparatus for optically measuring the degree of aggregation of a reaction mixture caused by a reaction, comprising: (i) physically and/or chemically applying a substance that is immunologically active against the analyte in the specimen to the surface; (11) means for injecting a dispersion medium into the measurement cell; (iii) injecting a specimen into the measurement cell; means, (iv) stirring means for stirring the contents of the measurement cell; (V) means for switching the stirring capacity of the stirring means; (Vi) optically measuring the degree of aggregation of the contents in the measurement cell. (Vii) means for determining the state of dispersion of the dry reagent fine particles in the dispersion medium from optical measurement data of the dry reagent fine particles and the dispersion medium mixture in the stirred measurement cell, and controlling the continuation/stop of stirring; A measuring device having , .

The present invention will be specifically explained 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 staphylococci and streptococci treated with red blood cell dispersion can be mentioned. Examples of the inorganic fine particles include:
Examples include silica, alumina, and bentonite. 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. Examples include fine particles of copolymers and butadiene copolymers such as methyl methacrylate-butadiene copolymers.

The binding of immunologically active substances to such microparticles can be done physically and/or chemically, as described below.
For physical bonding, it is preferable that the particle surface is hydrophobic, and styrene homopolymer particles, vinyl copolymer particles containing styrene as the main component, or styrene-butadiene copolymer containing styrene as the main component are used. Particularly preferred. The particle diameter of the above-mentioned fine particles is 0.0 regardless of whether they are biologically derived fine particles, inorganic fine particles, or organic fine particles.
5 μm to 5 μm is preferred, and 0.1 μm to 2 μm is particularly preferred. If the particle size is around 0.05 μm, it will be difficult to disperse the dry reagent, and if the particle size is around 5 μm, the stability of the dispersed reagent will be poor.

The immunologically active substances to be bound to the surface of the solid fine particles used in the present invention include IgG, Ig
M.

Immunoglobulins such as IgE - Plasma proteins such as complement, CRP, ferritin, α1 microglobulin, β2 microglobulin, and their antibodies α-fetoprotein, carcinoembryonic antigen (CEA), prostatic acid phosphatase (PAP), CA-19 -9, CA-125 and other tumor markers and their antibodies.

Luteinizing hormone (LH), follicle stimulating hormone (FSH)
, human ciliary gonadotropin (hcc), estrogen, insulin, and other hormones and their antibodies: H
BV-related antigens (HBs, HBe, HBc), HIV
Substances related to virus infection such as ATL and their antibodies; Bacteria such as Nidifthilia, Clostridium botulinum, Mycoplasma pallidum, and Treponema pallidum; and protozoa such as Toxoplasma gondii, Trichomona threishmania, Trivanosoma, and Plasmodium; and their antibodies: Drugs such as anti-epileptic drugs such as phenytoin and phenobarbital, cardiovascular drugs such as quinidine and digoxinin, anti-asthmatic drugs such as theophylline, antibiotics such as chloramphenicol and gentamicin, and their antibodies; other enzymes, bacteria There are exotoxins and their antibodies, and a substance that causes an antigen-antibody reaction with a substance to be measured in a specimen is appropriately selected and used depending on the type of specimen.

The binding of microparticles and immunologically active substances has been described, for example, in JP-A-5
This can be carried out by known physical and/or chemical bonding methods described in Japanese Patent Publication No. 3-52620, Japanese Patent Publication No. 53-12966, and the like. The binding reaction between the fine particles and the immunologically active substance is preferably carried out in water or a mixed solvent of water and an organic solvent such as an alcohol or a ketone that is compatible with water. In addition, a phosphate buffer solution is added to the reaction system for the purpose of stabilizing the particles and preventing non-specific aggregation.
It is preferable to add physiological saline, a buffer such as Tris-HCf buffer, an inert protein such as bovine serum albumin, a surfactant, and the like. The pH of the reaction solution is usually 6 to I
O1 is preferably 7-9. Further, the concentration of fine particles in the reaction solution is usually 0.01 to 2.0% (by weight).

In order to chemically bond microparticles with immunologically active substances, for example, amino groups, carboxyl groups, hydroxyl groups, oxirane groups, etc. are oriented on the surface of the microparticles, and polyamide compounds, polyimide compounds, polyaldehyde compounds, polyoxirane compounds, etc. There are two methods: a method of reacting with an immunologically active substance via microparticles, and a method of aligning aldehyde groups, oxirane groups, etc. on the surface of fine particles and reacting with an immunologically active substance.

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

It is advantageous to remove the dispersion medium at a temperature below 60°C, preferably below 30°C, in order to maintain the activity of the immunologically active substance. A particularly preferred embodiment for removal of the dispersion medium is removal by lyophilization, in which case the sensitivity of the reagent remains consistently high. When introducing a dry immunoreagent into a measurement cell, a dispersion of fine particles bound with a predetermined amount of an immunologically active substance may be placed in the measurement cell and dried as described above to remove the dispersion medium. Alternatively, a predetermined amount of dried immunoreagent from which the dispersion medium has been removed may be placed in the measurement cell.

As the measurement cell, one made of transparent glass or plastic (for example, polystyrene, polymethyl methacrylate, polyvinyl chloride, polycarbonate, polysulfone, etc.) is used.

A dispersion medium for dispersing the dried immunoreagent in the measurement cell is water or a mixed solvent of water and an organic solvent compatible with water such as alcohols or ketones.

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

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; for example, a phosphate, Tris, or HCI buffer is used. Proteins are added for the purpose of preventing non-specific reactions, such as bovine serum albumin and gelatin.

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 polyacrylates. , and droxyethylcellulose 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. The solid content concentration varies depending on the type or size of the measurement cell used, but is generally preferably 0.01 to 5%, more preferably 0.05%.
It is adjusted in the range of ~2%.

To disperse the dry immune reagent in the dispersion medium, a method of injecting a predetermined amount of the dispersion medium into a measurement cell containing the dry immunoreagent, inserting and stirring a stirring tool, or a method of shaking the measurement cell can be selected as appropriate.

Among these, dispersion by ultrasonic stirring is preferred as it is the most effective method for dispersing fine particles. The ultrasonic waves used for ultrasonic stirring vary depending on the type and size of the measurement cell, but generally, ultrasonic waves with a vibration frequency of 15 Hz to 50 Hz are 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 transmitted through the reaction cell decreases and becomes almost constant in a uniformly dispersed state.

In addition, a preferred method for determining the dispersion state was derived from the results of the experimental examples described below, and this method is based on the measurement cell containing a complete dispersion of dry reagent fine particles in a dispersion medium measured in advance. Manipulates the intensity of the incident light when monochromatic light passes through it. , the intensity of transmitted light and/or scattered light is ■, and IogI. /I=To the index indicated by Ao For 〇,
I is the intensity of the incident light when the monochromatic light passes through the measurement cell containing the dry reagent fine particle dispersion obtained in step (ii) of the present invention. This method is performed by setting the intensity of transmitted light and/or scattered light to 1 and confirming that the index A expressed as 'OgIo/I=A is within the following range.

A/A o ≦1.1 When dispersing dry reagent fine particles in a dispersion medium by stirring,
By completing the dispersion process when A/A o satisfies the above range while performing optical measurement and proceeding to the next reaction process, trace components can be detected regardless of the type of substance to be measured. It also allows for stable measurements with little variation in measured values.

Next, the index A of a complete dispersion of dry reagent microparticles.

The method of determining the After combining the composition of the dispersion medium of the reagent latex suspension with the composition of the dispersion medium used for redispersing the dried reagent fine particles,
Preferably, it is determined by optical measurement.

Furthermore, in the dispersion process of the dry reagent, in the present invention, the dispersion state is checked by optical measurement as described above, and the obtained measurement result of the dispersion state is compared with optical data indicating the dispersion state set in advance. Continue, stop, or control the stirring intensity.

By the above-mentioned control, a highly sensitive dispersion reagent with a uniform dispersion state can be obtained. This dispersion reagent is subsequently mixed and reacted with the specimen.

In other words, a substance (substance to be measured) that is reactive with an immunologically active substance bound to the surface of fine particles in the reagent in the sample.
When the analyte is contained, the analyte reacts with the immunologically active substance, causing an antigen-antibody reaction, and aggregation progresses depending on the concentration of the analyte in the sample. Mixing during the reaction is performed by inserting a stirrer into the measurement cell or by shaking the measurement cell.

Note that the purpose of stirring during this reaction is to uniformly mix the dispersion reagent and the specimen, and as a result of mixing, the reaction immediately starts and aggregation progresses. Therefore, stirring is continued or stopped within a range in which the formed aggregates do not dissociate. Also,
This stirring is preferably performed with a weaker stirring force than the stirring for dispersing the dry reagent described above.

Methods for measuring the aggregation state by irradiating the reaction mixture in the measurement cell with light include, for example, a method of measuring transmitted light intensity, a method of measuring scattered light intensity, a method of combining these methods, and a method of measuring integrating sphere turbidity. There are ways to do this.

The concentration of the substance to be measured in the specimen can be calculated from the obtained measurement data using, for example, the known rate assay method, end point method, etc. [Immunology Experimental Procedures ■, Bunkodo, P2401 (19
79)].

A flowchart showing the basic principle of the measuring method according to the present invention is shown in FIG.

In the present invention, the stirring capacity can be changed even with the same type of stirring means, and the stirring capacity can be changed in each of the two steps described above.

In other words, by providing a stirring device that can vary the stirring force, the same device can serve as both the stirring for dispersing the dry reagent (strong stirring) and the stirring during the reaction (weak stirring). For example, when using ultrasonic agitation with adjustable output in two processes, the output in the process of dispersing dry reagents is 10 W to 90 W (20 K), depending on the cell material, capacity, and effective efficiency.
Hz), and in the reaction process the output is 0.1 to 10 Watts (20
KHz) is preferable. Further, the shaking ability may be varied.

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 FIG. 1, an optical cell 2 made of acrylic resin or (quartz) glass containing a dry latex reagent has a barcode 12 pasted on top thereof indicating optical data at the time of dispersion of the latex reagent. It is. Optical cell 2 is a cell holder and constant temperature bath 1
Set to 0. The constant temperature bath can have a variable output in order to have a stirring function. A stirring device 11 consisting of an ultrasonic vibrator is attached. The barcode pasted on the optical cell 2 is read by the barcode reader 13,
The data is sent to the data processing device 14 and stored in memory. A fixed amount of dispersion medium is injected into the optical cell from a dispersion medium container 8 in a constant temperature bath 7 through a liquid supply valve 17. The inside of the optical cell is ultrasonically stirred at 10 in a constant temperature bath. In the stirring process, the light beam emitted from the light source 1 is introduced into the optical cell 2. When the light source 1 emits coherent light, it is a He-Ne gas laser (wavelength: 632.8% m) or a semiconductor laser (wavelength: 780 nm).

830 nm) is used.

Further, 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 optical 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.

Furthermore, 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. Detection signals from the photodetectors 3 and 4 are also sent to the data processing device 14, input from the A/D conversion circuit to the comparison calculation circuit, and compared with 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 stirring control device 15 to stop the ultrasonic stirring,
Continuation or control of dispersion intensity, the stirring process is finished,
A sample is injected into the optical cell 2 from the sample container 9 through the liquid feed pump 18 . The inside of the optical cell 2 is ultrasonically stirred for a certain period of time (3 to 5 seconds) in an IO in a constant temperature bath, and after stopping, optical measurement is performed again. After a certain period of time (20 seconds to 2 minutes) has elapsed, optical measurement is performed again, and these signals are sent to the data processing device 14, where the A/D conversion circuit and the measurement calculation circuit also process the calibration curve data input in advance. The concentration data is then subjected to arithmetic processing, and the results are displayed on the display device 16.

〔Example〕

Hereinafter, the present invention will be explained in more detail using Examples.

1-Preparation of hCG antibody-sensitized latex suspension: Add hCG antibody (rabbit) (Bio Ma (manufactured by k or) 8mf
was added, stirred thoroughly, and then heated at 40°C for 2 hours to effect sensitization.

After centrifugally washing the above sensitized latex, 1% bovine serum albumin and 5% sucrose were added to make a 1% suspension.
7.2 phosphate buffer-physiological saline (hereinafter referred to as PBS) was added to prepare an hCG antibody-sensitized latex suspension.

Drying of reagent: The hCG antibody-sensitized latex suspension prepared above was freeze-dried in liquid nitrogen under reduced pressure to obtain dry reagent microparticles-1.

Measurement/Reproducibility Evaluation: PBS was added to a glass optical cell (optical path length: 2 mm) containing 11.2 mg of the above-mentioned dry reagent fine particles, and the reagent solid content concentration was adjusted to 0.2%. 20 cells above
Ultrasonic stirring treatment at KHz, 15W output, and the dispersion
The intensity of the incident light is I. , the intensity of transmitted light is 1, and Lo
gI. Find the index A shown by /I=A (measurement wavelength 6
33 nm).

On the other hand, PBS was added to the hCG antibody-sensitized latex obtained in advance (preparation of antibody-sensitized latex suspension) to adjust the reagent solid content concentration to 0.2%, and the transmitted light I was adjusted in the same manner as above. Measure A.

(= L o g I o / I ), and the ultrasonic stirring process was stopped when the above A satisfied A/Ao≦1.1 (stirring time 160 seconds), and 10 IU/I was added to the optical cell. mI! 100 μf of standard hCG solution adjusted to
and ultrasonic stirring (20K Hz 2 W) for 3 seconds.
) L, changes in absorbance were measured after 20 seconds and 200 seconds. The above measurement was continued a total of 10 times. The results are shown in Table 1.

°-() Table [Summary of Effects of the Invention] As explained above, the present invention can quantify immunologically active substances such as antigens and antibodies in a specimen by using 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 reagent storage is relaxed (conventional reagents cannot be frozen and must be stored with care).

3. Dry reagents are stable and can be stored for a longer period of time.

Using a dry immunoreagent with the above advantages, the dispersion state is optically measured in the process of redispersing the fine particles, and the subsequent agglutination reaction is carried out smoothly by controlling the stirring means, and the data obtained. This makes it possible to significantly improve reproducibility and reliability.

Furthermore, since the stirring time can be controlled to the minimum in the reagent dispersion process, it is possible to prevent a decrease in sensitivity and at the same time to shorten the measurement time. Furthermore, since the same stirring device can perform stirring in the reagent dispersion step and stirring in the reaction step, the device can be made smaller.

[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. In FIG. 1, 1... light source, 2... optical cell, 3
~5...Photodetector, 6...Half mirror 17...
Dispersion medium constant temperature bath, 8... Dispersion medium container, 9... Sample container, 10... Cell holder and constant temperature bath, 11... Stirring device (ultrasonic vibrator) with variable output, 12. ... Barcode, 13... Barcode reader, 14.
...Data processing device, 15...Control device for stirring device,
16...Display device, 17.18...Liquid feeding valve, 1
9...Liquid feeding tube No.

Claims (1)

[Claims] (1) Physically and/or chemically bind a substance that is immunologically active to the analyte in the specimen to the surface of the solid fine particles, and transfer the specimen to the bound immunologically active substance in a liquid medium. A device for chemically measuring the degree of aggregation of a reaction mixture generated by reacting in a sample, the device comprising: (i) physically and/or physically adding a substance immunologically active to the analyte in the sample to the surface; means for fixing a measurement cell containing chemically bonded and dried solid fine particles (hereinafter referred to as dry reagent fine particles); (ii) means for injecting a dispersion medium into the measurement cell; (iii) a means for injecting a sample into the measurement cell. (iv) Stirring means for stirring the contents of the measuring cell; (V) Means for switching the stirring capacity of the stirring means; (Vi) Optically measuring the degree of aggregation of the contents in the measuring cell. (vii) means for 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 measurement cell. A device for measuring immunologically active substances.