JPS6281567A - Quantification method using particle agglutination reaction - Google Patents

Abstract

PURPOSE:To make it possible to simultaneously quantify a plurality of substances, in a latex agglutination method, by allowing fine particles different in particle size to respectively support different responsive substances. CONSTITUTION:For example, particles are classified into two kinds in particle size and two kinds in the adhesion of a fluorescent dye and, further, ones having no dye adhered thereto are considered to form 6 groups in total. Reagents prepared by respectively supporting six kinds of antibodies by the particles of the groups are mixed with a specimen solution to prepare measuring solutions which are, in turn, measured by a flow sight meter together having colter volume measuring function and fluorescent intensity measuring function to obtain particle size distribution at every group. The concn. at every substance to be measured can be calculated by this particle size distribution and a plurality of the substances to be measured contained in a specimen can be simultaneously and accurately quantified.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention relates to a quantitative method that utilizes specific binding between substances, such as antigen-antibody reaction.

(Prior Art) Immunoassay methods that utilize immunological reactions have excellent specificity and sensitivity, and are frequently used in the medical field. Among them, radioimmunoassay (RIA) and endeimmunoassay (EIA) have excellent sensitivity and accuracy, and are representative in the field of trace analysis. In 1959, Pearn and Yarrow used RIA as a method for measuring insulin, and it has spread all over the world, and is now widely applied not only to proteins but also to hormones, viruses, drugs, etc. However, on the other hand, there are problems such as the need for special equipment for RI, the problem of disposal of RI contaminated materials, and the attenuation of RI labeled materials, and research on non-radioactive labeled materials has been actively conducted. EIA was born out of this research, and at the time it was first announced, it was said to be a method that would completely replace RIA in a few years. It is true that it is safe, the stability of the labeled substance is relatively good, and it has the advantage of being able to be measured in a homogeneous system that does not require separation of antigen-antibody bound form and free form, so-called B/P separation. There are also unstable factors such as the use of biological enzyme reactions for detection. In terms of sensitivity, there are cases where the sensitivity is higher than that of RIA depending on the substance to be measured, but in general it is said to be lower than RIA, and the reality is that it cannot even replace RIA.

On the other hand, there is a method called "latex agglutination method" in which the agglutination that occurs when an antibody (or antigen) is applied to microparticles carrying an antigen (or antibody), such as latex particles, is visually determined on a plate. Due to its simplicity and speed, it has been frequently used in recent years.It is said that it is not suitable for trace analysis due to the qualitative aspect of relying on visual perception and the fact that sensitivity is low. Recently, a measurement method for quantifying latex aggregation has been developed and is attracting attention.

For example, L
PIA (Latex Photometric Im
muno-assay), “Inspection and Technology”
Magazine Vol. 12 No. 7 No. 581 (1984), LA
(LatexAgglupination Immun
oassay) was introduced in the Journal of Medical Electronics and Bioengineering, Vol. 22, No. 4, Page 39 (1984), and PACIA (Par
Tickle Counting Immunoassay
) is r J, of Immun, Meth, J
It is introduced in Volume 18, Page 33 of the magazine (1977).

Others, JP-A-58-190760. Japanese Unexamined Patent Publication 1986-17
Techniques using latex have also been disclosed in JP-A No. 3759-151357, etc., and these techniques are said to be comparable in sensitivity to RIA, depending on the object to be measured.

In the medical field, lesions and the like are often detected based on changes in substances within a living body, but in this case it is often difficult to make accurate judgments based solely on changes in one parameter. For example, CEA, which is a typical tumor marker, is said to have unresolved problems such as organ specificity and false positives in benign diseases. In order to solve these problems, it is essential to look at several nosolameters and make a comprehensive judgment. However, at present, it is necessary to repeat measurements for as many parameters as you want to know.
It is only possible to capture changes as a sum total of each parameter, and there is no example in which it has been possible to measure multiple items at the same time. Therefore, not only the labor of the doctor but also the burden on the patient due to blood sampling, etc., and the time required for results to be obtained cannot be ignored. Furthermore, when the target to be measured is blood components, sensitivity is often reduced due to the effects of impurities, interfering substances, etc.

(Problems to be Solved by the Invention) The present invention has been made to solve the above-mentioned problems. Another object of the present invention is to provide a method for simultaneously, accurately and quickly measuring the amount of a plurality of Sakakidame I nitrogen substances in a single compact by improving a method for measuring the agglomeration state of fine particles carrying a sensitive substance.

(Means and effects for solving the problem) The outline of the present invention for achieving the above object is to reduce the amount of each of the n types of analyte substances in a sample by specifically binding to each of the analyte substances. This method uses microparticles of uniform particle size that support n types of sensitive substances, and the n Using the fine particles that can be distinguished into species, each type of particle is loaded with the n types of sensitive substances that specifically bind to the respective analyte as a reagent, and the reagent and the specimen are brought into contact with each other, The particle aggregation reaction is characterized in that the agglomeration state of the resulting particles is measured, the measurement results are grouped according to the type of particles, and the amounts of each of the n types of substances to be measured are measured. This is the quantitative method used. Here, n is a natural number of 2 or more.

A typical specific binding reaction between substances is the antigen-antibody reaction, but other reactions such as those between hormones and receptors, sugars and lectins, etc. can also be used.

The content of the present invention will be explained in more detail by taking an antigen-antibody reaction as an example. If the object to be measured can be an antigen (including Hashiran, etc.), the corresponding antibody is supported on the fine particles. Depending on the object to be measured, the combination of antigen and antibody may be reversed.

In the present invention, in order to quantify a plurality of substances at the same time, it is configured in advance so that fine particles (with uniform particle sizes of about 0.1 to 30 μm) can be classified into a plurality of groups. For example, the particles can be made to have multiple sizes, or they can be labeled with a fluorescent substance or dye. Further, by combining these, more groupings can be made. When particles are labeled with a fluorescent substance, the presence/absence or concentration of the fluorescent substance, the type of fluorescence (for example, fluorescein)
and PI (Propidium Iodide)), it can be distinguished into multiple groups. Furthermore, by combining particle sizes as described above, it is possible to discriminate even more particle groups.

Examples of fine particles that can be used include cells such as red blood cells, metals, microcapsules such as liposomes, and latex particles such as polystyrene.

In order to make microparticles support a predetermined sensitive substance, such as an antibody, there are known methods such as physical adsorption, chemical bonding using functional groups on the microparticles, and the like.

The sensitive substance refers to a substance that reacts specifically with a substance to be measured, and when the substance to be measured is an antigen, it is an antibody that specifically reacts with the antigen. As mentioned above, depending on the type of substance to be measured, hormones, receptors, sugars, lectins, etc. can also be used.

The present invention will be explained in detail by taking the use of an antibody-antigen reaction as an example.

When a reagent containing fine particles carrying antibodies is brought into contact with a specimen containing an antigen, the particles aggregate due to an antigen-antibody reaction. The higher the concentration of the antigen (i.e., test substance) in the specimen, the greater the chance of agglutination, and the degree of agglutination will vary. Therefore, by measuring the degree of agglutination, that is, the state of agglutination, it is possible to know the amount of antigen, ie, the substance to be measured, in the specimen. However, when the concentration of the test substance increases beyond a certain level, a so-called zone phenomenon occurs and the degree of aggregation becomes low. Therefore, for accurate measurement, it is necessary to appropriately control the concentration of the specimen.

Methods for measuring the state of aggregation include a method of visually observing and counting the agglomerated particles by turning them into plano or lard, or taking microscopic photographs, and a method of measuring the absorbance and scattered light of a solution containing the agglomerated particles. can give. However, these methods have problems with accuracy and it is generally not easy to distinguish between particle types.

Therefore, a preferred embodiment is a method in which these particles or aggregated particles are measured one by one by flow cytometry.

Flow cytometry is mainly related to optical instrument analysis, and it allows particles to be flowed through one by one, illuminated with a laser beam, etc., and measured the scattered light. In this method, the particles are labeled in advance with a fluorescent substance, etc., and the characteristics of the particles are measured by measuring the intensity of the fluorescent light. In addition, based on the so-called Coulter's principle, a method of electrically measuring the capacitance of particles (called a Coulter gorium) and a method combining this and optical measurement are also used.

The agglomeration state can be determined by measuring the size of each particle or agglomerated particle agglomerate, or the fluorescence intensity if the particle is fluorescent or has fluorescent activity, and determining the aggregation state ( That is, the measurement is performed by determining whether the particles are non-agglomerated or 2.3, 4, 5... particles are aggregated. FIG. 1 schematically shows an example of measuring the particle size distribution of a certain type of particle group in an agglomeration reaction. When measuring n types of particle groups (n types of substances to be measured), n graphs corresponding to FIG. 1 are obtained.

To distinguish particles into n groups, for example, by using two groups based on particle diameter and two types of fluorescent dyes, four groups of particles can be prepared. Furthermore, by adjusting particles to which no fluorescent dye is attached, the total group of particles can be adjusted. In this case, a measurement solution in which six types of antibodies are supported on each group of particles is mixed with a sample solution using a known flow cytometer that has both a Coulter & Lium measurement function and a fluorescence intensity measurement function. When measured, particles can be divided into groups based on the particle size and the presence or absence of fluorescence at a specific wavelength, and based on this, the particle size distribution for each group can be obtained.

The concentration of each substance to be measured can be calculated from the particle size distribution.

For this grouping, the discrimination circuit shown in Japanese Patent Application No. 130882/1982 can be used.

Here, a reference example is shown in which a calibration curve for measuring human serum albumin (ISA) was created using one type of particles.

(1) Adjustment of reagent Polystyrene beads (solid content: 1 m9) containing a fluorescent dye with a particle size of 2.02 μm were mixed in a boric acid isotonic buffer (BBS).
After washing and centrifugation at pH 8.2), add 5 ml of 10-fold diluted anti-H8A goat antiserum and suspend. The beads were stirred for 2 hours at room temperature and overnight at 4°C until they did not precipitate, and the antibodies were thoroughly adsorbed onto the beads. After washing, the beads were washed with phosphate buffered saline (PBS) at pH 7.4 (11 as a protective colloid).
1.25 ml (containing BSA). After applying ultrasonic waves for about 20 minutes to promote redispersion, a 1% suspension is applied to the reagent.

(11) Creation of calibration curve Standard i containing different concentrations of ISA (PBS solution) 5
Mix 0 μl with 50 μl of the above reagent, let stand for a while, then add 5 ml of PBS to dilute. This will be analyzed using a flow cytometer. The analysis results are shown in Figure 2.

As a method for evaluating the aggregation state, the parameter a...
・There is a method of evaluation by calculating the average particle volume, b...number of monomers/number of analyzed particles (h), C...number of dimers/number of monomers (%), etc. However, the one with the highest accuracy can be selected depending on the measurement conditions. It can be seen from Figure 2 that any parameter can be quantified, but a has the largest rate of change and is suitable for this measurement.

(Example) Simultaneous measurement of human IgG, IgM (i) Preparation of reagents Polystyrene beads containing (2,98 μm) and polystyrene beads (2,95 μm) without fluorescent dye were each incubated at 4°C in BBS (PH8,2). Affinity-purified anti-human IgG and anti-human IgM antibodies are separately loaded at ℃. After sufficient support, wash and add PBS (1% BSA
(containing), sonicate, and mix each in the same conditions. One suspension of each of the beads thus obtained is used as a reagent.

(11) Creating a calibration curve Mix 50 μl of the standard solution containing each concentration of IgG and IgM with 50 μl of the above reagent, let stand for a while, and then mix 5 ml.
dilute with PBS. The aggregation state of each particle, which is distinguished by the presence or absence of fluorescent dye, is analyzed using a flow cytometer. Figure 3 shows the analysis results.
It can be seen that accurate quantification is possible at the same time.

(Effects) According to the method of the present invention, a plurality of substances to be measured contained in a specimen can be accurately quantified simultaneously.

The present invention utilizes a measurement method that utilizes specific binding between substances, such as antigen-antibody reactions, that is, the phenomenon of particle aggregation that occurs when antibodies (antigens) come into contact with particles carrying antigens (antibodies). Concerning immunoassays, in particular by analyzing individual particles or aggregates,
Existing methods can detect slight changes such as momme, which increases measurement sensitivity and makes it possible to measure multiple items at the same time. This method can be used in a wide range of fields, including microanalysis for various research purposes, clinical testing, and drug quantification. It can be applied to any field as long as the measurement target is something that can be used as an antigen (including Hashiten, etc.) or an antibody.In addition to antigens and antibodies, it can also be applied to hormones, receptors, sugars, and lectins. can. To analyze individual particles, it is possible to use a microscope, but flow cytometry is preferred.)
This is an application of 7-(FCM). By analyzing particle aggregation due to antigen-antibody reactions using FCM, it is possible to determine the number of unaggregated particles, particle size distribution, average particle volume, etc. in a homogeneous system.
IP parameters can be measured, LPIA.

Compared to LA, PACIA, etc., it has a larger number of parameters and has better accuracy and sensitivity. In addition, because it uses the measurement of fluorescence activity, which is a characteristic of FCM, it is possible to distinguish between particles based on the presence or absence of pigment, changes in amount, type (difference in fluorescence wavelength), etc. It is also easy to measure. It is thought that this method will greatly simplify and speed up the diagnosis of diseases that require comprehensive judgment based on multiple items, such as heart disease, liver disease, kidney disease, abnormalities in the coagulation and fibrinolytic system, and malignant tumors.

[Brief explanation of drawings]

Figure 1 shows the particle size distribution curve of the agglutination reaction. Figure 2 shows the H8A concentration and a...average particle volume, b...
・Calibration curve showing the relationship between number of monomers/number of analyzed units, C... number of dimers/number of monomers, Figure 3 shows the concentrations of IgM and IgG mentioned in the examples. A calibration curve showing the relationship between and average particle volume is shown.

Claims (1)

[Scope of Claims] 1. Particles of uniform size each carrying n types of sensitive substances that specifically bind to each of the n types of analyte substances in the sample. A method that uses microparticles to measure particles, by particle size and/or by labeling with fluorescent substances or dyes.
Using the fine particles that can be distinguished into n types, each type of particle supports each of the n types of sensitive substances that specifically bind to each analyte as a reagent, and the reagent and the sample are brought into contact. , a particle aggregation reaction characterized by measuring the agglomeration state of the resulting particles, grouping the measurement results by type of particles and totaling them, and measuring the amount of each of the n types of substances to be measured. quantitative method using 2. The method according to claim 1, wherein the specific binding is an antigen-antibody reaction. 3. The method according to claim 1 or 2, wherein the aggregation state of the fine particles is measured by flow cytometry.