JPS6281566A - Quantification method by measurement of fluorescent intensity of fine particle - Google Patents

Abstract

PURPOSE:To make it possible to simultaneously quantify a plurality of substances, in a fluorescent immunoassay using a solid phase, by allowing fine particles different in a particle size to respectively support different responsive substances. CONSTITUTION:Particles are classified, for example, into three kinds in a particle size and three kinds in the presence and absence of the adhesion of a fluorescent dye to form 6 kinds of groups in total. Reagents prepared by respectively supporting 6 kinds of antibodies by the particles of six groups are contacted with a mixture of a specimen solution and a definite amount of a standard substance labelled with a substance different from the aforementioned fluorescent dye and competition reaction is allowed to generate to prepare measuring solutions which are, in turn, measured by a known flow sight meter together having colter volume measuring function and fluorescent intensity measuring function to make it possible to discriminate the groups. The quantity of a substance to be measured is measured by measuring the fluorescent intensity of the fluorescent substance labelled with the standard substance. By this method, the concn. of the substance to be measured at every group can be measured.

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) Measuring methods using immunological reactions have already been established as microanalysis methods in the medical field, and the mainstream among them is radioimmunoassay (RIA). RIA
It was first developed in 1959 by Persson, Yarrow et al. as a method for measuring insulin, and has since become explosively popular. However, issues such as the handling of radioeintozo, dedicated equipment, safety during disposal, and the lifespan of the labeled compound have long been closed-offs, and research into non-radioactive labeling methods has been actively conducted. It's here. Enzyme immunoassay (EIA) was introduced in 1971 as a method comparable in sensitivity, and RIA
It has attracted attention as an alternative to It is certainly reported that it has the same or higher sensitivity than RIA for some measurement items, and unlike RIA, it is a homogeneous method that does not require separation of the antigen-antibody bound form and the free form. Although it has the advantage of being able to perform measurements using a biological system, there are problems such as instability due to the use of biological reactions, relative sensitivity, and complexity, and there is no suitable alternative to RIA. That would be the current situation. Fluorescent immunoassay (FIA) uses a fluorescent probe instead of radiointosol.

Although it is not as good as RIA in terms of sensitivity, there is no problem in terms of safety, it can be used on the same principle as RI, and B
/'F It is widely used in Western countries because it can be measured in a homogeneous system that does not require separation.

In the medical field, changes in specific biological substances (markers) are extremely important in understanding the occurrence, progression, and therapeutic effects of diseases. For example, if it is a tumor marker, CA
A number of markers are known, including -125, CA-19-9, AFP, and CEA, but to date, there are no known markers that are common to all tumors and that can help in early detection. It has not been done. Therefore, in order to accurately grasp the possibility of the presence of a tumor, its site of occurrence, etc., the only way to perform an examination is to examine multiple markers and make a comprehensive judgment. However, currently, if you want to obtain multiple pieces of data, you have no choice but to measure that number, which causes pain to the patient when blood is drawn, time required for testing, and
It was a fool's errand as time increased.

(Problems to be Solved by the Invention) The present invention has been made to solve the above-mentioned problems, and is capable of accurately measuring the amount of a substance to be measured in a sample, which has been difficult with conventional FIA. The purpose is to provide a method.

(Means and effects for solving the problem) The present invention is an improvement of the competitive method of FIA using a solid phase, and involves using fine particles as the solid phase and analyzing the fluorescent activity of each particle. The main feature is that the n contained in the sample
A method for measuring the amount of each of the various analyte substances using microparticles as carriers carrying n types of sensitive substances that specifically bind to each of the analyte substances. and/or by labeling with a fluorescent substance, the fine particles can be distinguished into n types as carriers, and each type of carrier carries each of the n types of sensitive substances that specifically bind to each analyte. The sample is used as a reagent, and the n types of analyte substances in the sample are used as standard substances and fluorescently labeled with a different type of fluorescent substance than the above-mentioned fluorescent substances, and each of the fluorescently labeled standard substances is contacting the mixture of the sample and the sample with the reagent to cause a competitive reaction between the analyte and the fluorescently labeled standard substance against the sensitive substance;
The object of the present invention is to provide a method for quantitative determination by measuring the fluorescence intensity of fine particles, which is characterized by measuring the fluorescence intensity of the fine particles.

Here, n is a natural number, and a particularly remarkable effect is exhibited when n is a natural number of 2 or more.

The typical specific binding reaction between substances is the antigen-antibody reaction, but there are also hormones, receptors,
Reactions such as sugar and lectin can also be used.

This will be explained in more detail using the antigen-antibody reaction as an example. If the target to be measured is something that can be an antigen (including Hashiten, etc.), the fine particles are made to carry the corresponding antibody. 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, the microparticles (with a uniform particle size of about 1 to 100 microns) are configured in advance so that they can be distinguished into a plurality of groups. One way to do this is to have multiple levels of particle size. Furthermore, when particles are labeled with a fluorescent substance, they can be classified into a plurality of groups based on the presence/absence or concentration of the fluorescent substance, the type of fluorescence, etc. By combining the two, it is possible to discriminate even more particle groups.

In addition, when labeling a standard substance with a fluorescent substance, multiple types of fluorescent substances (for example, FITC (Fluoresc
ein l5othicyanate) and PI (P
ropidiumIodide) etc.),
This makes it possible to identify and quantify the type of substance to be measured, and it goes without saying that such a method also falls within the technical scope of the present invention.

As the fine particles, for example, cells such as red blood cells, metals, microcapsules such as liposomes, latex particles such as polystyrene, etc., and particles having a particle size of about 1 to 100 μm can be used.

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.

A sensitive substance is a substance that specifically reacts with a substance to be measured, and when the substance to be measured is an antigen.

An antibody that specifically reacts with that 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 using the antibody-antigen reaction as an example. To facilitate understanding, explanation will be given first using n==1 as an example.

A specific antibody is supported on microparticles of a certain size. On the other hand, if the same substance as the analyte in the specimen is used as the standard substance, for example, if the analyte in the specimen is immunoglobulin A,
(IgA is used as a standard substance), prepare a certain amount of IgA with a fluorescent label attached, and mix this with the sample. In this mixed solution, a substance to be measured (hereinafter referred to as A) and a certain amount of a fluorescently labeled standard substance (hereinafter referred to as A*) coexist.

A and A* cause a similar antigen-antibody reaction to antibodies supported on microparticles (hereinafter referred to as B).

Therefore, when the mixed solution and the reagent are brought into contact, A and A* compete with each other to cause an antigen-antibody reaction with B. Since A* and B are constant amounts, the smaller the amount of A, the more A* will be combined with B, and the amount of fluorescence of the particles will increase. On the contrary, A
When the amount of is large, the amount of fluorescence of the particles is small. Based on this principle, the amount of A can be determined by measuring the amount of fluorescence of particles.

When there are n types of substances to be measured, they are expressed as (A,...An), and the corresponding fluorescently labeled standard substances and antibodies are expressed as (A, *...An*) (B,...An), respectively.・・B
, ), then (A, , A1*, B,) - (An
, An*, Bn), and the amount of (A1...An) can be measured using the same principle as above.

However, depending on the substance to be measured, the relationship between antigen and antibody may be reversed.

The grouping of A,...,A is B1. ...Bn
The particles carrying 1 n of particles are configured in advance so that they can be classified into a plurality of groups. One way to do this is to have multiple levels of particle size.

In the present invention, since the fluorescence intensity of each particle is measured, it is necessary to make the diameters of the fine particles the same. The particle size of the fine particles is, for example, 5μ (a glue f) and 1
If 0μ (group b) is used, the a group and b group can be sufficiently distinguished by a known method. Furthermore, when particles are labeled with a fluorescent substance, they can be classified into a plurality of groups based on the presence/absence or concentration of the fluorescent substance, the type of fluorescence, etc. Furthermore, if a plurality of fluorescent substances are used when fluorescing the standard substance, fine particles can ultimately be distinguished. And with these 5 combinations,
It is possible to distinguish even more particle groups.

In addition, in the present invention, it is necessary to take as much time as possible to prevent nonspecific aggregation of particles or aggregation due to antigen-antibody reaction, and for this purpose monoclonal antibodies are used. It is preferable to use relatively large particles, or to keep the particle concentration dilute.

Although microscopic photometry can be used to distinguish groups of fine particles and measure fluorescence intensity, one preferred embodiment is to measure these particles one by one using flow cytometry. can be mentioned.

Flow cytometry is mainly concerned with optical instrument analysis, and particles are passed through one by one.

The size and color of the particles can be determined by shining a radar light on the particles and measuring the scattered light, or by labeling the particles with a fluorescent substance in advance and measuring the fluorescence intensity. It measures the characteristics of particles. In addition, methods that electrically measure the capacitance of particles (called Coulter's volume) based on the so-called Coulter principle, and methods that combine this and optical measurements are also used.

In order to classify particles into n groups, for example, by adjusting three groups based on particle size, particles with fluorescent dye attached and particles without fluorescent dye, a total of six groups of particles can be prepared. In this case, a reagent in which six types of antibodies are supported on each group of particles is brought into contact with a mixture of a sample solution and a fixed amount of a standard substance labeled with a type different from the fluorescent substance, When the measurement solution in which the competitive reaction has occurred is measured using a known flow cytometer that has both a Coulter Delium measurement function and a fluorescence intensity measurement function, the particles can be detected based on the particle size and the presence or absence of fluorescence attached to the particles. can be divided into groups.

The amount of the substance to be measured is determined by measuring the fluorescence intensity of a fluorescent substance labeled with a standard substance. Double-walled substances can be distinguished by selecting the wavelength of fluorescence intensity measurement, and the concentration of the tag can be measured for each group of particles, ie, for each substance to be measured.

Furthermore, this grouping is based on the patent application No. 130882/1986.
The method shown in can be used.

(Example 1) (1) Measurement of human serum albumin (H8A) (i) Preparation of reagent Polystyrene beads (average particle size 5.29 μm) 5 to 5 ml of phosphate buffered saline (PBSPH7) as solid content
, 4). Add 5 ml of 10-fold diluted anti-H3A goat antiserum to the mixture, stir gently at room temperature for 6 hours, and then gently stir at 4°C overnight.

After washing by centrifugation, it is suspended in 5 ml of PBS containing 1 g of bovine serum albumin as a protective colloid, and used as a latex reagent with a solid content of 0.1% for testing.

(11) Creating a calibration curve Add FITC to 100 μl of standard solution containing H8A at different concentrations.
Labeled 1(SA (10til/ml) at 100p
Add H at a time. After adding 100 μl of the reagent to this mixed solution, the mixture was gently shaken for about 1 hour. Thereafter, the reaction solution was completely removed by centrifugation and suspended in 3 ml of PBS. This suspension was analyzed using a cell analyzer to obtain a calibration curve as shown in FIG. The vertical axis represents the most common fluorescence intensity (peak value) when ioo and ooo particles were analyzed, and the average value of the overall fluorescence activity.

(Example 2) Simultaneous measurement of human IgG and IgM (1) Preparation of reagents a. Reagent for human IgG analysis 5 mg of solid content of polystyrene beads (5.29 μm) was added to borate buffered saline (BBS pH 8.2) 2 .5
ml and mixed with 2.5 ml of 0.2 ml of affinity purified anti-human IgG antibody (goat). After gently stirring at room temperature for 2 hours and overnight at 4°C, centrifuge and add 5 ml of PBS (1% BSA).
to obtain a 0.1% latex reagent.

b. Human IgG analysis reagent Perform the same procedure as a using polystyrene beads (10 μm) and anti-human IgM affinity purified antibody (goat) to finally obtain 5 ml of 0.2 latex reagent.

(11) Creation of standard curve ■ FITC-labeled IgG, IgM, 30 pg each
/ml ■ (i) Isometric mixture of the reagents prepared in a and b.

■Standard solutions containing different concentrations of IgG and IgM.

Mix 100 μl of ①, ① and 200 μl of ②, and after 2 hours of reaction, centrifuge, resuspend in PBS, and analyze with a cell analyzer. That is, according to Coulter's principle, 5.
Beads of 29 .mu.m and 10 .beta.m are completely differentiated, and the fluorescent activity of each bead is examined. The analysis results are shown in Figure 2.

(Effects) According to the method of the present invention, one or more substances to be measured contained in a specimen can be simultaneously and accurately measured.

A preferred embodiment of this analysis is a flow cytometer capable of simultaneously measuring particle size and fluorescence activity. J
- (FCM). By using FCM, fine particles can be differentiated based on differences in particle size, type and amount of fluorescent dye contained in the particles, etc., and the fluorescent activity caused by antigen-antibody reactions can be measured for each particle. As a result, it becomes possible to measure multiple items simultaneously.

If it becomes possible to measure multiple items simultaneously in this way, it will be of great help in making a comprehensive diagnosis, and it will greatly contribute to the reduction of misdiagnoses such as false positives and false negatives, as well as the early detection of various diseases. Probably.

[Brief explanation of drawings]

FIG. 1 is a graph of a measurement example showing the relationship between the concentration of ISA and fluorescence intensity, and FIG. 2 is a graph showing the relationship between the concentration of IgG and IgM and fluorescence intensity.

Claims (1)

[Scope of Claims] 1. The amount of each of the n types of analyte contained in the sample is determined by a carrier comprising fine particles carrying n types of sensitive substances that specifically bind to each of the analyte substances. In this method, the microparticles, which can be distinguished into n types by particle size and/or labeling with a fluorescent substance, are used as carriers, and each type of carrier is loaded with each substance to be measured. The above-mentioned n types of sensitive substances that specifically bind to the above-mentioned fluorescent substances are used as reagents, and the n types of analyte substances in the specimen are used as standard substances and fluorescent substances of a different type from the above-mentioned fluorescent substances are used as reagents. A mixture of a predetermined amount of each of the optically labeled and fluorescently labeled standard substances and the sample is brought into contact with the reagent to cause a competitive reaction between the analyte and the fluorescently labeled standard substance against the sensitive substance, and A quantitative method by measuring the fluorescence intensity of fine particles, characterized by measuring the fluorescence intensity of the particles. 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 fluorescence intensity of the fine particles is measured by flow cytometry.