JPH0627112A - Immunological measurement method - Google Patents

Abstract

PURPOSE:To obtain an immunological measurement method which can clearly distinguish two or more kinds of measurement substances in solution to be examined and highly detect them as well as which can increase the number of items to be simultaneously measured. CONSTITUTION:Reagent obtained by making an antibody or antigen respectively coupled to measurement substances a solid phase on two or more kinds of particles having different refraction factors is mixed with solution to be examined and a fluorescent marker antibody to have them reacted, and then fluorescent intensity of the particles is measured by a flowcytometer.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an immunological measuring method, and more specifically, it uses a flow cytometer to measure the fluorescence intensity of particles bound with a complex of a substance to be measured and a fluorescent labeled antibody. The present invention also relates to an immunological measurement method capable of simultaneously quantifying two or more kinds of measurement substances in a test liquid with high accuracy.

[0002]

2. Description of the Related Art In recent years, in medical fields, biochemistry, hygiene, immunology, and other fields, it has become increasingly important to quantitatively and accurately measure trace substances in blood and the like. . As a measuring method for detecting an antigen or an antibody in a test liquid such as serum using an antigen-antibody reaction, RIA, E have been conventionally used.
IA, FIA, etc. are known. EIA is a method that utilizes the fact that the amount of an antigen or antibody in a test solution is proportional to the enzyme activity, and that the color development due to the reaction between the enzyme-labeled substance and the substrate is measured by a spectrophotometer or the like. . FIA is, for example, after the antigen in a test solution and a fluorescent-labeled antigen are competitively bound to a fixed amount of antibody, then irradiated with excitation light, and the fluorescence emitted at that time is measured with a fluorometer. To do.

Recently, a substance to be measured is bound to a minute particle such as latex particles having an antibody or an antigen immobilized thereon by an antigen-antibody reaction, and further, a fluorescent label is reacted therewith, and then the fluorescence intensity of the particle is increased. A method for quantifying a substance to be measured has been proposed by measuring a substance with a flow cytometer.
According to the flow cytometer, by detecting the scattered light when the laser light is irradiated, it is possible to obtain the information on the size of each substance passing through the laser light irradiation part, and to detect the size of each substance different in size. The fluorescence intensity of each substance can be measured. Therefore, since the unbound excess fluorescent label present in the mixed reaction solution of the antibody or the antigen-immobilized particles, the test solution, and the fluorescent label can be measured separately from the particles, the flow site can be measured. It is not necessary to perform B / F separation of excess fluorescent label prior to measurement with a meter. Furthermore, the following multi-item simultaneous measurement has been proposed by utilizing the fact that information on particle size can be obtained from scattered light intensity.

That is, an antibody or an antigen that specifically binds to each of a plurality of measurement substances present in a test liquid is solid-phased into a group of particles having different particle diameters, and the different particle diameters are obtained. It is possible to detect a plurality of substances to be measured in a test solution at the same time by measuring a mixed reaction solution of a reagent composed of particles, a test solution, and a fluorescent label only once with a flow cytometer. (JP-A-52-15
No. 815).

However, when such a measurement is carried out, the reagent particles are usually placed in a buffer solution containing salts and the like.
Aggregation of particles often occurs. In addition, particles to which the substance to be measured is bound may aggregate using the fluorescent label as a bridge. In this way, when particles agglomerate, the flow cytometer apparently detects them as particles with a large particle size, so it cannot be distinguished from another group of particles with different particle sizes, and as a result, There is a problem that it is not possible to perform simultaneous multi-item simultaneous measurement.

Further, particles having different particle diameters can be measured because they are aligned only on the upper right line in the forward scattered light intensity (horizontal axis) / side scattered light intensity (vertical axis) dot plot diagram of the flow cytometer. The number of types of particles having different particle diameters is reduced, so that the number of items that can be simultaneously measured is limited.

[0007]

SUMMARY OF THE INVENTION The present invention has been made to solve the problem in the multi-item simultaneous measurement using the conventional flow cytometer as described above, and comprises two or more kinds in the test liquid. Clearly distinguishing the measured substances of
It is an object of the present invention to provide an immunological measurement method capable of detecting with high accuracy and increasing the number of items that can be simultaneously measured.

[0008]

The immunological assay method according to the present invention comprises a reagent in which two or more kinds of particles having different refractive indexes are immobilized with an antibody or an antigen that binds to different assay substances, respectively. And the fluorescence-labeled antibody are mixed and reacted, and the fluorescence intensity of the particles is measured with a flow cytometer.

The particles used in the present invention are not particularly limited as long as they can be measured by a flow cytometer, and for example, synthetic high polymer particles, red blood cells, gelatin particles and the like can be used. Among these,
In the present invention, water-insoluble, water-dispersible synthetic polymer particles are preferably used because the refractive index of the particles can be easily changed and adjusted.

Generally, water-insoluble water-dispersible synthetic polymer particles are prepared by emulsion (co) polymerization or suspension (co) of appropriate monomers.
It is already known that it can be obtained by polymerization, etc., but for particles having different refractive indices, the composition of the monomer used is appropriately selected in the production of particles by (co) polymerization of an appropriate monomer. It can be obtained by doing. For example,
As high refractive index particles, polystyrene particles (refractive index 1.
59) can be used, while trifluoroethyl methacrylate (refractive index 1.4
(Co) polymer particles of a methacrylic acid fluoroalkyl ester derivative such as 5) can be used. If necessary, when preparing the particles, as a comonomer, for example, by using a polymerizable unsaturated carboxylic acid such as acrylic acid, to introduce a carboxyl group on the surface of the resulting particles Such functional groups are advantageously used to covalently attach the antibody or antigen to the particles. Further, the particles used in the present invention can be adjusted in refractive index by encapsulating an inorganic substance, a metal or the like inside the above-mentioned synthetic high molecular weight polymer particles. The refractive index of the particles used in the present invention is not particularly limited as long as it can be measured with a flow cytometer.

In the present invention, the particle size of the particles used is
There is no problem as long as it can be measured by a flow cytometer, but particles of 1 to 20 μm are usually preferably used. Moreover, particles having different particle diameters may be used as the particles having different refractive indexes. In the present invention, an antibody or an antigen that binds to a different substance to be measured is immobilized on particles having different refractive indexes, and this is mixed and used as a reagent. Here, the mixing ratio of the particles having different refractive indexes is not particularly limited, however, when the particle diameter is different between the particles having different refractive indexes, in order to match the detected number of each particle, It is preferable to mix the particles according to their numbers.

In the present invention, the method of immobilizing the antibody or antigen on the particles is not particularly limited, and any conventionally known method can be used. As a typical example of such a method, for example, a method of physically adsorbing an antibody or an antigen to a particle, or as described above, a carbodiimide or the like is used for a particle having a carboxyl group to immobilize the antibody or the antigen by covalent bonding. And the like.

Next, the immunological measurement method according to the present invention will be described. According to the method, as described above, first, two or more kinds of particles having different refractive indices are mixed with solid-phased antibodies or antigens that bind to different measurement substances to prepare a particle mixing reagent. Then, a predetermined amount of the test solution such as serum is added to a predetermined amount of the particle-mixed reagent, and the mixture is reacted for several minutes to several hours. Next, FITC (fluoresc
A predetermined amount of an antibody labeled with a fluorescent substance such as ein isothiocyanate) or PE (phycoerythrin) is added. Alternatively, the test liquid and the fluorescent label may be simultaneously added to the particle-mixed reagent and reacted. As a result, when the test substance is present in the test liquid, the fluorescent substance is bound to the particles by using the test substance as a bridge. Next, this reaction solution is measured by a flow cytometer, but it can be measured without B / F separation, or after removing excess fluorescent label by centrifugation or the like. , Can also be measured.

When measuring the above reaction liquid with a flow cytometer, each particle flows through a narrow tube and is irradiated with laser light. Forward scattered light at a laser irradiation angle of 180 °
Side scattered light is detected at a laser irradiation angle of 90 °. Furthermore, the side scattered light, after passing through the filter,
Detect fluorescence of a specific wavelength. For example, FITC is 53
0 nm, PE can be detected at a wavelength of 575 nm.

FIG. 9 shows the forward scattered light intensity / side scattered light intensity dot plot measured by a flow cytometer.
A1, A2 and A3 represent particle groups having the same refractive index but different particle diameters. A1 ′ and A2 ′ represent a particle group in which two particles A1 and A2 are aggregated. If each particle is a completely monodisperse particle group, it can be separated on the dot plot, but when the particles are agglomerated, the particle groups of A1 'and A2, A2' and A3 overlap, Accurate measurement is not possible. Further, A1, A2, and A3 appear only on the upper right straight line, and many kinds of particles cannot be used in order to prevent the particle groups from overlapping with each other.

On the other hand, B1 represents a particle group having a lower refractive index than A1 to A3 and the same particle size as A1, and C1 represents B1.
The particle group has a lower refractive index and the same particle size as A1. In B1 and C1, since the dot plot appears at a position different from the above-mentioned straight line on the upper right side, more kinds of particles can be separated and measured. Also, B
1'and C1 'represent a particle group in which two particles of B1 and C1 are aggregated, but since the particle groups of A1, B1, C1, A1', B1 'and C1' do not overlap, Accurate measurement is possible.

In the measurement with the flow cytometer, the forward scattered light intensity, the side scattered light intensity, and the fluorescence intensity are measured for each particle. For example, in order to analyze the fluorescence intensity of the A1 particle group, only the particles in the A1 particle group on the dot plot are taken out and calculated as the average value of the fluorescence intensity of these particles. Therefore, since the fluorescence intensity of each of the particles sorted on the dot plot can be obtained separately, multi-item simultaneous measurement can be performed.

To obtain the concentration of the substance to be measured in the test liquid, it is well known that if a calibration curve is prepared in advance,
It can be calculated immediately from the fluorescence intensity.

[0019]

As described above, according to the method of the present invention,
Since the fluorescence intensity is measured by a flow cytometer using a reagent obtained by immobilizing antibodies or antigens that specifically bind to different measurement substances to particles having different refractive indexes, the appearance position of scattered light dot plots can be measured. Due to the difference, it is possible to accurately measure two or more kinds of measurement substances simultaneously.

[0020]

The present invention will be described below with reference to examples.
The present invention is not limited to these examples. Example 1 (a) Immobilization of antibody on particles 5 ml of 5% distilled water dispersion of carboxylated polystyrene particles (particle diameter 2.5 μm, refractive index 1.59) prepared by seed polymerization method, borate buffer solution (0.1 M, pH 7.5) 2 ml and distilled water 11 ml were mixed, and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride aqueous solution (2
2 ml of (mg / ml) was added, and 10 minutes later, 5 ml of an anti-human AFP antibody (mouse IgG) aqueous solution (2 mg / ml) was added, and the mixture was reacted at 10 ° C. for 2 hours. Then borate buffer (0.01M,
After centrifugation and washing at pH 8) three times, the particles were redispersed in the same buffer to prepare an anti-AFP antibody-immobilized particle dispersion.

Next, carboxylated (styrene / 2,2,2-trifluoroethylmethacrylate) copolymer particles (styrene / 2,2,2-trifluoroethylmethacrylate copolymerization weight ratio 3) prepared by the seed polymerization method. / 1, particle size 2.
5 μm, refractive index 1.55) in the same manner as above, anti-human C
A particle dispersion liquid in which an EA antibody (mouse IgG) was immobilized was prepared.

Furthermore, carboxylation (styrene / 2,2,2
-Trifluoroethylmethacrylate) copolymer particles (styrene / 2,2,2-trifluoroethylmethacrylate copolymerization weight ratio 1/1, particle diameter 2.5 μm, refractive index 1.5)
In the same manner as in 1) above, an anti-human β ₂ -microglobulin antibody (mouse IgG) (hereinafter, also simply referred to as β ₂ -antibody) was solid-phased to prepare a particle dispersion liquid. (b) Preparation of FITC-labeled antibody An anti-human AFP antibody (rabbit IgG) was added to a carbonate buffer solution (0.5
3 ml of this antibody (1 mg / ml) after dialysis against M, pH 9.5)
FITC (0.2 mg / ml) (0.6 ml) was added to and the mixture was allowed to stand at 25 ° C. for 3 hours. Then, excess FITC in the above reaction solution was removed with Sephadex G-25, and FITC-labeled anti-human AFP was added.
An antibody (phosphate buffer, pH 8, F / P = 3) was obtained.

Then, in the same manner as above, the FITC-labeled anti-human CEA antibody (phosphate buffer, pH 8, F / P = 3) and the FITC-labeled anti-human β ₂ -microglobulin antibody (phosphate buffer, pH 8) were used. , F / P = 3) was obtained. (c) Flow cytometer measurement Anti-human AFP antibody-immobilized particle dispersion prepared in (a) (0.
02%) 100 μl of human AFP solution of known concentration 100
Then, 100 μl of FITC-labeled anti-human AFP antibody (0.02 mg / ml) prepared in (b) was added, and the mixture was added at 37 ° C.
And reacted for 3 hours. The mixed solution was measured by a flow cytometer (FACScan, manufactured by Becton Ditzkinson). The obtained forward scattered light intensity / side scattered light intensity dot plots are shown in FIG.
Table 1 shows the fluorescence intensity at the FP concentration.

Next, 100 μl of the human CEA solution having a known concentration was added to 100 μl of the anti-human CEA antibody-immobilized particle dispersion (0.02%) prepared in (a), and then the FITC label prepared in (b) was added. Anti-human CEA antibody (0.02mg / ml) 10
0 μl was added, and the mixture was reacted at 37 ° C. for 3 hours. The mixed solution was measured by a flow cytometer (FACScan, manufactured by Becton Ditzkinson). Figure 2 shows the obtained forward scattered light intensity / side scattered light intensity dot plot.
And the fluorescence intensity at each human CEA concentration is shown in Table 2.

Next, 100 μm of the anti-human β ₂ -microglobulin antibody-immobilized particle dispersion liquid (0.02%) prepared in (a).
100 μl of human β ₂ -microglobulin solution of known concentration
100 μl of FITC-labeled anti-human β ₂ -microglobulin antibody (0.02 mg / ml) prepared in (b) was added.
1 was added and the mixture was reacted at 37 ° C. for 3 hours. This mixed solution was applied to a flow cytometer (Becton Ditzkinson, F
ACS Scan). The obtained forward scattered light intensity / side scattered light intensity dot plot is shown in FIG. 3, and the fluorescence intensity at each human β ₂ -microglobulin concentration is shown in Table 3.

Next, the anti-human AFP antibody-immobilized particle dispersion liquid (0.06%) prepared in (a), the anti-human CEA antibody-immobilized particle dispersion liquid (0.06%) and the anti-human β ₂ -Equivalent amounts of microglobulin antibody-immobilized particle dispersion (0.06%) were mixed to prepare a particle dispersion for simultaneous measurement of AFP, CEA and β ₂ -microglobulin. Next, F prepared in (b)
ITC-labeled anti-human AFP antibody (0.06 mg / ml), FIT
C-labeled anti-human CEA antibody (0.06 mg / ml), FITC-labeled anti-human β ₂ -microglobulin antibody (0.06 mg / ml)
Were mixed in equal amounts to prepare a labeled antibody solution for simultaneous measurement of AFP, CEA and β ₂ -microglobulin.

Next, the particle dispersion 100 for simultaneous measurement described above.
100 μl each of human AFP solution, human CEA solution and human β ₂ -microglobulin solution of known concentration were added to μl,
Further, 100 μl of the FITC-labeled antibody for simultaneous measurement described above was added, and the mixture was reacted at 37 ° C. for 3 hours. A flow cytometer measurement was performed on this mixed solution. Figure 4 shows the obtained forward scattered light intensity / side scattered light intensity dot plot.
Table 4 shows the fluorescence intensity at each concentration of the measurement substance.

[0028]

[Table 1]

[0029]

[Table 2]

[0030]

[Table 3]

[0031]

[Table 4]

As is clear from the results shown in the table, even in the simultaneous measurement, almost the same fluorescence intensity as in the case of measuring each independently could be obtained.

Comparative Example 1 (a) Immobilization of Antibody on Particles Carboxylated polystyrene particles (particle diameter 3.1 μm, refractive index 1.59) prepared by the seed polymerization method were used in the same manner as in Example 1. Thus, an anti-human CEA antibody-immobilized particle dispersion liquid was prepared.

Next, using carboxylated polystyrene particles (particle diameter 4.2 μm, refractive index 1.59), Example 1
In the same manner as above, an anti-human β ₂ -microglobulin antibody-immobilized particle dispersion liquid was prepared. (b) Measurement by flow cytometer Human AFP solution 1 of known concentration in 100 μl of anti-human AFP antibody-immobilized particle dispersion liquid (0.04%) prepared in Example 1
00 μl was added, 100 μl of FITC-labeled anti-human AFP antibody (0.02 mg / ml) prepared in Example 1 was added, and 37
The reaction was carried out at 0 ° C for 3 hours. This mixed solution was measured by a flow cytometer. The obtained forward scattered light intensity / side scattered light intensity dot plot is shown in FIG. 5, and the fluorescence intensity at each human AFP concentration is shown in Table 5.

100 μl of the anti-human CEA antibody-immobilized particle dispersion (0.04%) prepared in (a) had a known concentration of human CE.
FITC prepared in Example 1 by adding 100 μl of solution A
100 μl of labeled anti-human CEA antibody (0.02 mg / ml) was added, and the mixture was reacted at 37 ° C. for 3 hours. This mixed solution was measured by a flow cytometer. The obtained forward scattered light intensity / side scattered light intensity dot plot is shown in FIG. 6, and the fluorescence intensity at each human CEA concentration is shown in Table 6.
Shown respectively.

Next, 100 μm of the anti-human β ₂ -microglobulin antibody-immobilized particle dispersion liquid (0.10%) prepared in (a).
100 μl of human β ₂ -microglobulin solution of known concentration
μl was added to the FITC-labeled anti-human β prepared in Example 1.
_2- microglobulin antibody (0.02 mg / ml) 100 μl
Was added and reacted at 37 ° C. for 3 hours. This mixed solution was measured by a flow cytometer. The obtained forward scattered light intensity / side scattered light intensity dot plot is shown in FIG. 7, and the fluorescence intensity at each human β ₂ -microglobulin concentration is shown in Table 7.

Next, the anti-human AFP antibody-immobilized particle dispersion liquid (0.06%) prepared in Example 1, the anti-human CEA antibody-immobilized particle dispersion liquid (0.12%) and anti-human β were prepared. Equal amounts of _2- microglobulin antibody-immobilized particle dispersion (0.30%) were mixed to prepare a particle dispersion for simultaneous measurement of AFP, CEA and β ₂ -microglobulin. Next, 100 μl of the particle dispersion for simultaneous measurement described above was added with human AF of known concentration.
100 μl each of P solution, human CEA solution and human β ₂ -microglobulin solution were added, and further 100 μl of FITC-labeled antibody for simultaneous measurement prepared in Example 1 was added, and the mixture was incubated at 37 ° C.
And reacted for 3 hours. This mixed solution was measured by a flow cytometer. The obtained forward scattered light intensity / side scattered light intensity dot plot is shown in FIG. 8, and the fluorescence intensity at each concentration of the measured substance is shown in Table 8.

[0038]

[Table 5]

[0039]

[Table 6]

[0040]

[Table 7]

[0041]

[Table 8]

As is clear from the results shown in the table, according to the simultaneous measurement, the fluorescence intensity at a low CEA concentration is abnormally high and the CEA concentration is high as compared with the single measurement. In this case, the fluorescence intensity at a low β ₂ -microglobulin concentration was abnormally high, and accurate simultaneous measurement could not be performed.

[0043]

[Brief description of drawings]

[Figure 1]

[Fig. 2]

FIG. 3 and

FIG. 4 is a diagram showing forward scattered light intensity / side scattered light intensity dot plots according to the method of the present invention.

[Fig. 5]

[FIG. 6]

FIG. 7 and

FIG. 8 is a diagram showing forward scattered light intensity / side scattered light intensity dot plots by a conventional method as a comparative example.

FIG. 9 is a diagram showing a forward scattered light intensity / side scattered light intensity dot plot measured by a flow cytometer.

Claims (1)

[Claims] 1. A reagent obtained by immobilizing an antibody or an antigen that binds to different measuring substances to two or more kinds of particles having different refractive indexes, is mixed with a test solution and a fluorescently labeled antibody, reacted, and then flowed. An immunological measurement method, which comprises measuring the fluorescence intensity of the particles with a cytometer.