JPS63214668A - Method for measuring cell - Google Patents

Abstract

PURPOSE:To exactly detect desired cells from miscellaneous cell groups by bringing a cell to be tested into reaction with an antibody labeled by a microcapsule or latex particle and detecting the cell to be tested by using light scattering, fluorescence, etc. CONSTITUTION:The antibody 3 is conjugated to the microcapsule or latex particle 1 contg. a light absorptive material or fluorescent material as a labeling compd. This labeled antibody 4 and the cell 5 to be tested are brought into reaction to induce an antigen-antibody reaction. The labeled cell 7 obtd. in such a manner is injected into an automatic analysis instrument for cells. The flow of the cells 21 in a sheath 2 in the analysis instrument flows into a flow cell 24. A light source 23 and a detector 26 as well as a signal processing circuit 27 detect the cells and make quantitative determination thereof from the quantity of fluorescence of the cells 21 and the quantity of the light absorption. The cells 24 past the flow cell 24 are sieved by an electric field 28 and the desired cells are taken into a sample collecting vessel 29.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a method for measuring cells, and particularly to flow cytometry that can accurately detect only target cells from a miscellaneous group of cells.

[Conventional technology]

The conventional cell measurement method involves flowing cells into a thin tube, irradiating the cells with a laser, and detecting the cells from the scattering of the cells.

Alternatively, by directly or indirectly binding a fluorescent marker to an antibody that specifically reacts with the test cell, and allowing the antibody labeled with the fluorescent marker to react with the test cell, it is possible to identify the target cell based on the amount of fluorescence. I am determining whether there is. In other words, according to the latter method, target cells are labeled with a fluorescent marker and can be distinguished from other unlabeled cells6. metrics (procedural and practical),
Kani Shobo, edited by Ota-O et al., April 21, 1984, No. 1
It is described on pages 10 to 121.

In addition, as a measuring device used for flow cytometry,
There is one described in Special rjM No. 60-209141.

[Problem that the invention seeks to solve]

However, in the conventional method of binding a fluorescent substance to an antibody, the absolute amount of the fluorescent substance that can be bound to the antibody is not sufficient. As a result, the amount of fluorescence is insufficient, making it impossible to accurately detect cells labeled with fluorescent substances, and furthermore, it is not possible to accurately quantify cells or clearly measure the amount of antigen on the cell surface from the amount of fluorescence. Ta.

Furthermore, in the conventional cell measurement method using light scattering,
There was a problem in that the scattering intensity was weak because the cells were not large enough.

Therefore, it was not possible to accurately detect cells from the scattering intensity.

In order to solve the above-mentioned problems, the present invention aims to provide a cell measurement method that can accurately detect target cells.

[Means for Solving the Problems] In order to achieve the above object, the present invention binds an antibody that specifically reacts with a test cell to a microcapsule or latex particle, and then combines the test cell with the microcapsule or latex particle. react with the antibody labeled with
This cell measurement method is characterized in that the test cells are detected using light scattering, light absorption, or fluorescence.

[Effect]

According to the present invention, since antibodies that specifically bind to test cells are bound to microcapsules or latex particles, the following effects are achieved. In other words, the intensity of the scattered light increases as the size of the cell to which the latex particles or microcapsules are bound becomes larger than that before binding. Moreover, by incorporating a light-absorbing substance or a fluorescent substance into the microcapsules, the absolute amount of the light-absorbing substance or fluorescent substance is increased. This increases the absorption or fluorescence signal.

〔Example〕

Next, embodiments of the present invention will be described in detail with reference to the accompanying drawings.

FIG. 1 shows the steps of one embodiment.

In FIG. 1, a labeled compound 2 is encapsulated within a microcapsule 1. The labeling compound may be a light-absorbing substance or a fluorescent substance. By encapsulating the fluorescent substance, the amount of fluorescent substance is 103 times greater than in the conventional method in which the fluorescent substance is bound to one antibody. As a result, the amount of signal for detecting cells increases by 103 times. As the signal amount increases, the sensitivity for accurately detecting target cells by distinguishing them from other cell groups increases.

In the case of an absorbing substance, as in the case of a fluorescent substance, the absolute amount of the absorbing substance increases, so the absorption intensity increases.

As a result, it is possible to accurately determine the absorbed amount of the target cells by distinguishing them from other cell groups, and it becomes possible to detect and quantify the target cells.

Next, it is made into microcapsules containing the labeling substance.

Bind antibody 3. This labeled antibody 4 is reacted with the test cell 5 to cause an antigen-antibody reaction. At this time, if an antibody that specifically reacts with the target cell (for example, a cancer cell) is used, the antigen of the target cell will react with the antibody, and the target cell will be labeled with a fluorescent marker or absorbing substance. It means that it was done.

The labeled cells 7 thus obtained are injected into an automatic cell analyzer (FIG. 2).

In FIG. 2, a flow of cells 21 is formed within the sheath 2, and an individual flow of cells is generated within the flow cell 24.

A light source 23 is provided in the middle of the flow cell, and a slit 5 is present at the other end of the light source.

The light passing through the slit 25 enters a detector 26, and a detection signal from the detector 6 is sent to a signal processing device 27.

The signal processing device 27 performs processing for detecting target cells and quantifying them based on the amount of fluorescence or light absorption.

The test cells flow through a small tube (flow cell) and are exposed to light source 2.
Laser from 3 is irradiated. It can be determined whether the cells are the target cells based on the amount of fluorescence absorbed by the laser irradiation. Quantification can also be performed. Furthermore, by examining scattering, cells can be detected and quantified.

A cell sorting device, that is, a cell sorter can be added to the automatic cell analysis device shown in FIG. The target cell descends on the jet stream, and just when it reaches the ground where the water stream turns into water droplets, a positive or negative charge is applied to the jet water column. If a cell becomes negatively charged,
When the target cell with a negative charge is charged, the water droplet containing the target cell with a negative charge is attracted to the positive electrode side while passing through the electric field 28 created by the high voltage. The falling direction of water droplets is bent toward the pole side. In this way, target cells can also be sorted into the sample collection container 29.

Next, a specific experimental example of the embodiment shown in FIG. 1 will be explained.

The method for preparing microcapsules is the method of Hsia et al. [Mezorad in Enzymeology.

(Methad j,n Enzymol,ogy,v
oQ, 74, p. 152)] can be applied. The labeling substance to be accommodated in the microcapsules may be any fluorescent substance, such as FIT C (fluorescein).
sce in 1 sotiocyonate).

RI T C (rhodamine 1 sothioc
yanate), 5RITC(substituted
rhoda＋＊ine 1sothiocyanat
a) Rhodamine B is used. As FITC becomes more precise, quenching (quenching phenomenon) occurs, so it is necessary to consider the concentration of preparation.

In addition, as a method for binding antibodies to ribosomes, a conventional method [Nature (1980) voQ
, 288, 602-604)). That is, antibodies and ribosomes are bonded using a crosslinking agent.

Next, a method for preparing a cell suspension from test cells, such as blood, will be explained. Collect 10 mQ of blood into anticoagulated tubes. This is centrifuged (700Xg) to separate plasma and bathycoat. Discard the plasma and collect the blood clot with a Pestool pipette. Suspension medium was added to the sample to make the volume 10 mQ.

Add 5 mfi Ficoll to this in a 15 mQ centrifuge tube.
- Overlay on Hypaque and centrifuge at 700Xg for 20 minutes. This allows the suspension medium layer and F
A mononuclear cell layer occurs between the icoll-Hypaque layers. The mononuclear cell layer was collected with a pipette, transferred to a 15 m12 test tube, suspended in suspension medium, and incubated for 10 min.
Perform centrifugal washing by centrifuging twice. 5-1OX10Bcell/mQ with suspension medium after washing
Prepare to. Add 55 μl of the monoclonal antibody stock solution labeled with microcapsules to 100 μQ of the prepared cell suspension.
Add ~10μQ. At this time, the above-mentioned fluorescent substance is contained in the microcapsule. The reaction temperature between the antibody and the antigen on the cell surface is 4°C, and the needle is left in place for 30 minutes. After antigen-antibody reaction, centrifugal washing with phosphate buffer for 3
Finally, add 4 to 500μQ of phosphate buffer.
The cells were resuspended and subjected to the automatic cell analyzer shown in Figure 2.

According to the method described above, membrane surface antigens of cancer cells were translocated in T cells. First, as a monoclonal antibody, anti-L eu 3a (Telper/1ndu
FITC was microencapsulated as a fluorescent substance using an antibody. The Leu3a monoclonal antibody was bound to the microcapsule containing FIT'C according to a conventional method. The FITC-labeled monoclonal antibody is reacted with the antigen and antibody according to the above-described method for preparing a cell suspension from blood to label the cell surface.

The cells whose cell surfaces have been labeled in this manner are injected into an automatic cell analyzer. Cells pass through the thin tube one by one at a constant speed. Specific wavelengths are applied to these cells from a light source. Since FITC is used as the IN-sensing compound, 488 nm of the argon ion laser is appropriate. The light excited by the laser beam is detected by the detector 6 in the perpendicular direction as an amount of fluorescent light. The signal reaching the detector 6 is converted into an electrical signal by a photomultiplier tube and amplified in the signal processing device 7. The number of times the signal enters the detector is the number of cells. The signals thus obtained can be expressed as a histogram by plotting the amount of fluorescence on the Y-axis and the number of cells on the Y-axis. A histogram of anti-Leu3a+ lymphocytes is shown in FIG. On the other hand, a histogram of a conventional method in which a fluorescent substance is directly bound to a motuclonal antibody is shown in FIG.

FIG. 4 is a histogram of a conventional method, which is obtained when a fluorescent substance is directly bound to a monoclonal antibody.

In the conventional method, there is approximately 10 fluorescence from cells that are not bound to the monoclonal antibody, that is, cells that do not have Leu3a antigen on the cell surface, and the fluorescence intensity differs by only one order of magnitude from the peak of labeled cells.

However, according to the method of preparing and labeling with microcapsules in this example, when comparing the fluorescence of cells that do not have Leu3a+ antigen, the amount of fluorescence is certainly more than two orders of magnitude higher. Thereby, cells can be detected and quantified with higher sensitivity than conventional methods. Furthermore, it is possible to detect even small amounts of antigens on the cell surface, making it effective for cell surface analysis.

Next, other embodiments of the present invention will be described in detail with reference to the accompanying drawings.

FIG. 5 shows a process diagram of this embodiment. In this example, microcapsules are labeled with multiple monoclonal antibodies that bind to different antigens.

A plurality of different antibodies are bound to the microcapsule 1. 31 is a microcapsule bound to a certain antibody, and 32 is a microcapsule bound to a different antibody. These labeled antibodies are combined with cells 3 having multiple antigens.

The antibody labeled in this reaction binds to each antigen site of the target cell. Since each antibody is bound to a microcapsule, the absolute amount of light-absorbing substance and fluorescent substance contained in the microcapsule is increased compared to the embodiment shown in FIG. As a result, the signal amount is further increased, and target cells can be detected and quantified with higher accuracy. In the case of scattering, many microcapsules bind to the cells to be measured, and as the cells grow larger, the scattering intensity also increases.

Next, a specific experimental example of this embodiment will be explained. A cell suspension is prepared in the same manner as in the experimental example shown in FIG. 1, and the volume is 100 μα. Add 5 to 10 μα of a solution containing multiple monoclonal antibodies and react at 4°C. 30
After a minute, add 2 mQ of phosphate buffer and perform centrifugal washing three times. After washing, resuspend at 4-500 μQ with phosphate buffer.

The plurality of monoclonal antibodies shown here may be any antibody as long as it can specifically react with the antigen on the surface of lymphocytes.For example, the 0KTs+Leul molecule is expressed on the membrane of all T cells, 0KT4 is a helper, inducer T cell, Leu2a, 0K
T8 is a suppressor, found on the surface of killer T cells. Functional T cells can be counted with high sensitivity by using multiple types of monoclonal antibodies against these antigens.

When using anti-Leul and ○KT3 monoclonal antibodies, add 10 ILQ of each antibody stock solution to 1 cell suspension.
00 μQ and react. FITC-containing microcapsules are bound to this monoclonal antibody in advance.

After reacting the monoclonal antibody-bound microcapsules with the cells, the same operations as in the embodiment shown in FIG. 1 above are performed, and the microcapsules are subjected to an automatic cell analyzer.

A histogram of the test cells is shown in FIG. The reason why the peak is divided into two in FIG. 6 is thought to be due to the difference in antibody titer. In FIG. 6, 1 is the fluorescence caused by the cells themselves, and 2 and 3 are the fluorescence caused by the labeled cells. According to FIG. 7, it can be seen that the number of cells corresponding to each fluorescence intensity is greater than the histogram of FIG. 3. This is the result of improved quantitative accuracy due to increased signal strength.

〔Effect of the invention〕

According to the present invention, the measurement accuracy of labeled cells is improved.

[Brief explanation of the drawing]

Figure 1 is a process diagram of an embodiment of the present invention, Figure 2 is a configuration diagram of an automatic cell analyzer, and Figure 3 is the fluorescence intensity and cell number of a specimen processed according to the embodiment of Figure 1. FIG. 4 is a graph showing the relationship between fluorescence intensity and cell number in the conventional method in which a fluorescent substance is directly bound to the test cells. FIG. 5 is a graph showing the relationship between fluorescence intensity and cell number in another embodiment of the present invention. FIG. 6, a process chart showing an example, is a graph showing the relationship between the fluorescence intensity and the number of cells determined according to the embodiment shown in FIG. 5 of the present invention. 1... Microcapsule, 2... Labeled compound, 3.
...Antibody, 4...Labeled antibody, 5...Cell, 6...
Antigen, 7...labeled cells.

Claims (1)

[Claims] 1. An antibody that specifically reacts with a test cell is bound to a microcapsule or latex particle, and then the test cell is reacted with the antibody labeled with the microcapsule or latex particle, and light scattering is performed. , a method for measuring cells, characterized in that the test cells are detected using light absorption or fluorescence. 2. The cell measuring method according to claim 1, characterized in that the microcapsules contain a light-absorbing substance or a fluorescent substance. 3. The cell measuring method according to claim 1, wherein the microcapsule is a ribosome.