JPH0412273A - Method for measuring propagatability of cell - Google Patents

Abstract

PURPOSE:To measure the propagatability of cells with a simple operation by bringing a DNA polymerase alpha monoclonal antibody labeled with a fluorescent dye into antigen-antibody reaction with the cells. CONSTITUTION:The DNA polymerase alpha monoclonal antibody labeled with the fluorescent dye is brought into the antigen-antibody reaction wit the DNA polymerase alpha positive cells which are in a period exclusive of the resting period GO and have the propagatability. For example, the suspended cells of, for example a human or the like are fractionated and fixed; therefore, the cells are mixed with the fluorescent dye labeled antibody formed by labeling the DNA polymerase alpha monoclonal antibody with the fluorescent dye. The suspended cells and the unreacted antibody mentioned above are then removed. Then, at a 2nd process, the respective cells are equally divided to individual liquid drops and the presence or absence of the fluorescence from the respective liquid drops is preferably examined by a flow site meter, by which the quantity of the DNA polymerase alpha positive cells is quantitativeyl detacted. This method is cable of measuring the suspended cells and does not require the taking of the cells on a slide glass. The quantitative measurement is easily, exactly and rapidly executed with a small amt. of the sample.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to a method for measuring the proliferation ability of cells, specifically, a method for measuring the proliferation ability of cells.
This invention relates to a method for measuring cell proliferation ability by detecting polymerase α.

[Prior Art] Since the multiple helix model of DNA was reported by Watson and Hayaick in 1953, there has been little research on DNA replication.
rg's "D N A Replication J is D
It is famous as the culmination of NA replication (Rep l i
cation, pp, 724 W, H, Fre
eman&co,, San Francisco
(1980), Supplement t.

DNA Replicatin, pp, 273
W-14, Freeman & C.

, San Francisco (1982)).

DNA polymerase α is an important enzyme that is directly responsible for the synthesis of chromosomal DNA in animal cells, and is mainly involved in the replication of chromosomal DNA. It is composed of polypeptides with a molecular weight of approximately 150,000 and 50,000 to 60,000, and exhibits molecular diversity through chemical modification. In addition, 2. It is thought that it forms a complex with other enzymes and protein factors to carry out DNA replication.

DNA polymerase α is closely related to the proliferation ability of cells, which is often required to be detected in cancer research and cell biology, and its presence is not observed in resting (GO) cells; G2. It is known to exist in the nucleus of cells in S phase and in the cytoplasm of cells in M phase. (Protein Nucleic Acid Enzyme 28, 242.1983
Experimental Ce1Research
h, 151.123.1984).

In this way, DNA polymerase α is involved in the proliferation ability of cells, but in the past, the proliferation ability of cells has not been measured as follows without utilizing this DNA polymerase α. Cells are cultured in culture medium, and isotope uptake into the nucleus is measured by taking advantage of the fact that thymidine is taken up into the nucleus during cell proliferation, or by taking advantage of the fact that the nucleus takes up bromodeoxyuridine during cell growth. The proliferation ability of cells was measured by measuring its uptake using an anti-bromodeoxyuridine antibody.

-3= These were all measurements of only cells in the S phase among proliferating cells.

In 1982, Masamoto et al. created a monoclonal antibody against DNA polymerase α (Nucleic Ac
1d Re5earch 104,703.1982
), and using this, Rokushika et al. recently (1988) reported a method to determine the proliferation ability by measuring DNA polymerase α using an immunohistological staining method (Cancer,
61.1182, 1988). This method allows cells to
A NA polymerase α antibody (consisting of IgG) is mixed, this antibody specifically binds to intracellular DNA polymerase α, and then an enzyme-labeled anti-1gG is bound to the antibody, and the enzyme produces a suitable compound. Using staining reaction,
This is a method for measuring DNA polymerase α.

[Problems to be Solved by the Invention] However, the method of measuring the proliferation ability of cells using thymidine or bromodeoxyuridine requires treatment to incorporate these substances. In addition, the cells and tissues used for culturing must be fresh, and the operation requires skill, which is another problem.Furthermore, methods that use radioisotopes require isotope equipment. However, the method of incorporating appropriate compounds into cells was not an easy measurement method.

On the other hand, with the method using DNA polymerase α antibodies, even if you try to measure intracellular DNA polymerase α and other intracellular antigens (hereinafter referred to as secondary antigens) at the same time, double staining will occur as shown below. As a result, it was difficult to carry out such simultaneous measurements.
gG) is also added to the cells, and both antibodies are reacted and bound to the cells.
Since G binds together, it was not possible to selectively measure DNA polymerase α and the second antibody.

An object of the present invention is to provide a method for measuring cell proliferation ability that can be carried out with simple operations and can be easily applied with double staining.

[Means and effects for solving the problem] The method for measuring the proliferation ability of cells of the present invention, which can achieve the above-mentioned objects, involves causing an antigen-antibody reaction with cells with a DNA polymerase α monoclonal antibody labeled with a fluorescent dye ( (first step), and then detects DNA polymerase α-positive cells using fluorescence (second step).

According to the first step of the present invention, DN labeled with a fluorescent dye
The A polymerase α monoclonal antibody is directed against DNA polymerase α positive cells (i.e. D
It specifically reacts with cells containing NA polymerase α. In addition, DNA polymerase α uses G other than resting GO.
l, G2. Since it exists in cells in the S and M phases, the DNA polymerase α-positive cells referred to in the present invention are
Gl other than telogen GO.

G2. It refers to cells in the S and M phases.

Specifically, in this first step, for example, floating cells such as human cells are fractionated and fixed, and then mixed with a fluorescent dye-labeled antibody formed by labeling a DNA polymerase α monoclonal antibody with a fluorescent dye. This can be carried out by removing floating cells and unreacted fluorescent dye-labeled antibodies.

The DNA polymerase α monoclonal antibody used here can be one obtained by fractionating mouse ascites containing this antibody with ammonium sulfate, followed by separating and purifying the IgG fraction using protein A Sepharose.

However, it is better to cultivate a cell line that produces this antibody in vitro and use an IgG fraction purified from the culture supernatant, since it contains fewer impurities and is free from non-specific reactions when measuring cell proliferation ability.
This is preferable because reactions other than the reaction between DNA polymerase α and DNA polymerase α monoclonal antibody can be removed. The DNA polymerase α monoclonal antibody is typically a mouse monoclonal antibody, but other antibodies can also be used.

In order to label the DNA polymerase α monoclonal antibody with a fluorescent dye, a known method for labeling other monoclonal antibodies with fluorescence may be followed.

Fluorescent dyes that can be used in this case include fluorescein isothiocyanate (FITC), tetramethylrhodamine isothiocyanate (TRITC), substituted rhodamine isothiocyanate (XRITC), rhodamine B isothiocyanate, and dichlorotriazine fluorescein. In (DTAF), phycoerythrin (P
E) can be exemplified.

After the first step of the present invention and the second step, that is, the step of detecting DNA polymerase α-positive cells using fluorescence, the proliferative ability of the cells can be measured.

The proliferative ability of cells can be used as an indicator of diagnostic and therapeutic effects not only for leukemia but also for various tumors and malignant tumors.

Detection of DNA polymerase α-positive cells in the second step is as follows:
Detection is preferably performed with a flow cytometer. A flow cytometer quantitatively determines D by distributing each cell into individual droplets and examining the presence or absence of fluorescence from each droplet.
The amount of NA polymerase α-positive cells can be determined.

According to the flow cytometer, floating cells can be measured, and the cells do not need to be taken on a glass slide as in the immunohistological staining method by Rokushika et al.

In addition, quantitative measurements can be performed with a small amount of sample.

Moreover, the measurement is simple, accurate, and quick.

In the measurement method of the present invention, in the first step, DNA polymerase α is an antibody against a cell antigen different from DNA polymerase α.
A fluorescent dye-labeled DNA polymerase α monoclonal antibody labeled with a second fluorescent dye different from the fluorescent dye that labeled polymerase α (second fluorescent dye-labeled antibody), or a reagent that specifically stains DNA. In addition, if cells are made to react against different antigens, that is, DN
Different colors are developed for A polymerase α and other cell antigens (including DNA), making double staining possible.

As a result, at the same time as the DNA polymerase α-positive cells in the cells, the second fluorescent dye-labeled antibody-positive cells or D
NA can be detected.

Examples of antibodies against cell antigens used for double staining include monoclonal antibodies that specifically react with human lymphocyte surface antigens. Furthermore, examples of dyes that specifically react with DNA include propium iodite, which is a fluorescent reagent.

"Effects of the Invention" According to the method for measuring cell proliferation ability of the present invention, the proliferation ability of not only fresh cells but also non-fresh cells can be measured without the need for treatment to incorporate compounds such as thymidine into the cells, and without the need for skilled manipulation or radiotherapy. It can be easily measured without requiring isotope equipment. In addition to this effect, you can enjoy the following advantages.

That is, the present invention utilizes an antigen-antibody reaction with excellent specificity, so the measurement sensitivity is extremely high, and therefore proliferating cells can be identified even when using a specimen containing a low percentage of DNA polymerase α-positive cells, such as a dying sample. In addition, it also enables double staining in which DNA ponomerase α and other cell antigens are detected simultaneously. Note that, as described above, the present invention enables highly sensitive measurement and is therefore suitable for clinical medical research.

Furthermore, the present invention is capable of detecting not only cells in stage 8 of proliferating cells but also cells in stages other than stage 00, and is therefore extremely suitable when such detection is required in terms of cell biology. be.

Furthermore, since the content of DNA polymerase α is proportional to the fluorescence intensity, according to the present invention using a flow cytometer, it is possible to determine not only the content of proliferating cells in a specimen but also the amount of DNA polymerase α possessed by proliferating cells. It is also possible to obtain relative and qualitative knowledge.

In the method of the present invention applying double staining, DNA
In addition to detecting polymerase α, other cell antigens can be detected simultaneously and easily. As a result, samples with the same amount of DNA polymerase α but different amounts of other cell antigens can be distinguished in a single measurement. It becomes possible. Therefore, D
Diseases that cannot be distinguished based on the amount of NA polymerase α, that is, cell proliferation ability alone, can be easily distinguished.

[Examples] Above, Examples will be given to explain the present invention in more detail, but the present invention is not limited to these Examples.

Examples] (1) Production and purification of monoclonal antibodies against DNA polymerase α (a) Antibody production method is Nucleic Ac1ds
It was carried out according to the method described in Re5earch 104, 703.1982.

(b) Purification of monoclonal antibodies ■ The cell line that produces the monoclonal antibodies obtained in (a) above was cultured in a culture medium (trade name: RPMI-1640) containing 10% fetal bovine serum (Fe2), The culture supernatant was collected and used as a material for antibody purification.

Antibody purification was carried out as follows by adding an equal volume of saturated ammonium sulfate to the culture supernatant and precipitating the IgG-containing fraction.
After dissolving in 50mM phosphate buffer (pH 8.5), the dialyzed solution was dialyzed overnight with the same buffer and added to a medium (Protein A Sepharose, manufactured by Pharmacia) equilibrated with the same buffer. The monoclonal antibody fraction, which was developed and washed with 10 column volumes of the same buffer, was added to 100 mM sodium phosphate-citrate buffer (p)13,0).
The monoclonal antibody was cultured using the following method, and the purified tumorigenesis promoter pristane (2, 6, 10
, 14-tetramethylpentadecane) was intraperitoneally administered to BALB/c mice at 1X106 per mouse.
~lXl0'' monoclonal antibody-producing cell line obtained in (a) above was administered, and the monoclonal antibody (1 to 1
Ascites containing 0 mg/ml) was obtained. Antibody was purified from this ascites according to the above method (2) Preparation of fluorescent dye-labeled antibody The antibody obtained by purification in (b)
After adjusting the concentration to L, it was dialyzed overnight against a sodium carbonate-sodium hydrogen carbonate buffer, and 1 mg of fluoroscene isothiocyanate (hereinafter referred to as FITC dihydrate) was added to the L-dialyzed solution.
1, B, S) were added and mixed, and the reaction products were allowed to react for 4 hours while stirring at room temperature.The reaction products were eluted with a phosphoric acid buffer using a gel filtration method to separate the unreacted FITC and the reaction products. (antibody-FITC conjugate) and target F
ITC-labeled DNA polymerase α mouse monoclonal antibody was obtained. (3) Quantification of DNA polymerase α in peripheral blood lymph nuclei by flow cytometer method. (a) Preparation of peripheral blood lymphocytes. Blood collected from humans is diluted 2 times by adding an equal amount of physiological saline to it, and then diluted by 2 times.
] (Product name x manufactured by Nyegaard),
18 to 20°CC1400X (centrifugal acceleration), 30
Lymphocytes were separated by centrifugation for minutes. Take this lymphocyte fraction into a centrifuge tube, and transfer it to a centrifuge tube that has been washed as follows.
After adding BS and suspending the cells, 4°C, 1500X
g. After centrifuging for 10 minutes to precipitate the lymphocytes, PBS was removed using a centrifuge tube. Repeat this washing procedure three times.
It was used as a lymphocyte fraction.

(b) Fixation of lymphocytes Add 2% paraformaldehyde to the lymphocyte fraction obtained in (a) above, leave at 4°C for 1 hour, then add PBS and perform the same procedure as in (a) above. The lymphocytes were washed with water and used as fixed lymphocytes.

(c) Reaction between FITC-labeled anti-DNA polymerase α mouse monoclonal antibody and lymphocytes. To the lymphocytes fixed in (b) above, an equal amount of the FITC-labeled antibody prepared in (2) above was added, and the mixture was heated at 4°C for 30 minutes. After leaving the lymphocytes to react with the labeled antibody, PBS was added and the lymphocytes were washed in the same manner as in (a) above. (d) Measurement using a flow cytometer. FITC labeling in (C) above. The lymphocyte fraction reacted with the antibody was subjected to a flow cytometer to measure DNA polymerase α-positive cells.
Table 1 shows the percentage of DNA polymerase α-positive cells in LT-4 cells (acute lymphocytic leukemia-derived T cell line) and bone marrow cells of acute myeloid leukemia patients (Percentage of DNA polymerase α-positive cells in peripheral blood lymphocytes) ) According to Example 1, the proliferation ability of various types of cells could be easily measured without requiring skilled operations or radioisotope equipment.

Furthermore, in this example, the measurement sensitivity was extremely high, and the proliferation ability of cells containing only a low percentage of DNA polymerase α-positive cells, such as Sample 2, could be measured.

In (d) of this example, the amount of DNA polymerase α possessed by individual proliferating cells was determined by detecting the fluorescence intensity of a flow side meter, based on the fact that the content of DNA polymerase α is proportional to the fluorescence intensity. We were able to obtain quantitative and qualitative knowledge.

Example 2 Simultaneous measurement method using double staining method (1) Double staining method After fixing MOLT-4 cultured cells in the same manner as in Example] (3), FITC-labeled anti-DNA polymerase α mouse monoclonal antibody and FITC-labeled anti-DNA polymerase α/mouse monoclonal antibody were used. coerythrin-labeled anti-Leu
3a antibody was added at the same time and left at 4°C for 30 minutes to react with lymphocytes.Then, the cells were washed in the same manner as in Example 1 (3).For MOLT-4 cells, the initial cell density was
The cells were set at 105 cells/ml, cultured for 4 days according to a conventional method, and measured over time.

(2) Measurement using a flow cytometer Example 1 A lymphocyte fraction reacted with two types of fluorescently labeled antibodies in the same manner as in (3) was subjected to a flow cytometer in the same manner as in Example 1 (3), and DNA polymerase α As shown in Table 2, in which positive cells and Lue3a-positive cells were measured, and Table 2 shows the positive rate of cells stained with each antibody, intracellular DNA polymerase α
and other cellular antigens could be measured simultaneously.

Table 2 (DNA polymerase α positive in MOLT 4 cultured cells/<ti
proportion of M and proportion of Leu3a positive cells)

Claims (1)

[Scope of Claims] 1. A method of detecting cells that is characterized by detecting DNA polymerase α-positive cells using fluorescence after causing a DNA polymerase α monoclonal antibody labeled with a fluorescent dye to cause an antigen-antibody reaction with the cells. Method for measuring proliferation ability. 2. When reacting the fluorescent dye-labeled monoclonal antibody with the cells, a second fluorescent dye-labeled antibody is obtained by labeling an antibody against a cell antigen different from the DNA polymerase α with a second fluorescent dye of a type different from the fluorescent dye. Alternatively, a reagent that specifically stains DNA is also reacted with the cells to perform double staining, and the second fluorescent dye antibody-positive cells or DNA are detected together with the DNA polymerase α-positive cells in the cells. The measuring method according to claim 1.