JP2008268167A - Protein quantitatively determining method by fluorescent multiple dyeing - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a protein quantitative determining method which can be carried out in a simpler manner. <P>SOLUTION: This protein quantitative determining method includes setting the ratio of the measured total fluorescent intensity of the object protein and the total fluorescent intensity of contrast protein as a fixed quantity value of the object protein, by measuring the respective total fluorescent intensities of the dyed object protein and contrast protein, by dyeing the object protein and the contrast protein of a fixed quantity in a sample, by using an antibody labeled by respectively different coloring matter. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a protein quantification method.

  As conventional protein quantification methods, quantitative reverse transcription PCR (qRT-PCR), enzyme-linked immunosorbent assay (ELISA) and the like have been established.

  However, in order to quantify proteins by these conventional methods, there is a problem that a considerable amount (for example, about 5 mm square) of a living tissue such as a tumor tissue is required as a sample. Since it is necessary to obtain a living tissue clinically by surgery or the like, it is relatively difficult to obtain it. For this reason, it is difficult to handle living tissue unless it is a university hospital grade facility.

  In contrast, an example of isolating cancer cells from a formalin-fixed paraffin-embedded tissue instead of live tissue using the laser capture microdissection method and quantifying proteins in cancer cells using qRT-PCR (Non-patent document 1: Patrick G. J et al. Cancer Res 1995, D. Edler et al. Euro J of Cancer 1997). Formalin-fixed paraffin-embedded tissue is produced during pathological examination of biopsy tissue, and is widely used in normal medical practice. Therefore, if the protein can be quantified using the formalin-fixed paraffin-embedded tissue, the one used in the pathological examination of the biopsy tissue can be used as it is. However, the above-described qRT-PCR protein quantification method is complicated and requires no clinical application.

Patrick G. J et al. Cancer Res 1995, D. Edler et al. Euro J of Cancer 1997

  Under such circumstances, there has been a demand for a protein quantification method that can be performed more easily.

  The present inventor has intensively studied to solve the above problems. In order to more easily quantify the protein in the sample, the quantified value is calculated from the ratio of the total fluorescence intensity of the target protein to be quantified in the sample and the total fluorescence intensity of the control protein using the multiple fluorescent antibody method. The inventors have found that it is sufficient to obtain the present invention and have completed the present invention.

That is, the present invention is as follows.
(1) The target protein and the control protein for quantification in the sample are stained with antibodies labeled with different fluorescent dyes,
Measure the total fluorescence intensity of each of the stained target protein and control protein,
A method for quantifying a protein, comprising setting a ratio between the measured total fluorescence intensity of a target protein and the total fluorescence intensity of a control protein as a quantitative value of the target protein.
(2) The method according to (1), wherein the control protein is a protein encoded by a housekeeping gene.
(3) The method according to (2), wherein the protein encoded by the housekeeping gene is β-actin.
(4) The method according to any one of (1) to (3), wherein the sample used for protein quantification is a formalin-fixed paraffin-embedded tissue.
(5) The method according to any one of (1) to (4), wherein the target protein is at least one selected from the group consisting of thymidylate synthase, dihydropyrimidine dehydrogenase, and orotate phosphoribosyltransferase.
(6) Fluorescene isothiocyanate, carboxymethyl indocyanine, tetramethylrhodamine isothiocyanate, phycoerythrin, green fluorescent protein, Alexa (registered trademark) 488, 4 ', 6-diamidine 2'- Any one of (1) to (5), which is at least two selected from the group consisting of phenylindole dihydrochloride, Texas Red, Cy2, Alexa (registered trademark) 543, Alexa (registered trademark) 548, Cy5, and ToTo3 The method according to one.
(7) The method according to any one of (1) to (6), wherein the quantitative value of the target protein is a quantitative value of the target protein of the target cell in the sample.
(8) The method according to (7), comprising a step of discriminating and labeling target cells in the sample with a fluorescent dye different from the fluorescent dye for labeling the target protein and the control protein.
(9) The method according to (7) or (8), wherein the target cell is a cancer cell.
(10) Identification markers for cancer cells are cytokeratin, epithelial cell membrane specific antigen, carcinoembryonic antigen, hepatocyte, α-fetoprotein, estrogen receptor, progesterone receptor, human epidermal growth factor receptor 2, thyroid transcription factor-1, The method according to (9), wherein the method is performed by staining at least one protein selected from the group consisting of prostate-specific antigen, CD10, thyroglobulin, P63, p53, Ki-67 and Cyclin-D1 with a fluorescent dye.
(11) Fluorescent dyes for identifying and labeling target cells are fluorescein isothiocyanate, carboxymethyl indocyanine, tetramethylrhodamine isothiocyanate, phycoerythrin, green fluorescent protein, Alexa (registered trademark) 488, 4 ′, 6- (8) to (10) which is at least one selected from the group consisting of diamidine 2′-phenylindole dihydrochloride, Texas Red, Cy2, Alexa (registered trademark) 543, Alexa (registered trademark) 548, Cy5 and ToTo3. ).
(12) An anticancer agent sensitivity prediction method that predicts anticancer agent sensitivity using as an index the quantitative value of the protein obtained by the method according to any one of (1) to (11).
(13) An anticancer agent effect prediction method for predicting the effect of an anticancer agent using the quantitative value of the protein obtained by the method according to any one of (1) to (11) as an index.
(14) From the quantitative value of thymidylate synthase, the quantitative value of dihydropyrimidine dehydrogenase, and the quantitative value of orotate phosphoribosyltransferase, “quantitative value of orotate phosphoribosyltransferase / quantitative value of dihydropyrimidine dehydrogenase”, “orotate phosphoribosyl” Quantitative value of transferase / quantitative value of thymidylate synthase ”and“ quantitative value of orotate phosphoribosyltransferase / (quantitative value of dihydropyrimidine dehydrogenase + quantitative value of thymidylate synthase) ” The method according to (12) or (13), wherein the ratio is obtained, and the sensitivity of the anticancer agent or the effect of the anticancer agent is predicted using the ratio of the obtained quantitative values as an index.
(15) A primary antibody that binds to the target protein, a primary antibody that binds to the control protein, a secondary antibody that binds to the complex of the target protein and the primary antibody, and a complex of the control protein and the primary antibody A protein quantification kit comprising a secondary antibody and a fluorescent dye for distinguishing and labeling each secondary antibody.
(16) Further, a primary antibody that binds to a predetermined protein of the target cell, a secondary antibody that binds to a complex of the predetermined protein of the target cell and the primary antibody, and the secondary antibody is combined with another secondary antibody. The kit according to (15), which comprises a fluorescent dye that is discriminated and labeled to identify and label a target cell.

  The present invention provides a novel protein quantification method. According to a preferred embodiment of the present invention, protein quantification can be performed more easily.

Hereinafter, the present invention will be described in detail. However, the scope of the present invention is not limited to these descriptions, and modifications other than the following exemplifications can be made as appropriate without departing from the spirit of the present invention.
It should be noted that all publications cited in the present specification, for example, prior art documents, and publications, patent publications, and other patent documents, are incorporated herein by reference in their entirety.

<Overview>
The protein quantification method of the present invention quantifies a protein contained in a sample such as a tissue or a body fluid collected from a living body using a multiple fluorescent antibody method. In the method of the present invention, a target protein and a control protein for quantification in a sample are stained with antibodies labeled with different fluorescent dyes, and the total fluorescence intensity of each of the stained target protein and control protein is measured, A ratio of the measured total fluorescence intensity of the target protein to the total fluorescence intensity of the control protein is used as a quantitative value of the target protein. That is, the present invention is a method for quantifying a target protein from the ratio of the total fluorescence intensity of a target protein to be quantified in a sample and the total fluorescence intensity of a control protein using a multiple fluorescent antibody method. In a preferred embodiment of the present invention, protein quantification can be performed more easily.

  Here, the “sample” refers to a material used for protein quantification. The “target protein” refers to a protein quantified by the method of the present invention. On the other hand, the “control protein” refers to a protein that serves as a reference when quantifying a target protein in a sample.

  “Quantitative” means to determine the amount of the target protein. In the present invention, the amount of the target protein of the sample (total fluorescence intensity) is determined by the ratio to the amount of the control protein (total fluorescence intensity) of the sample. The “ratio” is, for example, the amount of target protein (total fluorescence intensity) / the amount of control protein (total fluorescence intensity). The determined ratio is defined as a “quantitative value”.

  “Staining the target protein and the control protein with antibodies labeled with different fluorescent dyes” means that the target protein and the control protein are stained with different fluorescent dyes so that they can be distinguished from each other. . “Staining with a fluorescent dye” refers to binding a fluorescently labeled antibody to a protein directly or indirectly, for example, via a primary antibody. The “fluorescence intensity” refers to the intensity or brightness of fluorescence observed when a protein stained with a fluorescent dye is observed with a fluorescence microscope. The “total fluorescence intensity” refers to, for example, a screen obtained by observing a stained sample, and a total or total of the fluorescence intensities in each section. The “section” is, for example, an image dot. The total fluorescence intensity is preferably approximately proportional to the amount of protein in the sample. “Measuring the total fluorescence intensity” means quantifying the total fluorescence intensity.

<Multiple fluorescent antibody method>
The fluorescent antibody method is also called an immunofluorescence method, and is a method that makes it possible to identify an antigen in a sample using a fluorescently labeled antibody. Examples of the method of “identification” include a method using a quantitative PCR apparatus such as a light cycler, a flow cytometry, and the like in addition to a method using a fluorescence microscope. In the present invention, it is preferable to identify an antigen by a method using a fluorescence microscope, which can be performed more easily. The multiple fluorescent antibody method is a method in which two or more antibodies labeled with different fluorescent dyes are used to detect a plurality of types of antigens separately in the same sample. Examples of the multiple fluorescent antibody method include a double fluorescent antibody method in which a target protein and a control protein are dyed with two different fluorescent dyes. In the multiple fluorescent antibody method, the time required until a detection result is obtained is approximately 1 to 2 days.

The fluorescent antibody method includes a direct method and an indirect method. The direct method is a method in which an antibody that specifically binds to an antigen is labeled with a fluorescent dye and used for visualization of the antigen. On the other hand, the indirect method is a method in which an antibody that specifically binds to an antigen is a primary antibody, an antibody that binds to the primary antibody is a secondary antibody, and the secondary antibody is labeled with a fluorescent dye and used for antigen visualization. It is. In a preferred embodiment of the invention, an indirect method is used. The indirect method has an advantage that, when the reaction of the primary antibody is weak, it is amplified by the secondary antibody and the detection sensitivity is increased.
In the present invention, an “antigen” is a target protein and a control protein.

For example, the staining is performed as follows.
In the direct method, when the sample is a living tissue, the sample is first fixed with acetone, and phosphate buffer (PBS) (pH 7.4, NaH ₂ PO ₄ · 2H ₂ O: 9.0 g, NaHPO ₄ · 12H ₂ O: 65.45 g, NaCl: 160 g, purified water: 20 L). When the sample is a formalin-fixed paraffin-embedded block, the sample is sliced into an appropriate thickness (for example, around 4 μm), deparaffinized with xylene (for example, 5 minutes × 3 (5 minutes 3 times)), dehydration ( For example, 100% ethanol 3 minutes twice, 75% ethanol 3 minutes twice), followed by washing with water (for example, 5 minutes), followed by antigen activation corresponding to each antibody. Next, an antibody labeled with a fluorescent dye is bound to the target protein. The sample is then washed again with PBS. Thereafter, an antibody labeled with another fluorescent dye is bound to the control protein, and the sample is washed again with PBS. Finally, a sample is sealed and used for measuring the total fluorescence intensity. In addition, the target protein may be stained in the reverse order of staining the target protein first, and then the control protein, and then the target protein may be stained. In addition, two antibodies can be mixed and the staining can be completed once without dividing the staining into two.

In the indirect method, the same sample preparation as in the direct method is performed, followed by washing with PBS. Next, the target protein and the control protein are stained as follows using a primary antibody and a secondary antibody labeled with a fluorescent dye, for example.
First, the primary antibody is bound to the target protein, and the sample is washed again with PBS. Next, the secondary antibody labeled with a fluorescent dye is bound to the complex of the target protein and the primary antibody. The sample is then washed again with PBS. Thereafter, similarly to the treatment applied to the target protein, a primary antibody and a secondary antibody labeled with another fluorescent dye are bound to the control protein. The sample is then washed again with PBS. Finally, a sample is sealed and used for measuring the total fluorescence intensity.

In addition, in the reverse order to the above staining operation, the control protein can be stained first, and then the target protein can be stained. Alternatively, two primary antibodies may be mixed and the binding of the primary antibody to the target protein and the control protein may be terminated by a single operation. Furthermore, two secondary antibodies are mixed, and the binding of the secondary antibody to the complex of the target protein and the primary antibody and the binding of the secondary antibody to the complex of the control protein and the primary antibody can be performed in one operation. It is also possible to end it.
In the case of the indirect method, a control stained with a secondary antibody alone may be prepared.

<Fluorescent dye>
The fluorescent dye used for labeling the antibody is not particularly limited as long as it is a dye that emits fluorescence when irradiated with excitation light and can be bound to the antibody. For example, fluorescein isothiocyanate (FITC), carboxymethyl indocyanine ( Cy3), tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin (PE), green fluorescent protein (GFP), Alexa ^TM 488, 4 ', 6-diamidine 2'-phenylindole dihydrochloride (DAPI), Texas Red , Cy2, Alexa ^™ 543, Alexa ^™ 548, Cy5 and ToTo3 (Morecular Probes). Since the present invention is a multiple fluorescent antibody method, two or more fluorescent dyes are used in combination. For example, in the double fluorescent antibody method, fluorescent dyes that develop different colors such as FITC and Cy3, FITC and TRITC, or Cy5 and FITC can be used in combination to stain the target protein and the control protein.

<Antibodies labeled with fluorescent dyes>
As the antibody labeled with a fluorescent dye, a commercially available one may be used, or it may be prepared by itself. Examples of commercially available products include Fluorescein isothiocyanate isomer 1 (FITC) conjugated anti-rabbit immunoglobulin (IgG) (DakoCytomation, CA, USA) and Cy3 conjugated secondary antibody (anti-mouse IgG, CHEMICON International Inc). The method for binding the fluorescent dye to the antibody is not particularly limited, and can be performed, for example, as follows (Immunochemical experiment method, 1992, Shunsuke Ueda et al., Nishimura Shoten). First, an antibody is dissolved in a carbonate buffer and dialyzed to prepare a solution having a predetermined antibody concentration (for example, 10 to 20 mg / ml). Next, a predetermined amount (for example, 1 mg) of fluorescent dye per 1 mg of antibody is added to the dialyzed solution and mixed at a predetermined temperature (for example, 4 ° C.) for a predetermined time (for example, overnight). Next, the mixture is applied to a gel filtration column and eluted with PBS. Then, the fraction of the antibody labeled with a fluorescent dye is collected.

<Antibody>
The antibody used in the present invention is an antibody capable of recognizing the target protein, the control target protein, the complex of the target protein and the primary antibody, or the complex of the control protein and the primary antibody (hereinafter referred to as the target protein). Either a polyclonal antibody or a monoclonal antibody may be used. However, it is more preferable to use a monoclonal antibody having high specificity. An antibody can be produced according to a known antibody or antiserum production method using a target protein, a control protein, a primary antibody, or the like as an antigen. Of course, commercially available products can also be used. Examples of commercially available antibodies include antibodies sold by DAKO, Roche, Chemicon international, Inc., Novocastra, Santa Cruz Biotechnology, Inc., and the like. Antibodies sold by DAKO include HER-2 (Hercep Test), c-kit, cyclin D1, ER, PGR, E-cadherin, Chromogranin A Thyroglobulin, Ki-67 and the like. Antibodies sold by Santa Cruz Biotechnology, Inc include thymidylate synthase (TS) MGMT, h-MLH1, HER-2, c-kit, Chromogranin A, and VEGF. The antibodies marketed by Novocastra include HER-2, c-kit, cyclinD1, ER, PGR, E-cadherin, ChromograninA, Thyroglobulin and the like. Examples of antibodies sold by Roche include dihydropyrimidine dehydrogenase (DPD). Examples of antibodies sold by Chemicon international, Inc. include MGMT and c-kit. The method of the present invention can be applied relatively easily as long as the antibody is a commercially available protein and immunostaining has been established.

Preparation of Monoclonal Antibody-Producing Cells First, the target protein or the like is administered to a mammal at a site where antibody production is possible by administration, or with a carrier and a diluent. Examples of mammals to be used include monkeys, rabbits, dogs, guinea pigs, mice, rats, sheep and goats, and mice and rats are preferably used. When producing monoclonal antibody-producing cells, select a warm-blooded animal immunized with an antigen, for example, an individual with a confirmed antibody titer from a mouse, collect spleen or lymph nodes, and use the antibody-producing cells contained therein as bone marrow. Monoclonal antibody-producing hybridomas can be prepared by fusing with tumor cells (Nature, 256, 495 (1975)). Known methods can be used to screen for monoclonal antibody-producing hybridomas. The selection of the monoclonal antibody can also be performed according to a known method or a similar method.

Purification of monoclonal antibodies Separation and purification of monoclonal antibodies can be carried out in the same way as separation and purification of ordinary polyclonal antibodies (eg, salting out, alcohol precipitation, isoelectric precipitation, electrophoresis, ion exchange) Absorbing and desorbing with a body (eg, DEAE), ultracentrifugation, gel filtration, or collecting an antibody only with an active adsorbent such as an antigen-binding solid phase, protein A or protein G, and dissociating the binding to obtain an antibody Specific purification method].

Preparation of polyclonal antibody Polyclonal antibodies can be produced according to known methods or modifications thereof. For example, a complex of an immunizing antigen (an antigen such as the protein of the present invention) and a carrier protein is prepared, a mammal is immunized in the same manner as the above-described monoclonal antibody production method, and the antibody against the target protein or the like is immunized from the immunized animal. It can be produced by collecting the contents and separating and purifying the antibody.

<Measurement method of total fluorescence intensity>
The method for measuring the total fluorescence intensity is not particularly limited as long as it is a method capable of measuring the total fluorescence intensity of each of the target protein and the control protein of the sample, and examples thereof include a method using a fluorescence microscope. In the method using a fluorescence microscope, the excitation light is incident on the sample under the microscope, the excitation light is removed by a filter, and the fluorescence emitted by the fluorescent dye is captured by the naked eye, a video camera, a CCD camera, etc. Is a method of measuring. As such a fluorescence microscope, for example, it is desirable to use a confocal laser microscope or a wavelength analysis confocal laser microscope. For example, if you already have a confocal laser microscope or a wavelength analysis confocal laser microscope, you can use it, which has the advantage that additional capital investment is relatively small. In a preferred embodiment of the present invention, fluorescence is captured using a high-sensitivity video camera, a high-sensitivity CCD camera, or the like to acquire an image, and the acquired image is analyzed using software or the like. taking measurement.

  More specifically, for example, the fluorescence intensity of each dot of an image captured by a high-sensitivity video camera or high-sensitivity CCD camera is digitized using software, and the digitized fluorescence intensity of each dot is integrated. By doing so, the total fluorescence intensity can be measured.

  Software for processing an image and measuring fluorescence intensity and software for accumulating numerical fluorescence intensity are not particularly limited, and may be configured as one piece of software or separately. It may be software. Moreover, as such software, you may utilize what is distribute | circulated in the market, or you may use what was produced by oneself or was commissioned and produced by others.

  Examples of such commercially available software include LSM 5 PASCAL (Carl Zeiss) and LSM image Examiner (Carl Zeiss).

<Sample>
The sample used for protein quantification may be appropriately selected according to the purpose and is not particularly limited, and examples thereof include living tissue, formalin-fixed paraffin-embedded tissue and the like. Samples derived from non-human mammals such as humans and non-human mammals such as cattle, monkeys, birds, cats, mice, rats, guinea pigs, hamsters, pigs, dogs, rabbits, sheep, and horses. Can be mentioned. Other vertebrates, invertebrates, viruses, bacteria, and the like can also be used. Examples of the tissue used as a sample include epithelial tissue, support tissue, muscle tissue, nerve tissue, cultured cells, and these cancerous tissues. Cancers include brain cancer, tongue cancer, pharyngeal cancer, lung cancer, breast cancer, esophageal cancer, stomach cancer, pancreatic cancer, biliary tract cancer, gallbladder cancer, duodenal cancer, colon cancer, liver cancer, uterine cancer, ovarian cancer, prostate cancer, renal cancer, Bladder cancer, rhabdomyosarcoma, fibrosarcoma, osteosarcoma, chondrosarcoma, skin cancer, and various leukemias (eg, acute myeloid leukemia, acute lymphocytic leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, adult T cell leukemia) , And malignant lymphoma).

  Live tissue can be collected from a human or the like by surgery, endoscopic surgery, transvascular surgery, endoscopic biopsy, percutaneous biopsy, and the like. In a preferred embodiment of the present invention, even if a living tissue is a trace amount (for example, several cells), the protein can be accurately quantified from the living tissue, and an endoscope can be used without relying on a surgical specimen. A lower biopsy specimen can be adequately performed. In the case of endoscopic biopsy, there are advantages such as less invasion when collecting a living tissue, and less burden on a human or the like when preparing a sample.

  The formalin-fixed paraffin-embedded tissue can be prepared by fixing the living tissue collected as described above with a formalin solution and embedding with paraffin according to a conventional method. In a preferred embodiment of the present invention, a formalin-fixed paraffin-embedded tissue prepared at the time of pathological examination of a biopsy tissue can be used as it is in a protein quantification method. In this way, it is not necessary to collect a new tissue from a patient or the like for protein quantification, so that the burden on the patient or the like can be reduced. In addition, since many formalin-fixed paraffin-embedded tissues are stored in medical institutions, etc., by opening the way to effectively use these tissues as research materials in the medical field or life science field, the medical field or life science field It is expected that further research will progress.

<Target protein and control protein>
The target protein and the control protein may be appropriately selected according to the purpose of protein quantification, and are not particularly limited.
Examples of the purpose of protein quantification include the purpose of predicting anticancer drug sensitivity, the purpose of predicting side effects of anticancer drug administration, the purpose of analyzing prognostic factors such as cancer, and the purpose of pathological diagnosis. According to a preferred embodiment of the present invention, anticancer drug sensitivity can be predicted, and as a result, improvement in treatment results can be expected by selecting an appropriate anticancer drug, useless anticancer drug administration is reduced, and not only is the prognosis improved for patients. It is also possible to reduce medical costs.

  For the purpose of predicting anticancer drug sensitivity, for example, a tissue containing cancer cells is used as a sample, a protein serving as an anticancer drug sensitivity marker is used as a target protein, and a protein encoded by a housekeeping gene of cells in the tissue is used. Control protein. For example, for the purpose of predicting the sensitivity of 5-FU anticancer drugs such as S-1 (Takuma Pharmaceutical Co., Ltd.), the target proteins include thymidylate synthase (TS), dihydropyrimidine dehydrogenase (DPD), orolo Examples include tate phosphoribosyltransferase (OPRT). In the case of Taxan anticancer agents such as paclitaxel (Taxol) (Bristol-Myers Squibb) and docetaxel (Taxotere) (Sanofi-Aventis), CHFR may be mentioned. In the case of an alkylated anticancer agent such as cyclophosphamide (endoxan), hMLH-1, MGMT and the like can be mentioned. Currently, HER-2 protein immunostaining is clinically applied to predict the sensitivity of the anticancer drug Herceptin to breast cancer, but it is thought that more detailed sensitivity prediction can be made by applying this method. Similarly, by applying this method to immunostaining of c-kit with anticancer agent imatinib (Gleevec) for Gastrointestinal stromal tumor (GIST) and EGF receptor with anticancer agent gefitinib (Iressa) for non-small cell lung cancer by applying this method Can be expected for further sensitivity prediction or further research progress. By applying this method to estrogen and progesterone receptors, which are considered to be indicators of the effects of hormone therapy for breast cancer, for example, even if they are not anticancer agents, it is possible to accumulate data such as “about what percentage can be expected” Become. Examples of the protein encoded by the housekeeping gene include β-actin and GAPDH.

  For the purpose of predicting the side effects of anticancer drug administration, the protein associated with the side effects of the anticancer drug is the target protein, and the protein encoded by the housekeeping gene of cells in the tissue is the control protein. In this case, as target proteins, for example, NF-kB transcription factor, iNOS nitric oxide inducing enzyme, COX-2 (cyclooxygenase), PGE2 (prostaglandin), IGF (insulin-like growth factor) and VEGF (vascular endothelial growth factor) ) And the like. Moreover, the measurement of these factors can be re-measured by an endoscopic biopsy specimen even in the middle of treatment, and can be used as an index for determining whether to continue or discontinue anticancer drug treatment. Examples of the protein encoded by the housekeeping gene include β-actin and GAPDH.

  For the purpose of predicting prognosis such as cancer, a protein serving as a marker of prognostic factors is used as a target protein, and a protein encoded by a housekeeping gene of a cell in a tissue is used as a control protein. In this case, p53, Ki-67, epidermal growth factor receptor (EGFR), angiogenesis factor (VEGF), Cyclin family, p21, pRB, Matrix metalloproteinase family, E-cadherin, And numerous proteins such as β-catenin. Examples of the protein encoded by the housekeeping gene include β-actin and GAPDH.

  For the purpose of pathological diagnosis, the diagnostic key protein (mainly secreted protein) is the target protein, and the protein encoded by the housekeeping gene of the cells in the tissue is the control protein. In this case, CEA, AFP, chromogranin A, synaptophysin, etc. are mentioned as diagnostic key proteins, and in the thyroid gland, thyroid hormone, thyroglobulin, thyroid stimulating hormone, calcitonin and the like can be mentioned. In the adrenal gland, ACTH and steroid hormones, in the gastrointestinal tract, somatostatin and gastrin, in the pancreas, amylase, glucagon, insulin, trypsin and the like can be mentioned. In addition, sex hormones can be quantified in the ovary and testis. In this way, it is possible to examine how much hormone is secreted in which tissue, and it can be expected to play a major role in pathological analysis and diagnosis. Examples of the protein encoded by the housekeeping gene include β-actin and GAPDH.

<Anticancer agent sensitivity prediction method and anticancer agent effect prediction method>
According to another aspect of the present invention, there is provided an anticancer agent sensitivity prediction method for predicting anticancer agent sensitivity using as an index the protein quantitative value obtained by the protein quantification method of the present invention. Also provided is an anticancer agent effect prediction method that predicts the effect of an anticancer agent using the protein quantification value obtained by the protein quantification method of the present invention as an index.

It can be said that when a cancer cell is killed when an anticancer agent is allowed to act on a certain cancer cell, the cancer cell is sensitive to the anticancer agent. When the anticancer drug is allowed to act on a cancer cell having such sensitivity to a certain anticancer drug, the protein that increases the expression level in the cancer cell, the protein that decreases the expression level, or the expression level falls within a predetermined range. There are proteins that are preserved. These proteins serve as indicators for predicting whether cancer cells are sensitive to the anticancer agent. In addition, in some anticancer agents, before the anticancer agent is allowed to act on cancer cells, if the expression level of a certain protein in the cancer cells is low, if it is high, or if the expression level is within a predetermined range, the cancer cells Is capable of predicting the presence or absence of sensitivity to anticancer drugs. Therefore, after the anticancer drug is allowed to act on cancer cells or before the anticancer drug is allowed to act on cancer cells, the expression level of these proteins in cancer cells should be quantified, and the quantitative value of the obtained protein should be used as an index. Thus, the sensitivity of the anticancer agent can be predicted, or the effect of the anticancer agent can be predicted.
Specifically, anticancer drug sensitivity and anticancer drug effect are predicted, for example, as follows using the obtained protein quantitative value as an index. That is, when using as an index a protein whose expression level in cancer cells decreases when the anticancer agent is allowed to act on cancer cells sensitive to a certain anticancer agent, or before an anticancer agent is allowed to act on cancer cells. In addition, if there is sensitivity to the anticancer agent and a protein whose expression level is low in the cancer cell is used as an indicator, when the quantitative value of the protein is not more than a predetermined reference value, the test cell is an anticancer agent Therefore, it can be expected that the effect of anticancer agents can be expected. On the other hand, when a protein that increases the expression level in a cancer cell is used as an index when the anticancer agent is allowed to act on a cancer cell sensitive to an anticancer agent, or before an anticancer agent is allowed to act on the cancer cell. If the protein is sensitive to the anticancer agent, and the protein expressed in the cancer cell is used as an indicator, the test cell is not sensitive to the anticancer agent when the quantitative value of the protein is not less than a predetermined reference value. Therefore, it can be expected that the sensitivity is high or the effect of the anticancer agent can be expected. In addition, when an anticancer drug is allowed to act on a cancer cell sensitive to a certain anticancer drug, a protein whose expression level in the cancer cell is within a predetermined range is used as an index, or a certain anticancer drug is used as a cancer. If there is a protein whose expression level in the cancer cell is within the predetermined range if it is sensitive to the anticancer agent before acting on the cell, the quantitative value of the protein is within the predetermined reference value range. When it is within the range, it can be predicted that the test cell is highly sensitive to the anticancer agent or that the effect of the anticancer agent can be expected.

In particular, it is preferable to calculate a quantitative value of two or more proteins from a sample, obtain a ratio from the calculated quantitative value of the protein, and predict the anticancer drug sensitivity and the anticancer drug effect using the ratio of the determined quantitative values as an index. . In other words, when a ratio that decreases when an anticancer agent is allowed to act on a cancer cell sensitive to an anticancer agent is used as an index, or before the anticancer agent is acted on, the cancer cell is sensitive to the anticancer agent. When the ratio of the quantitative value obtained is less than a predetermined reference value, the test cell is highly sensitive to the anticancer agent or the effect of the anticancer agent. Can be expected. On the other hand, when a ratio that increases when an anticancer agent is allowed to act on a cancer cell sensitive to an anticancer agent is used as an index, or before the anticancer agent is acted on, the cancer cell is sensitive to the anticancer agent. When the ratio of the determined quantitative value is greater than or equal to a predetermined reference value, the test cell is highly sensitive to the anticancer agent or the effect of the anticancer agent. Can be expected. Furthermore, when the ratio is within a predetermined range when the anticancer agent is allowed to act on cancer cells sensitive to an anticancer agent, or before the anticancer agent is allowed to act, If the ratio is within a predetermined range as long as it is sensitive to the anticancer agent, the test cell is an anticancer agent when the ratio of the determined quantitative values is within the predetermined reference value range. Therefore, it can be expected that the effect of anticancer agents can be expected.
For example, regarding the prediction of the effects of 5-FU anticancer drugs, including S-1, which is the most widely used and reported in Japan, in gastric cancer, the quantitative value of thymidylate synthase, the quantitative value of dihydropyrimidine dehydrogenase and From the quantitative value of orotate phosphoribosyltransferase, "quantitative value of orotate phosphoribosyltransferase / quantitative value of dihydropyrimidine dehydrogenase (OPRT / DPD ratio)", "quantitative value of orotate phosphoribosyltransferase / quantitative value of thymidylate synthase (OPRT / TS ratio) ”and“ quantitative value of orotate phosphoribosyltransferase / (quantitative value of dihydropyrimidine dehydrogenase + quantitative value of thymidylate synthase) (OPRT / (DPD + TS) ratio) ” Determine at least one ratio and determine the quantitative value The ratio of the above is used as an index to predict the sensitivity of the anticancer agent or the effect of the anticancer agent.

  The predetermined reference value can be set by conducting experimental research on cancer such as each organ and accumulating data. For example, in a study conducted by the present inventor regarding the prediction of the effect of a 5-FU anticancer agent on gastric cancer, an OPRT / DPD ratio of 1 or higher, or an OPRT / TS ratio of 1 or higher, an effect of an anticancer drug (PR (effective) or higher) ) Can be expected. In particular, the OPRT / (TS + DPD) ratio is most useful, and when the OPRT / (DPD + TS) ratio is 0.5 or more, the effect of an anticancer agent (effect more than PR (effective)) can be expected.

  In this way, by predicting anticancer drug sensitivity or anticancer drug effect, improvement in treatment results can be expected by selecting appropriate anticancer drugs, reducing unnecessary anticancer drug administration, not only improving prognosis to patients, but also reducing medical expenses Is also possible

<Microscopic tissue separation method>
A sample of cancerous tissue contains collagen fibers and other cells in addition to cancer cells. When it is intended to quantify protein only for cancer cells using such a sample, if it is a biopsy tissue, cancer cells, collagen fibers and other cells are frayed in the tissue. Only cancer cells can be distinguished relatively easily with a fluorescence microscope or the like. However, in tissues such as wet parts and metastatic parts, collagen fibers and other cells are complicatedly entangled with cancer cells, and it may be difficult to distinguish only cancer cells with a microscope or the like.
In such a case, for example, a target cell such as a cancer cell in a sample can be identified using a microscopic tissue separation method, and a quantitative value of a protein is obtained only for the target cell such as a cancer cell. It is preferable. When using the microscopic tissue separation method, the target protein and the control protein may be stained and then subjected to the microscopic tissue separation method so that the target cells can be identified, or the microscopic tissue separation method may be performed before the target protein and the control protein are stained. The target cells may be identified, or the target cells may be identified by performing microtissue separation simultaneously with staining of the target protein and the control protein. The “microdissection method” is a method that allows only target cells such as cancer cells to be distinguished from tissues in tissue observation under a microscope or the like. The “microscopic tissue separation method” includes, for example, a method using a needle, a laser capture microdissection method, a fluorescent tissue separation method, and the like. The “method using a needle or the like” is a method of separating a target cell in a sample by tearing the target cell from another tissue using a needle or the like. The “Laser capture microdissection method” is a method of punching out a tissue with a laser and separating target cells in a sample.

In a preferred embodiment of the present invention, a fluorescent tissue separation method is used as the microscopic tissue separation method. “Fluorescent tissue separation (fluorescential microdissection)” is a method in which target cells in a sample can be distinguished from tissues by identifying and labeling the target cells with a fluorescent dye. In a preferred embodiment of the present invention, a predetermined protein of a target cell in a sample is stained with a fluorescent dye. And, it is relatively easy to distinguish cancer cells that are difficult to distinguish in a sample, such as cancer cells with complex collagen fibers and other cells, or poorly differentiated cancer cells, from tissues by fluorescent tissue separation. Can do. Here, the “predetermined protein” is preferably a protein that is specifically expressed in the target cell. When the target cell is a cancer cell, the predetermined protein is, for example, cytokeratin specifically expressed in the cancer cell, epithelial cell membrane specific antigen, carcinoembryonic antigen, hepatocyte, α-fetoprotein, estrogen receptor, progesterone receptor, human epithelium Cell growth factor receptor 2, thyroid transcription factor-1, prostate specific antigen, CD10, thyroglobulin, P63, p53, Ki-67 and Cyclin-D1. Here, cytokeratin is an intermediate filament in epithelial cells and is a protein suitable for staining cancer cells derived from epithelial cells. There are more than 20 subtypes of cytokeratin including CK 1 to CK20. Since the type of cytokeratin expressed differs depending on the organ, the type of cytokeratin suitable for the identification marker of each cancer cell may be stained according to the organ in which the cancer cell has developed. Of course, multiple types of cytokeratins may be stained using a cocktail of several types of antibodies such as CK AE1, CK AE3, and CAM 5.2. Endothelial cell membrane specific antigen (EMA) is also a protein suitable for staining epithelial cell-derived cancer cells. Carcinoembryonic antigen (CEA) is a protein suitable for staining adenocarcinoma cells. Hepatocyte and alpha-fetoprotein (AFP) are suitable proteins for staining liver cancer cells. Estrogen receptor, progesterone receptor and human epidermal growth factor receptor 2 (HER2) are proteins suitable for staining breast cancer cells. Thyroid transcription factor-1 (TTF-1) is a protein suitable for staining lung cancer cells. Prostate-specific antigen (PSA) is a protein suitable for staining prostate cancer cells. CD10 is a protein suitable for staining renal cancer cells. Thyroglobulin is a suitable protein for staining thyroid cancer cells. P63 is a protein suitable for staining squamous cell carcinoma cells. Cytokeratin, epithelial cell membrane specific antigen, carcinoembryonic antigen, hepatocyte, α-fetoprotein, estrogen receptor, progesterone receptor, human epidermal growth factor receptor 2, thyroid transcription factor-1, prostate specific antigen, CD10, thyroglobulin and P63 is a protein that is specifically expressed in cancer cells in each organ, and is a protein suitable for identifying and labeling metastatic cancer cells. In addition, proteins such as p53, Ki-67, and Cyclin-D1, which are used to classify the malignancy of cancer, are proteins that can be used for identifying and labeling cancer cells. In a preferred embodiment of the invention, at least one of these proteins is labeled with a fluorescent dye. In the present invention, the protein to be stained with a fluorescent dye for identifying and labeling a target cell is not limited to the above-exemplified proteins, and may be appropriately selected in consideration of cancer cell-derived cells, biological characteristics, and the like. You can do it. Specific examples of proteins that are stained with a fluorescent dye to identify and label target cells are also described in, for example, Pathology and Clinical 2007 Vol. 25 Special Issue “Immunohistochemistry Useful for Diagnosis” p325-p367 “Antibody Index” Has been.
In the present invention, the fluorescent dye for labeling a predetermined protein of the target cell is different from the fluorescent dye for labeling the target protein and the control protein, and more preferably has a different color. Examples of fluorescent dyes include fluorescein isothiocyanate (FITC), carboxymethyl indocyanine (Cy3), tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin (PE), green fluorescent protein (GFP), Alexa ^TM 488, 4 Examples include ', 6-diamidine 2'-phenylindole dihydrochloride (DAPI), Texas Red, Cy2, Alexa ^™ 543, Alexa ^™ 548, Cy5, and ToTo3 (Morecular Probes). In a further preferred embodiment of the present invention, among these fluorescent dyes, at least one fluorescent dye different from the fluorescent dye used for labeling the target protein and the control protein is used for labeling a predetermined protein of the target cell.

The target cells can be stained by the same method as described above using the same antibody as that described above. The antibody used for staining the target cell is, for example, an antibody that can recognize a protein for identifying and labeling the target cell, or an antibody that can recognize a complex of the protein and the primary antibody.
More specifically, in order to identify the target cell from the sample by the fluorescent tissue separation method, for example, cytokeratin specifically expressed in cancer cells derived from epithelial cells is labeled with Cy5 (blue). By this labeling, only the target cancer cell glows blue when observed under a fluorescence microscope. For example, the target protein is labeled with FITC (green) and the control protein such as β-actin is labeled with Cy3 (red). That is, the sample is stained using a triple fluorescent antibody method using three different dyes. Then, only cancer cells that glow blue are extracted using the same image analysis software as described above, and the fluorescence intensity is analyzed, whereby the protein can be quantified only for the cancer cells.
The target cell may be stained before the target protein and the control protein are stained, or after the target cell and the control protein are stained, or the target cell and the target protein are stained. It may be performed simultaneously.

<Protein determination kit>
According to another aspect of the present invention, a protein quantification kit is provided. The kit includes a primary antibody that binds to the target protein, a primary antibody that binds to the control protein, a secondary antibody that binds to the complex of the target protein and the primary antibody, and a complex of the control protein and the primary antibody. And a fluorescent dye that distinguishes and labels each secondary antibody.
According to a preferred embodiment of the present invention, the kit further comprises a primary antibody that binds to a predetermined protein of the target cell, a secondary antibody that binds to a complex of the predetermined protein of the target cell and the primary antibody, and The secondary antibody is labeled separately from other secondary antibodies, and a fluorescent dye that distinguishes and labels the target cell is included.
The kit may contain auxiliary agents, special containers, other necessary accessories, instructions, etc., as necessary.
By using the kit of the present invention, the protein in the sample can be quantified more easily.

  Hereinafter, the present invention will be described in more detail with reference to examples. However, the present invention is not limited to this example.

  In this example, thymidylate synthase (TS), dihydropyrimidine dehydrogenase (DPD), and orotate phosphoribosyltransferase (OPRT) were used for the purpose of predicting the sensitivity of 5-FU anticancer agent S-1 (Takuma Pharmaceutical Co., Ltd.). ) Was quantified.

<S-1 metabolic pathway>
S-1 is a ternary oral anticancer drug consisting of tegafur (FT), gimeracil (CDHP) and oteracil potassium (Oxo). TS is an enzyme required to synthesize DNA by converting dUMP to dTMP, but S-1 inhibits the activity of this TS and inhibits the synthesis of DNA in cancer cells. Indicates.
The metabolic pathway of S-1 is shown in FIG. 1 (Takeuchi H et al. J Clin Oncol 16 (8), 1998).
FT is gradually converted into 5-FU in the body, further phosphorylated by OPRT in cancer cells, and converted into 5-fluoronucleotides (FdUMP, etc.). When FdUMP forms a complex with TS and the like necessary for DNA synthesis, TS in cancer cells is depleted and DNA synthesis of cancer cells is inhibited.
CDHP also increases the concentration of 5-FU converted from FT by selectively inhibiting dihydropyrimidine dehydrogenase (DPD), which inactivates and degrades 5-FU in cancer cells.
Oxo is mainly distributed in the digestive tract tissue by oral administration, selectively inhibits OPRT, and inhibits the conversion from 5-FU to FUMP. This is believed to reduce gastrointestinal disorders.
Therefore, when (a) TS activity is low, (b) DPD activity is low, or (c) OPRT activity is high, it can be determined that sensitivity to S-1 is high. On the other hand, when (a) TS activity is high, (b) DPD activity is high, or (c) OPRT activity is low, it can be judged that sensitivity to S-1 is low.

<Sample>
A biopsy formalin-fixed paraffin-embedded specimen (formalin-fixed paraffin-embedded block) of a gastric cancer tissue collected under an endoscope before the start of treatment of a stomach cancer patient was used as a sample. Note that “before treatment” refers to before the start of any treatment including administration of S-1 or other anticancer agents.
Gastric cancer patients corresponding to biopsy formalin-fixed paraffin-embedded specimens have undergone chemotherapy after biopsy, and their therapeutic effects have been confirmed. That is, as a treatment, neoadjuvant chemotherapy (NA Chemotherapy: NAC) was performed, and after that, some patients performed resection. In neoadjuvant chemotherapy, some patients receive only S-1, other patients receive S-1 and CDDP (Randa Note: Nippon Kayaku Co., Ltd.), and the rest The patient received S-1 and Tax (paclitaxel; taxol, Bristol Pharmaceutical Co., Ltd.).
Gastric cancer patients corresponding to biopsy formalin-fixed paraffin-embedded specimens are treated with chemotherapy, complete response (CR: markedly effective), partial response (PR: effective), no change (NC: unchanged) and progressive disease ( PD: Progression) (Table 1). Furthermore, gastric cancer patients were classified into two groups, an effective case group (Responder (CR + PR)) and an ineffective case group (Non-responder (NC + PD)) (Table 2). The classification is based on the criteria of the Japanese Cancer Treatment Society.

Specifically, the meanings of CR, PR, NC, and PD are as follows.
CR: Measurable lesions, evaluable lesions, and secondary lesions caused by tumors disappeared and no new lesions persisted for 4 weeks or longer.
PR: the reduction rate of the bilaterally measurable lesion is 50% or more, and the state where the evaluable lesion and the secondary lesion caused by the tumor are not exacerbated and no new lesion appears is maintained for at least 4 weeks, or For unidirectionally measurable lesions, the reduction rate obtained by each calculation formula is 30% or more, and the state in which the evaluable lesion and the secondary lesion caused by the tumor are not hated and no new lesion appears is at least 4 weeks or more If persisted.
NC: The reduction rate of the bi-directionally measurable lesion is less than 50%, and in the unidirectionally measurable lesion, the reduction rate is less than 30%, or the increase is within 25% of each. If you are not hateful and have no new lesions for at least 4 weeks.
PD: If the product or diameter sum of measurable lesions increases by 25% or more, or other lesions hate or new lesions appear.

  As shown in Table 1 and Table 2 below, there is no significant difference between groups in age, sex, chemotherapy regimen, and tissue type in each group divided by the therapeutic effect by chemotherapy. That is, Tables 1 and 2 show that the cases used in this example are not statistically and artificially selected populations. Statistical analysis used Student's t test and chi-square test.

<Measurement of staining and fluorescence intensity>
First, formalin-fixed paraffin-embedded tissue was treated as follows.
The specimen was sliced 4 μm thick, stretched at 50 ° C., baked (insulated) at 37 ° C. for 2 hours, and fixed on a coated glass slide. At this time, it was not baked for a long time at high temperature. Next, deparaffinization with xylene (5 minutes X3) and dehydration (100% ethanol 3 minutes, twice, 75% ethanol 3 minutes, twice) were performed. Thereafter, it was thoroughly washed with water (5 minutes). Then, the next antigen activation treatment was performed only with TS. That is, 10 mM, pH 6.0 citric acid buffer was used and treated with microwave for 10 minutes, and then allowed to stand at room temperature for 2 hours to be sufficiently cooled. Then, after washing with PBS and blocking nonspecific reaction with 5% skim milk for 10 minutes, TS (anti-rabbit, 100-fold diluted), DPD (provided by Taiho Pharmaceutical Co., Ltd.) as the primary antibody. Anti-rabbit, diluted 100-fold) and OPRT (anti-rabbit, diluted 500-fold) polyclonal antibody were reacted at room temperature for 2 hours. Next, after washing the tissue again with PBS, FITC-labeled secondary antibodies (anti-rabbit, octopus) were reacted as 100-fold dilutions for 30 minutes, respectively. Thus, first, the target proteins TS, DPD and OPRT were stained with each sample. Next, the control protein β-actin was stained. First, an anti-β-actin antibody (Mouse, monoclonal, SIGMA, Saint Louis, USA) diluted 500 times as a primary antibody was reacted for 2 hours. Thereafter, Cy3-labeled antibody (anti-mouse: Chemicon) was reacted at room temperature for 30 minutes. Thereafter, it was washed with water and sealed.

  Here, FIG. 2 is an image photograph in which TS and β-actin of moderately differentiated adenocarcinoma are stained by the double fluorescent antibody method as described above. (A) is a stained image of β-actin, and (b) is a stained image of TS. (C) is an image obtained by combining (a) and (b). TS was visualized using FITC, and β-actin was visualized using Cy3. As shown in FIG. 2, TS appears green and β-actin appears red.

  Next, the fluorescence intensity was measured as follows. First, using Laser Scanning Microscope LSM5 Pascal (Carl Zeiss Microimaging: Jena Germany), CH1 of Absolute Frequency is set to Cy3 (β-actin), CH2 of Absolute Frequency is set to FITC (target protein), and possible at 400 times magnification As much as possible, images of double fluorescence immunostaining were obtained only with cancer cells so that no stroma or blood cells could enter. Next, fluorescence intensity was measured using the attached image analysis software (LSM image Examiner). At this time, the Reuse function was used to set the LSM so that all image acquisition conditions were the same. In addition, the same ZERO reference point in the background of all images was taken into consideration so that no artifacts were applied to the quantitative values. FIG. 3 is a photograph of the analysis screen at this time.

The image of moderately differentiated adenocarcinoma visualized by the double fluorescent antibody method is an 8-bit image, and the fluorescence intensity is indicated by 0 to 256 gradations (b (Intensity) in FIG. 3). The number of dots in the image is 512 × 512. The distribution of the fluorescence intensity of β-actin is shown as a1 (CH1 of Absolute Frequency) in FIG. 3, and the distribution of the fluorescence intensity of TS is shown as a2 (CH2 of Absolute Frequency) in FIG. The total fluorescence intensity of β-actin (control protein) was calculated by Σa1xb (0x0 + 1x0 + 2x0 + 3x33 + 4x6853 + ...). Similarly, the total fluorescence intensity of TS was calculated by Σa2xb (0x0 + 1x0 + 2x0 + 3x16416 + 4x17064 + ...). The quantitative value of TS (target protein) is a value obtained by dividing the total fluorescence intensity of TS by the total fluorescence intensity of β-actin.
Thus, the quantitative value was calculated | required about each of TS, DPD, and OPRT.

<Result>
The measurement results are shown in FIGS. 4 to 9, the significant difference was tested by Student's t-test and Kruskal-Wallis' test. 4 to 9, “*” indicates p <0.05, and “**” indicates p <0.01.
FIG. 4 is a graph comparing the average value of TS quantitative values in four groups of Complete Response (CR), Partial Response (PR), No Change (NC), and Progressive Disease (PD). FIG. 5 is a graph comparing the average value of the quantitative values of DPD in four groups of CR, PR, NC, and PD. Furthermore, FIG. 6 is a graph in which the average value of OPRT quantitative values is compared in four groups of CR, PR, NC, and PD. As shown in FIGS. 4 to 6, it can be seen that quantitative values of TS, DPD and OPRT can be obtained by the multiple fluorescence staining method. These quantitative values tend to be similar to the theoretical values. That is, in CR and PR, the activity of TS and DPD was low and the activity of ORPD was high compared to NC and PD.
Therefore, when quantifying TS, DPD, or OPRT contained in a sample using cancer cells collected from a patient before the start of treatment, if (a) the amount of TS is small, (b) the amount of DPD When the amount of OPRT is small or (c) the amount of OPRT is large, it can be determined that the sensitivity to S-1 or the effect of S-1 is high. Such patients can be expected to be classified as CR or PR in the future by administering S-1. On the other hand, when (a) the amount of TS is large, (b) the amount of DPD is large, or (c) the amount of OPRT is small, it is judged that the sensitivity to S-1 or the effect of S-1 is low. it can. Such patients may be classified as NC or PD in the future, even if they are administered S-1, and should not consider S-1 and consider other anticancer drugs. It is thought that.
The reference value that determines whether the amount of TS, DPD, or OPRT contained in the sample is large or small can be set by performing the above experiment and accumulating data.

Further, FIG. 7 is a graph comparing the average values of OPRT / DPD, OPRT / TS, and OPRT / (DPD + TS) in four groups of CR, PR, NC, and PD based on the determined quantitative values. When the sensitivity to S-1 is low, it is expected that (a) OPRT / DPD is small, (b) OPRT / TS is small, or (c) OPRT / (DPD + TS) is small. On the other hand, when sensitivity to S-1 is high, it is expected that (a) OPRT / DPD is large, (b) OPRT / TS is large, or (c) OPRT / (DPD + TS) is large. The Since S-1 contains a DPD inhibitor, OPRT / TS is more strongly correlated with the therapeutic effect than OPRT / DPD, and OPRT / (TS + DPD considering the action mechanism of the three proteins. ) Value is expected to correlate most with the therapeutic effect. Fig. 7 shows that using OPRT / DPD, OPRT / TS, and OPRT / (DPD + TS), the effective case group (Responder) and the ineffective case group (Non-Responder) can be used even if the number of cases is small. It can be seen that a significant difference appears.
Therefore, when cancer cells collected from patients before the start of treatment are used as samples, TS, DPD and OPRT are quantified, and the ratio of OPRT / DPD, OPRT / TS and OPRT / (DPD + TS) is determined ( When a) OPRT / DPD is large, (b) OPRT / TS is large, or (c) OPRT / (DPD + TS) is large, the sensitivity to S-1 or the effect of S-1 is high. I can judge. Such patients can be expected to be classified as CR or PR in the future by administering S-1. On the other hand, when (a) OPRT / DPD is small, (b) OPRT / TS is small, or (c) OPRT / (DPD + TS) is small, the sensitivity to S-1 or the effect of S-1 Can be judged to be low. Such patients may be classified as NC or PD in the future, even if they are administered S-1, and should not consider S-1 and consider other anticancer drugs. It is thought that.
The reference value that determines whether the ratio of OPRT / DPD, OPRT / TS, and OPRT / (DPD + TS) is large or small can be set by performing the above experiment and accumulating data. In this case, the reference values are, for example, an OPRT / DPD ratio of 1, an OPRT / TS ratio of 1, and an OPRT / (DPD + TS) ratio of 0.5.

FIG. 8 is a graph comparing the mean values of TS, DPD, and OPRT in two groups, an effective case group (Responder (CR + PR)) and an ineffective case group (Non-Responder (NC + PD)). is there. Comparing the two groups, it can be seen that in the effective case group, the TS and DPD activities were low and the ORPD activity was high as compared with the ineffective case group.
FIG. 9 is a graph comparing the mean values of OPRT / DPD, OPRT / TS, and OPRT / (DPD + TS) in two groups, an effective case group (CR + PR) and an ineffective case group (NC + PD). As shown in FIG. 9, a strong significant difference was recognized between the effective case group and the ineffective case group. That is, in the effective case group, as expected, it was confirmed that (a) OPRT / DPD was large, (b) OPRT / TS was large, and (c) OPRT / (DPD + TS) was large. In particular, a stronger difference was observed in (c) OPRT / (DPD + TS). Moreover, OPRT / TS is more strongly correlated with the therapeutic effect than OPRT / DPD, and OPRT / (TS + DPD) values taking into account the action mechanism of the three proteins are most correlated with the therapeutic effect. Indicated.

  In addition to the fact that the protein quantification method using the multiple fluorescent antibody method of the present invention is a method capable of accurately and easily quantifying the expressed protein, the quantitative values determined using the method of the present invention It is shown that it is possible to predict the effects of anticancer agents by using. If the effect of an anticancer agent can be predicted, administration of useless anticancer agents can be reduced, and not only can the patient's prognosis be improved, but also a large amount of medical expenses can be reduced. For example, although the response rate of 5-FU anticancer drugs varies depending on reports, it has been reported that gastric cancer alone is about 14.3 to 44.6% (Japan Clinical Special Issue 4, 2001, “Diagnosis and Treatment of Gastric Cancer” p393 -397). Moreover, anticancer drugs are much more expensive than common drugs. By increasing the response rate to, for example, about 70-80% by predicting sensitivity, huge medical costs can be reduced in Japan as a whole.

<Fluorescent tissue separation method>
(1) A biopsy formalin-fixed paraffin-embedded specimen (formalin-fixed paraffin-embedded block) of gastric mucosal tissue collected under an endoscope before the start of treatment of a stomach cancer patient was used as a sample. The target protein OPRT was stained with FITC and β-actin was stained with Cy3 in the same manner as in Example 1 except that cytokeratin was stained with Cy5. Specifically, staining was performed as follows. First, in the same manner as in Example 1, OPRT, which is the target protein, was stained with FITC, and β-actin was stained with Cy3. At this time, sealing after washing with water was not performed. Next, the specimen stained with OPRT and β-actin was subjected to microwave treatment with citrate buffer for 5 minutes. This microwave-treated specimen was reacted with an anti-cytokeratin antibody (histfine anti-keratin / cytokeratin monoclonal antibody manufactured by Nichirei Co., Ltd.) as a primary antibody for 1 hour at room temperature. Thereafter, the reaction was carried out for 30 minutes using a 100-fold diluted Cy5-labeled antibody (anti-mouse: Chemicon). Finally, the specimen was washed and sealed. That is, the above staining is triple fluorescence staining using three fluorescent dyes of Cy5, FITC and Cy3. Here, the present inventors stained normal gastric mucosal gland duct as a pilot, not a cancer tissue.

  FIG. 10 is an image photograph showing a state in which normal gastric mucosal gland ducts in a sample are stained by the fluorescent antibody method. (a) is a stained image of the target protein, and (b) is a stained image of β-actin. (C) is a stained image of cytokeratin, and (d) is an image obtained by combining (a), (b), and (c). As shown in FIG. 10, the target protein appears green, β-actin appears red, and cytokeratin appears blue. Since cytokeratin is a protein that is specifically expressed in epithelial cells, by staining cytokeratin, epithelial cells contained in a biopsy tissue can be identified and labeled as shown in FIG.

  In FIG. 10, normal ductal epithelial cells are identified and labeled. By staining cytokeratin specifically expressed in epithelial cells, cancer cells derived from epithelial cells are visually recognized as inflammatory cells and stromal cells. Can also be separated (distinguishable). Therefore, when it is assumed that the cells stained in blue in the image of FIG. 10 are cancer cells derived from epithelial cells rather than normal glandular epithelial cells, only the cancer cells stained in blue are image analysis software. The target protein can be quantified only for the cancer cells by analyzing the fluorescence intensity in the same manner as in Example 1 for only the extracted cancer cells. According to this method, not only cancer cells can be reliably selected, but also simple and low cost. In addition, since a confocal laser microscope is used in this embodiment, there is an advantage that the overlap of samples can be ignored.

(2) A biopsy formalin-fixed paraffin-embedded specimen (formalin-fixed paraffin-embedded block) of gastric cancer tissue with inflammatory cell wetting collected under an endoscope before the start of treatment of a stomach cancer patient was used as a sample. The target protein OPRT was stained with FITC, β-actin with Cy3, and cytokeratin with Cy5 by the method described in Example 2 (1) above. Here, the present inventors stained cancer tissue.

  FIG. 11 is an image photograph showing a state where moderately differentiated adenocarcinoma tissue in a sample is stained by the triple fluorescent antibody method. Referring to FIG. 11, it can be seen that cells identified and labeled with Cy5 (blue) are cancer cells derived from epithelial cells. In addition, since only cancer cells were stained with Cy5 (blue), the cancer cells could be visually separated from inflammatory cells and stromal cells. Therefore, cancer cells were identified based on blue fluorescence using image analysis software. FIG. 12 is an image photograph after cancer cells are identified and labeled. In FIG. 12, (a) is a stained image of the target protein, and (b) is a stained image of β-actin. (C) is a stained image of cytokeratin, and (d) is an image obtained by combining (a), (b), and (c). As shown in FIG. 12, in the cancer cells, the target protein appears green and β-actin appears red.

  Therefore, using the same software as in Example 1, CH1-T1 is set to FITC (target protein), CH1-T2 is set to Cy3 (β-actin), and Ch1-T3 is set to Cy5. Staining images were acquired. Next, the fluorescence intensity was measured in the same manner as in Example 1. FIG. 13 is a photograph of the analysis screen at this time.

  As is clear from the analysis screen shown in FIG. 13, it can be seen that the fluorescence intensity of the target protein and the control protein can also be calculated from the cancer cell image separated by the microscopic tissue separation method. That is, by staining cytokeratin in cancer cells with Cy5 and identifying and labeling cancer cells in the sample, a quantitative value of the target protein in the identified and labeled cancer cells can be obtained.

It is a figure which shows the metabolic pathway of S-1. It is an image photograph in which TS and β-actin of moderately differentiated adenocarcinoma are stained by the double fluorescent antibody method. It is an image photograph which shows the mode of data analysis. It is the graph which compared the quantitative value of TS in 4 groups of Complete Response (CR: remarkable effect), Partial Response (PR: effective), No Change (NC: unchanged), and Progressive Disease (PD: progression). It is the graph which compared the average value of the quantitative value of DPD in 4 groups, CR, PR, NC, and PD. It is the graph which compared the average value of the quantitative value of OPRT in 4 groups, CR, PR, NC, and PD. It is the graph which compared the average value of OPRT / DPD, OPRT / TS, and OPRT / (DPD + TS) in 4 groups of CR, PR, NC, and PD. It is the graph which compared the average value of the quantitative value of TS, DPD, and OPRT in two groups, an effective case group (Responder (CR + PR)) and an invalid case group (Non-Responder (NC + PD)). A graph comparing the mean values of OPRT / DPD, OPRT / TS, and OPRT / (DPD + TS) in two groups, the effective case group (Responder (CR + PR)) and the ineffective case group (Non-Responder (NC + PD)) is there. It is the image photograph which dye | stained OPRT of the ductal epithelial cell of normal gastric mucosa, (beta) -actin, and cytokeratin by the fluorescent antibody method. It is the image photograph which dye | stained OPRT, (beta) -actin, and cytokeratin of the gastric adenocarcinoma cell by the fluorescent antibody method. It is an image photograph which shows a mode that only the cancer cell was isolate | separated from the image of FIG. 11 by the fluorescence tissue separation method. It is an image photograph which shows the mode of the data analysis using the image of FIG.

Claims (16)

The target protein for quantification in the sample and the control protein are stained with antibodies labeled with different fluorescent dyes,
Measure the total fluorescence intensity of each of the stained target protein and control protein,
A method for quantifying a protein, comprising setting a ratio between the measured total fluorescence intensity of a target protein and the total fluorescence intensity of a control protein as a quantitative value of the target protein.   The method according to claim 1, wherein the control protein is a protein encoded by a housekeeping gene.   3. The method according to claim 2, wherein the protein encoded by the housekeeping gene is β-actin.   The method according to any one of claims 1 to 3, wherein the sample used for protein quantification is a formalin-fixed paraffin-embedded tissue.   The method according to any one of claims 1 to 4, wherein the target protein is at least one selected from the group consisting of thymidylate synthase, dihydropyrimidine dehydrogenase, and orotate phosphoribosyltransferase.   Different fluorescent dyes are fluorescein isothiocyanate, carboxymethyl indocyanine, tetramethylrhodamine isothiocyanate, phycoerythrin, green fluorescent protein, Alexa® 488, 4 ', 6-diamidine 2'-phenylindole di It is at least 2 chosen from the group which consists of hydrochloride, Texas Red, Cy2, Alexa (registered trademark) 543, Alexa (registered trademark) 548, Cy5, and ToTo3, It is any one of Claims 1-5 Method.   The method according to any one of claims 1 to 6, wherein the quantitative value of the target protein is a quantitative value of the target protein of the target cell in the sample.   The method according to claim 7, comprising the step of discriminating and labeling the target cells in the sample with a fluorescent dye different from the fluorescent dye for labeling the target protein and the control protein.   The method according to claim 7 or 8, wherein the target cell is a cancer cell.   Identification markers for cancer cells include cytokeratin, epithelial cell membrane specific antigen, carcinoembryonic antigen, hepatocyte, α-fetoprotein, estrogen receptor, progesterone receptor, human epidermal growth factor receptor 2, thyroid transcription factor-1, prostate specific antigen The method according to claim 9, wherein the method is performed by staining at least one protein selected from the group consisting of CD10, thyroglobulin, P63, p53, Ki-67 and Cyclin-D1 with a fluorescent dye.   Fluorescent dyes for identifying and labeling target cells are fluorescein isothiocyanate, carboxymethyl indocyanine, tetramethylrhodamine isothiocyanate, phycoerythrin, green fluorescent protein, Alexa (registered trademark) 488, 4 ', 6-diamidine 2' The phenylindole dihydrochloride, Texas Red, Cy2, Alexa (registered trademark) 543, Alexa (registered trademark) 548, Cy5, and ToTo3 are at least one selected from the group consisting of any one of claims 8 to 10. The method according to item.   The anticancer agent sensitivity prediction method which estimates anticancer agent sensitivity using the quantitative value obtained by the method of any one of Claims 1-11 as a parameter | index.   The anticancer agent effect prediction method which estimates the effect of an anticancer agent using the quantitative value of the protein obtained by the method of any one of Claims 1-11 as a parameter | index.   From the quantitative value of thymidylate synthase, quantitative value of dihydropyrimidine dehydrogenase and quantitative value of orotate phosphoribosyltransferase, "quantitative value of orotate phosphoribosyltransferase / quantitative value of dihydropyrimidine dehydrogenase", "quantitative value of orotate phosphoribosyltransferase" Value / quantitative value of thymidylate synthase ”and“ quantitative value of orotate phosphoribosyltransferase / (quantitative value of dihydropyrimidine dehydrogenase + quantitative value of thymidylate synthase) ”. The method according to claim 12 or 13, wherein the sensitivity of the anticancer agent or the effect of the anticancer agent is predicted using the ratio of the determined quantitative values as an index.   A primary antibody that binds to the target protein, a primary antibody that binds to the control protein, a secondary antibody that binds to the complex of the target protein and the primary antibody, and a secondary antibody that binds to the complex of the control protein and the primary antibody And a fluorescent dye for distinguishing and labeling each secondary antibody.   Furthermore, a primary antibody that binds to a predetermined protein of the target cell, a secondary antibody that binds to a complex of the predetermined protein of the target cell and the primary antibody, and the secondary antibody is labeled separately from other secondary antibodies. And a fluorescent dye for discriminating and labeling a target cell.

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