Quantification and labeling methods

Abstract

A quantification method according to the present invention is for quantifying three or more quantification-target substances contained in a plurality of samples collected from a single specimen, and comprises a quantification step for quantifying the three or more quantification-target substances, in each of the plurality of samples, on the basis of a detection value derived from a first labeling substance for labeling a reference substance contained in all of the plurality of samples. In the present invention, three or more observation-target substances can be observed or quantified without the need of multiple labeling substances, even when staining conditions are different from each other. Thus, the observation-target substances (a reference substance and quantification-target substances other than the reference substance) can each be observed or quantified using a smaller number of labeling substances than the number of observation-target substances.

Description

[Technical field]

[0001] The present invention relates to a quantification method for quantifying a plurality of types of observation target substances, and a labeling method for labeling observation target substances. [Background technology]

[0002] From among candidate compounds that can become medicines, only those compounds that are evaluated as effective are selected through screening. Medicines often bind to target substances in the body, such as receptors, enzymes, ion channels, and transporters. After reaching cells and binding to the target substance, the medicine exerts its medicinal effects by acting on the cells. Therefore, determining whether the medicine has reached the target substance or whether the drug that has reached it exerts its medicinal effects is an important criterion in screening. The above-mentioned determination is made by utilizing immunostaining methods using tissue sections (see, for example, Patent Documents 1 and 2).

[0003] Patent Document 1 describes a method for predicting pathological complete response by immunostaining tissue sections taken from cancer patients. The method described in Patent Document 1 predicts pathological complete response by labeling and detecting a cancer marker contained in a tissue section taken from a cancer patient. In addition, a method using fluorescent nanoparticles is also described as a labeling method. Furthermore, as another labeling method, it is also described that two types of cancer markers are stained on one tissue section using two types of dyes with different emission wavelengths.

[0004] Furthermore, Patent Document 2 describes a method for identifying cancer patients by identifying chromosomal abnormalities using FISH (Fluorescence In Situ Hybridization). In the method described in Patent Document 2, the ratios of multiple probes to markers are calculated, and the values ​​of the probes in tissue sections are compared to identify cancer patients. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] JP 2018-084568 A [Patent Document 2] JP 2013-046620 A Summary of the Invention [Problem to be solved by the invention]

[0006] Here, in the methods described in Patent Documents 1 and 2, in order to observe a plurality of types of observation substances in one tissue section, it is considered to increase the number of types of labeling substances including fluorescent dyes. However, when using a plurality of types of labeling substances (fluorescent dyes), the excitation wavelength and emission wavelength of each fluorescent dye must be separate from the excitation wavelength and emission wavelength of the other fluorescent dyes. In addition, when observing with a fluorescent microscope, it is also necessary to select a filter corresponding to each fluorescent dye. For these reasons, it is difficult to observe an observation substance using three or more types of fluorescent dyes, and it is not realistic to observe an observation substance using four or more types of fluorescent dyes in the first place. On the other hand, if different types of observation substances are stained in each of the tissue sections, the problem of the number of types of labeling substances (fluorescent dyes) is eliminated, but it is difficult to completely equalize the staining conditions and observation conditions between the tissue sections, and it is not easy to accurately compare the fluorescence intensity, etc.

[0007] Depending on the expression state of the observation substance, it may be preferable to change the staining conditions for each observation substance. When observing multiple types of observation substances in one tissue section, multiple types of staining conditions cannot be adopted, so some observation substances (e.g., observation substances with lower expression levels) cannot be stained appropriately. On the other hand, when staining multiple tissue sections, it is possible to change the staining conditions for each tissue section, but doing so still makes it impossible to accurately compare the fluorescence intensity between tissue sections.

[0008] The object of the present invention is to provide a quantification method and a labeling method that do not require three or more types of labeling substances (fluorescent dyes) and can more accurately measure three or more types of quantified substances even if the staining conditions are different from each other. [Means for solving the problem]

[0009] A quantitative method according to one embodiment of the present invention is a quantitative method for quantifying three or more types of target substances contained in multiple samples collected from one specimen, and includes a quantitative step of quantifying the three or more types of target substances in each of the multiple samples based on detection values ​​derived from a first labeling substance that labels a reference substance that is commonly contained in the multiple samples.

[0010] A labeling method according to one embodiment of the present invention is a method for labeling a plurality of types of observed substances including a reference substance contained in each of a plurality of observation tissues collected from a single specimen and three or more types of quantified substances contained in at least one of the plurality of observation tissues, the method comprising the steps of: preparing the plurality of observation tissues; and labeling each of the plurality of observed substances contained in the plurality of observation tissues with a different type of labeling substance. Effect of the Invention

[0011] According to the present invention, it is possible to provide a quantification method and a labeling method that do not require four or more types of labeling substances (fluorescent dyes) and that can measure the relative amounts of three or more types of quantification substances even if the staining conditions are different from each other. [Brief description of the drawings]

[0012] [Figure 1] FIG. 1 is a flowchart of the quantification method according to the first embodiment of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] The quantification method and labeling method according to one embodiment of the present invention will be described below.

[0014] [Embodiment 1] FIG. 1 is a flowchart of the quantification method according to the first embodiment of the present invention.

[0015] The quantification method according to the first embodiment of the present invention is a quantification method for measuring the relative amounts of a plurality of types of observation substances, including a reference substance contained in each of a plurality of observation tissues (samples) collected from one specimen, and three or more types of quantified substances contained in at least one of the plurality of observation tissues. As shown in Fig. 1, the quantification method according to the present embodiment may include a step of preparing a plurality of observation tissues collected from one specimen, a step of labeling the plurality of types of observation substances contained in the plurality of observation tissues with different types of labeling substances, a step of determining detection values ​​derived from the labeled plurality of types of observation substances, and a step of calculating the ratio of the detection values ​​of the quantified substances to the detection values ​​of the reference substance in the plurality of observation tissues.

[0016] In the step of preparing the tissue for observation, the tissue for observation to be used for quantification is prepared (S110). In this embodiment, in order to compare the amount of the observation substance between multiple tissues for observation, the tissue for observation is collected from one specimen. Here, the specimen may be cell tissue collected from the same individual (e.g., human or animal) or cultured cells. The type of the tissue for observation is not particularly limited as long as the observation substance can be detected. Examples of the tissue for observation include pathological tissue, tissue slices such as CDX (cell lone-derived xenograft) and PDX (patient-derived xenograft), and slide specimens from cultured cells. The number of tissues for observation to be prepared is set according to the type of the substance to be quantified. By preparing multiple tissues for observation, the quantitative relationship between multiple observation substances and the localization of the observation substances can be observed. In particular, adjacent slices (adjacent slices) and cell tissues of the same passage are particularly preferable as multiple tissues for observation, since they more accurately reflect the quantitative relationship and localization of the observation substances in the specimen.

[0017] The observed substance includes a reference substance and a substance to be quantified. The reference substance is a substance that is commonly contained in each of the multiple tissues for observation and can be stably observed. For example, it is preferable that the reference substance is contained in approximately the same amount in each of the multiple tissues for observation, or that the amount contained in each of the multiple tissues for observation can be known in advance. The reference substance functions as a reference marker. The reference substance may be the substance to be quantified, or may be a substance different from the substance to be quantified. Furthermore, the type of reference substance may be one type, or two or more types. In this embodiment, the effect is achieved as long as there are three or more types of observed substances.

[0018] A quantified substance is a substance to be quantified. A quantified substance can be quantified relatively by comparing it with a reference substance. In addition, for example, as described below, if a table or calibration curve for converting a detection value corresponding to each quantified substance into its absolute amount is prepared, the quantified substance can be absolutely quantified.

[0019] The type of the reference substance and the substance to be quantified is not particularly limited.Examples of the reference substance and the substance to be quantified include nucleic acid (DNA, RNA, polynucleotide, oligonucleotide, PNA (peptide nucleic acid), etc., which may be single-stranded or double-stranded, or nucleoside, nucleotide and their modified molecules); protein (polypeptide, oligopeptide, receptors present in the cell membrane of target cells, etc.); amino acid (including modified amino acid); carbohydrate (oligosaccharide, polysaccharide, sugar chain, etc.); lipid; exosome; or their modified molecules and complexes. Further, more specific examples of the reference substance and the substance to be quantified include 5T4, AXL, BCMA, C4.4A, CA6, Cadherin3, Cadherin6, CEACAM5, CD16, CD19, CD22, CD37, CD56, CD71, CD138, CD142, CD352, DLL3, EphA2, EphrinA4, ETBR, FcγRIII, FOLR1, FGFR2, FGFR3, GCC, HER1 (EGFR), HER2, HER3, HER4, IntegrinαV, LAMP1, LIV1, Mesothelin, MUC1, MUC16, NaPi2B, Nectin4, NOTCH3, PD-1, PD-L1, PSMA, PTK7, SLAMF7, SLITRK6, STEAP1, TROP2, Ki67, HER4, ER, and PR.

[0020] In the labeling step, the reference substance and the substance to be quantified are labeled (S120). The method of labeling the reference substance and the substance to be quantified is not particularly limited. Examples of the method of labeling the reference substance and the substance to be quantified include an immunostaining method using an antibody or an antibody fragment, and a staining method using a molecular recognition group similar to an antibody.

[0021] In the immunostaining labeling method, a tissue for observation (specimen) containing a reference substance and a substance to be quantified is immunostained to obtain an immunostained image in which the reference substance and the substance to be quantified are visualized by fluorescent labeling.

[0022] In addition, in the secondary reaction of the immunostaining method, a labeling substance containing fluorescent substance-accumulating particles (fluorescent dye-accumulating particles) can be used. Fluorescent substance-accumulating particles (fluorescent dye-accumulating particles) as a labeling substance are particularly preferable because they have high brightness per particle and allow the observed substance to be observed or quantified more accurately. For example, the reference substance is labeled with a first labeling substance containing fluorescent dye-accumulating particles, and the substance to be quantified is labeled with a second labeling substance containing fluorescent dye-accumulating particles that have an emission wavelength (color) different from that of the first labeling substance and that have different emission wavelengths from each other. Also, for example, the reference substance is labeled with two types of first labeling substances containing fluorescent dye-accumulating particles with two different emission wavelengths, and the substance to be quantified is labeled with two types of second labeling substances containing fluorescent dye-accumulating particles that have a color different from that of the first labeling substance and that have different emission wavelengths from each other. If the fluorescent dye-accumulating particles contained in the first labeling substance and the fluorescent dye-accumulating particles contained in the second labeling substance have the same emission wavelength, the two cannot be distinguished from each other. For example, the reference substance can be labeled with one type of first labeling substance, and three or more types of the substance to be quantified can be labeled with the other two types of second labeling substances. The number of types of labeling substances, which is a combination of the first labeling substance and the plurality of second labeling substances, is preferably smaller than the number of types of observation substances, which is a combination of the reference substance and the substance to be quantified. This allows all of the observation substances to be observed or quantified with fewer types of labeling substances than the types of the observation substances.

[0023] The phosphor-aggregated particles are nano-sized particles having a structure in which a plurality of phosphors (e.g., fluorescent dyes and semiconductor nanoparticles) are encapsulated therein and / or adsorbed to the surface of a particle made of an organic or inorganic substance. Examples of the fluorescent dyes constituting the phosphor-aggregated nanoparticles include rhodamine-based dyes, Cy-based dyes, Alexa Fluor (registered trademark)-based dyes, BODIPY-based dyes, squarylium-based dyes, cyanine-based dyes, aromatic ring-based dyes, oxazine-based dyes, carbopyronine-based dyes, and pyrromethene-based dyes. Examples of the materials of the semiconductor nanoparticles constituting the phosphor-aggregated nanoparticles include II-VI group semiconductors, III-V group semiconductors, and IV group semiconductors. The phosphor-aggregated particles can be prepared according to a known method (see, for example, JP 2013-57937 A).

[0024] In the method of labeling a reference substance and a substance to be quantified by a staining method using a molecular recognition group similar to an antibody, for example, an aptamer or a SNAP-tag is used as the molecular recognition group.

[0025] In the step of obtaining the detection value, the detection value derived from the labeled multiple types of observation target substances is obtained (S130). The method of obtaining the detection value is appropriately selected based on the labeling substance that labeled the observation target substance. For example, the observation tissue labeled with the reference substance and the quantified substance is irradiated with excitation light to obtain a fluorescent image, and then the image is processed by a predetermined image processing software. In this way, for example, when phosphor-accumulating particles (fluorescent dye-accumulating particles) are used in the secondary reaction of the immunostaining method, a value (detection value) obtained by summing up the luminance values ​​derived from the reference substance and a value (detection value) obtained by summing up the luminance values ​​derived from the quantified substance can be obtained. The luminance value may be a total value of the number of particles whose luminance is equal to or greater than a predetermined value. The obtained luminance value may be divided by the luminance per phosphor-accumulating particle (fluorescent dye-accumulating particle) to convert it into the number of particles. The detection value may be the number of bright spots reflecting the emission of the phosphor-accumulating particles (fluorescent dye-accumulating particles) that labeled the observation target substance.

[0026] In the step of calculating the ratio of the detection value of the quantified substance, the multiple types of observed substances are quantified based on the detection value (S140). Specifically, the multiple types of quantified substances are quantified based on the reference substance. For example, the ratio of the quantified substance to the reference substance can be obtained by dividing the brightness value derived from the quantified substance by the brightness value derived from the reference substance. Next, for example, the amount of the quantified substance can be quantified based on a calibration curve obtained in advance. As will be shown in the following examples, when multiple reference substances are used, it is confirmed whether the reference substances are functioning properly. Specifically, the expression levels of multiple reference substances are compared between multiple observation tissues to confirm whether the reference substances are functioning properly. For example, the fluorescent dye-aggregated nanoparticle scores based on the brightness values ​​of two types of reference substances are compared between observation tissues to determine whether they are suitable as reference substances.

[0027] (effect) As described above, in the present invention, a reference substance contained in a plurality of tissues for observation is labeled with a first labeling substance, and a plurality of substances to be quantified (other than the reference substance) contained in a plurality of tissues for observation is labeled with a second labeling substance, so that three or more types of substances to be observed can be observed or quantified without the need for many types of labeling substances, even if the staining conditions are different from one another. In other words, according to the present invention, each substance to be observed can be observed or quantified with fewer types of labeling substances than the number of substances to be observed (the reference substance and the substances to be quantified other than the reference substance). Specifically, the relationship between the types of substances to be observed (N) and the minimum number of types of labeling substances required (n) can be expressed by the following formula. N=nm-X(m-1) m: Number of tissues (samples) to be observed X: Type of reference material Here, as long as the labeling substances have different main emission wavelengths (colors), they can be regarded as different types of substances, as long as they do not interfere with the detection of the detection value. In particular, when there are four or more observation substances, it is very difficult to label each of them with labeling substances having different main emission wavelengths (colors). However, according to the present invention, more observation substances can be observed or quantified with a smaller number of labeling substances.

[0028] [Embodiment 2] Next, a labeling method according to a second embodiment of the present invention will be described.

[0029] A labeling method according to embodiment 2 of the present invention includes the steps of preparing a plurality of observation tissues collected from one specimen, each containing a plurality of types of observation substances, and labeling the plurality of types of observation substances contained in each of the collected observation tissues with a plurality of labeling substances, wherein the observation substances include a reference substance contained in each of the observation tissues, and three or more types of quantified substances contained in at least one of the observation tissues.

[0030] The step of preparing a tissue for observation can be performed in the same manner as the "step of preparing a tissue for observation" in the first embodiment.

[0031] The labeling step can be carried out in the same manner as the "labeling step" in the first embodiment.

[0032] (effect) As described above, in the labeling method according to the present embodiment, a fluorescently labeled observation tissue that can be used in the quantification method according to the first embodiment can be prepared. EXAMPLES

[0033] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0034] 1. Preparation of fluorescent dye-integrated nanoparticles (first and second labeled substances) 14.4 mg of red fluorescent dye Sulforhodamine 101 (Texas Red) was added to 22 mL of water and dissolved. Next, 2 mL of a 5% aqueous solution of polyoxyethylene oleyl ether (Emulgen (registered trademark) 430; Kao Corporation), an emulsifier for emulsion polymerization, was added to this solution. Next, the solution was heated to 70°C while stirring with a hot stirrer, and 0.65 g of a melamine resin raw material (Nikarak (registered trademark) MX-035; Nippon Carbide Industries Co., Ltd.) was added. Next, 1000 μL of a 10% aqueous solution of dodecylbenzenesulfonic acid (Kanto Chemical Co., Ltd.) was added to this solution as a reaction initiator, and the solution was heated and stirred at 70°C for 50 minutes. After that, the solution was heated to 90°C and heated and stirred for 20 minutes to obtain a dispersion of red fluorescent dye-aggregated nanoparticles.

[0035] The obtained nanoparticle dispersion was washed with pure water to remove impurities such as excess melamine resin raw material and red fluorescent dye. Specifically, it was centrifuged at 20,000G for 15 minutes in a centrifuge (Micro refrigerated centrifuge Model 3740; Kubota Shoji Co., Ltd.), the supernatant was removed, and then ultrapure water was added and ultrasonically irradiated to redisperse. The washing by centrifugation, removal of the supernatant, and redispersion in ultrapure water was repeated five times. Through the above process, red fluorescent dye-aggregated nanoparticles (excitation wavelength 590 nm, emission wavelength 620 nm) were produced.

[0036] In the preparation of red fluorescent dye-aggregated nanoparticles, Pyrromethene 556 dye was used instead of Sulforhodamine 101 (Texas Red) dye to prepare green fluorescent dye-aggregated nanoparticles (excitation wavelength 490 nm, emission wavelength 520 nm).

[0037] In the preparation of red fluorescent dye-integrated nanoparticles, Cy5 dye was used instead of Sulforhodamine 101 (Texas Red) dye to prepare near-infrared fluorescent dye-integrated nanoparticles (excitation wavelength 643 nm, emission wavelength 647 nm).

[0038] 2. Modification of fluorescent dye-containing nanoparticles After introducing maleimide to the ends of the red fluorescent dye-holding nanoparticles using an NHS-PEG (N-hydroxysuccinimide-polyethylene glycol)-maleimide reagent, a thiolated antibody was bound to the nanoparticles. This was designated "immunostaining agent A (first labeling substance)."

[0039] After introducing maleimide to the terminus of the green fluorescent dye-holding nanoparticles, a thiolated antibody was bound to it. This was designated "immunostaining agent B (second labeling substance)."

[0040] After introducing maleimide to the end of the near-infrared fluorescent dye-holding nanoparticles, a thiolated antibody was bound to it. This was named "immunostaining agent C (second labeling substance)."

[0041] [Example 1] In this example, three sections were processed using the same protocol, and seven observed substances (HER2, EGFR, HER3, Ki67, HER4, ER, PR), including one reference substance (HER2) and six quantified substances (EGFR, HER3, Ki67, HER4, ER, PR), were quantified.

[0042] Tissue was collected from a breast cancer patient, and a formalin-fixed paraffin-embedded tissue block was prepared according to a standard method. Sections 1 to 3 were then prepared by serially cutting the tissue using a microtome.

[0043] (Specimen pre-treatment process) Each section was deparaffinized and then washed with water. The washed sections were boiled in 0.1 M citrate buffer (pH 6.0) at 95°C for 40 minutes to activate the antigens. After the activation process, each section was washed and blocked for 15 minutes with PBS containing 1% BSA.

[0044] In section 1, HER2, EGFR, and HER3 were observed.

[0045] In section 2, HER2, Ki67, and HER4 were observed.

[0046] In section 3, HER2, ER, and PR were observed.

[0047] As mentioned above, HER2 is the reference substance. EGFR, HER3, Ki67, HER4, ER and PR are the substances to be quantified.

[0048] Table 1 shows the combinations of each section, immunostaining agent, antibody, and fluorescent dye-integrated nanoparticles.

[0049] [Table 1]

[0050] The methods for staining fluorescent dye-accumulating nanoparticles and obtaining fluorescent dye-accumulating nanoparticle scores were as follows.

[0051] (Immunostaining process) A reaction treatment solution was prepared using PBS containing 1% (w / w) BSA, containing 0.1 nM of each of immunostaining agents A, B, and C. Each section that had undergone the specimen pretreatment process was immersed in this reaction treatment solution and allowed to react at room temperature for 2 hours.

[0052] (Fixation process) After each immunostained section was washed with PBS, each section was fixed with 4% neutral paraformaldehyde solution for 10 minutes.

[0053] (Staining process for morphological observation) After the fixation step, each section was stained with Mayer's hematoxylin solution for 1 minute to carry out hematoxylin staining, and then washed with running water for 5 minutes.

[0054] (Specimen post-processing process) Each section (stained section) after the staining process for morphological observation was dehydrated by immersing in pure ethanol for 5 minutes four times. Then, the section was immersed in xylene for 5 minutes three times. Finally, each stained section was placed in a mounting medium (Marinol; Muto Chemicals Co., Ltd.) and covered with a cover glass to prepare the stained section for observation.

[0055] (Fluorescence microscope observation and photography process) A fluorescence microscope (BX-63; Olympus Corporation) and a microscope digital camera (DP80; Olympus Corporation) attached to the fluorescence microscope were used to obtain immunostained images (400x) of each stained section after the specimen post-processing process. The excitation light and fluorescence wavelengths for observing and photographing the fluorescent dye-aggregated nanoparticles of each color were as shown in Table 2, and were set using the excitation light optical filter and fluorescence optical filter equipped in the fluorescence microscope. The exposure time during image capture was adjusted within a range such that the brightness of the image was not saturated, and was set to, for example, 30 ms.

[0056] Table 2 shows the excitation light wavelength and fluorescence wavelength for each immunostaining agent.

[0057] [Table 2]

[0058] Photographs were taken of hematoxylin-stained images for observing cell morphology in the bright field of a fluorescence microscope, and of immunostained images of each color in the same field of view, with five fields per stained section.

[0059] (Image processing and measurement process) For image processing, the image processing software "ImageJ" (open source) was used. The shape of the cells (position of the cell nucleus) was identified by image processing using the stained image for morphological observation, and the image was overlaid with the immunostained image to extract bright spots representing the fluorescent dye-aggregated nanoparticles of each color that labeled each substance to be observed. Next, bright spots with a brightness equal to or greater than a predetermined value were extracted, and the total brightness of these bright spots was divided by the brightness of one fluorescent nanoparticle to calculate the number of particles per field of view. The number of bright spots of each substance to be observed was then measured in five fields of view per stained section, and the number of bright spots per unit area (100 μm 2 ) and the average was calculated to be the "fluorescent dye-accumulating nanoparticle score" for that section.

[0060] The calculated fluorescent dye-integrated nanoparticle scores are shown in Table 3.

[0061] [Table 3]

[0062] Table 4 shows the ratio of the fluorescent dye-integrated nanoparticle score of each marker to the fluorescent dye-integrated nanoparticle score of HER2 stained with immunostaining agent A.

[0063] [Table 4]

[0064] As shown in Table 4, the expression ratio was HER2:EGFR:HER3:Ki67:HER4:ER:PR=1:0.83:0.62:0.17:0.39:0.09:0.23. In this way, the relative amounts of seven types of substances could be measured using three color particles (fluorescent dyes).

[0065] Finally, the amount of the observed substance was determined based on a previously prepared calibration curve.

[0066] [Example 2] In this example, three sections were processed using different protocols to quantify four observed substances (HER2, EGFR, Ki67, ER), including one reference substance (HER2) and three quantified substances (EGFR, Ki67, ER).

[0067] Tissue was collected from a breast cancer patient, and a formalin-fixed paraffin-embedded tissue block was prepared according to a standard method. Sections 1 to 3 were then prepared by serially cutting the tissue using a microtome.

[0068] Section 1 was activated by boiling in 0.1 M citrate buffer (pH 6) at 95° C. for 40 minutes. In section 1, HER2 and EGFR were used as the substances to be observed.

[0069] Section 2 was activated by autoclaving with 0.1 M citrate buffer (pH 6) at 121° C. for 5 minutes. In section 2, HER2 and Ki67 were used as the substances to be observed.

[0070] Section 3 was activated by autoclaving in 1 M Tris-HCl buffer (pH 9) at 121° C. for 5 minutes. In section 3, HER2 and ER were used as the observed substances.

[0071] Table 5 shows the combinations of each section, immunostaining agent, antibody, and fluorescent dye-integrated nanoparticles.

[0072] [Table 5]

[0073] The methods for staining the fluorescent dye-holding nanoparticles and obtaining the fluorescent dye-holding nanoparticle scores were the same as in Example 1.

[0074] The calculated fluorescent dye-integrated nanoparticle scores are shown in Table 6.

[0075] [Table 6]

[0076] Table 7 shows the ratio of the fluorescent dye-integrated nanoparticle score of each marker to the fluorescent dye-integrated nanoparticle score of HER2 stained with immunostaining agent A.

[0077] [Table 7]

[0078] As shown in Table 7, the expression ratio was HER2:EGFR:Ki67:ER = 1:0.84:0.19:0.09. Thus, even when different activation treatments were performed for each section, the relative amounts of the four types of substances could be measured using two-color particles (fluorescent dyes).

[0079] Finally, the amount of the observed substance was determined based on a previously prepared calibration curve.

[0080] [Example 3-1] In this example, three sections were processed using different protocols to quantify five observed substances (HER2, EGFR, HER3, HER4, PR), including two reference substances (HER2, EGFR) and three quantified substances (HER3, HER4, PR).

[0081] Tissues were collected from breast cancer patients, and formalin-fixed paraffin-embedded tissue blocks were prepared according to standard methods. These were then serially sliced ​​using a microtome to prepare sections 1 to 3. Each section was deparaffinized and then washed with water.

[0082] Section 1 was activated by boiling in 0.1 M citrate buffer (pH 6) at 95° C. for 40 minutes. In section 1, HER2, EGFR, and HER3 were used as the observed substances.

[0083] Section 2 was activated by autoclaving with 0.1 M citrate buffer (pH 6) at 121° C. for 5 minutes. In section 2, HER2, EGFR, and HER4 were used as the observed substances.

[0084] Section 3 was activated by autoclaving in 1 M Tris-HCl buffer (pH 9) at 121° C. for 5 minutes. In section 3, HER2, EGFR, and PR were used as the substances to be observed.

[0085] Table 8 shows the combinations of each section, immunostaining agent, antibody, and fluorescent dye-integrated nanoparticles.

[0086] [Table 8]

[0087] The methods for staining the fluorescent dye-holding nanoparticles and obtaining the fluorescent dye-holding nanoparticle scores were the same as in Example 1.

[0088] The calculated fluorescent dye-integrated nanoparticle scores are shown in Table 9.

[0089] [Table 9]

[0090] Table 10 shows the ratio of each marker to HER2 stained with immunostaining agent A (upper row) and the ratio of each marker to EGFR stained with immunostaining agent B (lower row).

[0091] [Table 10]

[0092] As shown in Table 10, no significant difference was observed in the ratios of HER3, HER4, and PR when HER2 stained with immunostain A was used as the reference substance, or when EGFR stained with immunostain B was used as the reference substance. From this, the ratios of HER3, HER4, and PR can be appropriately calculated when HER2 or EGFR is used as the reference substance. In this way, even when different activation treatments were performed for each section, the relative amounts of five types of substances could be measured using three color particles (fluorescent dyes).

[0093] Finally, the amount of the observed substance was determined based on a previously prepared calibration curve.

[0094] [Example 3-2] In this example, three sections were processed with different protocols to compare five observed substances (HER2, EGFR, HER3, HER4, PR), including two reference substances (HER2, EGFR) and three quantified substances (HER3, HER4, PR).

[0095] Tissues were collected from breast cancer patients, and formalin-fixed paraffin-embedded tissue blocks were prepared according to standard methods. These were then serially sliced ​​using a microtome to prepare sections 1 to 3. Each section was deparaffinized and then washed with water.

[0096] Section 1 was activated by boiling in 0.1 M citrate buffer (pH 6) at 95° C. for 40 minutes. In section 1, HER2, EGFR, and HER3 were used as the substances to be observed.

[0097] Section 2 was activated by autoclaving in 1 M Tris-HCl buffer (pH 9) at 121° C. for 5 minutes. In section 2, HER2, EGFR, and HER4 were used as the observed substances.

[0098] Section 3 was activated by autoclaving in 1 M Tris-HCl buffer (pH 9) at 121° C. for 15 minutes. In section 3, HER2, EGFR, and PR were used as the substances to be observed.

[0099] Table 11 shows the combinations of each section, immunostaining agent, antibody, and fluorescent dye-integrated nanoparticles.

[0100] [Table 11]

[0101] The methods for staining the fluorescent dye-holding nanoparticles and obtaining the fluorescent dye-holding nanoparticle scores were the same as in Example 1.

[0102] The calculated fluorescent dye-integrated nanoparticle scores are shown in Table 12.

[0103] [Table 12]

[0104] Table 13 shows the ratio of the fluorescent dye-aggregated nanoparticle score of each marker to the fluorescent dye-aggregated nanoparticle score of HER2 stained with immunostain A (top row), and the ratio of the fluorescent dye-aggregated nanoparticle score of each marker to the fluorescent dye-aggregated nanoparticle score of EGFR stained with immunostain B (bottom row).

[0105] [Table 13]

[0106] As described above, when HER2 and EGFR are used as the reference substances, the fluorescent dye-aggregated nanoparticle scores of HER2 and EGFR are correlated (see Example 3-1). However, in this Example, as shown in Table 13, when the reference marker is HER2, the expression level of EGFR relative to HER2 was 0.86 in section 1, 0.85 in section 2, and 1.02 in section 3. On the other hand, when the reference marker is EGFR, the expression level of HER2 relative to EGFR was 1.2 in section 1, 1.2 in section 2, and 0.98 in section 3. That is, in sections 1 and 2, the fluorescent dye-aggregated nanoparticle scores of HER2 and the fluorescent dye-aggregated nanoparticle scores of EGFR are suitable as reference substances, but in section 3, the fluorescent dye-aggregated nanoparticle scores of HER2 and the fluorescent dye-aggregated nanoparticle scores of EGFR are not suitable as reference substances.

[0107] In segment 3, the HER2 fluorescent dye-aggregated nanoparticle score and the EGFR fluorescent dye-aggregated nanoparticle score are almost the same value, so the EGFR fluorescent dye-aggregated nanoparticle score is considered to have reached the upper limit. Therefore, in cases like this where the reference material is inappropriate, the value of that reference material is not used.

[0108] That is, in this embodiment, it is found that the expression ratio is HER2:EGFR:HER3:HER4:PR=1.2:1:0.69:0.49:0.26. In this way, by using two or more types of reference substances, inappropriate values ​​can be eliminated, so that the substance to be quantified can be quantified more accurately. Furthermore, even when different activation treatments were performed for each section, the relative amounts of five types of substances could be measured using three color particles (fluorescent dyes).

[0109] Finally, the amount of the substance to be quantified was determined based on the calibration curve prepared in advance. This makes it possible to determine the absolute amount of the substance to be quantified. In the above examples, observation and quantification were performed using one or more types of the substance to be quantified as a reference substance, but the same effect can be obtained by using a substance different from the substance to be quantified as a reference substance, and each substance to be quantified can be observed or quantified with fewer types of labeling substances than the substances to be observed (reference substances and substances to be quantified).

[0110] This application claims priority from Japanese Patent Application No. 2021-100808, filed on June 17, 2021. The contents of the specification and drawings of that application are incorporated herein by reference in their entirety. [Industrial Applicability]

[0111] The present invention is useful, for example, for screening medicines, proving the mechanism of action of medicines, and evaluating the toxicity of medicines.

Claims

1. A method for quantifying three or more types of target substances contained in a plurality of samples collected from one specimen, comprising the steps of: A quantification step of quantifying the three or more target substances in each of the plurality of samples based on a detection value derived from a first labeling substance that labels a reference substance commonly contained in the plurality of samples. Quantification method.

2. The quantification method according to claim 1 , wherein the reference substance is one or more of the three or more types of target substances.

3. The method according to claim 1 , wherein in the quantifying step, the three or more types of target substances are quantified based on detection values ​​derived from a plurality of second labeling substances that respectively label target substances other than the reference substance.

4. The method according to claim 1 , wherein the reference substance is a substance different from the three or more substances to be quantified.

5. The quantification method according to claim 1 , wherein in the quantification step, the three or more target substances are quantified based on detection values ​​derived from a plurality of second labeling substances that respectively label the three or more target substances.

6. the first labeling substance includes a fluorescent dye-holding particle, the second labeling substance has an emission wavelength different from that of the first labeling substance and includes fluorescent dye-accumulating particles having emission wavelengths different from each other; The quantitative method according to claim 3 or claim 5.

7. The quantification method according to claim 6 , wherein the number of types of labeled substances, which is a combination of the first labeled substance and the plurality of second labeled substances, is smaller than the number of types of observed substances, which is a combination of the reference substance and the substance to be quantified.

8. 1. A method for labeling a plurality of types of observation substances, including a reference substance contained in each of a plurality of observation tissues collected from one specimen, and three or more types of quantified substances contained in at least one of the plurality of observation tissues, comprising: preparing the plurality of tissues for observation; labeling the plurality of types of observation target substances contained in the plurality of observation tissues with different types of labeling substances; The labeling method according to claim 1,

9. the reference substance is labeled with a first labeling substance including a fluorescent dye-integrated particle; the three or more types of quantified substances are labeled with a second labeling substance having a color different from that of the first labeling substance and containing fluorescent dye-accumulating particles having colors different from each other; The labeling method according to claim 8.

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