Quantitative method for the transcription factor GATA3
The method of immobilizing GATA3 antibodies with plasmon metal complexes and fluorescent labeling enhances detection of early-stage breast cancer by quantifying GATA3, addressing the limitations of existing screening methods.
Patent Information
- Application Number
- JP2024137579
- Authority / Receiving Office
- JP · JP
- Patent Type
- Applications
- Current Assignee / Owner
- Filing Date
- 2024-08-19
- Publication Date
- 2026-03-04
AI Technical Summary
Current breast cancer screening methods, such as mammography and ultrasound, are inadequate for detecting early-stage breast cancer, and there is a lack of a simple and rapid liquid biopsy method for quantifying the transcription factor GATA3, which is crucial for ultra-early cancer detection.
A method involving immobilization of GATA3 antibodies using plasmon metal complexes, followed by fluorescent labeling and surface plasmon enhancement, allowing for the quantification of GATA3 through fluorescent puncta counting.
Enables ultra-early detection of stage 0 or Tis breast cancer by quantifying GATA3 in blood samples, overcoming the limitations of imaging diagnostics.
Smart Images
Figure 2026034912000001_ABST
Abstract
Description
[Technical Field]
[0001] The present invention relates to a method for quantifying the transcription factor GATA3 in blood by fluorescent dot counting using immunofluorescent staining. [Background technology]
[0002] Early detection of cancer is crucial for improving the effectiveness of cancer treatment. Prostate cancer prediction has been improved through PSA testing, a cancer marker, and this has increased the cure rate. However, there are no tumor markers useful for early detection of breast cancer. A combination of CEA and CA15-3 is often used to monitor recurrence during postoperative follow-up, and the 1CTP bone destruction marker has been used to monitor breast cancer metastasis. In recent years, genetic mutations in the transcription factor GATA3, a key tumor marker comparable to PSA in prostate cancer, have been attracting attention due to their involvement in diseases such as breast cancer (Non-Patent Documents 1 and 2). This finding is expected to be useful in elucidating the causes and establishing treatments for these diseases. However, breast cancer screening, when a possible breast cancer diagnosis is made through mammography or ultrasound, is limited to immunohistochemistry (IHC) testing, where cells or tissues are collected by cytology or histology and then immunostained to confirm the diagnosis. A simple and rapid liquid biopsy method using blood or other samples has yet to be developed. [Prior art documents] [Non-patent literature]
[0003] [Non-Patent Document 1] A study of immunohistochemical differential expression in pulmonary and mammary carcinomas: MODERN PAGTHOLOGFY (2010) 23,654-661 [Non-patent document 2] GATA3 expression in breast carcinomas: utility in triple-negative, sarcomatoid, and metastic carcinomas: HumPathol. 2013 July;44(7):1341-1349 [Non-patent document 3] Interaction of the pioneer transcription factor GATA3 with nucleosomes: Nature Communications Author Hiroki Tanaka et al. DOI number 10.1038 / s41467-020-17959-y Summary of the Invention [Problem to be solved by the invention]
[0004] However, when breast cancer is detected by mammography or ultrasound images, as shown in Figure 6, it is often too late because the size detectable by imaging diagnostics is only 1-2 cm. Even though the survival rate is high (over 95%) when breast cancer is detected at "non-invasive cancer (very early stage)" (Tis) that remains within the milk duct, or at stage 0 where no lumps or abnormal shadows are detected by imaging diagnostics (Figure 7), the current situation in Japan is that the rate of breast cancer screening is low compared to Europe and the United States (Figure 5). This is because breast cancer screening imaging tests have difficulty detecting Tis or very early stage cancer (stage 0), and breast cancer cytology, as shown in Figure 4, is painful. Therefore, an objective of the present invention is to provide a liquid biopsy method that enables the quantitative determination of the transcription factor GATA3, which is necessary for realizing ultra-early cancer detection that cannot be detected by diagnostic imaging. [Means for solving the problem]
[0005] The inventors discovered that the aggregation action of plasmon metal complexes enables the immobilization of GATA3 antibodies, and that this immobilized substrate can be used to capture GATA3 (Figure 1). Furthermore, when this GATA3 is fluorescently labeled, the surface plasmon effect of the plasmon metal complex is enhanced, allowing detection as fluorescent puncta, and that GATA3 can be quantified by counting the fluorescent puncta (Figure 2). The present invention relates to a method for quantifying GATA3 based on such findings, and comprises the steps of: A) immobilizing GATA3 antibodies on a metal substrate using plasmon metal complex quantum crystals by agglutinating plasmon metal complexes onto a metal substrate having a more noble electrode potential using an electrode potential difference; B) fluorescently labeling sample GATA3 with a labeled antibody to produce fluorescently labeled GATA3; C) dropping a fluorescently labeled GATA3 solution onto the GATA3 antibody-immobilized substrate to capture the fluorescently labeled GATA3 on the plasmon metal complex quantum crystal; D) irradiating the fluorescently labeled GATA3 captured on the plasmon metal complex quantum crystal with excitation light to generate fluorescence, and enhancing the fluorescence through the surface plasmon enhancement effect of the plasmon metal complex quantum crystal; and E) observing with a fluorescence microscope and collecting and counting fluorescent spots above a certain threshold, thereby quantifying GATA3 by counting the fluorescent spots. [Effects of the Invention]
[0006] According to the present invention, 1) GATA3, which has previously been confirmed by immunoenzymatic staining in cytology, can be extracted from cells and captured with a GATA3 antibody immobilized by the aggregation action of a plasmonic metal complex quantum crystal, and 2) the GATA3 captured by the immobilized GATA3 antibody is observed as fluorescent spots because the fluorescent material used for labeling is enhanced by the surface plasmon effect obtained by irradiating the plasmonic metal with excitation light. Quantification of GATA3 is possible by counting these fluorescent spots. This enables the ultra-early detection of Tis or stage 0 cancer, which cannot be detected by imaging diagnostics. It has been reported that GATA3 binds to specific sites on nucleosomes, as shown in Figure 3, through in vitro analysis and cryo-electron microscopy (Non-Patent Document 3). Therefore, it is believed that GATA3 is incorporated into nucleosomes. The inventors used silver peroxide mesocrystals made from plasmonic metal quantum crystals to detect positively charged methylated nucleosomes in blood by charge capture and then confirmed the capture of nucleosomes through an autofluorescence test of the captured nucleosomes (WO 2021 / 132576). However, they were unable to detect the fluorescence of the labeled GATA3 antibody from the nucleosomes. Furthermore, they successfully immobilized the GATA3 antibody and confirmed the presence of labeled GATA3 through an antigen-antibody reaction (WO 2021 / 221142) (Figure 2(b)). However, the fluorescence of the labeled antibody was not observed in GATA3-bound nucleosomes. Therefore, the present inventors have demonstrated that GATA3 can be detected in nucleosomes using a solid-phase antibody by pre-treating nucleosomes to decompose or disassemble GATA3. This pre-treating method allows GATA3 to be exposed or released from nucleosomes, making it possible to immunofluorescently stain GATA3. This pre-treating method uses buffers that are used to disassemble chromatin and nucleosomes in chromatin immunoprecipitation and next-generation sequencing, such as Triton-X and high-salt buffer. (PBS, TBS, BSA) and blocking buffers containing them It is preferable to use a blocking buffer. [Brief explanation of the drawings]
[0007] [Figure 1] FIG. 1 is a process diagram showing a specific example of the method of the present invention. [Figure 2] FIG. 1 is a comparative diagram of blank (a) and GATA3 (b) observed by the method of the present invention. [Figure 3] This is a predicted diagram of GATA3 binding to DNA in a nucleosome. [Figure 4] 1A and 1B are schematic diagrams showing cell extraction methods for cytological diagnosis of breast cancer. [Figure 5]This is a graph showing breast cancer screening rates in each country. [Figure 6] FIG. 1 is an explanatory diagram showing the progression of breast cancer cells. [Figure 7] This is a graph showing the stage of breast cancer detection and its survival rate. DETAILED DESCRIPTION OF THE INVENTION
[0008] As shown in Figure 1, the present invention comprises A) a GATA3 antibody immobilization step, B) a GATA3 fluorescent labeling step, C) a step of capturing the fluorescently labeled GATA3 on the GATA3 antibody immobilization layer, D) a step of surface plasmon enhancement of the fluorescently labeled GATA3, and E) a step of counting the number of fluorescent spots above a certain threshold observed with a fluorescence microscope. In this specification, plasmonic metals are metals in which surface plasmons occur, and typical metals are silver and gold, but metals such as copper, titanium, and chromium are also used.
[0009] A) GATA3 antibody immobilization step A-1) To immobilize the GATA3 antibody, a GATA3 antibody, an aqueous solution of a plasmon metal complex, a metal substrate having an electrode potential more noble than that of the plasmon metal complex, and a buffer solution for diluting the GATA3 antibody are prepared. As the GATA3 immobilized antibody, NOV's "Anti-GATA3: Rabbit Polyclonal" is used, which is diluted with buffer to a concentration of 100 μg / ml. A 2000 ppm aqueous solution of silver thiosulfate is prepared as a plasmon metal complex aqueous solution. A phosphor bronze plate is prepared as the metal substrate. A-2) Mix equal amounts of the GATA3 solid-phase antibody dilution and a 2000 ppm aqueous solution of silver thiosulfate to prepare a solution, drop it onto a metal substrate, and after 1 minute, blow air over it to solidify the GATA3 antibody with silver thiosulfate quantum crystals, so that the GATA3 antibody is essentially dispersed on the metal substrate together with the plasmon metal complex quantum crystals, creating a GATA3 solid-phase substrate.
[0010] B) Fluorescent labeling process of GATA3 NOV's "Anti-GATA3: Rabbit Polyclonal" is fluorescently labeled using Dojindo Laboratories' "Fluorescein Labeling Kit-NH2" to adjust the FITC antibody concentration to 20 μg / ml. This is then mixed with Abnava's GATA3 protein "GATA3 Recombinant Protein" at a concentration of 0.02 μg / ml and diluted with the above buffer to prepare fluorescently labeled GATA3, a complex of FITC antibody and GATA3.
[0011] C) Fluorescently labeled GATA3 capture process The above-mentioned fluorescently labeled GATA3 solution is dropped onto a GATA3 antibody solid-phase substrate, which is a measurement chip, and the fluorescently labeled GATA is captured on the substrate. GATA3 is then sandwiched between the GATA3 antibody on the substrate and the fluorescently labeled GATA3 antibody, washed with a buffer solution, etc., and prepared for fluorescent staining measurement (sandwich method).
[0012] D) Surface plasmon enhancement process Fluorescently labeled GATA3 captured in a silver thiosulfate complex quantum crystal is irradiated with 460-495 nm excitation light to generate fluorescence. This fluorescence is enhanced by several hundred times or more due to the surface plasmon enhancement effect of the plasmonic metal complex quantum crystal, making it observable with a fluorescence microscope.
[0013] E) Fluorescent dot counting process E) The above fluorescent image is observed under a fluorescent microscope, and a fluorescent image is acquired in the fluorescent range of 510-550 nm. The fluorescent image is measured twice horizontally and twice vertically in one field of view, and then stitched together to create a single image. FITC fluorescent puncta with brightness values above a certain threshold in the fluorescent image are counted using Evident's software "CellSens."
[0014] (result) When the threshold was set to 45,000 or more and the fluorescent images of the blank (buffer solution) and the GATA3 antigen were compared (Fig. 2 a, b), 8 fluorescent points were observed in the blank and 53 fluorescent points were observed in the GATA3 antigen. This demonstrates that, according to the present invention, GATA3 can be quantified by immunofluorescence staining.
[0015] (Detection of GATA3 in nucleosomes) As shown in Figure 3, fluorescent labeling of GATA3 is difficult when it is incorporated into chromatin and nucleosomes. Therefore, it is preferable to pretreat the chromatin and nucleosomes to expose or disassemble GATA3 prior to labeling. For this pretreatment, a buffer solution used to disassemble chromatin and nucleosomes is used. Examples of such buffers include the high-salt buffer mentioned above, as well as the following: 1. Low Salt Buffer: Example ingredients: 10 mM Tris-HCl (pH 7.4), 10 mM NaCl, 3 mM MgCl2, 0.1% NP-40 Uses: Used to partially disassemble chromatin and release DNA from nucleosomes while preserving its structure. 2. High Salt Buffer: Example ingredients: 10 mM Tris-HCl (pH 7.4), 400 mM NaCl, 1 mM DTT Uses: Used to completely disassemble nucleosomes and disrupt histone-DNA interactions. 3. Sarkosyl buffer: Example ingredients: 50 mM Tris-HCl (pH 8.0), 0.5% Sarkosyl Uses: A strong detergent used to disrupt chromatin structure and separate DNA from nuclei. 4. RIPA Buffer (Radio Immunoprecipitation Assay Buffer): Example ingredients 50mM Tris-HCl (pH 7.4), 150mM NaCl, 1% NP-40, 0.5% Na-deoxycholate, 0.1% SDS Usage: Widely used as a pretreatment for chromatin immunoprecipitation (ChIP) and protein-DNA interaction analysis, effectively disaggregating chromatin and nucleosomes. The salt concentrations are as follows: low salt: 0.01 M to 0.05 M NaCl, medium salt: 0.1 M to 0.2 M NaCl, high salt: 0.3 M to 0.5 M NaCl, and ultra-high salt: 1 M or more.
[0016] In general, nucleosome disassembly should be performed as follows: 1. Mix the buffer and blood sample. 2. Allow to react for a certain period of time to disassemble the nucleosomes. 3. Separate using a centrifuge. 4. Remove the supernatant and measure. 5. In the GATA3 process, serum or plasma that has already been centrifuged is used, so it is preferable not to add anticoagulants (such as EDTA) to the buffer. 6. Although TBS is a suitable buffer, PBS can also be used. Triton X-100 (a surfactant) and protease can also be added.
[0017] Although blood, which is easy to collect, and plasma obtained by centrifugation are preferred as specimens, other body fluids, such as urine and cell-containing mammary gland fluid, may also be used by collecting other specimens using various dissection methods that liberate or expose GATA3 from nucleosomes so that GATA3 can be labeled. Therefore, the gist of the present invention is to collect GATA3 in nucleosomes using a liquid biopsy method, enhance the fluorescence intensity using surface plasmon enhancement by immunofluorescence staining, and quantify GATA3 by counting the fluorescent spots.
[0018] Although the present invention has been described in detail using the direct sandwich method as an embodiment, it will be readily understood by those skilled in the art that the present invention does not exclude the so-called indirect sandwich method using a secondary antibody or the sensitization method. [Explanation of symbols]
[0019] A) GATA3 antibody immobilization step B) GATA3 labeling step C) Fluorescently labeled GATA3 capture process D) Fluorescence enhancement process E) Fluorescent dot counting process
Claims
1. A) a step of aggregating an aqueous solution of the plasmon metal complex on a metal substrate having a more noble electrode potential in the presence of a buffer solution by an electrode potential difference, thereby immobilizing the GATA3 antibody together with the plasmon metal complex quantum crystal in a substantially dispersed state on the metal substrate; B) fluorescently labeling a sample GATA3 with a labeled antibody to produce fluorescently labeled GATA3; C) dropping the fluorescently labeled GATA3 onto a substrate in the presence of a buffer solution on which a GATA3 antibody has been substantially dispersed and immobilized, thereby capturing the fluorescently labeled GATA3 in a plasmon metal complex quantum crystal; D) irradiating the fluorescently labeled GATA3 captured in the plasmon metal complex quantum crystal with excitation light to generate fluorescence, and enhancing the fluorescence by the surface plasmon enhancement effect of the plasmon metal complex quantum crystal; E) A method for quantifying the transcription factor GATA3 in a sample by counting the number of fluorescent spots collected, which comprises observing the sample with a fluorescence microscope and collecting and counting fluorescent spots that are equal to or greater than a certain threshold.
2. The method according to claim 1, wherein the sample is a patient's blood, mammary gland fluid, urine, or cell lysate, and contains nucleosomes.
3. The method of claim 1, wherein when GATA3 is collected in a state bound to nucleosomes, a pretreatment is performed to disassemble the nucleosomes to which GATA3 is bound to expose GATA3 or to release GATA3, and the labeled GATA3 is captured on a GATA3 antibody solid-phase layer.
4. 4. The method according to claim 3, wherein a high-salt buffer (including PBS, TBS, and BSA) used in chromatin immunoprecipitation or next-generation sequencing is used in the pretreatment step.