Use of a kit for the diagnosis or the aid to the diagnosis of SPN

Abstract

The present application relates to the use of a diagnostic or auxiliary diagnostic kit for SPN, in particular to the use of ABCD1 as a new marker for the diagnosis or auxiliary diagnosis of SPN. The present application also relates to the use of ABCD1 as a new marker for the development of a diagnostic or auxiliary diagnostic kit for SPN.

Description

Technical Field

[0001] This application belongs to the biomedical field and relates to the application of ABCD1 as a novel biomarker in the development of reagents for the diagnosis or auxiliary diagnosis of solid pseudopapillary tumors (SPN) of the pancreas. Background Technology

[0002] A tumor is a new growth formed by the proliferation of local tissue cells under the influence of various carcinogenic factors. Because these new growths often present as space-occupying, mass-like protrusions, they are also called neoplasms. Based on their biological characteristics and the different levels of harm they cause to the body, tumors can be divided into two main categories: benign tumors and malignant tumors. The former grows slowly, has clear boundaries with surrounding tissues, does not metastasize, and poses little threat to human health. The latter grows rapidly, can metastasize to other parts of the body, and can produce harmful substances, damage normal organ structure, cause bodily dysfunction, and threaten life.

[0003] Solid pseudopapillary neoplasm of the pancreas (SPN) is a rare, low-grade malignant tumor, accounting for 0.9-2.7% of all pancreatic tumors. SPN typically occurs in women aged 20-30, with a lower incidence in men. SPN usually progresses slowly and has a long course. In most cases, it is asymptomatic, but a few cases may present with nonspecific symptoms such as abdominal discomfort, tenderness in the liver area, and diarrhea. In some cases, the tumor may compress adjacent organs, leading to pancreatitis, hypersplenism, and hydrocholecystitis.

[0004] The diagnosis of SPN is mainly based on imaging examinations and histopathological features. Imaging examinations are the preferred method for diagnosing SPN. Commonly used imaging examinations include abdominal ultrasound, abdominal CT scan, and MRI. According to the guidelines of the International Association of Pancreatology (IAP), the following conditions must be met to diagnose SPN: (1) Imaging examination shows that the lesion is a solid pancreatic lesion; (2) The size of the lesion is usually between 2 and 10 cm; (3) The lesion has both cystic and solid structures; (4) The lesion margin is clear; (5) The lesion shows significant enhancement on contrast-enhanced scans. Clinically, histological examination is used to confirm the diagnosis of SPN and differentiate it from other tumors. Typical histological features of SPN include: (1) Tumor cells are arranged in cystic or pseudopapillary patterns; (2) The cysts are filled with mucus or bloody material; (3) There is fibrous tissue and fat deposits between the cysts and tumor cells; (4) The tumor cells have a relatively heterogeneous appearance, with large nuclei, obvious atypia, and few or no mitotic figures. The pathological markers of SPN are positive for multiple lineage markers, including epithelial, mesenchymal, and neuroendocrine markers. The main markers include: (1) β-catenin: β-catenin is usually present in the nuclear positivity of SPN tumor cells, with a positive rate of up to 95%, which is an important marker for distinguishing SPN from other tumors; (2) CD10: CD10 has a high positive rate in SPN and can be used to distinguish SPN from other tumors, such as pancreatic neuroendocrine tumors and pancreatic ductal adenocarcinoma; (3) vimentin: vimentin is a marker of mesodermal cells. SPN tumor cells usually express vimentin, while other tumors do not; (4) α1-antitrypsin: α1-antitrypsin has a high positive rate in SPN tumor cells, while other tumors usually do not express it.

[0005] SPNs typically exhibit typical histological morphological features, but some SPNs can be confused with other tumors, a notable example being non-functional pancreatic neuroendocrine tumors (NF-PanNETs). NF-PanNETs are a relatively rare type of pancreatic tumor, accounting for approximately 5%-10% of all pancreatic tumors. They are characterized by the absence of significant excessive hormone secretion. Both SPNs and NF-PanNETs are generally well-defined, large masses within the pancreas, which can be solid or cystic-solid, prone to hemorrhage, calcification, and necrosis. The morphological features of SPNs and NF-PanNETs can be quite similar in some cases; under a microscope, tumor cells can be arranged in solid, trabecular, or acinar patterns. The cell size of both types of tumors is relatively consistent, often showing tightly packed round or polygonal cells, with a relatively small ratio of nucleus to cytoplasm, making morphological differentiation difficult. SPN and NF-PanNET may show similar immunohistochemical staining results, expressing varying degrees of vimentin, synaptophysin, CD200, CD99, Syn, and NSE. In certain situations, the clinical manifestations of SPN and NF-PanNET can be very similar. Both can occur in young women, often as a side effect, and may present with no obvious features or only nonspecific symptoms such as abdominal pain or upper abdominal discomfort, without signs of excessive hormone secretion. Furthermore, their imaging findings are somewhat similar. Therefore, accurate diagnosis of SPN and NF-PanNET requires a combination of clinical manifestations, imaging examinations, and pathological examinations.

[0006] Pancreatic neuroendocrine tumors (SPNs) share many overlapping morphological features with other pancreatic tumors, including non-functional pancreatic neuroendocrine tumors (NF-PanNETs), acinar cell carcinomas, and pancreatoblastomas. In the absence of typical clinical and histomorphological manifestations, immunohistochemistry plays a crucial role in the differential diagnosis of SPNs from other tumors. Although β-catenin is considered an important diagnostic marker for SPNs, typically exhibiting abnormal nuclear aggregation and marked nuclear positivity, it is not irreplaceable in differentiating SPNs from other tumors. Reports have shown positive expression of β-catenin in pancreatic neuroendocrine tumors and pancreatoblastomas. Undoubtedly, no immunohistochemical profile is completely specific for SPNs, and existing immunohistochemical markers show expression overlap in these tumors.

[0007] Furthermore, under normal circumstances, non-mutated β-catenin is also expressed to some extent in the cell nucleus. Simultaneously, β-catenin also exhibits diffuse positive expression in the cytoplasm, which can affect the interpretation of nuclear expression, easily leading to false positives or false negatives in SPN (sporadic nuclei). Whether it's a primary or metastatic SPN lesion, when the tissue cell morphology is similar, relying solely on existing immunohistochemical markers is insufficient for accurate diagnosis. In practical applications, a comprehensive evaluation combining other clinical, imaging, and pathological features is necessary.

[0008] In summary, SPN shares certain similarities with various other pancreatic tumors, including non-functional pancreatic neuroendocrine tumors, acinar cell carcinomas, and pancreatoblastomas, in terms of gross appearance, morphological features, and immunohistochemical staining. For cases where differential diagnosis of SPN is difficult, new specific pathological diagnostic markers are needed to confirm the diagnosis. Summary of the Invention

[0009] To address the aforementioned technical problems, this application provides a novel biomarker, ABCD1 (ATP binding cassette subfamily D member 1), which can be used to diagnose or assist in the diagnosis of solid pseudopapillary tumors of the pancreas (SPN), and has significant application value. In some embodiments, the biomarker ABCD1 can be used to differentiate or assist in the differentiation of SPN from other non-SPN tumors. In some embodiments, the biomarker ABCD1 can be used to screen or assist in the screening of SPN patients.

[0010] In one aspect, this application provides an antibody or antigen-binding fragment thereof that binds to ABCD1 (ATP-binding cassette subfamily Dmember 1), wherein the complementarity-determining region (CDR) has the following amino acid sequence: heavy chain CDR1 is selected from at least one of the amino acid sequences shown in SEQ ID NOs: 1-8; heavy chain CDR2 is selected from at least one of the amino acid sequences shown in SEQ ID NOs: 9-17; heavy chain CDR3 is selected from at least one of the amino acid sequences shown in SEQ ID NOs: 18-24; light chain CDR1 is selected from at least one of the amino acid sequences shown in SEQ ID NOs: 25-30; light chain CDR2 is selected from at least one of the amino acid sequences LVS, WAS, RMS or YAS; and light chain CDR3 is selected from at least one of the amino acid sequences shown in SEQ ID NOs: 31-35.

[0011] In one aspect, this application provides a polynucleotide, characterized in that the polynucleotide encodes the antibody or antigen-binding fragment thereof described in this application.

[0012] In one aspect, this application provides a recombinant vector, characterized in that the recombinant vector comprises the polynucleotide described in this application.

[0013] In one aspect, this application provides a host cell, characterized in that the host cell comprises the polynucleotides described in this application and / or the recombinant vectors described in this application.

[0014] In one aspect, this application provides a pharmaceutical composition, characterized in that the pharmaceutical composition comprises the antibody or antigen-binding fragment thereof described in this application.

[0015] In one aspect, this application provides a kit, characterized in that the kit comprises the antibody or antigen-binding fragment thereof described in this application.

[0016] In one aspect, this application provides a reagent or kit comprising a substance for detecting ABCD1, said reagent or kit being used in any one or more of the following (a)-(c): (a) said reagent or kit is used for diagnosing or assisting in the diagnosis of SPN; (b) said reagent or kit is used for differentiating or assisting in the differentiating of SPN from other non-SPN tumors; and / or (c) said reagent or kit is used for screening or assisting in the screening of SPN patients.

[0017] In another aspect, this application provides a system for diagnosing or assisting in the diagnosis of SPN, comprising a detection system and an interpretation system, wherein the detection system is used to perform ABCD1 immunostaining on tumor tissue or tumor cells of a subject; and the interpretation system is used to convert the result of the ABCD1 immunostaining into an interpretation result, wherein the interpretation result refers to SPN or non-SPN.

[0018] In another aspect, this application provides a system for identifying or assisting in the identification of SPN from other non-SPN tumors, including a detection system and an interpretation system, wherein the detection system is used to perform ABCD1 immunostaining on tumor tissue or tumor cells of a subject; and the interpretation system is used to convert the results of the ABCD1 immunostaining into an interpretation result, wherein the interpretation result refers to SPN or other non-SPN tumors.

[0019] In one aspect, this application provides the use of ABCD1 as a detection biomarker in any one or more of the following (a)-(c): (a) use in the development of a diagnostic reagent for the diagnosis or auxiliary diagnosis of SPN; (b) use in the development of a differential diagnostic reagent for the differentiation or auxiliary differentiation of SPN from other non-SPN tumors; and / or (c) use in the development of a screening reagent for the screening or auxiliary screening of patients with SPN.

[0020] In another aspect, this application provides the use of a substance for detecting ABCD1 in any one or more of the following (a)-(c): (a) use in the preparation of a diagnostic reagent for the diagnosis or auxiliary diagnosis of SPN; (b) use in the preparation of a differential reagent for the differentiation of SPN from other non-SPN tumors; and / or (c) use in the preparation of a screening reagent for the screening or auxiliary screening of patients with SPN.

[0021] In another aspect, this application provides the use of a substance for detecting ABCD1 in the preparation of a kit, wherein the kit functions as any one or more of the following (a)-(c): (a) for the diagnosis or auxiliary diagnosis of SPN; (b) for the differentiation or auxiliary differentiation of SPN from other non-SPN tumors; and / or (c) for the screening or auxiliary screening of patients with SPN.

[0022] In one aspect, this application provides a method for diagnosing or assisting in the diagnosis of SPN, the method comprising using ABCD1 as a marker to diagnose or assist in the diagnosis of SPN by detecting said marker.

[0023] In another aspect, this application provides a method for identifying or assisting in the identification of SPN from other non-SPN tumors, the method comprising using ABCD1 as a marker to identify or assist in the identification of SPN from other non-SPN tumors by detecting said marker.

[0024] In another aspect, this application provides a method for screening or assisting in the screening of SPN patients, the method comprising using ABCD1 as a biomarker to screen or assist in the screening of SPN patients by detecting said biomarker. Attached Figure Description

[0025] Figure 1 These are representative transmission electron microscope images and statistical results from step one of Example 1.

[0026] Figure 2 These are representative images and statistical results of immunofluorescence staining in step one of Example 1.

[0027] Figure 3 The differential gene expression results of pancreatic tumor tissue from SPN patients and adjacent normal pancreatic tissue, and pancreatic tumor tissue from SPN patients and pancreatic tumor tissue from NF-PanNET patients, as shown in the volcano plot in step two of Example 1, are presented.

[0028] Figure 4 The results of Western blot analysis of pancreatic tumor tissue and adjacent normal pancreatic tissue from SPN patients in step two of Example 1.

[0029] Figure 5 These are representative photographs of paraffin sections of pancreatic tumor tissue and adjacent normal pancreatic tissue from SPN patients in step two of Example 1, stained with HE and IHC for ABCD1.

[0030] Figure 6 These are representative photos of each score in Example 2.

[0031] Figure 7 The scores are for pancreatic tumor tissue and adjacent normal pancreatic tissue in 50 SPN patients in Example 2, and for metastatic tumor tissue in 16 SPN patients.

[0032] Figure 8 The ROC curve is shown in step two of Example 3.

[0033] Figure 9 The ROC curve is shown in step three of Example 3.

[0034] Figure 10 This is a representative image of IHC staining of pancreatic tumor tissue from a patient with pancreatic neuroendocrine tumor in Example 4.

[0035] Figure 11 This is a representative image of IHC staining of pancreatic tumor tissue from a patient with pancreatic acinar cell carcinoma in Example 4.

[0036] Figure 12 This is a representative image of IHC staining of pancreatic tumor tissue from pancreatoblastoma in Example 4.

[0037] Figure 13 This is a representative image of IHC staining of pancreatic tumor tissue from pancreatic ductal adenocarcinoma in Example 4.

[0038] Figure 14 The scoring results are for 99 SPN patients in Example 3, 91 NF-PanNET patients in Example 4, 10 insulinoma patients in Example 4, 9 acinar cell carcinoma patients in Example 4, 3 pancreatoblastoma patients in Example 4, and 10 pancreatic ductal adenocarcinoma patients in Example 4.

[0039] Figure 15 and Figure 16 The image shows the ELISA results of the binding activity of antibodies 07PC09, 07PC17, 07PC27, 07PC28, 07PC44, 07TE02, 07TE04, 07TE08, 07PC03, and 07PC08 prepared in Example 5 to the ABCD1 protein.

[0040] Figure 17This is a graph showing the scoring results of 30 patients with solid pseudopapillary tumors of the pancreas, 20 patients with pancreatic neuroendocrine tumors, 10 patients with pancreatic intraepithelial neoplasia, 20 patients with pancreatic ductal adenocarcinoma, 4 patients with serous cystadenoma, 4 patients with mucinous cystadenoma, 3 patients with acinar cell carcinoma, and 3 patients with pancreatoblastoma, using the antibody prepared in Example 5 in Example 6. The vertical axis of the graph represents the percentage of the number of samples with each score in the group out of the total number of samples in the group.

[0041] Figure 18 The images shown in Example 6 are representative IHC staining images of pancreatic solid pseudopapillary tumor, pancreatic neuroendocrine tumor, pancreatic intraepithelial neoplasia, pancreatic ductal adenocarcinoma, serous cystadenoma, mucinous cystadenoma, acinar cell carcinoma, and pancreatoblastoma tissues prepared in Example 5. Detailed Implementation

[0042] Unless otherwise stated, the terminology used herein has the common meaning understood by one of ordinary skill in the art. It may vary for those skilled in the art depending on the desired properties and effects sought through this application, and each numerical parameter should be interpreted according to the number of significant figures and conventional rounding methods or as understood by one of ordinary skill in the art. Generally, the nomenclature used herein and the experimental procedures in organic chemistry, medicinal chemistry, and biology described herein are well-known and commonly used in the art. Unless otherwise defined, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains. Where multiple definitions exist for terms used herein, the definitions in this section shall prevail unless otherwise stated.

[0043] In this document, terms such as “comprising,” “containing,” “including,” and “having” should be understood as open-ended, meaning “including but not limited to,” encompassing components other than those explicitly stated. Terms such as “composed of” and “consisting of” are closed-ended, containing only explicitly stated components.

[0044] When used in this document, the expression “A and / or B” includes three cases: (1) A; (2) B; and (3) A and B. The expression “A, B and / or C” includes seven cases: (1) A; (2) B; (3) C; (4) A and B; (5) A and C; (6) B and C; and (7) A, B and C. The meanings of similar expressions can be deduced by analogy.

[0045] In this article, ABCD1 is referred to as ATP-binding cassette sub-family D member 1. The ABCD1 gene is located on the long arm of the human X chromosome (Xq28). The NCBI accession number for ABCD1 (Homo sapiens) is NP_000024.2 (12-MAR-2023). The NCBI Gene ID for the ABCD1 gene (Homo sapiens) is 215, and its cDNA accession number is NM_000033.4 (12-MAR-2023).

[0046] In this study, the DAB channel staining intensity reflects the ABCD1 staining intensity of tumor cells, and the hematoxylin staining area reflects the number of tumor cell nuclei.

[0047] In this context, a "probe" refers to a single-stranded DNA or RNA sequence used to locate its complementary sequence within a sample's genome. The probe is brought into contact with the sample under conditions that allow hybridization with its complementary sequence to identify the target sequence. The probe is tagged with a radioactive or chemical label to make it visible to the target sequence. "ABCD1-specific probe" in this context refers to a probe that is complementary to the ABCD1 target sequence, and under specific conditions can hybridize and identify the ABCD1 target sequence.

[0048] In this article, "primer" refers to a short, single-stranded DNA fragment that can be used in PCR to hybridize with sample DNA and amplify the target gene region, generating numerous copies of the target gene fragment in a short time. "ABCD1 gene-specific primers" refer to primers used to amplify the target sequence of the ABCD1 gene.

[0049] In this context, "diagnosis" refers to the process of determining the nature of a disease or condition and distinguishing it from other conditions.

[0050] Application of ABCD1 as a marker

[0051] This article discloses a novel biomarker, ABCD1 (ATP-binding cassette subfamily Dmember 1), for the diagnosis or auxiliary diagnosis of solid pseudopapillary tumors of the pancreas (SPN), for the differentiation or auxiliary differentiation of SPN from other non-SPN tumors, or for the screening or auxiliary screening of SPN patients. In some embodiments, ABCD1 is combined with one or more biomarkers selected from the following for the diagnosis or auxiliary diagnosis of SPN, for the differentiation or auxiliary differentiation of SPN from other non-SPN tumors, or for the screening or auxiliary screening of SPN patients: β-catenin, CD10, vimentin, and α1-antitrypsin.

[0052] This document discloses a method for diagnosing or assisting in the diagnosis of spastic pancreatic neoplasms (SPN). The method includes using ABCD1 as a biomarker to diagnose or assist in the diagnosis of SPN by detecting said biomarker. In some embodiments, the method includes diagnosing or assisting in the diagnosis of SPN by performing ABCD1 proteomic analysis, qPCR, transcriptome sequencing, or ABCD1 immunostaining (e.g., immunohistochemical staining, immunocytochemical staining, immunofluorescence staining, multiplex immunofluorescence staining) on ​​clinical samples, such as tumor samples, preferably pancreatic tumor samples. In some embodiments, the method includes diagnosing or assisting in the diagnosis of SPN by performing ABCD1 immunostaining on clinical samples and analyzing the staining results. In some embodiments, the method includes diagnosing or assisting in the diagnosis of SPN by performing ABCD1 immunostaining on paraffin sections or frozen tissue sections prepared from clinical tissue samples and analyzing the staining results manually or with software. In the above methods, ABCD1 can be combined with one or more biomarkers selected from the following for diagnosing or assisting in the diagnosis of SPN: β-catenin, CD10, vimentin, and α1-antitrypsin. In some embodiments, the ABCD1 immunostaining is performed using the antibody or its antigen-binding fragment provided in this application.

[0053] This document discloses a method for identifying or assisting in the identification of pancreatic acinar neoplasms (SPNs) from other non-SPN tumors. The method includes using ABCD1 as a biomarker to identify or assist in the identification of SPNs from other non-SPN tumors by detecting this biomarker. In some embodiments, the method includes identifying or assisting in the identification of SPNs from other non-SPN tumors by performing ABCD1 proteomic analysis, qPCR, transcriptome sequencing, or ABCD1 immunostaining on clinical samples, such as pancreatic tumor samples. In some embodiments, the method includes identifying or assisting in the identification of SPNs from other non-SPN tumors by performing ABCD1 immunostaining on clinical samples and analyzing the staining results. In some embodiments, the method includes identifying or assisting in the identification of SPNs from other non-SPN tumors by performing ABCD1 immunostaining on paraffin sections or frozen tissue sections prepared from clinical tissue samples and analyzing the staining results manually or using software. In some embodiments, other non-SPN tumors include pancreatic acinar cell carcinoma, pancreatoblastoma, pancreatic neuroendocrine tumors (including non-functional pancreatic neuroendocrine tumors (NF-PanNETs) and insulinomas), and pancreatic ductal adenocarcinoma. In some embodiments, commonly used immunostaining methods include immunohistochemical staining, immunocytochemical staining, immunofluorescence staining, and multiplex immunofluorescence staining. In the above methods, ABCD1 can be combined with one or more markers selected from the following for the identification or auxiliary identification of SPN: β-catenin, CD10, vimentin, and α1 antitrypsin. In some embodiments, the ABCD1 immunostaining is performed using the antibody or its antigen-binding fragment provided in this application.

[0054] This document discloses a method for screening or assisting in the screening of SPN patients. The method includes using ABCD1 as a biomarker to screen or assist in the screening of SPN patients by detecting this biomarker. In some embodiments, the method includes screening or assisting in the screening of SPN patients by performing ABCD1 proteomic analysis, qPCR detection, transcriptome sequencing, or immunostaining on clinical samples, such as tumor samples from pancreatic tumor patients (e.g., tumor tissue samples and tumor cell samples). In some embodiments, the method includes screening or assisting in the screening of SPN patients by analyzing the detection results after performing ABCD1 proteomic analysis, qPCR detection, transcriptome sequencing, or immunostaining on clinical samples. In the above method, ABCD1 can be combined with one or more biomarkers selected from the following for screening or assisting in the screening of SPN patients: β-catenin, CD10, vimentin, and α1 antitrypsin. In some embodiments, commonly used immunostaining methods include immunohistochemical staining, immunocytochemical staining, immunofluorescence staining, and multiplex immunofluorescence staining. In some embodiments, the ABCD1 immunostaining is performed using the antibody or its antigen-binding fragment provided in this application.

[0055] This document discloses the use of ABCD1 as a detection biomarker in any one or more of the following (a)-(c): (a) in the development of diagnostic reagents for the diagnosis or auxiliary diagnosis of SPN; (b) in the development of differential diagnostic reagents for the differentiation or auxiliary differentiation of SPN from other non-SPN tumors; and / or (c) in the development of screening reagents for the screening or auxiliary screening of patients with SPN.

[0056] This document discloses the use of a substance for detecting ABCD1 in any one or more of the following (a)-(c): (a) use in the preparation of a diagnostic reagent for the diagnosis or auxiliary diagnosis of SPN; (b) use in the preparation of a differential reagent for the differentiation of SPN from other non-SPN tumors; and / or (c) use in the preparation of a screening reagent for the screening or auxiliary screening of patients with SPN.

[0057] This document discloses the use of a substance for detecting ABCD1 in the preparation of a kit, wherein the kit functions as one or more of the following (a)-(c): (a) for the diagnosis or auxiliary diagnosis of SPN; (b) for the differentiation or auxiliary differentiation of SPN from other non-SPN tumors; and / or (c) for the screening or auxiliary screening of patients with SPN.

[0058] In some embodiments, the substance used to detect ABCD1 includes an anti-ABCD1 antibody, an ABCD1 gene-specific primer, and an ABCD1 gene-specific probe. In some preferred embodiments, the substance used to detect ABCD1 includes the antibody or its antigen-binding fragment provided in this application.

[0059] In some embodiments, the other tumors not of the SPN include pancreatic acinar cell carcinoma, pancreatoblastoma, pancreatic neuroendocrine tumors (including non-functional pancreatic neuroendocrine tumors and insulinomas), and pancreatic ductal adenocarcinoma.

[0060] In some embodiments, the diagnostic or auxiliary diagnostic target of the diagnostic reagent and the identification or auxiliary identification target of the differential reagent are tumors, preferably pancreatic tumors. In some embodiments, the screening or auxiliary screening target of the screening reagent is a tumor patient, preferably a tumor sample from a pancreatic tumor patient.

[0061] In some embodiments, the diagnostic reagent, identification reagent, or screening reagent further comprises a substance for detecting one or more of the following markers: β-catenin, CD10, vimentin, and α1-antitrypsin.

[0062] Antibodies or antigen-binding fragments that bind to ABCD1

[0063] This document discloses an antibody or antigen-binding fragment thereof that binds to ABCD1, characterized in that its complementarity-determining region (CDR) has the following amino acid sequence: heavy chain CDR1 is selected from at least one of the amino acid sequences shown in SEQ ID NOs: 1-8; heavy chain CDR2 is selected from at least one of the amino acid sequences shown in SEQ ID NOs: 9-17; heavy chain CDR3 is selected from at least one of the amino acid sequences shown in SEQ ID NOs: 18-24; light chain CDR1 is selected from at least one of the amino acid sequences shown in SEQ ID NOs: 25-30; light chain CDR2 is selected from at least one of the amino acid sequences LVS, WAS, RMS, or YAS; and light chain CDR3 is selected from at least one of the amino acid sequences shown in SEQ ID NOs: 31-35. In some embodiments, the amino acid sequence of the complementarity-determining region of the antibody or antigen-binding fragment thereof is selected from at least one of the following groups (1)-(10):

[0064] (1) The heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 are the amino acid sequences shown in SEQ ID NO: 1, 9, 18, 25, RMS, and 31, respectively;

[0065] (2) The heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 are the amino acid sequences shown in SEQ ID NO: 2, 10, 19, 26, YAS, and 32, respectively;

[0066] (3) The heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 are the amino acid sequences shown in SEQ ID NO: 3, 11, 20, 27, WAS, and 33, respectively;

[0067] (4) The heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 are the amino acid sequences shown in SEQ ID NO: 1, 12, 18, 25, RMS, and 31, respectively;

[0068] (5) The heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 are the amino acid sequences shown in SEQ ID NO: 1, 13, 18, 25, RMS, and 31, respectively;

[0069] (6) The heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 are the amino acid sequences shown in SEQ ID NO: 4, 14, 21, 25, RMS, and 31, respectively;

[0070] (7) The heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 are the amino acid sequences shown in SEQ ID NO: 5, 15, 22, 28, RMS, and 31, respectively;

[0071] (8) The heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 are the amino acid sequences shown in SEQ ID NO: 6, 16, 23, 29, LVS, and 34, respectively;

[0072] (9) The heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 are the amino acid sequences shown in SEQ ID NO: 7, 17, 24, 30, WAS, and 35, respectively;

[0073] (10) Heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 are the amino acid sequences shown in SEQ ID NO: 8, 16, 23, 29, LVS, and 34, respectively.

[0074] In some embodiments, the heavy chain variable region of the antibody or its antigen-binding fragment contains an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to the sequence shown in any one of SEQ ID NO:36-45, and the light chain variable region of the antibody or its antigen-binding fragment contains an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to the sequence shown in any one of SEQ ID NO:46-55.

[0075] In some embodiments, the amino acid sequences of the heavy chain variable region and the light chain variable region of the antibody or its antigen-binding fragment are selected from at least one of the following groups (1)-(10):

[0076] (1) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:36, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:46;

[0077] (2) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:37, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:47;

[0078] (3) The heavy chain variable region contains an amino acid sequence as shown in SEQ ID NO:38, and the light chain variable region contains an amino acid sequence as shown in SEQ ID NO:48;

[0079] (4) The heavy chain variable region contains an amino acid sequence as shown in SEQ ID NO:39, and the light chain variable region contains an amino acid sequence as shown in SEQ ID NO:49;

[0080] (5) The heavy chain variable region contains an amino acid sequence as shown in SEQ ID NO:40, and the light chain variable region contains an amino acid sequence as shown in SEQ ID NO:50;

[0081] (6) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:41, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:51;

[0082] (7) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:42, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:52;

[0083] (8) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:43, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:53;

[0084] (9) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:44, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:54;

[0085] (10) The heavy chain variable region contains an amino acid sequence as shown in SEQ ID NO:45, and the light chain variable region contains an amino acid sequence as shown in SEQ ID NO:55.

[0086] This application also provides a polynucleotide, characterized in that the polynucleotide encodes the antibody or its antigen-binding fragment described in this application.

[0087] This application also provides a recombinant vector, characterized in that the recombinant vector comprises the polynucleotide described in this application.

[0088] This application also provides a host cell, characterized in that the host cell comprises the polynucleotides described in this application and / or the recombinant vectors described in this application.

[0089] This application also provides a pharmaceutical composition, characterized in that the pharmaceutical composition comprises the antibody or its antigen-binding fragment described in this application.

[0090] This application also provides a kit, characterized in that the kit includes the antibody or antigen-binding fragment thereof described in this application.

[0091] ABCD1 test reagent or kit

[0092] This document discloses a reagent comprising a substance for detecting ABCD1, said reagent being used in any one or more of the following (a)-(c): (a) said reagent being used to diagnose or assist in the diagnosis of SPN; (b) said reagent being used to differentiate or assist in the differentiation of SPN from other non-SPN tumors; and / or (c) said reagent being used to screen or assist in the screening of SPN patients.

[0093] This article discloses a kit containing a substance for detecting ABCD1, wherein the kit functions as one or more of the following (a)-(c): (a) for the diagnosis or auxiliary diagnosis of SPN; (b) for the differentiation or auxiliary differentiation of SPN from other non-SPN tumors; and / or (c) for the screening or auxiliary screening of SPN patients.

[0094] In some implementations, the substances used to detect ABCD1 include anti-ABCD1 antibodies, ABCD1 gene-specific primers, and ABCD1 gene-specific probes.

[0095] In some embodiments, the reagent may further comprise a substance for detecting one or more of the following markers: β-catenin, CD10, vimentin, and α1-antitrypsin.

[0096] In some implementations, the object of diagnosis or assisted diagnosis, identification or assisted identification is a tumor, preferably a pancreatic tumor, and the object of screening or assisted screening is a tumor patient, preferably a tumor sample from a pancreatic tumor patient.

[0097] In some embodiments, any of the substances described above for detecting ABCD1 are reagents or combinations of reagents for immunostaining clinical tissue samples (e.g., prepared paraffin sections or frozen sections) with ABCD1.

[0098] For example, the substance used to detect ABCD1 described above is an anti-ABCD1 antibody. The anti-ABCD1 antibody may be selected from recombinant anti-ABCD1 / ALD antibody (rabbit monoclonal antibody [EPR15929], purchased from Abcam, catalog number ab197013). In some preferred embodiments, the substance used to detect ABCD1 includes the antibody or its antigen-binding fragment provided in this application.

[0099] Specifically, any of the above-mentioned image analysis using FIJI software includes the following steps:

[0100] (1) Import the image obtained in step 1 into the FIJI software, click process-subtract background-rolling ball radius 50.0 pixels-check light background-click OK; click image-color-colour-deconvolution-select H DAB-click OK.

[0101] (2) Select the DAB image (color 2) obtained in step (1) - click image-type-8 bit - click analyze-calibrate - select uncalibrate OD - click OK; click image-adjust-threshold; click analyze-set measurements - check limit to threshold, integrated density, display label - click OK; click analyze-measure, and the integrated density is the DAB staining intensity of this screenshot.

[0102] (3) Select the H image (color 3) obtained in step (1) - click image-type-8 bit - click analyze-calibrate - select uncalibrate OD - click OK; click image-adjust-threshold; click analyze-set measurements - check limit to threshold, area, display label - click OK; click analyze-measure, and the area obtained is the hematoxylin staining area of ​​this screenshot.

[0103] Systems for diagnosing or identifying SPN

[0104] This document discloses a system for diagnosing or assisting in the diagnosis of pancreatic tumor neoplasms (SPN), comprising a detection system and an interpretation system. The detection system is used to perform ABCD1 immunostaining on tumor tissue or tumor cells of a subject. The interpretation system is used to convert the results of the ABCD1 immunostaining into an interpretation result, which indicates SPN or non-SPN. This interpretation system can convert the immunostaining results into an interpretation result based on manual scoring or FIJI scoring, etc. In some embodiments, the tumor tissue of the subject is paraffin-embedded or frozen sections of pancreatic tumor tissue. Commonly used immunostaining methods include immunohistochemical staining, immunocytochemical staining, immunofluorescence staining, and multiplex immunofluorescence staining. In some embodiments, the immunostaining is immunohistochemical staining or immunocytochemical staining, and the interpretation result is based on the expression intensity and / or distribution of ABCD1 in pancreatic tumor tissue or tumor cells. In some embodiments, the immunostaining is immunohistochemical staining or immunocytochemical staining, and the interpretation result is based on the ratio of immunotumor histochemical staining intensity to hematoxylin staining area or the ratio of immunotumor cytochemical staining intensity to hematoxylin staining area, respectively. In some preferred embodiments, the ABCD1 immunostaining is performed using the antibody or its antigen-binding fragment provided in this application.

[0105] In some implementations, the interpretation system may specifically be a system that performs the following operations:

[0106] After ABCD1 immunohistochemical staining, the brown color of the cytoplasm and cell membrane is used as a marker. The staining intensity and proportion are distinguished as follows: no staining or staining proportion <5% is judged as negative (-); staining proportion ≥5% and <50%, weak and incomplete staining is judged as weak positive (+); staining proportion ≥50% and <80%, moderate or local intact cell membrane staining is judged as moderate positive (++); staining proportion ≥80%, strong and intact staining is judged as strong positive (+++).

[0107] A score of - or + indicates a non-SPN (Special Persistent Number), while a score of ++ or +++ indicates an SPN.

[0108] The interpretation system may specifically be a system that performs the following operations:

[0109] 1. After ABCD1 immunohistochemical staining, the slides were scanned into digital slides using a NanoZoomer S360 digital slide scanning device in bright field, ×40, single layer, and autofocus mode. The slides were then imported into NDP.view2 viewing software. Five tumor observation areas were randomly selected under the objective lens (20x) and exported (format: JPEG; resolution: 300dpi).

[0110] 2. For each slice, the five screenshots obtained in step 1 were analyzed using FIJI software. The score result = DAB channel staining intensity (reflecting the ABCD1 staining intensity of tumor cells) / hematoxylin staining area (reflecting the number of tumor cell nuclei). The average score result of the five screenshots was used for statistical analysis. 0.1089 was used as the threshold. If the score result was ≤ the threshold, it was judged as non-SPN. If the score result was > the threshold, it was judged as SPN.

[0111] This document discloses a system for identifying or assisting in the identification of SPNs (spinal tumors) from other tumors, comprising a detection system and an interpretation system. The detection system is used to perform ABCD1 immunostaining on tumor tissue or tumor cells of a subject. The interpretation system is used to convert the results of the ABCD1 immunostaining into an interpretation result, which refers to SPNs or other non-SPN tumors. The interpretation system can convert the immunostaining results into interpretation results based on manual scoring or FIJI scoring, etc. In some embodiments, the identification or assistance system targets tumors, preferably pancreatic tumors. In some embodiments, the other non-SPN tumors include pancreatic acinar cell carcinoma, pancreatoblastoma, pancreatic neuroendocrine tumors (including non-functional pancreatic neuroendocrine tumors), and pancreatic ductal adenocarcinoma. In some embodiments, the tumor tissue of the subject is a paraffin section or frozen section of pancreatic tumor tissue. In some embodiments, the immunostaining is immunohistochemical staining or immunocytochemical staining, and the interpretation result is based on the expression intensity and distribution of ABCD1 in the tumor tissue or cells. In some embodiments, the immunostaining is immunohistochemical staining or immunocytochemical staining, and the interpretation results are based on the ratio of immunotumor histochemical staining intensity to hematoxylin staining area or the ratio of immunotumor cytochemical staining intensity to hematoxylin staining area, respectively. In some embodiments, the interpretation results are based on the ratio of DAB channel staining intensity to hematoxylin staining area. In some preferred embodiments, the ABCD1 immunostaining is performed using the antibody or its antigen-binding fragment provided in this application.

[0112] In some implementations, the interpretation system may specifically be a system that performs the following operations:

[0113] After ABCD1 immunohistochemical staining, the brown color of the cytoplasm and cell membrane is used as a marker. The staining intensity and proportion are distinguished as follows: no staining or staining proportion <5% is judged as negative (-); staining proportion ≥5% and <50%, weak and incomplete staining is judged as weak positive (+); staining proportion ≥50% and <80%, moderate or local intact cell membrane staining is judged as moderate positive (++); staining proportion ≥80%, strong and intact staining is judged as strong positive (+++).

[0114] A score of - or + indicates a tumor other than SPN, while a score of ++ or +++ indicates SPN.

[0115] The interpretation system may specifically be a system that performs the following operations:

[0116] 1. After ABCD1 immunohistochemical staining, the slides were scanned into digital slides using a NanoZoomer S360 digital slide scanning device in bright field, ×40, single layer, and autofocus mode. The slides were then imported into NDP.view2 viewing software. Five tumor observation areas were randomly selected under the objective lens (20x) and exported (format: JPEG; resolution: 300dpi).

[0117] 2. For each slice, the five screenshots obtained in step 1 were analyzed using FIJI software. The score result = DAB channel staining intensity (reflecting the ABCD1 staining intensity of tumor cells) / hematoxylin staining area (reflecting the number of tumor cell nuclei). The average score result of the five screenshots was used for statistical analysis. 0.1089 was used as the threshold. If the score result was ≤ the threshold, it was judged as other tumors that were not SPN. If the score result was > the threshold, it was judged as SPN.

[0118] Example

[0119] The following description, in conjunction with the accompanying drawings, illustrates exemplary embodiments of this application, including various details of these embodiments to aid understanding. It should be understood that these are merely exemplary and are in no way intended to limit the scope of protection of this application. The scope of protection of this application is defined only by the claims. Therefore, those skilled in the art will recognize that various changes and modifications can be made to the embodiments described herein without departing from the scope of this application. Similarly, for clarity and brevity, descriptions of well-known functions and structures are omitted in the following description.

[0120] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0121] Example 1: Discovery of ABCD1 as a diagnostic biomarker

[0122] I. Increased number of peroxisomes in pancreatic tumor tissue of SPN patients

[0123] Peroxisomes are intracellular organelles enclosed in a single membrane, primarily responsible for biochemical reactions such as lipid metabolism, redox reactions, and the metabolism of reactive oxygen species. The size, morphology, and number of peroxisomes vary across different cell types and species. The formation and maintenance of peroxisomes require the synergistic action of a series of peroxisome membrane proteins (including channel proteins) and enzyme proteins. Currently known peroxisome membrane proteins and channel proteins include, but are not limited to: membrane proteins (PEX1, PEX3, PEX5, PEX7, PEX10, PEX11, PEX13, PEX14, PEX16, PEX19, PEX22, PEX26), and channel proteins (ABCD1, ABCD2, ABCD3, ABCD4), etc. Currently known peroxisome enzymes include, but are not limited to: fatty acyl-CoA oxidase (ACOX), catalase (CAT), fibronectin α (α-oxidation), fibronectin β (β-oxidation), D-hydroxyacyl-CoA lyase (D-3-Hydroxyacyl-CoA dehydrogenase (HSD17B4), and D-2,4-dienoyl-CoA reductase (DECR).

[0124] Transcriptome data from pancreatic tumor tissues of SPN patients were compared with those from adjacent normal pancreatic tissues, as well as with those from NF-PanNET patients. Differentially expressed genes were statistically analyzed using software such as DEseq2 / edgeR and Limma / DEP, and functional annotation was performed using GO and KEGG. The analysis revealed that peroxisome membrane protein and channel protein-related genes (PEX1, PEX3, PEX10, PEX11B, PEX13, PEX26, ABCD1, ABCD3, ABCD4) and enzyme protein-related genes (ACOX1, ACOX3) were upregulated in pancreatic tumor tissues of SPN patients, suggesting that the number or function of peroxisomes in pancreatic tumor tissues of SPN patients may be abnormal.

[0125] Based on the foregoing, the inventors collected pancreatic tumor tissue and adjacent normal pancreatic tissue from SPN patients, and performed transmission electron microscopy (TEM) observation and catalase immunofluorescence staining (catalases are enzymes on peroxisomes, reflecting the number of peroxisomes). Representative TEM images and statistical results are shown below. Figure 1 . Figure 1In the middle: SPN represents pancreatic tumor tissue from SPN patients, and normal pancreas represents adjacent normal pancreatic tissue from SPN patients; the vertical axis of the right figure represents the average number of peroxisomes per cell. Representative images and statistical results of immunofluorescence staining are shown below. Figure 2 . Figure 2 In the diagram, SPN represents pancreatic tumor tissue from patients with SPN, and normal pancreas represents adjacent normal pancreatic tissue from patients with SPN. The vertical axis of the right figure represents the average number of catalytic enzyme-positive spots per cell. Compared to adjacent normal pancreatic tissue, the number of peroxisomes is increased in the pancreatic tumor tissue of patients with SPN.

[0126] II. Increased abundance of ABCD1 in pancreatic tumor tissue of SPN patients

[0127] ABCD1, short for ATP-binding cassette sub-family D member 1, is a member of the ABC transporter superfamily. ABC transporters are divided into seven distinct subfamilies (ABC1, MDR / TAP, MRP, ALD, OABP, GCN20, White). ABCD1 is a member of the ALD subfamily and is involved in the peroxisome transport of long-chain fatty acids and / or fatty acyl-CoA in the cytoplasm. All known peroxisome ABC transporters are hemitransporters, requiring binding to another hemitransporter molecule to form a functional homodimeric transporter. The ABCD1 gene is located on the long arm of the human X chromosome (Xq28). ABCD1 (Homo sapiens) is registered in NCBI as NP_000024.2 (12-MAR-2023). The ABCD1 gene (Homo sapiens) has a Gene ID of 215 in NCBI, and its cDNA is numbered NM_000033.4(12-MAR-2023) in NCBI.

[0128] Transcriptomic data analysis of pancreatic tumor tissues from SPN patients and adjacent normal pancreatic tissues, as well as pancreatic tumor tissues from SPN patients and NF-PanNET patients, showed that ABCD1 expression was upregulated in pancreatic tumor tissues from SPN patients, with Log2FC=1.622 compared to adjacent normal pancreatic tissues and Log2FC=2.057 compared to pancreatic tumor tissues from NF-PanNET patients. The differential gene expression results between pancreatic tumor tissues from SPN patients and adjacent normal pancreatic tissues, and between pancreatic tumor tissues from SPN patients and NF-PanNET patients, are shown in the volcano plot. Figure 3 . Figure 3In Chinese: SPN refers to pancreatic tumor tissue in patients with SPN, normal pancreas refers to normal pancreatic tissue adjacent to the cancer in patients with SPN, and NF-PanNET refers to pancreatic tumor tissue in patients with NF-PanNET.

[0129] Pancreatic tumor tissue and adjacent normal pancreatic tissue were collected from SPN patients and subjected to Western blotting (using ABCD1 antibody as the primary antibody; antibody information is given in Example 3). Results are shown in […]. Figure 4 . Figure 4 In the diagram: -1, -2, -3, and -4 represent four SPN patients; SPN indicates pancreatic tumor tissue in SPN patients, and normal indicates normal pancreatic tissue adjacent to the cancer in SPN patients.

[0130] Paraffin sections (adjacent sections) of pancreatic tissue from SPN patients were subjected to HE staining and IHC staining for ABCD1 (the method for IHC staining for ABCD1 is described in Example 3). Representative photographs are shown below. Figure 5 (The top image shows HE staining, and the bottom image shows IHC staining for ABCD1). Figure 5 In Chinese: SPN refers to pancreatic tumor tissue in patients with SPN, and Normal pancreas refers to normal pancreatic tissue adjacent to the tumor in patients with SPN.

[0131] Example 2: Confirmation of ABCD1 as a diagnostic marker

[0132] Paraffin sections of pancreatic tumor tissue and adjacent normal pancreatic tissue were collected from 50 patients with SPN (sporanic pancreatitis) who underwent surgery and were pathologically diagnosed at Peking Union Medical College Hospital from January 2016 to April 2022. IHC staining was performed on ABCD1, and then scoring was performed (staining and scoring methods were the same as in Example 3). Representative images of each score are shown below. Figure 6 . Figure 6 In Chinese: SPN refers to pancreatic tumor tissue in patients with SPN, and normal pancreas refers to normal pancreatic tissue adjacent to the cancer in patients with SPN.

[0133] Paraffin-embedded tissue samples and corresponding clinicopathological data of SPN (spinal parasite) from sites other than the pancreas, surgically treated and pathologically confirmed at Peking Union Medical College Hospital from January 2016 to April 2022, were collected. Sixteen patients with metastatic SPN were enrolled. Relevant information included: one patient had two liver metastases, and another patient had both a retroperitoneal mass and liver metastases, totaling 18 metastatic lesions (8 liver metastases, 2 retroperitoneal masses, 4 intraperitoneal masses, 3 lymph node metastases, and 1 ovarian metastasis). Paraffin sections of the metastatic tissue were obtained and IHC stained against ABCD1, then scored (staining and scoring methods were the same as in Example 3). All metastatic lesions were strongly positive (+++).

[0134] The scoring results of pancreatic tumor tissue and adjacent normal pancreatic tissue in the above 50 SPN patients and the scoring results of metastatic tumor tissue in the above 16 SPN patients are shown in the figure. Figure 7 . Figure 7 In the middle: SPN primary lesion refers to the primary pancreatic tumor tissue of SPN patients (50 cases), normal pancreas refers to the normal pancreatic tissue adjacent to the cancer of SPN patients (50 cases), and SPN metastatic lesion refers to the metastatic pancreatic tumor tissue of SPN patients (18 cases); the vertical axis represents the percentage of the sample number of each score out of the total sample number.

[0135] Evidence shows that ABCD1 expression is upregulated in the primary tumor lesions or metastases of SPN patients.

[0136] Example 3: Performance evaluation of ABCD1 as a diagnostic biomarker

[0137] Paraffin-embedded tissue samples and corresponding clinicopathological data were collected from patients with spastic paraneoplastic neuralgia (SPN) and NF-PanNET who underwent surgical treatment and were pathologically diagnosed at Peking Union Medical College Hospital from December 2016 to June 2022. Ninety-nine SPN patients were included (20 males, 79 females; median age 31 years); and 91 NF-PanNET patients were included (39 males, 52 females; median age 52 years).

[0138] I. Immunohistochemical staining of pancreatic tumor tissue with ABCD1

[0139] 1. Take paraffin-embedded tissue samples and prepare paraffin tissue sections with a thickness of 4μm.

[0140] 2. The paraffin tissue sections obtained in step 1 are successively baked, dewaxed, and hydrated.

[0141] Baking: Place in a 60℃ oven for at least 60 minutes.

[0142] Dewaxing: xylene for 20 min, xylene for 20 min.

[0143] Hydration: 100% ethanol for 10 min, 100% ethanol for 10 min, 95% ethanol solution for 5 min, 90% ethanol solution for 5 min, 85% ethanol solution for 5 min, 70% ethanol solution for 5 min, PBS buffer rinse.

[0144] 3. Perform antigen retrieval on the paraffin tissue sections obtained in step 2, and then rinse with PBS buffer.

[0145] Antigen retrieval: Add sodium citrate antigen retrieval solution to a pressure cooker, immerse the rinsed paraffin slices in the sodium citrate antigen retrieval solution, place the pressure cooker in a microwave oven and heat until boiling, open the lid to check for bubbles (bubbles indicate that the sodium citrate antigen retrieval solution has boiled), close the lid and continue heating for 5 minutes, then open the lid and let it cool naturally at room temperature.

[0146] 4. Take the paraffin tissue sections from step 3, block endogenous catalase, and wash with PBS buffer.

[0147] Blocking endogenous catalase: Paraffin sections were completely immersed in 3% hydrogen peroxide solution and blocked at room temperature in the dark for 30 min.

[0148] 5. Take the paraffin tissue sections from step 4 and block them with BSA antigen blocking solution at room temperature for 60 minutes.

[0149] BSA antigen blocking solution: contains 5% BSA, with the remainder being 0.1% PBST solution.

[0150] 6. Take the paraffin tissue sections from step 5, incubate them in the primary antibody working solution for 2 hours, and then rinse them with PBS buffer.

[0151] Primary antibody working solution: Dilute the ABCD1 antibody to 100-fold volume with PBS buffer. ABCD1 antibody: Recombinant Anti-ABCD1 / ALD antibody, rabbit monoclonal antibody [EPR15929] to ABCD1 / ALD, Abcam, catalog number ab197013.

[0152] 7. Take the paraffin tissue sections from step 6, incubate them in the secondary antibody working solution for 1 hour, then add biotin substrate and react at room temperature for 30 minutes, then rinse with PBS buffer.

[0153] Secondary antibody working solution: Dilute the enzyme-labeled secondary antibody to 1000 times its original volume with PBS buffer. Enzyme-labeled secondary antibody: Goat anti-rabbit IgG H&L (HRP), Abcam, catalog number ab6721.

[0154] 8. Take the paraffin tissue section from step 7, add 1×DAB staining solution, observe the staining under a microscope (stop staining with tap water in time), and rinse with tap water.

[0155] 9. Take the paraffin tissue sections from step 8, immerse them in hematoxylin staining solution for 10-20 seconds, then rinse with tap water, and then soak them in PBS buffer at pH 7.2-7.4 for 10 minutes.

[0156] 10. Take the paraffin tissue sections from step 9 and perform tissue dehydration, paraffin clearing, and mounting in sequence.

[0157] Tissue dehydration: 70% ethanol solution for 5 min, 85% ethanol solution for 5 min, 90% ethanol solution for 5 min, 95% ethanol solution for 5 min, 100% ethanol for 10 min, 100% ethanol for 10 min.

[0158] Transparent paraffin: xylene 20 min, xylene 20 min.

[0159] II. Manual scoring

[0160] The pathological tissue sections prepared in step one were manually scored (positive was indicated by brown markings on the cytoplasm and cell membrane, differentiated according to staining intensity and proportion): no staining or staining proportion <5% was considered negative (-); staining proportion ≥5% and <50%, weak and incomplete staining was considered weakly positive (+); staining proportion ≥50% and <80%, moderate or partially intact cell membrane staining was considered moderately positive (++); staining proportion ≥80%, strong and intact staining was considered strongly positive (+++). The evaluation was conducted by two qualified pathologists to reach a consensus.

[0161] The results of manual scoring showed that among the 99 SPN patients, 96 were rated 3+ (i.e., +++), 2 were rated 2+ (i.e., ++), and 1 was rated 1+ (i.e., +); among the 91 NF-PanNET patients, 7 were rated 1+ (i.e., +), and 84 were rated negative (i.e., -).

[0162] Patients with a score of - and a score of + were classified as non-SPN patients, while patients with a score of ++ and a score of ++++ were classified as SPN patients. See ROC curves. Figure 8 The sensitivity was 97.98%, the specificity was 100%, and the Youden index was 0.9798. This demonstrates that ABCD1 IHC staining combined with manual scoring can effectively differentiate between SPN and NF-PanNET patients.

[0163] III. FIJI Rating

[0164] Method for FIJI scoring of pathological tissue sections prepared in step one:

[0165] 1. The NanoZoomer S360 digital slide scanning device (C13220-01, Hamamatsu) was used to scan the slides into digital slides in bright field, ×40, single layer, and autofocus mode. The slides were then imported into NDP.view2 viewing software. Five tumor observation areas were randomly selected under the objective lens (20x) and exported (format: JPEG; resolution: 300dpi).

[0166] 2. The five screenshots obtained in step 1 for each slice were analyzed using FIJI software.

[0167] (1) Import the image obtained in step 1 into the FIJI software, click process-subtract background-rolling ball radius 50.0 pixels-check light background-click OK; click image-color-colour-deconvolution-select H DAB-click OK.

[0168] (2) Select the DAB image (color 2) obtained in step (1) - click image-type-8 bit - click analyze-calibrate - select uncalibrate OD - click OK; click image-adjust-threshold; click analyze-set measurements - check limit to threshold, integrated density, display label - click OK; click analyze-measure, and the integrated density is the DAB staining intensity of this screenshot.

[0169] (3) Select the H image (color 3) obtained in step (1) - click image-type-8 bit - click analyze-calibrate - select uncalibrate OD - click OK; click image-adjust-threshold; click analyze-set measurements - check limit to threshold, area, display label - click OK; click analyze-measure, and the area obtained is the hematoxylin staining area of ​​this screenshot (the hematoxylin staining area reflects the number of cells).

[0170] The scoring result is calculated as DAB staining intensity divided by hematoxylin staining area.

[0171] Statistical analysis was performed using the average score of five screenshots from each pathological tissue section.

[0172] Using 0.1089 as the threshold, patients with a score ≤ the threshold were classified as non-SPN patients, and those with a score > the threshold were classified as SPN patients. ROC curves are shown below. Figure 9The sensitivity was 97.98%, the specificity was 94.51%, and the Youden index was 0.9249. This demonstrates that ABCD1IHC staining combined with the FIJI score can effectively differentiate between SPN and NF-PanNET patients.

[0173] Example 4: Abundance of ABCD1 in Patients with Various Other Pancreatic Tumors

[0174] Paraffin-embedded tissue samples and corresponding clinicopathological data were collected from patients with pancreatic neuroendocrine tumors (including NF-PanNET and insulinoma) who underwent surgical treatment and were pathologically confirmed at Peking Union Medical College Hospital from August 2011 to November 2021. Ninety-one NF-PanNET patients were included, with 39 males and 52 females, and a median age of 52 years. Ten insulinoma patients were included, with 8 females and 2 males, and a median age of 56 years. Paraffin-embedded tissue samples and corresponding clinicopathological data were also collected from patients with pancreatic acinar cell carcinoma who underwent surgical treatment and were pathologically confirmed at Peking Union Medical College Hospital from August 2011 to November 2021. Nine pancreatic acinar cell carcinoma patients were included, with 4 females and 5 males, and a median age of 58 years. Paraffin-embedded tissue samples and corresponding clinicopathological data were collected from patients with pancreatoblastoma who underwent surgical treatment and were pathologically diagnosed at Peking Union Medical College Hospital from August 2011 to November 2021. Three patients with pancreatoblastoma were included (1 female and 2 males); the median age was 32 years. Paraffin-embedded tissue samples and corresponding clinicopathological data were also collected from patients with pancreatic ductal adenocarcinoma who underwent surgical treatment and were pathologically diagnosed at Peking Union Medical College Hospital from August 2011 to November 2021. Ten patients with pancreatic ductal adenocarcinoma were included (1 female and 9 males); the median age was 61 years.

[0175] I. Immunohistochemical staining of pancreatic tumor tissue with ABCD1

[0176] Same as step one in Example 3.

[0177] II. Manual scoring

[0178] Same as step two in Example 3.

[0179] Representative images of IHC staining of pancreatic tumor tissue from patients with pancreatic neuroendocrine tumors (including NF-PanNET and insulinoma) are shown below. Figure 10 Representative images of IHC staining of pancreatic tumor tissue from patients with pancreatic acinar cell carcinoma are shown below. Figure 11 Representative images of pancreatic tumor tissue from pancreatoblastoma with IHC staining are shown below. Figure 12 Representative images of pancreatic tumor tissue stained with IHC from patients with pancreatic ductal adenocarcinoma are shown below. Figure 13The scoring results of 99 SPN patients in Example 3, 91 NF-PanNET patients in Example 4, 10 insulinoma patients in Example 4, 9 acinar cell carcinoma patients in Example 4, 3 pancreatoblastoma patients in Example 4, and 10 pancreatic ductal adenocarcinoma patients in Example 4 are shown in the figure. Figure 14 . Figure 14 The vertical axis represents the percentage of samples with each score in that group relative to the total sample size. Of the 99 SPN patients, 96 were rated +++, 2 were rated ++, and 1 was rated +. Of the 91 NF-PanNET patients, 7 were rated + and 84 were rated -. Of the 10 insulinoma patients, 9 were rated - and 1 was rated +. Of the 9 pancreatic acinar cell carcinoma patients, 3 were rated -, 5 were rated +, and 1 was rated ++. Of the 3 pancreatoblastoma patients, 2 were rated - and 1 was rated ++. Of the 10 pancreatic ductal adenocarcinoma patients, 9 were rated - and 1 was rated +. It is evident that ABCD1 IHC staining combined with manual scoring can effectively differentiate between SPN, NF-PanNET, insulinoma, pancreatic acinar cell carcinoma, pancreatoblastoma, and pancreatic ductal adenocarcinoma.

[0180] Example 5: Preparation and activity identification of anti-ABCD1 antibody

[0181] 1. Animal immunization

[0182] The antigen protein ABCD-1 (amino acid sequence as shown in SEQ ID NO:56, recombinantly expressed using the E. coli prokaryotic system) and a synthetic polypeptide (amino acid sequence as shown in SEQ ID NO:57, synthesized using chemical methods) purified from prokaryotic expression were used as immunogens, and Balb / c mice were immunized at a dose of 50 μg / mouse. A second immunization was performed half a month after the first immunization, followed by immunizations every half month thereafter. Negative serum was collected from mice 3 days before immunization, and 50 μL of blood was collected by tail clipping 6 days after each immunization. The negative and immune sera were diluted proportionally (1:0.2K, 1:0.4K, 1:0.8K, 1:1.6K, 1:3.2K, 1:6.4K, 1:12.8K). The serum titer of the ABCD-1 antigen protein was detected using Cell-ELISA. When the titer met the requirements and anti-human ABCD-1 antibody was detected at a dilution >1:12.8K, the rat spleen and lymph nodes were harvested.

[0183] 2. Cell fusion

[0184] (1) Gently blow healthy sp2 / 0 cells off the culture flask wall and aspirate them into a 50ml centrifuge tube.

[0185] (2) Blood was collected from the eyes of the mice, and they were then euthanized by pulling their necks. The mice were then soaked in 75% alcohol for 5 minutes.

[0186] (3) Pour a small amount of serum-free IMDM into a petri dish, and place the cell strainer and syringe core into the petri dish. Remove the spleen from the mouse with scissors and tweezers and place it on the cell strainer. Gently crush the spleen thoroughly with the syringe core, and aspirate the crushed cells into a centrifuge tube containing sp2 / 0. Centrifuge at 1500 rpm for 5 min.

[0187] (4) Remove the mouse thymus with scissors and tweezers and crush it. Put the crushed thymus cells into a 15ml centrifuge tube, add 2ml of HAT and 1ml of HT and place it in an incubator for later use.

[0188] (5) After centrifuging, discard the supernatant, carefully and gently mix the cells with serum-free IMDM, and centrifuge (1500 rpm, 5 min).

[0189] (6) Discard as much of the cell supernatant as possible after centrifugation. Tap the bottom of the centrifuge tube to fully suspend the cells, place the centrifuge tube in 37°C warm water, and slowly add 1 ml of PEG over about 1 minute. After adding the PEG, let it stand in the warm water for 1 minute. Then, slowly add 2 ml of serum-free IMDM over 2 minutes, followed by 8 ml of serum-free IMDM over 2 minutes. Centrifuge at 1000 rpm for 5 minutes.

[0190] (7) Discard the supernatant, add 10 ml of serum, carefully blow the cells evenly, and pour them into the prepared thymocytes. Add sterile semi-solid culture medium to a final volume of 50 ml and mix thoroughly. Then pour evenly into 30 cell culture dishes. Place the cell culture dishes in a humidified box and then incubate them in an incubator.

[0191] 3. Preliminary screening of positive clones using ELISA method.

[0192] (1) Dilute “Project 2023-601-ABCD1 protein” (amino acid sequence as shown in SEQ ID NO:56, recombinant expression using E. coli prokaryotic system) with coating buffer to a final concentration of 2ug / ml, 100ul / well, 4℃, overnight, and then wash 3 times with washing buffer.

[0193] (2) Block with blocking solution (2% skim milk powder), 200ul / well, incubate at 37℃ for 2h, then wash 3 times with washing solution.

[0194] (3) Add primary antibody (cell culture supernatant), negative control (SP2 / 0 culture supernatant), blank control (PBS), and positive control (positive serum diluted 1000 times with PBS), all at 100 μL / well. Incubate at 37°C for 1 h, then wash 3 times with washing buffer.

[0195] (4) Add PBS to dilute the secondary antibody 20,000 times, 100 μL / well, incubate at 37°C for 1 h, and wash 3 times with washing buffer after taking it out.

[0196] (5) Add 100ul of colorimetric solution per well and develop the color for about 10 minutes.

[0197] (6) Add 50 μL of stop solution to each well to terminate the process.

[0198] (7) Measure absorbance at dual wavelengths (450, 630 nm) and record the data. Read the absorbance at 450 nm on a microplate reader and analyze the data. Select cell lines with OD450 > 0.2 in the test supernatant as initial screening candidate positive cell lines, discard the culture supernatant of the positive cell lines, and add fresh HAT complete medium.

[0199] 4. Construction of antibody plasmids and in vitro recombinant expression

[0200] The antibodies screened in step 3 were sequenced in hybridoma cells. The antibody fragments obtained from the sequencing were used for gene synthesis and constructed into mouse IgG1, IgG2a, or IgG2b frameworks. Then, using molecular cloning technology, the antibody fragments were inserted into the PTT5 vector (Anhui Global Gene Technology Co., Ltd.) to construct a mammalian cell expression plasmid. The plasmid was then introduced into the host cell line CHO cells (Mab Select Sure LX, Cytiva, 17547403) using liposome transfection. The supernatant of the fermentation broth was purified by affinity chromatography (purification was performed using the protein A purification method, the expression system was CHO-S, and the chromatography column was Mab Select Sure LX (Cytiva, 17547403)). The purified recombinant antibody was finally obtained, and its CDR and variable region amino acid sequences are shown in Table 1 below.

[0201] Table 1. CDR sequences of antibodies

[0202]

[0203] 5. Purify the binding activity of the antibody.

[0204] The binding activity of the antibodies shown in Table 1 was detected using the ELISA method. Goat anti-mouse secondary antibody was diluted with HRP (Abcam; Ab205719), and the detection procedure was the same as the ELISA method in step 3. The results are as follows. Figure 15 and Figure 16 As shown in Table 1, all 10 antibodies disclosed in the table exhibit good binding activity to the ABCD1 protein.

[0205] Example 6: Immunohistochemical staining of pancreatic tumor tissue using anti-ABCD1 antibody

[0206] The antibody 07TE02 prepared in Example 5 was used to perform immunohistochemical staining on pancreatic tumor tissue. The clinical sample information and immunohistochemical staining method used were the same as in Example 3.

[0207] The scoring results for 30 patients with solid pseudopapillary tumors of the pancreas, 20 patients with pancreatic neuroendocrine tumors, 10 patients with pancreatic intraepithelial neoplasia, 20 patients with pancreatic ductal adenocarcinoma, 4 patients with serous cystadenoma, 4 patients with mucinous cystadenoma, 3 patients with acinar cell carcinoma, and 3 patients with pancreatoblastoma are shown in [the table below]. Figure 17 As shown.

[0208] Representative images of IHC staining of pancreatic solid pseudopapillary tumors, pancreatic neuroendocrine tumors, pancreatic intraepithelial neoplasia, pancreatic ductal adenocarcinoma, serous cystadenoma, mucinous cystadenoma, acinar cell carcinoma, and pancreatoblastoma tissues are available in the following images. Figure 18 As shown.

[0209] Figure 17 In the figure, the vertical axis represents the percentage of samples with each score in that group out of the total sample size. Specifically, among the 30 cases of solid pseudopapillary tumors of the pancreas, 14 were rated ++ and 16 were rated +. Among the 20 cases of pancreatic neuroendocrine tumors, 12 were rated - and 8 were rated +. Among the 10 cases of pancreatic intraepithelial neoplasia, 3 were rated -. Among the 20 cases of pancreatic ductal adenocarcinoma, all 20 were rated -. Among the 4 cases of serous cystadenoma, all 4 were rated -. Among the 4 cases of mucinous cystadenoma, all 4 were rated -. Among the 3 cases of acinar cell carcinoma, all 3 were rated -. Among the 3 cases of pancreatoblastoma, all 3 were rated -.

[0210] Figure 17 and Figure 18 The results showed that the anti-ABCD1 antibody prepared in Example 5, when used for IHC staining combined with artificial scoring, could effectively distinguish between solid pseudopapillary tumors of the pancreas and pancreatic neuroendocrine tumors, pancreatic intraepithelial neoplasia, pancreatic ductal adenocarcinoma, serous cystadenoma, mucinous cystadenoma, acinar cell carcinoma, pancreatoblastoma, etc.

[0211] The present application has been described in detail above. Those skilled in the art will recognize that the present application can be implemented in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments are given in this application, it should be understood that further modifications can be made to the present application. In summary, in accordance with the principles of this application, this application is intended to include any changes, uses, or improvements to the present application, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. The use of a substance for detecting ABCD1 (ATP-binding cassette subfamily D member 1) in the preparation of reagents or kits, characterized in that, The reagent or kit is used in any one or more of the following (a)-(c): (a) The reagents or kits described herein are used for the diagnosis or auxiliary diagnosis of solid pseudopapillary tumors (SPN) of the pancreas; (b) The reagents or kits described herein are used to identify or assist in the identification of SPN from other pancreatic tumors that are not SPN; and / or (c) The reagents or kits described are used for screening or assisting in the screening of SPN patients.

2. The use according to claim 1, wherein the substance for detecting ABCD1 comprises an antibody or antigen-binding fragment thereof that binds to ABCD1.

3. The use according to claim 2, wherein the amino acid sequence of the complementarity-determining region of the antibody binding to ABCD1 or its antigen-binding fragment is selected from at least one of the following groups (1)-(10): (1) The heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 are the amino acid sequences shown in SEQ ID NO:1, 9, 18, 25, RMS, and 31, respectively. (2) The heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 are the amino acid sequences shown in SEQ ID NO:2, 10, 19, 26, YAS, and 32, respectively. (3) The heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 are the amino acid sequences shown in SEQ ID NO:3, 11, 20, 27, WAS, and 33, respectively. (4) The heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 are the amino acid sequences shown in SEQ ID NO:1, 12, 18, 25, RMS, and 31, respectively; (5) The heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 are the amino acid sequences shown in SEQ ID NO:1, 13, 18, 25, RMS, and 31, respectively. (6) The heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 are the amino acid sequences shown in SEQ ID NO:4, 14, 21, 25, RMS, and 31, respectively. (7) The heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 are the amino acid sequences shown in SEQ ID NO:5, 15, 22, 28, RMS, and 31, respectively; (8) The heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 are the amino acid sequences shown in SEQ ID NO:6, 16, 23, 29, LVS, and 34, respectively; (9) The heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 are the amino acid sequences shown in SEQ ID NO:7, 17, 24, 30, WAS, and 35, respectively. (10) Heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 are the amino acid sequences shown in SEQ ID NO:8, 16, 23, 29, LVS, and 34, respectively.

4. In the use according to claim 2 or 3, the amino acid sequences of the heavy chain variable region and the light chain variable region of the antibody binding to ABCD1 or its antigen-binding fragment are selected from at least one of the following groups (1) to (10): (1) The heavy chain variable region is the amino acid sequence shown in SEQ ID NO:36, and the light chain variable region is the amino acid sequence shown in SEQ ID NO:46; (2) The heavy chain variable region is the amino acid sequence shown in SEQ ID NO:37, and the light chain variable region is the amino acid sequence shown in SEQ ID NO:47; (3) The heavy chain variable region is an amino acid sequence as shown in SEQ ID NO:38, and the light chain variable region is an amino acid sequence as shown in SEQ ID NO:48; (4) The heavy chain variable region is an amino acid sequence as shown in SEQ ID NO:39, and the light chain variable region is an amino acid sequence as shown in SEQ ID NO:49; (5) The heavy chain variable region is an amino acid sequence as shown in SEQ ID NO:40, and the light chain variable region is an amino acid sequence as shown in SEQ ID NO:50; (6) The heavy chain variable region is the amino acid sequence shown in SEQ ID NO:41, and the light chain variable region is the amino acid sequence shown in SEQ ID NO:51; (7) The heavy chain variable region is the amino acid sequence shown in SEQ ID NO:42, and the light chain variable region is the amino acid sequence shown in SEQ ID NO:52; (8) The heavy chain variable region is an amino acid sequence as shown in SEQ ID NO:43, and the light chain variable region is an amino acid sequence as shown in SEQ ID NO:53; (9) The heavy chain variable region is the amino acid sequence shown in SEQ ID NO:44, and the light chain variable region is the amino acid sequence shown in SEQ ID NO:54; (10) The heavy chain variable region is an amino acid sequence as shown in SEQ ID NO:45, and the light chain variable region is an amino acid sequence as shown in SEQ ID NO:

55.

5. The use according to claim 1, further comprising a substance for detecting one or more of the following markers: β-catenin, CD10, vimentin, α1-antitrypsin.

6. The use according to claim 1, wherein the other pancreatic tumors not of SPN include pancreatic acinar cell carcinoma, pancreatoblastoma, pancreatic neuroendocrine tumor, and pancreatic ductal adenocarcinoma; wherein the pancreatic neuroendocrine tumor includes non-functional pancreatic neuroendocrine tumors and insulinomas.

7. A device for diagnosing or assisting in the diagnosis of SPN, characterized in that, Including detection systems and interpretation systems, The detection system is used to perform ABCD1 immunostaining on the subject's tumor tissue or tumor cells; The interpretation system is used to convert the results of the ABCD1 immunostaining into interpretation results, which refer to SPN or non-SPN.

8. A device for identifying or assisting in the identification of SPN (spinal necrotic neoplasm) from other pancreatic tumors, characterized in that, Including detection systems and interpretation systems, The detection system is used to perform ABCD1 immunostaining on the subject's tumor tissue or tumor cells; The interpretation system is used to convert the results of the ABCD1 immunostaining into interpretation results, which refer to SPN or other pancreatic tumors that are not SPN.

9. The device of claim 8, wherein the other pancreatic tumors not of the SPN include pancreatic acinar cell carcinoma, pancreatoblastoma, pancreatic neuroendocrine tumor, and pancreatic ductal adenocarcinoma; wherein the pancreatic neuroendocrine tumors include non-functional pancreatic neuroendocrine tumors and insulinomas.

10. The apparatus according to any one of claims 7-9, wherein the immunostaining comprises immunohistochemical staining, immunocytochemical staining, and immunofluorescence staining.

11. The apparatus according to claim 7 or 8, wherein the interpretation result is based on the expression intensity and / or distribution of ABCD1 in tumor tissue and / or tumor cells.

12. The apparatus of claim 7 or 8, wherein the ABCD1 immunostaining is performed by using an antibody that binds to ABCD1 or an antigen-binding fragment thereof.

13. The apparatus according to claim 12, wherein the amino acid sequence of the complementarity-determining region of the antibody binding to ABCD1 or its antigen-binding fragment is selected from at least one of the following groups (1) to (10): (1) The heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 are the amino acid sequences shown in SEQ ID NO:1, 9, 18, 25, RMS, and 31, respectively. (2) The heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 are the amino acid sequences shown in SEQ ID NO:2, 10, 19, 26, YAS, and 32, respectively. (3) The heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 are the amino acid sequences shown in SEQ ID NO:3, 11, 20, 27, WAS, and 33, respectively. (4) The heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 are the amino acid sequences shown in SEQ ID NO:1, 12, 18, 25, RMS, and 31, respectively; (5) The heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 are the amino acid sequences shown in SEQ ID NO:1, 13, 18, 25, RMS, and 31, respectively. (6) The heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 are the amino acid sequences shown in SEQ ID NO:4, 14, 21, 25, RMS, and 31, respectively. (7) The heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 are the amino acid sequences shown in SEQ ID NO:5, 15, 22, 28, RMS, and 31, respectively; (8) The heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 are the amino acid sequences shown in SEQ ID NO:6, 16, 23, 29, LVS, and 34, respectively; (9) The heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 are the amino acid sequences shown in SEQ ID NO:7, 17, 24, 30, WAS, and 35, respectively. (10) Heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 are the amino acid sequences shown in SEQ ID NO:8, 16, 23, 29, LVS, and 34, respectively.

14. The apparatus of claim 12, wherein the amino acid sequences of the heavy chain variable region and the light chain variable region of the antibody binding to ABCD1 or its antigen-binding fragment are selected from at least one of the following groups (1) to (10): (1) The heavy chain variable region is the amino acid sequence shown in SEQ ID NO:36, and the light chain variable region is the amino acid sequence shown in SEQ ID NO:46; (2) The heavy chain variable region is the amino acid sequence shown in SEQ ID NO:37, and the light chain variable region is the amino acid sequence shown in SEQ ID NO:47; (3) The heavy chain variable region is an amino acid sequence as shown in SEQ ID NO:38, and the light chain variable region is an amino acid sequence as shown in SEQ ID NO:48; (4) The heavy chain variable region is an amino acid sequence as shown in SEQ ID NO:39, and the light chain variable region is an amino acid sequence as shown in SEQ ID NO:49; (5) The heavy chain variable region is an amino acid sequence as shown in SEQ ID NO:40, and the light chain variable region is an amino acid sequence as shown in SEQ ID NO:50; (6) The heavy chain variable region is the amino acid sequence shown in SEQ ID NO:41, and the light chain variable region is the amino acid sequence shown in SEQ ID NO:51; (7) The heavy chain variable region is the amino acid sequence shown in SEQ ID NO:42, and the light chain variable region is the amino acid sequence shown in SEQ ID NO:52; (8) The heavy chain variable region is an amino acid sequence as shown in SEQ ID NO:43, and the light chain variable region is an amino acid sequence as shown in SEQ ID NO:53; (9) The heavy chain variable region is the amino acid sequence shown in SEQ ID NO:44, and the light chain variable region is the amino acid sequence shown in SEQ ID NO:54; (10) The heavy chain variable region is an amino acid sequence as shown in SEQ ID NO:45, and the light chain variable region is an amino acid sequence as shown in SEQ ID NO:55.

Images