An akrlc1 immunohistochemistry antibody and uses thereof

Abstract

The application discloses an AKR1C1 immunohistochemical antibody and application thereof. The AKR1C1 immunohistochemical antibody provided by the application comprises a heavy chain variable region and a light chain variable region, the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO: 2, the amino acid sequence of the light chain variable region is shown as SEQ ID NO: 4, or the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO: 6, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO: 8. The antibody provided by the application can be used for identifying normal cells and tumor cells related to AKR1C1, identifying each subtype of breast cancer, and identifying tissue tumor subtypes including liposarcoma, leiomyosarcoma, gastrointestinal stromal tumor and rhabdomyosarcoma, and is an immunohistochemical detection antibody with great application potential.

Description

Technical Field

[0001] This invention relates to an AKR1C1 immunohistochemical antibody and its application, belonging to the field of biomedical technology. Background Technology

[0002] Immunohistochemistry is a technique that utilizes the principle of specific binding between antigens and antibodies and special labeling techniques to locate, qualitatively identify, or quantitatively detect specific antigens or antibodies within tissues and cells. Immunohistochemistry is characterized by high specificity, high sensitivity, accurate localization, and the integration of morphology and function, which is beneficial for in-depth research in the field of pathology and plays an important role in modern pathological diagnosis.

[0003] Pathology departments routinely use hundreds of immunohistochemical antibodies, playing a vital role in the diagnosis and treatment of human diseases. As our understanding of diseases continues to improve, the types of antibodies used in pathology are also gradually increasing. With the completion of the Human Genome Project and the advancement of the Proteome Project in the past decade, the potential value of more and more antibodies awaits discovery, and they await continuous expansion and addition to the catalog of antibodies used in pathology departments.

[0004] Antibodies used in immunohistochemistry, also known as primary antibodies, can be classified into monoclonal antibodies and polyclonal antibodies based on their preparation method. Monoclonal antibodies are widely favored by immunohistochemistry users due to their high specificity and account for the vast majority of antibodies used in pathology departments.

[0005] The application value of immunohistochemical antibodies encompasses multiple aspects: First, immunohistochemical antibodies can assist in diagnosis and differential diagnosis, providing objective evidence for pathological and differential diagnoses. For infectious diseases, immunohistochemistry can identify specific bacteria, viruses, and other microorganisms, improving the accuracy of pathological diagnosis. For neoplastic diseases, immunohistochemistry also provides objective diagnostic evidence to assist in pathological diagnosis through various pathways: such as determining tumor benignity or malignancy; tumor cell origin / differentiation direction; helping pathologists determine primary or metastatic tumors; differentiating tumor types; tumor grading; and detecting and identifying micrometastases. Second, it provides objective guidance for disease treatment, namely companion diagnostics: determination of tumor targets for molecularly targeted drugs; prediction of tumor chemotherapy response; and assessment of tumor prognosis. Due to the broad-spectrum clinical applications of immunohistochemistry, research on antibody applications is flourishing.

[0006] Chinese patent CN 114874318 A discloses an NG-2 immunohistochemical antibody, mainly used for the identification of NG-2-related normal cells and tumor cells, as well as the differential diagnosis of esophageal squamous cell carcinoma and esophageal basal squamous cell carcinoma; Chinese patent CN116217721A discloses a C14orf78 immunohistochemical antibody and its preparation method and application, wherein the C14orf78 immunohistochemical antibody is mainly used for the identification of C14orf78-related normal cells and tumor cells, as well as the differential diagnosis of thyroid tumors, liver tumors and kidney tumors.

[0007] However, there are currently no studies or reports on immunohistochemical antibodies related to the AKR1C1 gene and protein. Summary of the Invention

[0008] The technical problem to be solved by this invention is how to prepare and apply the AKR1C1 immunohistochemical antibody.

[0009] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0010] In a first aspect, the present invention provides an AKR1C1 immunohistochemical antibody, the antibody comprising a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 2; the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 4; or, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 6; and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 8.

[0011] In a second aspect, the present invention provides a gene encoding the heavy chain variable region and the light chain variable region of an AKR1C1 immunohistochemical antibody, wherein the gene sequence encoding the heavy chain variable region is shown in SEQ ID NO: 1 and the gene sequence encoding the light chain variable region is shown in SEQ ID NO: 3; or, the gene sequence encoding the heavy chain variable region is shown in SEQ ID NO: 5 and the gene sequence encoding the light chain variable region is shown in SEQ ID NO: 7.

[0012] A third aspect of the present invention provides the use of an AKR1C1 immunohistochemical antibody in the preparation of a kit for identifying normal cells and tumor cells associated with AKR1C1, said antibody comprising a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 2 and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 4; or, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 6 and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 8.

[0013] In a fourth aspect, the present invention provides the application of an AKR1C1 immunohistochemical antibody in the preparation of a diagnostic kit for identifying breast cancer subtypes, the antibody comprising a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 2 and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 4; or, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 6 and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 8.

[0014] Preferably, the breast cancer subtype includes any one of the following: apocrine carcinoma of the breast, invasive ductal carcinoma of the breast, non-specific invasive carcinoma of the breast, mucinous carcinoma of the breast, solid papillary carcinoma of the breast, micropapillary carcinoma of the breast, lobular carcinoma of the breast, neuroendocrine carcinoma, squamous cell carcinoma, fibroadenoma, and phyllodes tumor.

[0015] In a fifth aspect, the present invention provides the application of an AKR1C1 immunohistochemical antibody in the preparation of a diagnostic kit for identifying tissue tumor subtypes, wherein the tissue tumor subtypes include any one of liposarcoma, leiomyosarcoma, gastrointestinal stromal tumor, and rhabdomyosarcoma; the antibody includes a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 2, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 4; or, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 6, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 8.

[0016] This invention prepares an AKR1C1 immunohistochemical antibody from a discovered candidate molecular library. This antibody specifically recognizes apocrine glands and apocrine carcinoma of the breast, and can differentiate between apocrine gland carcinoma, invasive ductal carcinoma, and other rare types of breast cancer. The positive rate is 98% in normal apocrine gland tissue, negative in normal breast ducts and alveoli, and 90% in apocrine gland carcinoma, mostly diffusely strong positive. The positive rate for other invasive ductal carcinomas of the breast is approximately 15%, often focally positive. It is negative for special types of breast cancer, such as mucinous carcinoma, solid papillary carcinoma, micropapillary carcinoma, and lobular carcinoma. Furthermore, the AKR1C1 immunohistochemical antibody of this invention can also differentiate between liposarcoma, leiomyosarcoma, rhabdomyosarcoma, and gastrointestinal stromal tumors. Therefore, the AKR1C1 immunohistochemical antibody of this invention is a highly promising diagnostic indicator for various subtypes of breast cancer and tissue tumors, and has further value for exploration as a potential identification, prediction, and target at the organ, system, tissue, and cellular levels.

[0017] Compared with the prior art, the present invention has the following advantages:

[0018] (1) The AKR1C1 immunohistochemical antibody of the present invention is an antibody for immunohistochemical staining of tissues and cells, used to display AKR1C1-related normal cells and tumor cells, so as to achieve the purpose of tissue and cell typing and tumor tissue differential diagnosis; In addition, based on the solution provided by the present invention, it can also provide directions for exploring the further application of AKR1C1 as a potential identification, prediction and target at the level of other organs, systems, tissues and cells;

[0019] (2) The AKR1C1 immunohistochemical antibody of the present invention can specifically recognize apocrine glands and apocrine gland carcinoma of the breast, and can differentiate between apocrine gland carcinoma of the breast and invasive ductal carcinoma, as well as other rare types of breast cancer. The positive rate of normal apocrine gland tissue is 98%, while that of normal breast ducts and alveoli is negative, and the positive rate of apocrine gland carcinoma of the breast is 90%. In addition, the AKR1C1 immunohistochemical antibody of the present invention can also differentiate between liposarcoma, leiomyosarcoma, rhabdomyosarcoma, and gastrointestinal stromal tumors. It has great application potential.

[0020] (3) There are many types of breast tumors, including invasive ductal carcinoma, invasive lobular carcinoma, apocrine carcinoma, metaplastic carcinoma, solid papillary carcinoma, mucinous carcinoma, neuroendocrine carcinoma, squamous cell carcinoma, as well as fibroadenoma, phyllodes tumor, etc. Detailed data on the expression of AKR1C1 in various subtypes of breast cancer can be obtained through immunohistochemical detection, and AKR1C1 can be effectively used for the identification of apocrine carcinoma of the breast and the differentiation of various subtypes. Attached Figure Description

[0021] Figure 1 : Immunohistochemical staining pattern of the immunohistochemical antibody AKR1C1-7B11A10 in normal tissue;

[0022] Figure 2 Immunohistochemical staining image of immunohistochemical antibody AKR1C1-7B11A10 in breast tumor tissue.

[0023] Figure 3 Immunohistochemical staining image of immunohistochemical antibody AKR1C1-7B11A10 in liposarcoma, leiomyosarcoma, gastrointestinal stromal tumor, and rhabdomyosarcoma. Detailed Implementation

[0024] To make the present invention more apparent and understandable, preferred embodiments are described in detail below with reference to the accompanying drawings.

[0025] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0026] Example 1: Preparation of Monoclonal Antibodies

[0027] 1. Antigen preparation

[0028] 1.1 Locate the target gene sequence in the database, synthesize it, and construct it into a prokaryotic expression vector;

[0029] 1.2 The plasmid was transformed into E. coli, and protein expression was induced by IPTG;

[0030] 1.3 Proteins were purified using affinity purification methods.

[0031] 2. Animal Immunization and Potency Determination

[0032] 2.1 The antigen and adjuvant were mixed and emulsified on an emulsifier. The emulsified antigen was injected into mice via intraperitoneal injection. A total of 5 mice were immunized. The second injection was given 3 weeks later, and the third injection was given 2 weeks later.

[0033] 2.2 Ten days after the third immunization, blood was collected from the tail of the mice and serum was separated. The serum titer of the mice was determined by indirect ELISA.

[0034] 2.3 Select the mouse with the best serum titer and give it a booster immunization three days before fusion, without adjuvant, only by injecting the antigen.

[0035] 3. Cell fusion

[0036] 3.1 Prematurely revive and expand the culture of SP2 / 0 cells;

[0037] 3.2 Mice were sacrificed, their spleens were harvested and ground to prepare a cell suspension. Myeloma cells and spleen cells were mixed in a specific ratio, and PEG was added to promote fusion. The fused cells were transferred to 96-well plates and cultured in HAT medium. After a period of time, the cells that did not fuse successfully died.

[0038] 4. Cell screening

[0039] 4.1 Collect cell supernatant and use ELISA + immunohistochemistry for dual screening. Transfer wells with high titers and clear localization to 24-well plates and place them in a 37℃ CO2 incubator for extended culture.

[0040] 4.2 Limiting dilution method for clonal culture: Cell suspensions from 24-well plates were transferred to new 96-well plates, ensuring each well contained only one cell, and incubated in a 37°C CO2 incubator. After 10 days, double screening was performed using ELISA and immunohistochemistry to select clones with high titers and clear localization for further culture.

[0041] 5. Subtype identification and antibody sequencing

[0042] 5.1 The light and heavy chain isotypes of the selected clones with high titers and clear localization were identified using ELISA. The two best positive subclones were selected for ascites production preparation; the positive subclones were designated 7B11A10 and 5C5G5. The 7B11A10 positive subclone cell line was used for further ascites production and purification, with a purified antibody concentration of 10 mg / clone.

[0043] 5.2 The RACE method was used to sequence the heavy and light chains of hybridoma cells separately. The specific steps were as follows: hybridoma cell culture; hybridoma cell mRNA extraction; RNA reverse transcription to obtain cDNA; cDNA PCR to obtain the antibody heavy / light chain variable region gene; antibody gene ligation into a T vector; selection of positive clones and sequencing; bioinformatics analysis of the sequencing results to obtain the antibody sequence. The specific sequencing results are as follows:

[0044] 7B11A10 clone:

[0045] Heavy chain variable region sequence (SEQ ID NO: 1)

[0046] H-:

[0047] CAGGGTGCAGCTGCAGCAGTCTGGGGCTGAGCTTGTGAGGCCAGGGGCCTTAGTCAAGTTGTCCTGCAAAGCTTCTGGCTTCAACATTAAAGACTACTAT ATTCACTGGGTGAAGCAGAGGCCTGAACAGGGCCTGGAGTGGATTGGATGGATTGATCCTGAGAATGGTCATACTGTATATGACCCGAAGTTCCAGGGCAAGGCCATTATAACAGCAGACACATCCTCCAAC ACAGCCTACCTGCAACTCAGCAGCCTGACATCTGAGGACACTGCCGTCTATTACTGTGCTAGCCCTTATGCTTACGACGGATTACTAGCTATGGACCACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA;

[0048] Heavy chain variable region amino acid sequence (Translated protein, SEQ ID NO: 2):

[0049] QVQLQQSGAELVRPGALVKLSCKASGFNIKDYYIHWVKQRPEQGLEWIG WIDPENGHTVYDPKFQGKAIITADTSSNTAYLQLSSLTSEDTAVYYCASPYAYD GLLAMDHWGQGTSVTVSS;

[0050] Light chain variable region sequence (SEQ ID NO: 3)

[0051] K-:

[0052] GATATTGTGATGACTCAGACTGCACCCTCTGTTCCTGTCACTCCTGGAGAGTCAGTATCCATCTCCTGCAGGTCTAGTAAGAGTCTCCTGCATAGTACTGGCAATACTTACTTGTATTGGTTCCTGCAGAGGCCAGGCCAGTCTCCTCAGCTCCTGATATATCGGATGTC CAACCTTGTCTCAGGAGTCCCAGACAGGTTCAGTGGCAGTGGGTCAGGAACTGCTTTCACACTGACAATCAGTAGATGGAGGCTGAGGATGTGGGTGTTTATTACTGTATGCAACATCTAGAATATCCTTTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAACGG;

[0053] Amino acid sequence of the light chain variable region (Translated protein, SEQ ID NO: 4)

[0054] DIVMTQTAPSVPVTPGESVSISCRSSKSLLHSTGNTYLYWFLQRPGQSPQL LIYRMSNLVSGVPDRFSGSGSGTAFTLTISRVEAEDVGVYYCMQHLEYPFTFG AGTKLELKR.

[0055] 5C5G5 clone:

[0056] Heavy chain variable region sequence (SEQ ID NO: 5)

[0057] H-:

[0058] CAAGTACAGCTGCAGGAGTCTGGAGGAGGCTTGGTGCAACCAGGAGGATCCATGAAACTCTCCTGTGTTGCCTCTGGATTCATTGTCAGTAACTATTGGATGAACTGGGTCCGCCAGTCTCCAGAGAAGGGGCTTGAGTGGGTTGCTGAAATTAGATTGAAATCTAATAATTATGCAACACATTATGCGGAGTCTGTGAAA GGGAGGTTCACCATCTCAAGAGATGATTCCAAAAGTAGTGTCTATCTGCAAATGAACAACTTAAGAGTTGAAGACACTGGCATTTATTACTGTACCATCATTAGTTATTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA;

[0059] Heavy chain variable region amino acid sequence (Translated protein, SEQ ID NO: 6)

[0060] QVQLQESGGGLVQPGGSMKLSCVASGFIVSNYWMNWVRQSPEKGLEWV AEIRLKSNNYATHYAESVKGRFTISRDDSKSSVYLQMNNLRVEDTGIYYCTIIS YWGQGTTLTVSS;

[0061] Light chain variable region gene sequence (Light chain variable region sequence, SEQ ID NO: 7)

[0062] K-:

[0063] GATATTGTGATGACGCAGGCTGCATTCTCCAATCCAGTCACTCTTGGAACATCAGCTTCCATCTCCTGCAGGTCTAGTAAGAGTCTCCTACATAGTAATGGCATCACTTATTTGTATTGGTATCTGCAGAAGCCAGGCCAGTCTCCTCAGCTCCTGATTTATCAGATGTC CAACCTTGCCCCAGGAGTCCCAGACAGGTTCAGTAGCAGTGGGTCAGGAACTGATTTCACACTGAGAATCAGCAGAGTGGAGGCTGAGGATGTGGGTGTTTATTACTGTGGTCAAAATCTAGAACTTCCGTGGACGTTCGGTGGAGGCACGAAGCTGGAAATCAAACGG;

[0064] Amino acid sequence of the light chain variable region (Translated protein, SEQ ID NO: 8)

[0065] DIVMTQAAFSNPVTLGTSASISCRSSKSLLHSNGITYLYWYLQKPGQSPQL LIYQMSNLAPGVPDRFSSSGSGTDFTLRISRVEAEDVGVYYCGQNLELPWTFG GGTKLEIKR.

[0066] 6. Monoclonal antibody production

[0067] 6.1 Animal induction method for ascites preparation: Healthy BALB / c mice aged 10 weeks were selected and intraperitoneally injected with 0.5 ml of incomplete adjuvant 3-7 days prior to the procedure. The selected high-titer, well-defined clone 7B11A10 was cultured, and 2 × 10⁻⁶ cells were collected. 6 After washing the cells with sterile PBS, they were injected into the peritoneal cavity of mice. Ascites was usually produced sufficiently in about 7-10 days, and the ascites could then be harvested.

[0068] 6.2 Antibody purification: Ascites fluid was centrifuged, the supernatant was collected, and the antibody was purified from the supernatant using Protein A / G affinity purification method and then verified by ELISA and immunohistochemistry.

[0069] Example 2

[0070] Immunohistochemical detection was performed on normal tissue microarrays such as skin and organs using the AKR1C1-7B11A10 antibody:

[0071] 1. Select tissue microarrays containing normal tissues such as skin, testes, prostate, ureter, tonsils, thyroid gland, appendix, gallbladder, abdominal soft tissue, spleen, kidney tissue, bone marrow, tendons, brain tissue, adrenal gland, lung, placenta, pancreas, submandibular gland, parotid gland, gastric mucosa, small intestinal mucosa, duodenal mucosa, and liver tissue.

[0072] 2. PH9.0 antigen retrieval.

[0073] 3. Perform fully automated immunohistochemical detection of AKR1C1-7B11A10 antibody on the Roche platform.

[0074] 4. The results showed that AKR1C1-7B11A10 was not expressed in the skin, testes, prostate, tonsils, spleen, nerve bundles, or brain tissue, nor in the lungs, placenta, submandibular gland, ovary, or parotid gland. However, it was strongly expressed in the ureteral epithelium, exhibiting organ specificity. Figure 1 As shown.

[0075] Example 3

[0076] Immunohistochemical detection was performed on a breast tumor tissue microarray using the AKR1C1-7B11A10 antibody:

[0077] 1. Select tissue microarrays containing breast tumors of different tissue types.

[0078] 2. PH9.0 antigen retrieval.

[0079] 3. Perform fully automated immunohistochemical detection of AKR1C1-7B11A10 on the Roche platform.

[0080] 4. Results: AKR1C1-7B11A10 was strongly positive in apocrine breast cancer (86% positivity rate), but negative in invasive carcinoma and other rare subtypes of breast cancer. It has differential diagnostic value in breast cancer subtypes, such as... Figure 2 As shown.

[0081] Example 4

[0082] Immunohistochemical detection was performed on tissue microarrays of liposarcoma and other partial soft tissue tumors using the AKR1C1-7B11A10 antibody.

[0083] 1. Select tissue microarrays containing liposarcoma, leiomyosarcoma, gastrointestinal stromal tumor, and rhabdomyosarcoma.

[0084] 2. PH9.0 antigen retrieval.

[0085] 3. Perform fully automated immunohistochemical detection of AKR1C1-7B11A10 on the Roche platform.

[0086] 4. Results: AKR1C1-7B11A10 was strongly positive in liposarcoma (90% positivity rate), but negative in leiomyosarcoma, gastrointestinal stromal tumors, and rhabdomyosarcoma, thus having diagnostic value in differential diagnosis. Figure 3 As shown.

Claims

1. An AKR1C1 immunohistochemical antibody, characterized in that, The antibody includes a heavy chain variable region and a light chain variable region, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 2; The amino acid sequence of the variable region of the light chain is shown in SEQ ID NO:

4.

2. A nucleic acid molecule encoding the heavy chain variable region and the light chain variable region of the AKR1C1 immunohistochemical antibody as described in claim 1, characterized in that, The gene sequence encoding the heavy chain variable region is shown in SEQ ID NO: 1, and the gene sequence encoding the light chain variable region is shown in SEQ ID NO:

3.

3. The application of an AKR1C1 immunohistochemical antibody in the preparation of a diagnostic kit for identifying breast cancer subtypes, characterized in that, The antibody comprises a heavy chain variable region and a light chain variable region. The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 2, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

4. The identification of breast cancer subtypes is to differentiate between apocrine carcinoma in situ and non-specific invasive carcinoma of the breast, solid papillary carcinoma of the breast, invasive lobular carcinoma of the breast, and apocrine carcinoma in situ of the breast mucinous carcinoma.

4. The application of an AKR1C1 immunohistochemical antibody in the preparation of a diagnostic kit for identifying breast cancer subtypes, characterized in that, The antibody comprises a heavy chain variable region and a light chain variable region. The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 2, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

4. The identification of breast cancer subtypes is to differentiate between invasive apocrine carcinoma of the breast and non-specific invasive carcinoma of the breast, solid papillary carcinoma of the breast, invasive lobular carcinoma of the breast, and invasive apocrine carcinoma of the breast mucinous carcinoma.

5. The application of an AKR1C1 immunohistochemical antibody in the preparation of a diagnostic kit for identifying tissue tumor subtypes, characterized in that, The identification of tissue tumor subtypes is to differentiate between liposarcoma and leiomyosarcoma, gastrointestinal stromal tumor, and liposarcoma among rhabdomyosarcomas; the antibody includes a heavy chain variable region and a light chain variable region, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 2, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 4.

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