A CPZ immunohistochemical antibody and its application

Abstract

The present application relates to the field of antibody technology and discloses a CPZ immunohistochemical antibody and its application. The antibody comprises a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 2 or SEQ ID NO: 6, and a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 4 or SEQ ID NO: 8. The antibody prepared in the present application can assist in the differential diagnosis of hepatocellular carcinoma and intrahepatic bile duct carcinoma, ultimately effectively using CPZ to differentiate between malignant and benign liver tumors, thereby improving the diagnostic accuracy of hepatocellular carcinoma.

Description

Technical Field

[0001] The present application relates to the field of antibody technology, and more specifically, to a CPZ immunohistochemical antibody and its application. Background Art

[0002] Immunohistochemistry utilizes the specific binding between antigens and antibodies and specialized labeling techniques to locate, qualitatively, or quantitatively detect specific antigens or antibodies within tissues and cells. Immunohistochemistry boasts strong specificity, high sensitivity, accurate localization, and the integration of morphology and function. It facilitates in-depth research in the field of pathology and plays a vital role in modern pathological diagnosis.

[0003] Pathology departments routinely use hundreds of immunohistochemical antibodies, which play a vital role in the diagnosis and treatment of human diseases. As our understanding of disease continues to improve, the variety of antibodies used in pathology has also gradually increased. Over the past decade, with the completion of the Human Genome Project and the advancement of protein profiling projects, the potential value of more and more antibodies is waiting to be discovered, expanding and adding to the catalog of antibodies used in pathology.

[0004] Antibodies used in immunohistochemistry, also known as primary antibodies, can be divided 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 majority of antibodies used in pathology departments.

[0005] During the production of monoclonal antibodies, many clones are generated, most of which lack strong specificity. Therefore, screening for clones with strong specificity is necessary. Clone screening is a highly specialized task, requiring not only experienced clinical pathologists but also a wealth of clinical specimens with varying positive intensities and ranges to select specific and stable subclonal cell lines.

[0006] Carboxypeptidases (CPs) are a class of peptidase enzymes that primarily act at the free carboxyl termini of peptide chains, releasing individual amino acids from the C-termini of proteins and polypeptides through hydrolysis. Based on their distinct catalytic mechanisms, carboxypeptidases can be divided into serine carboxypeptidases (acid carboxypeptidases), cysteine ​​carboxypeptidases (cathepsin X), and metallocarboxypeptidases (MCPs). The former two are relatively less studied. MCPs are primarily reported in insects such as the silkworm, where they play a crucial role in the digestion and absorption of dietary proteins. Carboxypeptidase Z (CPZ), a new member of the metallocarboxypeptidase family, also known as metallocarbonyl peptidase Z, is also a Zn-dependent enzyme. This enzyme was first cloned in humans in 1997 and in rats in 1998. The predominant form of CPZ mRNA in human salivary glands encodes a protein with an open reading frame of 641 amino acids, and three variants have been identified. In addition to the carboxypeptidase homology domain, CPZ also contains a 120-residue cysteine-rich domain (CRD), which has 20%-30% identity with the amino acid sequence of the frizzled protein (i.e., the Wnt receptor family) of Drosophila and mammals. The Wnt signaling pathway plays an important role in antagonizing or synergistically regulating tumor proliferation, migration, and invasion. It is speculated that CPZ may act as a regulator of the Wnt signaling pathway. Previous reports have shown that CPZ mRNA is abundant in rat placenta and has low to moderate levels in rat brain, lungs, thymus, and kidneys. Looking back at domestic and foreign literature reports, this patent explores for the first time the expression of CPZ in normal tissues and various tumor tissues throughout the body, and explores its potential differential diagnostic value in clinical applications, and has broad application prospects. Therefore, the present invention intends to prepare and screen this candidate molecule in order to identify clones with high sensitivity and excellent specificity to assist clinical diagnosis and explore more potential values. Summary of the Invention

[0007] The purpose of the present invention is to explore the expression of CPZ in normal tissues and various tumor tissues throughout the body, and to explore its potential differential diagnostic value in clinical applications to assist clinical diagnosis.

[0008] The positive rate in normal liver tissue is 100%, the positive expression rate in hepatocellular carcinoma is 91%, and the expression is mostly diffuse and strongly cytoplasmic. Intrahepatic cholangiocarcinoma has a positive expression rate of 28%, often with focal cytoplasmic positivity or a small amount of linear positivity at the luminal margin. Other primary liver tumors, such as hepatocellular adenoma, biliary adenoma / hamartoma, diffuse large B-cell lymphoma, and liver metastases, have negative positivity rates. In summary, CPZ is a highly promising differential diagnostic marker for hepatocellular carcinoma and has the potential to be further explored as a potential identification, prediction, and target for other organs, systems, tissues, and cells. Therefore, this research group intends to prepare and screen this candidate molecule.

[0009] In order to achieve the above-mentioned purpose of the invention, in the first aspect, the present invention provides a CPZ immunohistochemistry antibody and its application, wherein the antibody comprises a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 2 or SEQ ID NO: 6, and a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 4 or SEQ ID NO: 8.

[0010] In a second aspect, the present invention provides genes encoding the heavy chain variable region and light chain variable region of the above-mentioned CPZ immunohistochemical antibody.

[0011] Furthermore, the coding gene includes:

[0012] The gene encoding the heavy chain variable region with the amino acid sequence shown in SEQ ID NO: 2 has the sequence shown in SEQ ID NO: 1;

[0013] a gene encoding the heavy chain variable region whose amino acid sequence is set forth in SEQ ID NO: 6, whose sequence is set forth in SEQ ID NO: 5;

[0014] The gene encoding the light chain variable region with the amino acid sequence shown in SEQ ID NO: 4 has the sequence shown in SEQ ID NO: 3;

[0015] The gene encoding the light chain variable region with the amino acid sequence shown in SEQ ID NO: 8 has the sequence shown in SEQ ID NO: 7.

[0016] In a third aspect, the present invention provides the use of the CPZ immunohistochemical antibody in the preparation of a product for differential diagnosis of hepatocellular carcinoma and / or intrahepatic bile duct carcinoma.

[0017] Furthermore, the product for differential diagnosis of hepatocellular carcinoma and / or intrahepatic bile duct carcinoma includes a detection kit.

[0018] Compared with the prior art, this application has the following beneficial effects:

[0019] 1. The antibodies prepared by the present invention can assist in the differential diagnosis of hepatocellular carcinoma and intrahepatic bile duct carcinoma, and ultimately effectively use CPZ in the differentiation of malignant and benign liver tumors, thereby improving the diagnostic accuracy of hepatocellular carcinoma.

[0020] 2. Utilizing high-yield and mass-produced tissue core libraries, this study first discovered that CPZ is primarily distributed in the liver in normal tissue. Compared with single-item detection of Arg-1 or Hep Par-1, both single-item detection of CPZ and combined detection of these three indicators demonstrate higher sensitivity and improved diagnostic accuracy for the diagnosis of hepatocellular carcinoma, and are more helpful in diagnosing poorly differentiated hepatocellular carcinoma. Therefore, this method offers complementary advantages for the differential diagnosis of hepatocellular carcinoma. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 : In intrahepatic cholangiocarcinoma lesions that should not express CPZ, some cells showed positive staining, with a heavy background, and no improvement was seen even with dilution, indicating that this clone preparation failed;

[0022] Figure 2 : The background coloring is obvious. After serial dilution, clones of different concentrations always show positive reactions in lymphocytes and interstitial cells and are localized in the nucleus, indicating that the preparation of this clone has failed.

[0023] Figure 3 :The positive reaction is localized in the small bile duct, indicating that the clone has failed to produce;

[0024] Figure 4 : CPZ expression in eight major organs of the body: no expression in the ureters, tonsils, alveolar epithelium, small bile ducts of the liver, testes, striated muscle, thyroid gland, intestinal mucosa, appendix, brain, and spleen, but diffuse strong expression in hepatocytes and a small amount of positive expression in the gallbladder gland epithelium; the images show that this clone is clearly localized, with no background staining, and the positive organs, tissues, and cells are clearly defined;

[0025] Figure 5 : CPZ expression in eight major organs of the body: no expression in smooth muscle, prostate, breast, nerve bundles, adrenal glands, renal tubules and glomeruli, esophagus, placenta, gastric mucosa, blood vessels, endometrium, pancreatic acini and ducts, but diffuse strong expression in pancreatic islets; images show clear localization of this clone, no background staining, and clear positive organs, tissues, and cells;

[0026] Figure 6 : Expression of CPZ in liver tumors. CPZ is diffusely and strongly positive in hepatocellular carcinoma, but negative in intrahepatic cholangiocarcinoma, hepatic diffuse large B-cell lymphoma, and cavernous hemangioma.

[0027] Figure 7: Expression of CPZ in liver tumors. CPZ is not expressed in liver-metastatic colorectal cancer, liver-metastatic breast cancer, liver-metastatic neuroendocrine tumors and liver-metastatic gastrointestinal stromal tumors. DETAILED DESCRIPTION

[0028] The technical solutions and effects of the present application are further described in detail below with reference to the embodiments. It should be understood that the specific embodiments described herein are only used to explain the invention, rather than to limit the invention.

[0029] Example 1: Preparation of monoclonal antibodies

[0030] The CPZ fragment was selected, a plasmid constructed, and prokaryotic expression and protein production were performed. Mice were immunized and antigenicity analyzed. Antigen was emulsified and immunized into five Balb / c mice. Blood was collected from the immunized mice to assess immune efficacy and serum titer analysis by ELISA. Pre-fusion booster immunizations were performed, and myeloma cell lines were revived and acclimated, completing pre-fusion preparations. Mice with optimal immune efficacy (titer levels meeting the required requirements) were selected for cell fusion in 96-well cell culture plates. The cell fusion rate was measured. Culture supernatants from parent clones were tested by ELISA, and positive parent clones were selected for immunohistochemical screening (serial limiting dilutions of positive parent clones were performed, and culture supernatants were tested by ELISA. ELISA-positive subclones were selected for immunohistochemical screening). The supernatants of the ten clones with the best immunohistochemical specificity were tested. The two best positive subclones were selected for ascites production and were designated 3C5A10 and 1B5B9. 3C5A10 and 1B5B9 positive subclone cell lines were provided for ascites production and purification, with a purified antibody yield of 10 mg per clone.

[0031] RNA was extracted from 3C5A10 and 1B5B9 hybridoma cells and reverse transcribed by 5' end RACE. cDNA PCR was used to obtain the antibody heavy chain / light chain variable region genes. The heavy chain and light chain variable region genes were cloned and sequenced. Bioinformatics analysis was performed. The sequencing results are as follows:

[0032] 3C5A10 clone:

[0033] Heavy chain variable region sequence (SEQ ID NO: 1):

[0034] H-

[0035] GATGTGCAGCTTCAGGAGTCAGGACCTGACCTGGTGAAACCTTCTCAGTCACTTTCACTCACCTGCACTGTCACTGGCTACTCCATCACCAGTGGTTATAGCTGGCACTGGATCCGGCAGTTTCCAGGAAACAAACTGGAATGGATGGGCTACATACACTACAATAATAACACTAACTACAACCCATCTCTCAAAAGTCGAATCTCTATCACTCGAGACACATCCAAGAACCAGTTCTTCCTGCAGTTGAATTCTGTGACTACTGAGGACACAGCCAAATATTACTGTGCAAGGGCCGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA;

[0036] Heavy chain variable region amino acid sequence (Translated protein, SEQ ID NO: 2):

[0037] DVQLQESGPDLVKPSQSLSLTCTVTGYSITSGYSWHWIRQFPGNKLEWM GYIHYNNNTNYNPSLKSRISITRDTSKNQFFLQLNSVTTEDTAKYYCARADYW GQGTTLTVSS;

[0038] Light chain variable region gene sequence (Light chain variable region sequence, SEQ ID NO: 3):

[0039] K-

[0040] GATGTTGTGATGACCCAGACTCCACTCACTTTGTCGGTTACCATTGGACAACCAGCCTCCATCTCTTGCAAGTCAAGTCAGAGCCTCTTAGATAGTTATGGAAAGACATATTTGAATTGGTTGTTGCAGAGGCCAGGCCAGTCTCCAAAGCGCCTAATCTATCTGGTGTCTAAACTGGACTCTGGAGTCCCTGACAGGTTCACTGGCAGTGGATCAGGGACAGACTTCACACTGAAAATCAGCAGAGTGGAGGCTGAGGATTTGGGAGTTTACTATTGCTGGCAAGGTACACATTTTCCTCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAACGG;

[0041] Amino acid sequence of the light chain variable region (Translated protein, SEQ ID NO: 4):

[0042] DVVMTQTPLTLSVTIGQPASISCKSSQSLLDSYGKTYLNWLLQRPGQSPK RLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPLTF GAGTKLELKR;

[0043] Clone 1B5B9:

[0044] Heavy chain variable region gene sequence (SEQ ID NO: 5):

[0045] H-

[0046] GAGGTTCAACTGCAGCAGTCTGGGGCAGAGTTTGTGAAGCCAGGGGCCTCAGTCAAGTTGTCCTGCACAGCTTCTGACTTCAACATTAAAGACTCCTATATGCACTGGGTGAAGCAGAGGCCTGAACTGGGCCTGGAGTGGATTGGAAGGATTGATCCTGCGAATGGTAATACTCAATATGACCCGAAGTTCCAGGTCAAGGCCACTATAACAGCAGACACATCCTCCAACACAGCCTACCTGCAGCTCAGCAGCCTGACATCTGAGGACACTGCCGTCTATTCCTGTAGTAGAGTACGACGGGGGGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA;

[0047] Heavy chain variable region amino acid sequence (Translated protein, SEQ ID NO: 6):

[0048] EVQLQQSGAEFVKPGASVKLSCTASDFNIKDSYMHWVKQRPELGLEWIG RIDPANGNTQYDPKFQVKATITADTSSNTAYLQLSSLTSEDTAVYSCSRVRRGA MDYWGQGTSVTVSS;

[0049] Light chain variable region gene sequence (Light chain variable region sequence, SEQ ID NO: 7):

[0050] K-

[0051] GACATTTGTGATGTCACAGTCTCCATCCTCCCTAACTGTGTCAGTTGGAGAAGATTACTATGAACTGCAAGTCCAGTCAGAGCCTTTTATATAGTAACAATCACGAGAACTACTTGGCCTGGTACCAGCAGAAACCAGGGCAGTCTCCTAAACTGCTGATTTACTGGGCA TCCACTAGGGAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTGTGAAGGCTGAAGACCTGGCAGTTTTATTACTGTCAGCAATATTATACCTATCCGCTCACGTTCGGTACTGGGACCAAGCTGGAGCTGAAACGG;

[0052] Light chain variable region amino acid sequence (Translated protein, SEQ ID NO: 8):

[0053] DIVMSQSPSSLTVSVGEKITMNCKSSQSLLYSNNHENYLAWYQQKPGQSP KLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYTYPLTF GTGTKLELKR.

[0054] In the present invention, the preparation of CPZ antibody has gone through multiple rounds of repeated screening, cloning and subcloning to obtain the above-mentioned ideal antibody. Figures 1 to 3 The immunohistochemical results of the clones that failed to be prepared are shown to illustrate the difficult clone selection process of CPZ antibody. Figure 1 It showed that in the intrahepatic cholangiocarcinoma lesion that should not express CPZ, some cells stained positively, with a heavy background, and no improvement was seen even with dilution, indicating that this clone preparation failed; Figure 2 The background coloring is obvious. After serial dilution, clones of different concentrations always show positive reactions in lymphocytes and interstitial cells, and are localized in the nucleus, indicating that the preparation of this clone has failed. Figure 3 The positive reaction was localized in the small bile ducts, suggesting that the clone had failed to produce.

[0055] Example 2: Immunohistochemical detection of CPZ-3C5A10 antibody on normal tissue microarray

[0056] Immunohistochemical detection was performed using CPZ-3C5A10 antibody on normal tissue microarrays including ureter, tonsil, gallbladder, alveolar epithelium, liver, testis, myocardium, thyroid, intestinal mucosa, appendix, brain, spleen, smooth muscle, prostate, breast, nerve bundles, adrenal gland, renal tubules and glomeruli, esophagus, placenta, gastric mucosa, blood vessels, pancreas, and endometrium.

[0057] 1. Select tissue microarrays containing multiple normal tissues.

[0058] 2. PH9.0 antigen retrieval.

[0059] 3. Perform fully automated immunohistochemistry detection of CPZ-3C5A10 antibody on the DAKO platform.

[0060] 4. No expression was found in ureters, tonsils, alveolar epithelium, small bile ducts, testicles, myocardium, thyroid, intestinal mucosa, appendix, brain, spleen, smooth muscle, prostate, breast, nerve bundles, adrenal glands, renal tubules and glomeruli, esophagus, placenta, gastric mucosa, blood vessels, pancreatic acinar and pancreatic ducts, endometrium, etc. However, it was strongly expressed in normal hepatocytes and pancreatic islets, and a small amount of positive expression was found in gallbladder gland epithelium (such as Figure 4 、 Figure 5 shown).

[0061] Example 3: Immunohistochemical detection of CPZ antibodies on tissue chips containing various liver tumors

[0062] Immunohistochemical detection of CPZ antibody was performed on tissue microarrays containing various liver tumors.

[0063] 1. Select liver tumor tissue microarrays containing different histological types.

[0064] 2. PH9.0 antigen retrieval.

[0065] 3. Perform fully automated immunohistochemistry detection of CPZ-3C5A10 antibody on the DAKO platform.

[0066] 4. The results showed that CPZ-3C5A10 was strongly positive in hepatocellular carcinoma and negative in intrahepatic bile duct carcinoma (e.g. Figure 6 、 Figure 7 shown).

[0067] This specific embodiment is merely an explanation of the present application and is not a limitation of the present application. After reading this specification, those skilled in the art may make non-creative modifications to the present embodiment as needed, but as long as they are within the scope of the claims of the present application, they are protected by the patent law.

Claims

1. A carboxypeptidase Z immunohistochemical antibody, characterized in that The antibody comprises a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 2, and a light chain variable region having an amino acid sequence as shown in SEQ ID NO:

4.

2. A gene encoding the heavy chain variable region and light chain variable region of the carboxypeptidase Z immunohistochemical antibody according to claim 1.

3. The coding gene according to claim 2, characterized in that include: a gene encoding the heavy chain variable region whose amino acid sequence is shown in SEQ ID NO: 2, whose sequence is shown in SEQ ID NO: 1; The gene encoding the light chain variable region with the amino acid sequence shown in SEQ ID NO: 4 has the sequence shown in SEQ ID NO:

3.

4. Use of the carboxypeptidase Z immunohistochemical antibody according to claim 1 in the preparation of an immunohistochemical product for distinguishing hepatocellular carcinoma from intrahepatic cholangiocarcinoma.

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