CPZ immunohistochemical antibody and application

Abstract

The invention relates to the technical field of antibodies, and discloses a CPZ immunohistochemical antibody and application, 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. The antibody prepared by the invention can assist in differential diagnosis of hepatocellular carcinoma and intrahepatic cholangiocarcinoma, and finally CPZ is effectively applied to identification of liver malignant tumors and benign tumors, so that the diagnosis accuracy of hepatocellular carcinoma is improved.

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 is a technique that uses the specific binding principle between antigens and antibodies and special labeling techniques to locate, qualitatively or quantitatively detect specific antigens or antibodies in tissues and cells. Immunohistochemistry has the characteristics of strong specificity, high sensitivity, accurate positioning, and the combination of morphology and function. It is conducive to in-depth research in the field of pathology and plays an important role in modern pathological diagnosis.

[0003] There are hundreds of immunohistochemical antibodies used in the daily work of the pathology department, which play an important role in the diagnosis and treatment of human diseases. As human understanding of diseases continues to improve, the types of antibodies used in the pathology department are gradually increasing. In the process of completing the human genome project and advancing the protein spectrum project in the past decade, the potential value of more and more antibodies is waiting to be discovered, waiting to be continuously expanded and added to the catalog of antibody applications in the pathology department.

[0004] The antibodies used in immunohistochemistry, also known as primary antibodies, can be divided into monoclonal antibodies and polyclonal antibodies according to their preparation methods. 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] During the preparation of monoclonal antibodies, many clones will be produced, most of which are not very specific. Therefore, it is necessary to screen clones with strong specificity. Clone screening is a very professional job that requires not only senior professional pathologists on the front line of clinical practice, but also a wealth of clinical specimens with different positive intensities and positive ranges to select specific and stable subclonal cell lines.

[0006] Carboxypeptidases (CPs) are a class of peptide chain end-lyases that mainly act on the free carboxyl termini of peptide chains, releasing single amino acids from the C-termini of proteins and polypeptides by hydrolysis. According to their different catalytic mechanisms, carboxypeptidases can be divided into serine carboxypeptidases (acid carboxypeptidases), cysteine ​​carboxypeptidases (cathepsin X) and metallocarboxypeptidases (MCPs), with the former two being relatively less studied. MCPs are mostly reported in insects such as silkworms, and play an important role in the digestion and absorption of their food proteins. Carboxypeptidase Z (CPZ) is a new member of the metallocarboxypeptidase family, also known as metallocarbonylpeptidase Z, and is also a Zn-dependent enzyme. The enzyme was first cloned in humans in 1997 and in rats in 1998. The main 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 found. 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 fruit flies 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 serve as a regulator of the Wnt signaling pathway. It has been previously reported that CPZ mRNA is abundant in rat placenta and has low to moderate content in rat brain, lung, thymus, and kidney. Tracing back domestic and foreign literature reports, the present invention patent explores the expression of CPZ in normal tissues and various tumor tissues throughout the body for the first time, 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 application to assist clinical diagnosis.

[0008] The positive rate of normal liver tissue is 100%, the positive expression rate of hepatocellular carcinoma is 91%, and most of them are diffuse strong cytoplasm positivity, while the positive expression rate of intrahepatic cholangiocarcinoma is 28%, often with focal cytoplasmic positive expression or a small amount of linear positive expression at the edge of the cavity. Other primary liver tumors such as hepatocellular adenoma, bile duct adenoma / hamartoma, diffuse large B-cell lymphoma, and liver metastatic tumors have negative positive rates. In summary, CPZ is a highly potential differential diagnostic indicator for hepatocellular carcinoma, and has the value of further exploration as a potential identification, prediction and target at the level of 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 whose amino acid sequence is shown in SEQ ID NO:2 or SEQ ID NO:6, and a light chain variable region whose amino acid sequence is 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 comprises:

[0012] A gene encoding a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 2, having a sequence as shown in SEQ ID NO: 1;

[0013] A gene encoding a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 6, having a sequence as shown in SEQ ID NO: 5;

[0014] A gene encoding a light chain variable region having an amino acid sequence as shown in SEQ ID NO:4, having a sequence as 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 cholangiocarcinoma.

[0017] Furthermore, the product for differential diagnosis of hepatocellular carcinoma and / or intrahepatic cholangiocarcinoma 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 cholangiocarcinoma, and ultimately effectively use CPZ in the differentiation of malignant tumors and benign tumors of the liver, thereby improving the diagnostic accuracy of hepatocellular carcinoma.

[0020] 2. The present invention uses high-yield and mass-produced tissue core libraries to discover for the first time that CPZ is mainly distributed in the liver in normal tissues. Compared with single detection of Arg-1 or Hep Par-1, whether it is single detection of CPZ or combined detection of the above three indicators, it has higher sensitivity and better diagnostic accuracy for diagnosing hepatocellular carcinoma, and is more helpful for diagnosing poorly differentiated hepatocellular carcinoma. Therefore, it is complementary to the differential diagnosis of hepatocellular carcinoma. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 : In the intrahepatic cholangiocarcinoma lesions that should not express CPZ, some cells were positively stained, the background was heavy, and there was no improvement even after dilution, indicating that the preparation of this clone failed;

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

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

[0024] Figure 4 :CPZ expression in the eight major organs of the body: no expression in ureter, tonsil, alveolar epithelium, small bile duct of liver, testis, striated muscle, thyroid gland, intestinal mucosa, appendix, brain, spleen, diffuse strong expression in liver cells, and a small amount of positive expression in gallbladder gland epithelium; the picture shows that this clone is clearly positioned, without background coloring, and the positive organs, tissues and cells are clear;

[0025] Figure 5 :CPZ expression in the eight major organs of the body: no expression in smooth muscle, prostate, breast, nerve bundle, adrenal gland, renal tubules and glomeruli, esophagus, placenta, gastric mucosa, blood vessels, endometrium, pancreatic acini and ducts, but diffuse strong expression in pancreatic islets; the image shows that this clone is clearly positioned, without background coloring, and the positive organs, tissues and cells are clear;

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

[0027] Figure 7:The 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 scheme and effects of the present application are further described in detail below in conjunction with 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] Select CPZ fragments, construct plasmids, prokaryotic expression and protein preparation. Immunize mice, analyze antigenicity; emulsify antigens and immunize 5 Balb / c mice; collect blood from immunized mice, detect immune effects and perform ELISA serum titer analysis, boost immunization before fusion, resuscitate and acclimate myeloma cell lines, and prepare for fusion. Select mice with ideal immune effects (titer can meet requirements) and perform cell fusion on 96-well cell culture plates; test cell fusion rate; perform ELISA test on the culture supernatant of the mother clone, select positive mother clones and perform immunohistochemical screening (perform serial limiting dilution of positive mother clones, perform ELISA test on the culture supernatant, select ELISA positive subclones for immunohistochemical screening), and select the supernatant of the 10 clones with the best immunohistochemical specificity for detection. Select the best two positive subclones for ascites production preparation, the positive subclone codes are: 3C5A10 and 1B5B9. Provide 3C5A10 and 1B5B9 positive subclone cell lines and perform ascites production and purification, and the purified antibody amount is 10 mg / clone.

[0031] RNA of 3C5A10 and 1B5B9 hybridoma cells was extracted, and 5' end RACE reverse transcription was performed. 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, and the sequencing results were as follows:

[0032] 3C5A10 clone:

[0033] Heavy chain variable region gene sequence (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] DVQLQESGPDLVKPSQSLSLTCTVTGYSITSGYSWHWIRQFPGNKLEWMGYIHY NNNTNYNPSLKSRISITRDTSKNQFFLQLNSVTTEDTAKYYCARADYWGQGTTLTVS S;

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

[0039] K-

[0040] GATGTTGTGATGACCCAGACTCCACTCACTTTGTCGGTTACCATTGGACAACCAGCCTCCATCTCTTGCAAGTCAAGTCAGAGCCTCTTAGATAGTTATGGAAAGACATATTTGAATTGGTTGTTGCAGAGGCCAGGCCAGTCTCCAAAGCGCCTAATCTATCTG GTGTCTAAACTGGACTCTGGAGTCCCTGACAGGTTCACTGGCAGTGGATCAGGGACAGACTTCACACTGAAAATCAGCAGAGTGGAGGCTGAGGATTTGGGAGTTTACTATTGCTGGCAAGGTACACATTTTCCTCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAACGG;

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

[0042] DVQLQESGPDLVKPSQSLSLTCTVTGYSITSGYSWHWIRQFPGNKLEWMGYIHY NNNTNYNPSLKSRISITRDTSKNQFFLQLNSVTTEDTAKYYCARADYWGQGTTLTVS S;

[0043] Clone 1B5B9:

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

[0045] H-

[0046] GAGGTTCAACTGCAGCAGTCTGGGGCAGAGTTTGTGAAGCCAGGGGCCTCAGTCAAGTTGTCCTGCACAGCTTCTGACTTCAACATTAAAGACTCCTATATGCACTGGGTGAAGCAGAGGCCTGAACTGGGCCTGGAGTGGATTGGAAGGATTGATCCTGCGAATGGTAATACTCAATATGACCCGAAGTTCCAGGTCAAGGCCACTATAACAGCAGACACATCCTCCAACACAGCCTACCTGCAGCTCAGCAGCCTGACATCTGAGGACACTGCCGTCTATTCCTGTAGTAGAGTACGACGGGGGGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA;

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

[0048] EVQLQQSGAEFVKPGASVKLSCTASDFNIKDSYMHWVKQRPELGLEWIGRIDPA NGNTQYDPKFQVKATITADTSSNTAYLQLSSLTSEDTAVYSCSRVRRGAMDYWGQGT SVTVSS;

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

[0050] K-

[0051] GACATTTGTGATGTCACAGTCTCCATCCTCCCTAACTGTGTCAGTTGGAGAAGATTACTATGAACTGCAAGTCCAGTCAGAGCCTTTTATATAGTAACAATCACGAGAACTACTTGGCCTGGTACCAGCAGAAACCAGGGCAGTCTCCTAAACTGCTGATTTAC TGGGCATCCACTAGGGAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTGTGAAGGCTGAAGACCTGGCAGTTTTATTACTGTCAGCAATATTATACCTATCCGCTCACGTTCGGTACTGGGACCAAGCTGG AGCTGAACGG;

[0052] Light chain variable region amino acid sequence (Translated protein, SEQ ID NO: 4):

[0053] DIVMSQSPSSLTVSVGEKITMNCKSSQSLLYSNNHENYLAWYQQKPGQSPKLLIY WASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYTYPLTFGTGTKLELK R.

[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 lesions that should not express CPZ, some cells were positively stained, the background was heavy, and there was no improvement even after dilution, indicating that this clone preparation failed; Figure 2 The background coloring was obvious. After multiple dilutions, clones of different concentrations always had positive reactions in lymphocytes and interstitial cells and were localized in the nucleus, indicating that the preparation of this clone failed. Figure 3 The positive reaction was localized in the small bile ducts, suggesting that the clone had failed in production.

[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 chips 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 chips 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, testes, 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 acini and pancreatic ducts, endometrium, etc. However, normal hepatocytes and pancreatic islets expressed strongly, and gallbladder gland epithelium expressed a small amount of positive expression (such as Figure 4 , Figure 5 shown).

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

[0062] Immunohistochemical detection of CPZ antibody on tissue microarray containing various liver tumors.

[0063] 1. Select liver tumor tissue chips 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 cholangiocarcinoma (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 modifications to the present embodiment without any creative contribution as needed. However, as long as it is within the scope of the claims of the present application, it shall be protected by the patent law.

Claims

1. A CPZ 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 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.

2. Genes encoding the heavy chain variable region and light chain variable region of the CPZ immunohistochemical antibody according to claim 1.

3. The coding gene according to claim 2, characterized in that include: A gene encoding a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 2, having a sequence as shown in SEQ ID NO: 1; A gene encoding a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 6, having a sequence as shown in SEQ ID NO: 5; A gene encoding a light chain variable region having an amino acid sequence as shown in SEQ ID NO:4, having a sequence as shown in SEQ ID NO:3; 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.

4. Use of the CPZ immunohistochemical antibody according to claim 1 in the preparation of a product for differential diagnosis of hepatocellular carcinoma and / or intrahepatic cholangiocarcinoma.

5. The use according to claim 4, characterized in that: The product for differential diagnosis of hepatocellular carcinoma and / or intrahepatic cholangiocarcinoma includes a detection kit.

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