Anti-IGF2 monoclonal antibody and application thereof as adrenal tumor diagnostic agent
By obtaining monoclonal antibodies against IGF2, the problem of difficulty in early diagnosis of adrenal tumors was solved, and specific detection of IGF2 was achieved, improving the accuracy of diagnosis.
Patent Information
- Application Number
- CN202411789717.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-06
- Publication Date
- 2025-03-07
AI Technical Summary
The prior art lacks effective early diagnosis markers, resulting in the fact that patients with adrenal tumors are mostly advanced at the time of diagnosis and have poor prognosis.
By immunizing rats with anti-IGF2, antiserum against IGF2 was obtained, and hybridoma cells were constructed using spleen cells, and monoclonal antibodies with high affinity and specificity were screened for detection of IGF2 and preparation of adrenal tumor diagnostic agents.
The monoclonal antibody can specifically bind IGF2 to early diagnosis of adrenal tumors, improving diagnosis accuracy and sensitivity.
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Figure CN119569876A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of biomedicine, and more particularly to an anti-IGF2 monoclonal antibody and its application as a diagnostic agent for adrenal tumors. Background Art
[0002] The adrenal gland consists of two parts: the cortex and the medulla. The cortex has the function of synthesizing and secreting hormones. Adrenal cortical carcinoma (ACC) originates from the cortex and is a malignant tumor with a low incidence and little research.
[0003] Adrenal gland cancer (ACC) is an adrenal gland tumor that usually has no obvious symptoms in the early stage and lacks effective early diagnostic markers. Many patients with adrenal tumors are already in the advanced stage when diagnosed, resulting in poor prognosis. Even if the tumor is completely removed, up to 25% of ACC patients will still experience local recurrence or distant metastasis.
[0004] Therefore, there is a need to develop molecular markers and diagnostic agents for early diagnosis of adrenal tumors.
[0005] Insulin-like growth factor 2 (IGF2) is one of the major oncogenes in ACC, and its overexpression in ACC has been widely studied. IGF2 activates tyrosine kinase receptors, which in turn activates MAPK and PI3K / Akt signaling pathways, which are involved in the proliferation, survival, and metastasis of cancer cells. Summary of the invention
[0006] The present invention obtains antiserum against IGF2 by immunizing rats with antigens, constructs hybridoma cells using spleen cells of rats, successfully screens out monoclonal antibodies with high affinity and specificity, and obtains the sequence of the antibody.
[0007] Based on the above research, the present invention provides a monoclonal antibody against IGF2, wherein the heavy chain variable region is shown in SEQ ID NO:1, and the light chain variable region is shown in SEQ ID NO:2.
[0008] The present invention also provides the use of the monoclonal antibody in detecting IGF2.
[0009] The present invention also provides the use of the monoclonal antibody in preparing an adrenal tumor diagnostic agent.
[0010] The present invention also provides an adrenal tumor diagnostic agent containing the above monoclonal antibody.
[0011] In a specific embodiment, the adrenal tumor diagnostic agent is used for one or more combinations of enzyme-linked immunosorbent assay, immunofluorescence assay, immunohistochemistry assay, immunoblotting assay, and flow cytometry assay.
[0012] The monoclonal antibody of the present invention can specifically bind to IGF2 with high affinity and good specificity, and can be used to detect IGF2 protein specifically expressed in adrenal tumors. Therefore, it can be used as an adrenal tumor diagnostic agent to diagnose adrenal tumors at an early stage. BRIEF DESCRIPTION OF THE DRAWINGS
[0013] Figure 1 The ELISA method was used to detect the titer of rat antiserum.
[0014] Figure 2 The ELISA method was used to detect the titer of monoclonal antibody IG-3#. DETAILED DESCRIPTION
[0015] 1. Obtaining antiserum
[0016] According to the sequence of IGF2, an expression vector was synthesized to obtain IGF2 protein, which was purified and used to immunize rats to obtain antiserum.
[0017] The method is as follows: 6-week-old rats were injected with antigen for immunization. For the first immunization, 100 μg of antigen was added to an equal volume of Freund's complete adjuvant; for the booster immunization, 50 μg of antigen was added to an equal volume of Freund's incomplete adjuvant. A total of 1 first immunization and 3 booster immunizations were performed.
[0018] Then, the tail vein blood was taken for the preparation of antiserum, and the antiserum titer was detected by indirect ELISA method. The ELISA detection method is as follows: 100 μl antigen (1 μg / ml purified protein or control protein) was used to coat the 96-well ELISA plate and incubate overnight. After washing with PBS, block with skim milk powder for 30 minutes; add 100 μl antiserum dilution for primary antibody incubation at 37°C for 2 hours; after washing with PBS, add 100 μl horseradish peroxidase-labeled goat anti-mouse IgG for secondary antibody incubation at 37°C for 2 hours; after washing with PBS, add color development solution for color development, terminate the reaction after color development, and measure OD on the ELISA reader. 450 .
[0019] The results are as follows Figure 1 As shown, the rat antiserum has a high titer and can be used for the next experiment.
[0020] 2. Preparation of hybridoma cells and acquisition of monoclonal antibodies
[0021] The spleen cells of the above rats were fused with myeloma cells SP2 / 0, and the positive hybridoma cells that were successfully fused were screened for expansion culture, and then injected into the mouse peritoneal cavity. After culture, the mouse ascites was obtained, and the supernatant was collected for testing the titer of the monoclonal antibody. The results showed that the monoclonal antibody IG-3# had a high titer, and its titer was as follows: Figure 2 shown.
[0022] The corresponding hybridoma cell lines were cultured, the culture was collected to extract total RNA, which was reverse transcribed into cDNA, and PCR amplification was performed to obtain the heavy chain and light chain variable region fragments, which were sequenced to obtain their sequences. The results showed that the amino acid sequence of the heavy chain variable region was as shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region was as shown in SEQ ID NO: 2.
[0023] 3. Detection of adrenal tumor tissue using monoclonal antibodies
[0024] Tumor tissue and adjacent tissue from patients with adrenal tumors were collected and total protein was extracted. The extracted protein samples were tested by West Blot. The method is as follows:
[0025] 1) SDS-PAGE electrophoresis: Load the protein sample into the electrophoresis well and perform electrophoresis to separate the protein bands.
[0026] (3) Transfer: The membrane was transferred using the wet transfer method. After the transfer, the membrane was blocked in skimmed milk powder for 2 h. The membrane was then washed three times with PBS on a shaker.
[0027] (4) Primary antibody incubation: Place the blocked membrane in monoclonal antibody dilution solution for primary antibody incubation in a shaking incubator at 4°C overnight.
[0028] 5) Secondary antibody incubation: After washing the membrane incubated with the primary antibody to remove the non-specifically bound primary antibody, add goat anti-mouse IgG for secondary antibody incubation, incubate at 37°C for 1 hour, and wash the membrane again with PBST solution.
[0029] (5) Color development: Add developer to develop the color. After color development, terminate the reaction and then photograph the membrane.
[0030] The results showed that the samples from tumor tissues showed positive reactions, while the samples from adjacent tissues showed negative reactions, which indicated that the monoclonal antibody of the present invention can effectively detect canceration of renal tissues, and thus can be used for the diagnosis of adrenal tumors.
[0031] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims
1. A monoclonal antibody against IGF2, characterized in that: The heavy chain variable region is shown in SEQ ID NO:1, and the light chain variable region is shown in SEQ ID NO:
2.
2. Use of the monoclonal antibody according to claim 1 in detecting IGF2.
3. Use of the monoclonal antibody according to claim 1 in the preparation of an adrenal tumor diagnostic agent.
4. An adrenal tumor diagnostic agent, characterized in that: Containing the monoclonal antibody according to claim 1.
5. The adrenal tumor diagnostic agent according to claim 4, characterized in that The adrenal tumor diagnostic agent is used for one or more combinations of enzyme-linked immunosorbent assay, immunofluorescence assay, immunohistochemistry assay, immunoblotting assay, and flow cytometry assay.