Anti-PGRMC1 monoclonal antibody and application thereof as adrenocortical carcinoma diagnostic agent
By developing high-affinity monoclonal antibodies against PGRMC1, the problem of difficulty in early diagnosis of adrenal cortical cancer was solved, and support for early diagnosis of adrenal cortical cancer was achieved, which significantly improved the accuracy and sensitivity of the diagnosis.
Patent Information
- Application Number
- CN202411789941.8
- 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 lack of effective early diagnosis markers in the prior art leads to adrenocortical carcinoma usually developing to a larger volume at the time of diagnosis and poor patient outcomes.
By obtaining high affinity and specific monoclonal antibodies against PGRMC1 and applying them to enzyme-linked immunosorbent assays, immunofluorescence assays, immunohistochemistry assays, immunoblotting assays, flow cytometry assays, etc., it is used to detect the PGRMC1 protein specifically expressed in adrenal cortical carcinoma.
Support for early diagnosis of adrenocortical carcinoma is achieved, and the accuracy and sensitivity of diagnosis are significantly improved by specifically binding to PGRMC1 protein.
Smart Images
Figure CN119569883A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of biomedicine, and more particularly to an anti-PGRMC1 monoclonal antibody and an application thereof as a diagnostic agent for adrenocortical carcinoma. Background Art
[0002] Adrenal cortical carcinoma (ACC) is a highly malignant tumor. There are usually no obvious symptoms in the early stages of adrenal cortical carcinoma, and there is a lack of effective early diagnostic markers. Many patients with adrenal cortical carcinoma have already formed a large volume 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.
[0003] Therefore, there is a need to develop molecular markers and diagnostic agents for early diagnosis of adrenocortical carcinoma.
[0004] Progesterone receptor membrane component 1 (PGRMC1) is a family of membrane-associated progesterone receptor proteins. Studies have shown that PGRMC1 protein is expressed in a variety of tumors including adrenal cortical carcinoma and can be used as a molecular marker for the diagnosis of adrenal cortical carcinoma. Summary of the invention
[0005] The present invention obtains antiserum against PGRMC1 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.
[0006] Based on the above research, the present invention provides a monoclonal antibody against PGRMC1, 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.
[0007] The present invention also provides the use of the monoclonal antibody in detecting PGRMC1.
[0008] The present invention also provides the use of the monoclonal antibody in preparing an adrenocortical carcinoma diagnostic agent.
[0009] The present invention also provides an adrenocortical carcinoma diagnostic agent containing the above monoclonal antibody.
[0010] In a specific embodiment, the adrenocortical carcinoma diagnostic agent is used for one or more combinations of enzyme-linked immunosorbent assay, immunofluorescence assay, immunohistochemistry assay, immunoblotting assay, and flow cytometry assay.
[0011] The monoclonal antibody of the present invention can specifically bind to PGRMC1 with high affinity and good specificity, and can be used to detect PGRMC1 protein specifically expressed in adrenal cortical carcinoma. Therefore, it can be used as an adrenal cortical carcinoma diagnostic agent to diagnose adrenal cortical carcinoma at an early stage. BRIEF DESCRIPTION OF THE DRAWINGS
[0012] Figure 1 The titer of rat antiserum was detected by ELISA.
[0013] Figure 2 The titer of monoclonal antibody PG-5# was detected by ELISA. DETAILED DESCRIPTION
[0014] 1. Obtaining antiserum
[0015] According to the sequence of PGRMC1, an expression vector was synthesized to obtain PGRMC1 protein, which was purified and used to immunize rats to obtain antiserum.
[0016] 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 5 booster immunizations were performed.
[0017] 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 .
[0018] The results are as follows Figure 1 As shown, the rat antiserum has a high titer and can be used for the next experiment.
[0019] 2. Preparation of hybridoma cells and acquisition of monoclonal antibodies
[0020] 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 the detection of the titer of the monoclonal antibody. The results showed that the monoclonal antibody PG-5# had a relatively high titer, and its titer was as follows: Figure 2 shown.
[0021] 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.
[0022] 3. Detection of adrenocortical carcinoma tissue using monoclonal antibodies
[0023] Tumor tissue and adjacent tissue from patients with adrenocortical carcinoma were collected and total protein was extracted. The extracted protein samples were subjected to West Blot detection. The method was as follows:
[0024] 1) SDS-PAGE electrophoresis: Load the protein sample into the electrophoresis well and perform electrophoresis to separate the protein bands.
[0025] (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.
[0026] (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.
[0027] 5) Secondary antibody incubation: After washing the membrane incubated with primary antibody to remove 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.
[0028] (5) Color development: Add developer to develop the color. After color development, terminate the reaction and then take a photo of the membrane.
[0029] 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 adrenocortical carcinoma.
[0030] 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 PGRMC1, 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 PGRMC1.
3. Use of the monoclonal antibody according to claim 1 in the preparation of a diagnostic agent for adrenocortical carcinoma.
4. A diagnostic agent for adrenocortical carcinoma, characterized in that: Containing the monoclonal antibody according to claim 1.
5. The adrenocortical carcinoma diagnostic agent according to claim 4, characterized in that The adrenocortical carcinoma diagnostic agent is used for one or more combinations of enzyme-linked immunosorbent assay, immunofluorescence assay, immunohistochemistry assay, immunoblotting assay, and flow cytometry assay.