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A hybridoma cell line secreting anti-chromaffin-a monoclonal antibody and its application

A monoclonal antibody, hybridoma cell line technology, applied in the direction of anti-receptor/cell surface antigen/cell surface determinant immunoglobulin, anti-animal/human immunoglobulin, analytical materials, etc., can solve the problem of anti-CgA antibody Patent application and other issues to achieve high specificity and sensitivity, improved accuracy, and good positioning

Active Publication Date: 2020-05-05
FUZHOU MAIXIN BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The anti-CgA antibodies currently available for immunohistochemical diagnosis, such as 5H7, DAK-A3 and LK2H10 antibodies, are all from abroad, but there is no relevant anti-CgA antibody patent application in China

Method used

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  • A hybridoma cell line secreting anti-chromaffin-a monoclonal antibody and its application
  • A hybridoma cell line secreting anti-chromaffin-a monoclonal antibody and its application
  • A hybridoma cell line secreting anti-chromaffin-a monoclonal antibody and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1 Preparation of recombinant CgA protein fragments

[0036] 1. Gene optimization and synthesis

[0037] According to the protein sequence with the accession number P10645 in the Uniprot database, CgA selects the protein fragment from the 19th to the 457th amino acid, and directly optimizes it into a gene fragment suitable for expression in Escherichia coli BL21 (DE3). In the process of PCR, the 5' and 3' ends of the gene were respectively added EcoR I and HindⅢ Restriction site. The PCR products were separated by agarose gel electrophoresis and recovered, and the recovered fusion protein gene and the plasmid vector pET-28a used for expression were analyzed respectively. EcoR I and Hind Digested with enzyme III, recovered by electrophoresis again, and ligated with T4 DNA ligase. The ligation product was transformed into Escherichia coli competent cell BL21(DE3), and the clones on the plate were picked and inoculated, and the bacteria liquid PCR was ident...

Embodiment 2

[0040] Example 2 Establishment of hybridoma cell lines

[0041] 1. Immunity

[0042] Mix the fusion protein purified in Example 1 and rapid adjuvant (Beijing Boaolong Company) according to the instructions, and immunize 4-6 week-old female BALB / c mice (purchased from Shanghai Wu’s Experimental Animal Center). Calf intramuscular injection, the dose is 50μg / head. On the 21st day, one shot of booster immunization was given in the same way. On the 35th day, indirect ELISA (wavelength 450 nm) was used to detect the polyantibody titer of the anti-immunogen in the mouse serum, and the mouse with the highest titer was immunized by intraperitoneal injection, and the antigen was mixed with normal saline, and the dose was 50 μg / mouse .

[0043] 2. Cell Fusion

[0044] Aseptically prepare the immune-qualified mouse splenocyte suspension, mix it with mouse myeloma cell SP2 / 0 (Shanghai Cell Bank, Chinese Academy of Sciences) at a volume ratio of 5:1, and centrifuge at 1500 rpm for 5 min...

Embodiment 3

[0047] Example 3 Preparation of monoclonal antibody by ascites induction method

[0048] 1. Ascites preparation

[0049] Cells in the logarithmic growth phase were washed with serum-free medium and suspended, counting 5×10 5 , 1ml. The suspended cells were injected intraperitoneally into mice previously sensitized with paraffin oil. Ascites collection was started 7 days later. The removed ascites was centrifuged at 4°C at 4000 rpm for 10 min. Carefully suck out the ascites in the middle and collect in a centrifuge tube, and store at 4°C or -20°C.

[0050] 2. Purification of monoclonal antibodies

[0051] Antibodies were purified from ascites using Protein G (Nanjing GenScript) affinity chromatography according to the instructions. The purity was identified by SDS-PAGE gel, and the concentration was determined by BCA method. Purified antibodies were stored at -20°C.

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Abstract

The invention provides a hybridoma cell line for secreting a monoclonal antibody against chromogranin A and application of the hybridoma cell line and also provides an application method of secreted monoclonal antibody capable of specifically recognizing human chromogranin A for immunological diagnosis on multiple kinds of tumors. CgA protein is a soluble acidic protein with highest content in human adrenal medulla, has the molecular weight being 48kD and extensively exists in neurons as well as neuroendocrine cells and tumor cells thereof. CgA protein expression is related to neuroendocrine tumors, and is used for marking the neuroendocrine tumors, thus being used for diagnosing the neuroendocrine tumors. An antigen for preparing the antibody is a recombinant protein which is expressed by escherichia coli and has immunogenicity activity, and the finally obtained antibody belongs to an IgG2b subtype. Discovered by immunohistochemistry assay on multiple kinds of tumor tissues, the antibody can well identify tumor occurrence and can be used for immunological diagnosis of the tumors.

Description

technical field [0001] The invention relates to a monoclonal antibody capable of recognizing human chromogranin A and a hybridoma cell line 4E5C6-11G6 capable of secreting the antibody. Specifically, the present invention provides an anti-tumor cell cytoplasmic antigen CgA monoclonal antibody, which can be used for immunohistochemical staining (IHC), enzyme-linked immunosorbent assay (ELISA) or Western Blot is used to detect the expression level of CgA in cells, so as to diagnose these tumors, which belongs to the field of biological detection. Background technique [0002] CgA protein is an acidic hydrophilic protein, a member of the chromaffin family, located in the chromaffin granules of neuroendocrine cells. Studies have found that almost all types of neuroendocrine tumors have upregulated expression of CgA. Therefore, CgA is considered as a non-specific marker of neuroendocrine tumors, and it is used for the diagnosis of such tumors, as well as the monitoring of tumor...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/20C07K16/30G01N33/574C12R1/91
CPCC07K16/30G01N33/57488
Inventor 汪世华杨清海谢成杰陈惠玲张丹萍贾坤志
Owner FUZHOU MAIXIN BIOTECH CO LTD
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