Monoclonal antibody for resisting cell surface ectopic expression, and preparation method and application thereof

A monoclonal antibody, cell surface technology, applied in the direction of antibodies, fusion cells, chemical instruments and methods, etc., can solve problems such as elevation

Active Publication Date: 2013-06-05
MABSTAR
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Hepatobiliary system diseases include hepatobiliary malignant tumors, abnormal bone development, pregnant women, etc. ALP will increase, but there are no reports of increased ALP in gastri...

Method used

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  • Monoclonal antibody for resisting cell surface ectopic expression, and preparation method and application thereof
  • Monoclonal antibody for resisting cell surface ectopic expression, and preparation method and application thereof
  • Monoclonal antibody for resisting cell surface ectopic expression, and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Four gastric cancer cell lines mixed in equal proportions (BGC823, MKN28, MKN45 and SGC7901, all from Shanghai Institute of Cell Biology, Chinese Academy of Sciences) were cultured in RPMI1640 medium containing 10% FBS, 5% CO 2 , After culturing at 37°C, the collected mixed living cells were used as immunogens in PBS buffer, in A / J-JAX mice (purchased from the Experimental Animal Model Center of Nanjing University, and the mice were from The Jackson Laboratory, the United States) Immunized by subcutaneous and intraperitoneal injection on the back, 1×10 per mouse each time 7 1 cell, immunized once every other week; one week after the third immunization, the mouse serum was collected, and the flow cytometry high-throughput system (FACS-HTS) was used to detect the reaction between the serum and 4 gastric cancer cell lines, and the PBMC of healthy volunteers As a control cell [Peripheral Blood Mononuclear Cells (PBMC) were separated from the peripheral blood of healthy volu...

Embodiment 2

[0049] Total RNA was extracted from the MS17-57 monoclonal antibody hybridoma cell line with the RNeasy kit from Qiagen (Valencia, California, USA), and the mRNA was reversed with the SuperScript III First-Strand kit from Invitrogen (Grand Island, New York, USA) The cDNA library of MS17-57 monoclonal antibody was recorded. Using the 23 primers of the "Mouse IgG Library Primer Set" (F2010) kit from ProgenBiotechnik in Germany, 21 PCR reactions (reactions not including the lambda light chain) were performed, and the specific light and heavy chain products generated were subjected to DNA sequencing and amino acid analysis. Translation of polypeptide sequences and identification of CDRs (epitope regions) and FWs (framework regions).

[0050]Table-1(a), the cDNA sequence of the variable region of the MS17-57 monoclonal antibody light chain (SEQ ID NO: 1):

[0051] 5'-atgtctgcatctccagggggaaaaggtcaccatgacctgcagggccagctcaagtat

[0052] aatttccagttacttgcactggttccagcagaagtcaggtgcccccc...

Embodiment 3

[0074] On a 96-well "U"-shaped plate, use 1% BSA / PBS to prepare an average of about 200,000 different gastric cancer cells / 100 microliters per well and add it to the U-shaped plate, and multiply the serum after immunization of living gastric cancer cells by 5 times. The titer was serially diluted, and then 100 μl was added to each well, mixed well and reacted on ice or at 4°C for 20 minutes, after washing twice, add 100 μL / well of goat anti-mouse IgG Fc-FITC diluted 1:333, 4°C After C reaction and washing, read the MFI value of each well on the LSR-II fluorescence flow cytometer-HTS machine of BD Company.

[0075] The results showed that the titer of the No. 1 mouse serum with high immunoreactivity combined with gastric cancer cells was significantly higher than that of normal human PBMC, and the splenocytes of this mouse were used for the production of MS17-57 monoclonal antibody hybridoma. fusion experiment. (Such as figure 1 shown).

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Abstract

The invention belongs to the field of biological pharmacy and provides a monoclonal antibody for resisting cell surface ectopic expression. The amino acid sequence of a light chain variable region at the Fab fragment antigen-binding site of an antibody molecule is shown as SEQ ID NO:2, and the amino acid sequence of a heavy chain variable region is shown as SEQ ID NO:4. The invention also provides application of the monoclonal antibody in preparation of medicaments for treating and preventing digestive tract tumors or digestive tract tumor metastasis. The invention also provides a detection kit and medicinal composition containing the monoclonal antibody. The invention also provides a preparation method of the monoclonal antibody. A series of in vivo and in vitro tests prove that the purified and degermed monoclonal antibody has high affinity for PALP and/or IALP antigen for resisting extracellular ectopic expression of tumor cells and has a biological activating function of inhibiting growth and migration of tumor cells.

Description

Technical field: [0001] The invention belongs to the field of biopharmaceuticals, and in particular relates to a monoclonal antibody, specifically a monoclonal antibody against alkaline phosphatase ectopically expressed on the surface of digestive tract tumor cells such as gastric cancer and its preparation method and application. Background technique: [0002] Gastric cancer (GC) is the most common digestive system malignancy and the second leading cause of cancer-related death worldwide. China is one of the countries with the highest incidence of gastric cancer in the world, with nearly 400,000 new cases each year, accounting for about 42% of the world's total, and many patients are already in the advanced stage when they seek treatment. At present, early screening and diagnosis of gastric cancer mainly rely on endoscopy combined with histopathological biopsy. Although the specificity, sensitivity, accuracy and safety of this early screening and diagnosis method have been ...

Claims

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Application Information

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IPC IPC(8): C07K16/40A61K39/395A61P35/00A61P35/04G01N33/577
CPCA61K39/395G01N33/577C12N5/16C07K16/40A61P35/00A61P35/04C07K16/3046C07K2317/34C07K2317/56G01N33/57446G01N33/57488
Inventor 陆梅生刘炳亚张冬青李明冯润华孙见宇陈雪华涂桂云高健鹏王玉苓冯振卿周亦凭杰弗瑞·李苏庆朱正纲
Owner MABSTAR
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