Monoclonal antibody (hybrid tumor) for neutralizing CYR61 and applications thereof

A monoclonal antibody and hybridoma technology, applied in the field of hybridoma, can solve the problems of inability to use in vivo, imperfect long-term transfection technology of SiRNA, etc.

Inactive Publication Date: 2010-06-23
SHANGHAI INST OF IMMUNOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, due to the incomplete long-term transfection technolo

Method used

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  • Monoclonal antibody (hybrid tumor) for neutralizing CYR61 and applications thereof
  • Monoclonal antibody (hybrid tumor) for neutralizing CYR61 and applications thereof
  • Monoclonal antibody (hybrid tumor) for neutralizing CYR61 and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1: Expression Pattern of CYR61 in Synovial Tissue of Rheumatoid Arthritis

[0047] 1. Materials and methods

[0048] 1 Experimental materials

[0049] 1.1 Clinical samples:

[0050] All samples in the study were from RA patients and OA patients undergoing knee replacement or synovectomy in clinical orthopedics department, and the diagnosis of all cases met the international diagnostic criteria. Two normal controls were from trauma patients. The patient's synovial tissue was used for cell culture in vitro, part of the tissue was fixed with 4% paraformaldehyde and reserved for immunohistochemistry, serum and synovial fluid (SF) were centrifuged at high speed to obtain supernatant, and stored at -80°C. All clinical samples used in this study were informed to the patients.

[0051] 2 Experimental methods

[0052] 2.1 Cell preparation

[0053] 2.1.1 Preparation of peripheral blood mononuclear cells:

[0054] Mononuclear cells were separated from heparin-antico...

Embodiment 2

[0080] Example 2: CYR61 in rheumatoid arthritis synovial fluid promotes the proliferation of FLS

[0081] 1. Materials and methods

[0082] 1.1 Synovial fluid stimulation experiment: Mix 3-5 parts of synovial fluid and add to FLS in logarithmic growth phase to continue culturing for 24 hours, then detect CYR61 mRNA and protein expression levels, or FLS proliferation (see below for details).

[0083] 1.2 Cell proliferation and antibody blocking experiments: take FLS in the logarithmic growth phase, digest with 0.25% trypsin and adjust the cell concentration to 1×10 4 cells / ml, inoculate in 96-well cell culture plate, add different concentrations of SF (the ratio of SF to culture medium is 1:32, 1:16, 1:8, 1:4, 1:2) or cytokines (IL-10, IL12, IFNγ, TNFa and IL-17), or pure CYR61 protein was co-cultured with FLS. 1 μCi of 3H (per well) was added 16 hours before the end of the culture, the cells were collected, and the proliferation was detected by a β liquid scintillation instr...

Embodiment 3

[0097] Example 3: Study on the mechanism of CYR61 promoting the proliferation of rheumatoid arthritis FLS

[0098] 1. Materials and methods

[0099] 1. Clinical samples, CYR61 gene and protein expression detection are the same as those described in the materials and methods of "Example 1: CYR61 expression pattern in rheumatoid arthritis synovial tissue".

[0100] 2. FLS culture and proliferation stimulation experiments are the same as those described in the materials and methods of "Example 2: CYR61 promotes the proliferation of FLS in rheumatoid arthritis synovial fluid".

[0101] 3. SiRNA interference experiment:

[0102] 3.1: SiRNA sequence and synthesis: Synthesize 3 siRNAs according to the siRNA design principle, the sequence is as follows:

[0103] S1: 5'-CCA GAA AUG UAU UGU UCA ATT-3-

[0104] 5’-UUG AAC AAU ACA UUU CUG GCC-3-

[0105] S2: 5’-UCA AGG UAU CAA UGU UUA ATT-3-

[0106] 5’-UUG CUC GCA ACA AAU CUG CTC-3-

[0107] S3: 5'-CAA CGA GGA CUG CAG CAA ATT-3-

...

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Abstract

The invention relates to a monoclonal antibody capable of neutralizing CYR61 and applications thereof and a hybrid tumor capable of generating the monoclonal antibody. The technical scheme adopted by the invention relates to the monoclonal antibody secreted by the mouse anti-human CYR61 monoclonal antibody hybrid tumor CGMCC No.2766 or CGMCC No.2767, and the applications of the monoclonal antibody to preparation of a medicament for treating rheumatic disease, wherein the rheumatic disease is the chronic infectious arthritis. The invention has the advantages that: (1) the self-prepared monoclonal antibody can research pathogenesis of CYR61 in rheumatoid arthritis and can accurately position the expression of CYR61 and detect the CYR61 level of the blood serum and the lesion sites (such as joints); (2) the monoclonal antibody can be used for preparing a detection kit so as to distinguish clinical diagnostics of osteoarthritis and rheumatoid arthritis; and (3) the monoclonal antibody also can search the combination epitope where the CYR61 on the surface of the synovium and in the synovial of the rheumatoid arthritis patient can be neutralized, and provides the screening basis for preparing a humanized antibody which can neutralize the CYR61 in the next step.

Description

【Technical field】 [0001] The invention relates to a monoclonal antibody, in particular to a monoclonal antibody capable of neutralizing CYR61, its application and a hybridoma capable of producing the monoclonal antibody. 【Background technique】 [0002] Rheumatoid arthritis (RA) is one of the most common rheumatic diseases. RA is one of the most common autoimmune diseases. The global prevalence of RA averages 0.5-1%. In my country, the prevalence rate of RA is 0.45%, and the total number of new patients is about 4 million each year. The onset of RA is mostly young adults. Since there is no specific treatment drug to control the development of the disease, most patients lose their ability to work due to disability around 6-10 years after the onset of the disease. The disability rate can reach 50% and 70%. Therefore, RA seriously affects people's life and health, and at the same time, it also brings a heavy burden to my country's developing and sound medical insurance, and ...

Claims

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Application Information

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IPC IPC(8): C07K16/18C12N5/20A61K39/395A61P19/02A61P29/00
Inventor 李宁丽沈佰华王利黄立东
Owner SHANGHAI INST OF IMMUNOLOGY
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