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Detection method of aav protein coat

A detection method and shell technology, applied in the direction of immunoglobulin, chemical instruments and methods, antiviral immunoglobulin, etc., can solve the problems of inaccurate quantitative detection and achieve high sensitivity

Active Publication Date: 2022-07-19
SHANGHAI MYGT BIOPHARMACEUTICAL LLC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no monoclonal antibody and detection method that specifically recognizes the protein capsid (AAVXL32.1), and it cannot be accurately quantitatively detected

Method used

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  • Detection method of aav protein coat
  • Detection method of aav protein coat
  • Detection method of aav protein coat

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Example 1: Production of Antigens

[0053] The intact hollow AAV protein capsid (AAVXL32.1) was produced as antigen by transfecting HEK293 cells with the two plasmids. AAV rep / cap plasmid: phelper: polyethyleneimine (PEI) = 1:1:2, mixed in Opti-MEM and left to stand for 12 min, the transfection solution was added to the suspended HEK293 cells. After 24 h in the carbon dioxide incubator, the old medium was discarded and new medium was added. After culturing for 24 hours, the supernatant and cells were collected. Add 2mM MgCl 2 , for ultrasonication. After 4 min of disruption at 300W, the cells were disrupted. Add 50 U / mL of Benzonase enzyme, incubate at 37 °C for 1 h, and centrifuge at 8000 × g for 15 min to remove cell debris. The supernatant was filtered sequentially using 0.8 μm and 0.45 μm filters.

[0054] Heparin-Sepharose HP (Heparin-Sepharose HP) was used for capture, and the heparin column was first equilibrated with equilibration solution (20mM Tris, 20...

Embodiment 2

[0055] Example 2: Acquisition of Hybridoma Cells

[0056] immunized animals

[0057] 6-8 week-old female Balb / c mice were immunized with the antigen obtained in Example 1, and the mice were made to generate sensitized B lymphocytes. Mice were killed by enucleation and exsanguination, and the spleen was taken out aseptically, squeezed and ground in a plate to prepare a spleen cell suspension.

[0058] Generation of hybridoma cells

[0059] Syngeneic myeloma cells were mixed 1:1 with mouse splenocytes and the fusogenic agent polyethylene glycol was added. Under the action of polyethylene glycol, B lymphocytes fuse with myeloma cells to form hybridoma cells. After fusion of B lymphocytes and myeloma cells, the following five cell types are generated: unfused spleen cells, unfused myeloma cells, spleen cell-spleen cell fusions, myeloma cells-myeloma cell fusions, and spleen cells-bone marrow Tumor cell hybrids (hybridoma cells). Then, using hybridoma cell screening tech...

Embodiment 3

[0062] Example 3: Production and Purification of Monoclonal Antibodies

[0063] Cells were harvested from the flasks and the number of viable cells was determined. If viability is above 80%, seed cells into roller bottles prefilled with 200 mL of antibody production medium (Hybridoma-SFM + 2.5% FBS (low IgG)) at an initial cell density of 0.25 x 10 5 cells / mL to 0.5 x 10 5cells / mL. After inoculation, incubate at 300 r / h in a roller bottle incubator without CO. 2 The cells were cultured at 37 °C for 14-16 days, and the cell culture supernatant was collected. Then, the cell suspension was transferred to a 350 ml centrifuge bottle and centrifuged at 3220 x g for 15 min at 4 °C, followed by filtration with a 0.45 μm filter to remove cells and cell debris.

[0064] The cell culture supernatant was loaded onto a pre-equilibrated Protein A affinity column, which was then washed with 10 CV of equilibration buffer (1x PBS, pH 7.2) until the OD of the flow-through sample became ze...

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Abstract

The present disclosure relates to a detection method of AAV protein coat and a kit that can be applied to the detection method. The detection method of the present disclosure can accurately quantify the concentration of the AAV protein coat with high sensitivity.

Description

technical field [0001] The present disclosure relates to a detection method of AAV protein coat and a kit that can be applied to the detection method. Background technique [0002] Adeno-associated virus (AAV) is a small, non-encapsulated virus with an icosahedral structure. Viral particles are between 20-26 nm in diameter and contain linear single-stranded DNA genomes around 4.7 kb in size. AAV was originally named after a contaminating component found in purified adenovirus fluid. Most adults have been infected with the AAV virus, but the virus has not been found to be a causative factor in any disease. [0003] Recombinant adeno-associated virus (rAAV) is a new gene vector that is further transformed on the basis of non-pathogenic wild-type AAV. Due to its good safety, wide range of host cells, low immunogenicity, and time to express foreign genes in vivo It is regarded as one of the most promising gene transfer vectors and has been widely used in gene therapy research...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/569G01N33/58G01N33/548G01N33/545C07K14/015C07K16/08
CPCG01N33/56983G01N33/581G01N33/545G01N33/548C07K14/005C07K16/081G01N2333/015G01N2469/10C12N2750/14122C07K2317/56C07K2317/565C07K2317/92
Inventor 王慧蒋威郑静肖啸吴侠杜增民王利群赵阳陈晨郑浩
Owner SHANGHAI MYGT BIOPHARMACEUTICAL LLC
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