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GM hybridoma, monoclonal antibody, kit, preparation method and application thereof

一种杂交瘤细胞、单克隆抗体的技术，应用在生物化学设备和方法、化学仪器和方法、抗真菌/藻类/地衣免疫球蛋白等方向，能够解决检测步骤繁琐、单克隆抗体制备成本高、不利于试剂盒推广和普及等问题，达到准确可靠检验结果、减少经验性抗真菌治疗、专一性高的效果

Active Publication Date: 2017-12-12
DYNAMIKER BIOTECH TIANJIN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The detection steps of this method are cumbersome, and the preparation cost of monoclonal antibody is high, which is not conducive to the promotion and popularization of the kit in clinical detection

Method used

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  • GM hybridoma, monoclonal antibody, kit, preparation method and application thereof
  • GM hybridoma, monoclonal antibody, kit, preparation method and application thereof
  • GM hybridoma, monoclonal antibody, kit, preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] The preparation of embodiment 1 Aspergillus galactomannan (GM) antigen

[0039] Aspergillus is used to prepare the GM antigen. The Aspergillus strain used in the present invention is purchased from the American Type Culture Collection (ATCC), and the preservation number is ATCC 1022.

[0040] Cultivate Aspergillus with solid medium until the medium is covered with green spores; filter to remove mycelium, inactivate the bacteria and spores; after centrifugation, collect the spores and wash; the spores are broken and filtered to remove spore fragments; the filtrate is subjected to alcohol precipitation 1. After washing, the GM antigen crude extract is obtained; the GM antigen crude extract is decolorized and ultrafiltered to obtain the GM antigen.

[0041] Wherein, the solid medium is selected from PDA medium, Sabouraud medium or Zabuba medium; preferably PDA medium. Specific steps are as follows:

[0042] 1. Preparation of GM crude product

[0043] Prepare PDA solid m...

Embodiment 2

[0064] Example 2 Modification of GM antigen

[0065] It is well known that molecular size can affect the formation of immunogenicity of substances, and the molecular weight of an effective immunogen is mostly above 10kD; the larger the molecular weight, the stronger the immunogenicity. This may be due to the fact that macromolecular substances are easy to form colloids in aqueous solution, stay in the body for a long time, and have more opportunities to contact immune cells, which is conducive to stimulating the body to produce an immune response. In addition, the chemical structure of macromolecular substances is relatively complex, and the types and quantities of effective antigen genes contained are relatively large.

[0066] The GM antigen obtained in Example 1 has a small molecular weight and poor immunogenicity, and needs to be coupled with related macromolecular substances to improve its immunogenicity. The macromolecules are one or more of latex microspheres, KLH (key...

Embodiment 3

[0076] The preparation of embodiment 3 anti-GM antigen monoclonal antibodies

[0077] 1. Preparation of monoclonal antibody against GM antigen

[0078] 1. Immunization of animals

[0079] Animals were immunized with GM antigen. Among them, the immunization can be performed by subcutaneous injection, foot pad injection, intrasplenic injection, intravenous injection or intraperitoneal injection; the animals can be rats, mice, guinea pigs, rabbits, chickens, sheep, horses, pigs or donkeys. In this embodiment, rabbits are used as immunized animals, and the specific steps are as follows:

[0080] Mix equal volumes of GM antigen and Freund's complete adjuvant to an appropriate volume, fully emulsify and inject New Zealand big-eared rabbits subcutaneously at multiple points, and the immunization dose of each rabbit is controlled at 0.01-0.1mg. Ear blood was collected 3 days before immunization, and serum was separated as a negative control. Immunize once every 2 weeks after the f...

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Abstract

The present invention provides a hybridoma having a deposit number of CGMCC No. 13827. The hybridoma cells are capable of producing monoclonal antibodies to Aspergillus oryzae galactomannan antigens and thus prepare kits. The kit provided by the invention can specifically bind GM antigens with the sensitivity and specificity of more than 95%, the detection limit is reduced from 1ng / mL of the prior product to 0.85ng / mL, and the result has higher coincidence rate than the reference reagent. The test result is more accurate and reliable, so that the IA can be detected early in the course of the disease so that the patient can be treated promptly and effectively to improve the patient's survival rate. Moreover, the kit is easy to operate, the detection is fast and sensitive, and the microplate reader is simple, universal and inexpensive, and the kit provides an effective tool for the quantitative determination of GM antigens.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a hybridoma cell producing a monoclonal antibody against Aspergillus galactomannan (GM) antigen, a monoclonal antibody, a kit, a preparation method and application thereof. Background technique [0002] Aspergillus (Aspergillus) is a saprophytic bacterium widely present in nature and a resident fungus of normal human skin and mucous membranes. Aspergillus spores are small, 2-3μm in diameter, can float in the air, and enter the human body through the respiratory tract. Since Aspergillus enters the human body mainly through the respiratory tract, Aspergillus infection mainly occurs in the lungs. [0003] The incidence of invasive aspergillosis (IA) in immunosuppressed patients has been increasing year by year due to the abuse of antibiotics, and it has become the main cause of death. Aspergillus fumigatus is the most common cause of severe deep Aspergillus infection in immunosuppresse...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/20C07K16/14G01N33/577G01N33/569
CPCC07K16/14G01N33/56961G01N33/577C07K16/44G01N2333/38C07K2317/31C07K2317/54C07K2317/55C07K2317/622C07K2317/626
Inventor 刘春龙彭洁张舟翟栓柱李宁粟艳周泽奇
Owner DYNAMIKER BIOTECH TIANJIN
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