A method for simultaneously preparing two monoclonal antibodies

A monoclonal antibody, hybridoma cell line technology, applied in microorganism-based methods, botanical equipment and methods, biochemical equipment and methods, etc., to achieve the effects of high specificity, low titer, rapid preparation and high efficiency

Active Publication Date: 2021-02-26
云南沃森生物技术股份有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Although there have been research reports on the preparation of polysaccharide monoclonal cell lines and antibodies for meningococcal meningitis with conjugated vaccines, there have been no reports that simultaneously consider the preparation of monoclonal cells for conjugated proteins and the preparation of monoclonal cells for polysaccharide antigens

Method used

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  • A method for simultaneously preparing two monoclonal antibodies
  • A method for simultaneously preparing two monoclonal antibodies
  • A method for simultaneously preparing two monoclonal antibodies

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038]In this example, both Pn33Fps (33F pneumococcal capsular polysaccharide) and HBs (hepatitis B surface protein) are from Yunnan Watson Biotechnology Co., Ltd.; Pn33Fps (10mg) standard was purchased from ATCC; The products were purchased from China National Institutes for Food and Drug Control. BALB / c mice (SPF grade): 6-8 weeks old female, 13-16g, purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. (experimental animal license number: SCXK (Beijing) 2016-0006); SP2 / 0 Myeloma cells were purchased from Kunming Institute of Zoology. Polyethylene glycol 4000 (PEG4000), HAT selection medium (H-Hypoxanthine hypoxanthine, A-Aminopterin aminopterin, T-Thymidine thymidine), Tween-20, and HRP-labeled goat anti-mouse IgG were all purchased From Sigma Company of the United States; RPMI-1640 modified medium (Thermo Fisher, the United States); paraffin oil (Sinopharm Chemical Reagent Co., Ltd., China); fetal bovine serum and neonatal bovine serum were purcha...

Embodiment 2

[0072] The preparation of embodiment 2 monoclonal antibody (mouse ascites)

[0073] 2.1 Pretreatment of mice

[0074] Three BALB / c mice aged 8 to 10 weeks were injected with sterile liquid paraffin, 0.5ml / mouse, and used to inoculate hybridoma cells 7 days later.

[0075] 2.2 Recovery, subcloning and expansion of hybridoma cells

[0076] Resuscitation is carried out according to the existing method. During the culture process, pay attention to observe the number and shape of cells. Subcloning is carried out according to the limited dilution method. After resuscitation, the positive rate of subcloned cells is 100%. Combined with observation under a microscope, select cells with better morphology to proceed. Expand cultivation.

[0077] 2.3 Mice were inoculated with hybridoma cells

[0078] When the cells cover 80% to 90% of the bottom of the plate, blow the cells down, centrifuge at 1200r / min for 5min, discard the supernatant, suspend the cells with RPMI-1640 medium, count t...

Embodiment 3

[0081] The establishment and application of embodiment 3 competition ELISA method

[0082] The indirect ELISA method was used to determine the working concentration of antigen and antibody, namely: Pn33Fps antigen coating concentration was 50ng / 0.1ml per well, antibody dilution was 1:1000; HBsAg antigen coating concentration was 50ng / 0.1ml per well, antibody dilution The degree is 1:2000.

[0083] Use above-mentioned antigen coating concentration and antibody dilution to carry out the detection of competition ELISA, draw Pn33F polysaccharide standard curve (detection range: 40ng-5 μ g, regression equation: y=-0.168ln(x)+1.5075) and HBs antigen standard curve ( Detection range: 8ng-1μg, regression equation: y=-0.128ln(x)+0.9952), the two standard curves show good regression within the detection range, and the regression coefficients are greater than 0.99 ( image 3 and Figure 4 ). Using the same method to detect the Pn33Fps and HBsAg samples of 3 batches prepared in additio...

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Abstract

The invention discloses a method for simultaneously preparing two kinds of monoclonal antibodies. In the method, the conjugate of 33F type pneumonia polysaccharide and hepatitis B surface protein is used as an antigen to immunize mice, and mouse splenocytes with higher antibody levels after immunization are selected. Fusion with SP2 / 0 myeloma cells, and then specific screening by 33F pneumonia polysaccharide, hepatitis B surface protein, and 33F pneumonia polysaccharide and hepatitis B surface protein conjugates as antigens to obtain hybridoma cells, and at the same time prepare a specific recognition of 33F pneumonia Monoclonal antibodies to polysaccharide and hepatitis B surface protein. The method of the present invention to simultaneously prepare monoclonal cell line cells and specific monoclonal antibodies for polysaccharides and proteins after immunization with pneumonia conjugates using hepatitis B surface protein as a carrier saves workload and improves screening efficiency. At the same time, the present invention The two monoclonal antibodies provided have the advantages of high specificity, high sensitivity and good passage stability.

Description

technical field [0001] The invention relates to the technical field of monoclonal antibodies, in particular to a method for simultaneously preparing two monoclonal antibodies. Background technique [0002] Pneumococcus and hepatitis B virus are two major pathogenic microorganisms that endanger human health. The successful marketing of vaccines against pneumococcus and hepatitis B virus provides effective means for the prevention and control of such diseases. In the process of the development and production of these two vaccines, quality control needs to be carried out through the determination of effective antigenic components. In the detection of antigen content, at present, the antigen content of pneumonia vaccine is mainly determined by chemical group method, and the content is judged by the theoretical ratio of chemical groups. Can not fully reflect its biological activity. Compared with traditional physical and chemical methods, the qualitative or quantitative detect...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/46C12N15/06C12N5/20C12R1/91
CPCC07K16/082C07K16/1275C07K16/46C07K2317/31C12N15/02
Inventor 钱雯陈南萍王丽丽陈玉秋吴凯陈敏赵志宏奚树花范荣坤
Owner 云南沃森生物技术股份有限公司
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