Monoclonal antibody capable of resisting group A meningococcal capsular polysaccharide conjugate, hybridoma cell strain and applications

A hybridoma cell line, meningococcal technology, applied in the direction of anti-bacterial immunoglobulin, microorganisms, immunoglobulin, etc., can solve the problems that need to be verified, and achieve the effect of high antibody titer, simple detection process, and high purity

Pending Publication Date: 2017-02-15
云南沃森生物技术股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

During the preparation process of meningococcal capsular polysaccharide-protein conjugated vaccine, there is a possibility of spatial shielding effect, so that some epitopes of polysaccharides are shielded by proteins; in addition, new epitopes are even produced after polysaccharides are combined with proteins, Therefore, the specificity of the monoclonal antibody provided by the patent to the polysaccharide conjugate vaccine and the ability to accurately detect the polysaccharide content need to be verified

Method used

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  • Monoclonal antibody capable of resisting group A meningococcal capsular polysaccharide conjugate, hybridoma cell strain and applications
  • Monoclonal antibody capable of resisting group A meningococcal capsular polysaccharide conjugate, hybridoma cell strain and applications
  • Monoclonal antibody capable of resisting group A meningococcal capsular polysaccharide conjugate, hybridoma cell strain and applications

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1: Establishment of anti-group A Neisseria meningitidis polysaccharide monoclonal antibody hybridoma cell line WV-A-01

[0035] Unless otherwise specified, the vaccines and polysaccharides involved in this example can be prepared according to Part Three of the Pharmacopoeia of the People's Republic of China 2015 Edition or obtained from the market.

[0036] 1.1 Source of antigen

[0037] 1.1.1 Group A meningococcal diphtheria toxoid non-toxic variant polysaccharide conjugate stock solution (batch number MenAps-CRM 197 20140201), provided by the Technology Center of Yunnan Watson Biotechnology Co., Ltd.;

[0038] 1.2 Source of mice

[0039] Female, 6-8-week-old BALB / c mice, SPF grade (Specific pathogen Free, SPF), 15-20 g, were purchased from Guangdong Medical Experimental Animal Center (permit number SCXK (Guangdong) 20130002).

[0040] 1.3 Source and maintenance of myeloma cells

[0041] Myeloma cells SP2 / 0 were purchased from the Kunming Cell Bank of the T...

Embodiment 2

[0082] Embodiment 2: Preparation of monoclonal antibody (mouse ascites)

[0083] 2.1 Pretreatment of mice

[0084] 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.

[0085] 2.2 Recovery, subcloning and expansion of hybridoma cells

[0086] Resuscitation was carried out according to the method in 1.6. During the culture process, pay attention to observe the number and shape of cells. Subcloning was carried out according to the limited dilution method. %, combined with observation under a microscope, select cells with better morphology for expanded culture (see figure 1 , figure 2 ).

[0087] 2.3 Mice were inoculated with hybridoma cells

[0088] 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 them, and adjust the number of cells to 5×10 5 ...

Embodiment 3

[0091] Example 3: Purification of monoclonal antibodies

[0092] 3.1 Use Protein A-Sepharose CL-4B (filler) affinity chromatography to purify the monoclonal antibody, monitor with AKTAexplorer100 (protein purification system), see the purification chromatography image 3 , the specific operation is as follows:

[0093] 3.1.1 Column packing: Dissolve 1.5g Protein A-Sepharose CL-4B dry powder in 6-7ml distilled water, soak in 0.02M, pH7.4 phosphate buffer for 15min, and then load it into the chromatography column.

[0094] 3.1.2 Equilibrium: pass through the column with 10 times of the column bed volume of the sample buffer, the flow rate is 1ml / min, and the pH of the effluent liquid tested by pH test paper is 7.4.

[0095] 3.1.3 Sample loading: Take 5ml of pretreated ascites, dilute to 50ml with loading buffer, filter with a 0.45μm filter membrane, and load the sample at a flow rate of 1ml / min.

[0096] 3.1.4 Elution: Wash with loading buffer, 10 times the column bed volume, ...

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Abstract

The invention discloses a monoclonal antibody capable of resisting group A meningococcal capsular polysaccharide conjugate, a hybridoma cell strain and applications. The hybridoma cell is named as WV-A-01, and is assigned with the accession number of CCTCC NO: C2015229. The monoclonal antibody is secreted by the hybridoma cell strain of claim 1. The monoclonal antibody can be used for the serotype typing diagnosis of group A epidemic cerebrospinal meningitis, and the quantitative determination for group A polysaccharide in the research and development processes of epidemic cerebrospinal meningitis multivalent polysaccharide vaccines and conjugate vaccines, is wide in applicability and high in specificity, and has a considerable practical value, and thus a foundation is laid for the quality control of epidemic cerebrospinal meningitis vaccines and the epidemiological investigation and prevention and control.

Description

technical field [0001] The invention belongs to the technical field of monoclonal antibodies, and in particular relates to an anti-meningococcal capsular polysaccharide monoclonal antibody of group A, a hybridoma cell line and applications. Background technique [0002] Meningitis is the abbreviation of epidemic cerebrospinal meningitis (Epidemic cerebrospinal meningitis), which is caused by Neisseria meningitidis and is extremely harmful to humans. In children, bacterial infection starts from the nasopharynx, then penetrates into the blood circulation, and ends in the meninges or other parts of the body to cause inflammatory damage. [0003] Neisserial meningitidis (N.meningitidis) is the pathogenic bacterium of meningitis, and its antigenic components mainly include capsular polysaccharides, outer membrane proteins, and lipopolysaccharides. These antigenic substances are also the basis for serological classification and typing of meningitis. Membrane polysaccharides are s...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/20C07K16/12G01N33/577G01N33/569
CPCC07K16/1217
Inventor 钱雯鲁卫东舒曼黄薇倪萍王丽丽陈玉秋施競杨晨
Owner 云南沃森生物技术股份有限公司
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