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Hepatitis-B-paratyphoid-fever-resistant O-SP monoclonal antibody, hybridoma cell strain and application

A hybridoma cell line, paratyphoid B technology, applied in the direction of anti-bacterial immunoglobulin, chemical instruments and methods, biochemical equipment and methods, etc., can solve the problem of inability to effectively distinguish between paratyphoid A and paratyphoid B , Monoclonal antibody screening is difficult, specific sites are not obvious, etc., to achieve the effect of high sensitivity, simple operation, and avoid complicated operation

Inactive Publication Date: 2016-09-28
云南沃森生物技术股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Also because the difference between the two types is so small, the use of traditional chemical group detection cannot effectively distinguish between paratyphoid A and paratyphoid B, and at the same time, it also makes the screening of monoclonal antibodies extremely difficult
Moreover, the molecular weight of paratyphoid A O-SP is small, and the specific site of exposure is not obvious, which will also affect the immune effect

Method used

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  • Hepatitis-B-paratyphoid-fever-resistant O-SP monoclonal antibody, hybridoma cell strain and application
  • Hepatitis-B-paratyphoid-fever-resistant O-SP monoclonal antibody, hybridoma cell strain and application
  • Hepatitis-B-paratyphoid-fever-resistant O-SP monoclonal antibody, hybridoma cell strain and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1: Establishment of anti-paratyphoid B O-SP monoclonal antibody hybridoma cell line WV-SPB-11

[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] Paratyphoid B O-SP conjugate vaccine (lyophilized) (batch number 20131015) was provided by Yuxi 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 Type Culture Collection Center of the Chinese Academy of Sciences (KCB92021YJ)

[0042] 1.4 Preparation of hybridoma cell...

Embodiment 2

[0072] Embodiment 2: Preparation of monoclonal antibody of type B paratyphoid hybridoma cell line

[0073] 2.1 Mice were inoculated with hybridoma cells

[0074] Normal 2-month-old BALB / c mice were selected and injected with liquid paraffin 0.5ml / mouse 1 day in advance. The cultured paratyphoid hybridoma cells were centrifuged to discard the supernatant, and the serum-free medium was added and injected into the peritoneal cavity of mice. Hybridoma cells proliferate in the peritoneal cavity of mice and produce and secrete monoclonal antibodies. About 1-2 weeks, the mouse abdomen can be seen to expand. A large amount of monoclonal antibodies can be obtained by extracting ascitic fluid with a syringe.

[0075] 2.2 Monoclonal antibody titer test Ascites titer test, double dilution of collected ascites: 1:10 3 , 1: 2×10 3 , 1:4×10 3 , 1:8×10 3 、1:1.6×10 4 、1:3.2×10 4 、1:6.4×10 4 、1:1.28×10 5 、1:2.56×10 5 , Each concentration was repeated twice, and the titer of the mono...

Embodiment 3

[0078] Example 3: Establishment of ELISA detection method for monoclonal antibody to paratyphoid B O-SP

[0079] 3.1 Configuration of the main solution

[0080] Coating buffer (0.1mol / L, hydrochloride buffer at pH 9.6): Na2CO3·10H2O0.86g; NaHCO30.586g; add sterilized water to 200ml.

[0081] PBS diluent (0.01mol / L, PBS buffer solution with pH7.4): 8g NaCl; 0.2g KCl; 2.9g Na2HPO4·12H2O; 0.2g KH2PO4; add sterilized water to 100ml.

[0082] Washing solution (0.01mol / L, PBST at pH7.4): 1000ml of 1PBS buffer; 0.5ml of Tween-20.

[0083] Blocking solution (5% skimmed milk powder PBS diluent): 1 g skimmed milk powder; 20 ml PBS diluent.

[0084] Chromogenic solution: substrate solution 10ml; TMB 0.5ml; H 2 o 2 32 μl.

[0085] Stop solution (2mol / L H2SO4): concentrated sulfuric acid (18mol / L 98%) 22.2ml; sterilized water 177.8ml.

[0086] 3.2 Elisa indirect method to detect monoclonal antibody titer

[0087] 3.2.1 Coating: SPB-O-SP-20130511 (1 mg / ml) was diluted to 500 ng / ml wit...

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Abstract

The invention discloses a hepatitis-B-paratyphoid-fever-resistant O-SP monoclonal antibody, a hybridoma cell strain and an application. The hybridoma cell strain is named WV-SPB-11, and the collection number is CCTCC NO:C2015228. The monoclonal antibody is secreted through the hybridoma cell strain in the claim 1. The monoclonal antibody is high in purity and antibody titer, and the following experimental requirement is met; the polysaccharide content of hepatitis-A paratyphoid fever O-SPO-SP is measured with the indirect competitive ELISA method built by the monoclonal antibody, the specificity and the sensitivity are high, and particularly, the monoclonal antibody and hepatitis-A paratyphoid fever O-SP polysaccharide, OSP polysaccharide combination and the like are not subjected to a cross reaction.

Description

technical field [0001] The invention belongs to the technical field of monoclonal antibodies, and in particular relates to an anti-paratyphoid B O-SP monoclonal antibody, hybridoma cell lines and applications. Background technique [0002] Salmonella typhi, Salmonella paratyphi type A and type B are a group of serious intestinal infectious diseases that cause bacteremia. With the wide application of typhoid Vi polysaccharide vaccine, the incidence of typhoid fever has gradually decreased, but in recent years, type A However, the incidence of paratyphoid fever B is constantly increasing, and the increasing number of patients has attracted more and more attention. Due to the migration of pathogenic strains, monovalent typhoid series vaccines can no longer provide long-term population protection, and the development of multivalent conjugate vaccines has become an inevitable choice. There have been typhoid, type A, and type B paratyphoid bacterial vaccines used to prevent typho...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/20C07K16/12G01N33/577G01N33/569C12R1/91
CPCC07K16/1235G01N33/56916G01N33/577G01N2400/10
Inventor 钱雯倪萍黄薇吴俊波熊应景王丽丽陈玉秋陈敏施競陈燕杨晨
Owner 云南沃森生物技术股份有限公司
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