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Tetanus toxoid resisting monoclonal antibody, hybridoma cell strain for producing antibody, and application of antibody

A tetanus toxoid, hybridoma cell line technology, applied in antibacterial immunoglobulins, microorganism-based methods, biochemical equipment and methods, etc., can solve the problems of quality control uncertainty, interference with test results, etc. The effect of improved sensitivity, simple operation and excellent performance

Inactive Publication Date: 2015-09-16
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

At the same time, other antibodies that may exist in the sample to be tested in this method will also interfere with the test results, which brings uncertainty to the quality control of the vaccine development process

Method used

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  • Tetanus toxoid resisting monoclonal antibody, hybridoma cell strain for producing antibody, and application of antibody
  • Tetanus toxoid resisting monoclonal antibody, hybridoma cell strain for producing antibody, and application of antibody
  • Tetanus toxoid resisting monoclonal antibody, hybridoma cell strain for producing antibody, and application of antibody

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Experimental program
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Effect test

Embodiment 1

[0020] Embodiment 1: the preparation of hybridoma cell line WV-TT-05

[0021] 1.1 Source of antigen

[0022] Tetanus toxoid antigen standard (TT standard) was purchased from China National Institutes for Food and Drug Control, and the other three batches of tetanus toxoid were prepared according to Part Three of the Pharmacopoeia of the People's Republic of China 2010 Edition.

[0023] 1.2 Source of mice

[0024] Female, 6-week-old BALB / c mice, SPF (Specific Pathogen Free, SPF) grade, 12-15 g, were purchased from the Experimental Animal Center of Kunming Medical University (permit number: SCXK Dian 2011-0004).

[0025] 1.3 Source of myeloma cells

[0026] Myeloma cells SP2 / 0 were purchased from the Kunming Cell Bank (KCB92021YJ) of the Type Culture Collection Center of the Chinese Academy of Sciences.

[0027] 1.4 Preparation of hybridoma cell lines

[0028] Monoclonal cells were prepared by in vivo immunization and polyethylene glycol (PEG) fusion.

[0029] 1.4.1 Mouse i...

Embodiment 2

[0051] Embodiment 2 hybridoma cell culture and the preparation of monoclonal antibody

[0052] 2.1 Paraffin injection

[0053] Two weeks before cell culture, take 3 BALB / c mice from 8 to 10 weeks old and inject paraffin, 0.5ml / mouse, to stimulate immune cells, cause granulocytes and macrophages to infiltrate inflammation, and produce a series of growth factors to facilitate the proliferation of hybridoma cells and the massive secretion of antibodies. Separate the cages, write the marks, and prepare to inject the hybridoma cells ten days later.

[0054] 2.2 Recovery and expansion of hybridoma cells

[0055] Take out the frozen cell tube from the liquid nitrogen tank, and quickly thaw the frozen hybridoma cells in a 37°C water bath. Put the thawed cells into the centrifuge, 800rpm, 8min, and centrifuge. Discard the supernatant, absorb 3ml of cell culture medium, mix well to precipitate the cells, aspirate the cell solution, mix well and inhale into a 6-well cell culture plat...

Embodiment 3

[0060] Embodiment 3 Monoclonal antibody titer determination of the present invention

[0061] 3.1 Ascites monoclonal antibody titer detection

[0062] 3.1.1 Indirect ELISA procedure

[0063] Dilute the TT standard to 0.5 μg / ml with the coating solution, coat the microtiter plate with 100 μl / well, and overnight at 4°C. Wash 3 times with washing solution the next day, block with 1% (volume ratio) bovine serum albumin in a 37°C incubator, wash 3 times with washing solution after 2 hours, and ascites with 0.01mol / l, pH 7.4 Dilute PBS according to the original times, 1:10, 1:100, 1:1000, 1:10000, 1:100000, 1:1000000, add 100 μl to each well, incubate in a 37°C incubator for 1 hour, wash the plate three times, add Horseradish peroxidase-labeled goat anti-mouse IgG enzyme-labeled secondary antibody (1:10000) diluted with 0.01mol / L, pH 7.4 PBS 100μl, incubated in a 37°C incubator for 1h, washed the plate 3 times, added 100μl substrate solution, measure A after color development 45...

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Abstract

The invention discloses a tetanus toxoid resisting monoclonal antibody, a hybridoma cell strain for producing the antibody, and an application of the antibody. The monoclonal antibody is secreted by the hybridoma cell strain WV-TT-05 with a preservation number of CCTCC NO:C2014200. The monoclonal antibody is used for specific detection of tetanus toxoid. The characteristics of being capable of removing interference of other proteins and detecting the tetanus toxoid specially and sensitively with high throughput are shown in the application of detecting the content of the tetanus toxoid in combined vaccines with the tetanus toxoid as a component and in coupling vaccines with the tetanus toxoid as a carrier, and the detection sensitivity reaches the nanogram level. Moreover, the operation is easy; the high specificity is achieved; and the detection sensitivity is improved.

Description

technical field [0001] The invention relates to a hybridoma cell strain producing specific antibody against tetanus toxoid, its monoclonal antibody and its application in the research, development and quality control of acellular DPT combined vaccine and conjugated vaccine. Background of the invention [0002] Tetanus is a highly fatal disease caused by Clostridium tetani, which mainly occurs in developing countries. It kills 800,000 to 1,000,000 people every year worldwide, including 500,000 neonatal tetanus deaths. Tetanus prevention is mainly achieved through active immunization (tetanus vaccine) or passive immunization (tetanus-specific immunoglobulin). Tetanus vaccine is made from tetanus toxoid (TT), which is a detoxified toxoid that can induce the body to produce protective antibodies after vaccination. The marketed vaccines containing tetanus toxoid components mainly include monovalent tetanus vaccine and pertussis-diphtheria-tetanus combined vaccine. In addition, ...

Claims

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Application Information

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IPC IPC(8): C12N5/20C07K16/12G01N33/577C12R1/91
Inventor 钱雯李作生马波黄薇张晨施競杨艳芬宋纯燕
Owner 云南沃森生物技术股份有限公司
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