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Tetanus toxoid monoclonal antibody and application thereof

A technology of tetanus toxoid and monoclonal antibody, which is applied in the field of immunology and vaccinology, can solve the problems of low detection efficiency and poor specificity, improve specificity and efficiency, reduce experimental costs, broad application prospects and market value Effect

Active Publication Date: 2015-11-11
SINOVAC RES & DEV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The purpose of the present invention is to break through the shortcomings of each step in the current tetanus vaccine production process and the finished product for the detection method of toxoid antigen content, which has poor specificity and low detection efficiency; Specific and low-cost anti-tetanus toxoid monoclonal antibody to achieve the goal of reducing costs and improving the specificity and efficiency of quality control

Method used

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  • Tetanus toxoid monoclonal antibody and application thereof
  • Tetanus toxoid monoclonal antibody and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Embodiment 1 immunogen animal immunization

[0037] (1) Mix equal volumes of tetanus toxoid and Freund's complete adjuvant to form an emulsion to immunize BALB / c mice on day 0, inject subcutaneously at multiple points on the back, 0.05ml per point, total 0.2ml, containing about 60 μg tetanus toxoid.

[0038] (2) Mix equal volumes of tetanus toxoid and Freund's incomplete adjuvant to form an emulsion to immunize BALB / c mice on day 14 and day 28, inject subcutaneously at multiple points on the back, 0.05ml per point, total amount 0.2ml contains about 50μg tetanus toxoid.

[0039] (3) One week after the third immunization, blood was collected from the tail vein of the mice to detect the antibody titer of the serum. If the antibody titer is higher than 10 3 1 week later, 0.2 ml of mixed emulsion of tetanus toxoid and incomplete adjuvant was used for intraperitoneal impact, containing about 60 μg of tetanus toxoid. After 3 days, the spleen of the mouse was taken for cell ...

Embodiment 2

[0040] Example 2 cell fusion

[0041] (1) Preparation of myeloma cells: resuscitate and culture SP2 / 0 cell line two weeks before cell fusion, expand culture 3 days before fusion, remove RPMI1640 cell culture medium (Gibco) one day before fusion, and add the same amount of culture medium again . On the day of fusion, the culture medium was collected in a 50ml centrifuge tube, centrifuged at 2000r / min for 5min, and the supernatant was discarded. Add incomplete IMDM culture solution to 50ml, centrifuge at 2000r / min for 5min, and discard the supernatant.

[0042] (2) Spleen cell preparation: sacrifice the mice for animal immunization, and prepare the mouse spleen cell suspension according to the conventional method.

[0043] (3) Add appropriate amount of incomplete IMDM medium (Gibco) to splenocytes and myeloma cell SP2 / 0 according to the counting results, shake and mix the SP2 / 0 cells, and pipette the splenocytes evenly.

[0044] (4) Mix splenocytes and SP2 / 0 cells in a 1:1 rati...

Embodiment 3

[0056] Example 3 Cloning of Hybridoma Cells (Limited Dilution Method) and Preparation of Monoclonal Antibody Cell Line Ascites

[0057] (1) Hybridoma cells were counted, and the hybridoma cells were diluted with HT medium containing 20% ​​serum.

[0058] (2) Diluted hybridoma cells were added to a 96-well plate for culture, 100 μl per well.

[0059] (3) 37°C, 5% CO 2 Wet culture for 8 days, when clones visible to the naked eye appear, detect antibody activity in time. Observe under an inverted microscope, mark the wells where only a single clone grows, and take the supernatant for antibody detection.

[0060] (4) Move the cells in the positive wells to a 24-well plate for expanded culture, and then transfer to a 25 or 175 cell bottle for expansion.

[0061] (5) Freeze the cell lines as soon as possible after amplification, number them, and store them in liquid nitrogen.

[0062] Resuscitate frozen monoclonal cells according to conventional methods, culture them, observe th...

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Abstract

The invention relates to a tetanus toxoid monoclonal antibody and application thereof, and belongs to the field of immunology. Mouse spleen cells and mouse myeloma cells are fused with one another after mice are subjected to tetanus toxoid immunization, tetanus-toxoid-resistant specific monoclonal antibody hybridoma cell strains can be generated by means of screening, and a preservation number of the hybridoma cell strains is CGMCC No.10590. The tetanus toxoid monoclonal antibody and the application have the advantages that the potency of the monoclonal antibody secreted by the hybridoma cell strains can reach 10<7>, an affinity constant of the monoclonal antibody is 1.24*10<9>M<-1>, and the tetanus toxoid monoclonal antibody can be used for preparing tetanus toxoid antigen detection reagent kits, also can be applied to detecting tetanus vaccine production procedures by the aid of enzyme-linked immunosorbent assay and detecting the content of toxoid in finished vaccine and has a broad application prospect and high market value.

Description

technical field [0001] The invention relates to the fields of immunology and vaccinology, in particular to a tetanus toxoid monoclonal antibody, a hybridoma cell line producing the antibody and the application of the antibody. Background technique [0002] Tetanus is a specific infection in which Clostridium tetani (Clostridium tetani) invades the human body through skin or mucous membrane wounds, grows and reproduces in an anoxic environment, produces tetanus toxin and causes paroxysmal muscle spasms. Tetanus is an infectious disease, not an infectious disease. It does not break out and spread among the crowd, and people cannot infect each other. The vast majority of cases are caused by traumatic infection, including neonatal tetanus and so on. Although tetanus morbidity and mortality have declined significantly in developed countries, it remains a major public health problem in most developing countries. 80% of the total number of deaths from tetanus are newborns, and th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/20C07K16/00G01N33/577A61K39/395A61P31/04C12R1/91
Inventor 刘一非陈磊童钦朱朗张星星蔡芳王治伟张立志高强尹卫东
Owner SINOVAC RES & DEV
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