Bordetella pertussis PT (pertussis toxin) antigen monoclonal antibody and application thereof

A monoclonal antibody and pertussis technology, applied in the field of immunology and vaccinology, can solve the problems of poor specificity and low detection efficiency of PT antigen content detection methods, and achieve the effects of reducing experimental costs, high titers, and high immunogenicity

Inactive 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

[0005] The purpose of the present invention is to break through the defects of poor specificity and low detection efficiency of PT antigen content detection methods in each step of the current DTaP vaccine production process and in the finished product, and provide a DTaP vaccine with high potency, high uniformity, good specificity and stability. Low-cost monoclonal antibody against PT antigen of Bordetella pertussis to achieve the goal of reducing costs and improving the accuracy and efficiency of quality control in the production process of pertussis vaccines

Method used

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  • Bordetella pertussis PT (pertussis toxin) antigen monoclonal antibody and application thereof
  • Bordetella pertussis PT (pertussis toxin) antigen monoclonal antibody and application thereof
  • Bordetella pertussis PT (pertussis toxin) antigen monoclonal antibody and application thereof

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Experimental program
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Effect test

Embodiment 1

[0040] Embodiment 1 immunogen preparation and animal immunization

[0041] (1) Bacteria pertussis 58003 strain (sourced from China Medical Bacteria Collection and Management Center) was fermented with ginger-packed medium.

[0042] (2) After fermentation, add formaldehyde solution to the final concentration of 0.5% in the fermented cells to sterilize.

[0043] (3) Centrifuge at 4000 rpm or more for more than 10 minutes, discard the supernatant, collect the bacteria, and blow the bacteria with PBS / 0.5% formaldehyde.

[0044] (4) Grinding the bacteria, resuspending and diluting with PBS / 0.5% formaldehyde after grinding.

[0045] (5) Mix the above-mentioned resuspended bacteria with Freund's complete adjuvant in equal volumes, and immunize BALB / c mice with the emulsion formed on day 0, day 14, and day 28, 0.2ml / mouse.

[0046] (6) One week after the last immunization, blood test antibody titer, if higher than 10 3 , 1 week after blood collection, 0.2 μg of emulsion was used for ...

Embodiment 2

[0047] Example 2 cell fusion

[0048] (1) Two weeks before cell fusion, the SP2 / 0 cell line was revived and cultured, expanded 3 days before fusion, and RPMI1640 cell culture medium (Gibco) was removed 1 day before fusion, and culture medium was added again.

[0049] (2) The mice were sacrificed after 3 days of intraperitoneal shock, and the mouse splenocyte suspension was prepared according to conventional methods.

[0050] (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.

[0051] (4) Mix splenocytes and SP2 / 0 cells in a 1:1 ratio in a 50ml centrifuge tube, and mix well.

[0052] (5) Add incomplete IMDM culture medium to 50ml, centrifuge for 5 minutes, pour off the supernatant as much as possible, and use a pipette to suck up the liquid from the nozzle.

[0053] (6) Lightly tap the bottom of the fusion tube to loosen the pre...

Embodiment 3

[0063] Cloning of embodiment 3 hybridoma cells (limiting dilution method)

[0064] (1) Hybridoma cells were counted, and the hybridoma cells were diluted with HT medium containing 20% ​​serum.

[0065] (2) Diluted hybridoma cells were added to a 96-well plate for culture, 100 μl per well.

[0066] (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.

[0067] (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.

[0068] (5) Freeze the cell lines as soon as possible after amplification, number them, and store them in liquid nitrogen.

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Abstract

The invention relates to a bordetella pertussis PT (pertussis toxin) antigen monoclonal antibody and application thereof, and belongs to the field of preparation of biological products. The bordetella pertussis PT antigen monoclonal antibody is secreted by hybridoma cell strains capable of stably secreting bordetella pertussis FT antigen monoclonal antibodies, and a preservation number of the hybridoma cell strains is CGMCC No.10588. The bordetella pertussis PT antigen monoclonal antibody and the application have the advantages that the monoclonal antibody is high in potency and good in specificity, and can be applied to monitoring the quality in bordetella pertussis vaccine production procedures, carrying out enzyme-linked immunosorbent assay on contents of PT components in finished vaccine and preparing bordetella pertussis PT antigen enzyme-linked immune monitoring reagents or enzyme-linked immune monitoring reagent kits.

Description

technical field [0001] The invention relates to the fields of immunology and vaccinology, in particular to an anti-Pertussis bacillus PT antigen monoclonal antibody, a hybridoma cell strain producing the antibody and the application of the antibody. Background technique [0002] Pertussis is a severe acute respiratory infectious disease caused by Bordetella Pertussis, which mainly infects children under 5 years old. For children who have not been immunized or have not completed the basic immunization program, pertussis is still the main cause of death. Pertussis is an infectious disease that can be prevented by vaccines. Since the widespread use of whole-cell pertussis vaccine (WP) in the 1950s and 1960s, especially after the global implementation of the Expanded Program on Immunization (EPI) in 1974, the prevalence of pertussis has been effectively controlled. Morbidity and mortality have dropped significantly. However, vaccination with whole-cell pertussis vaccine (WP) ma...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/20C07K16/12G01N33/68G01N33/577A61K39/40A61P31/04C12R1/91
Inventor 陈磊童钦雷永红刘一非张星星蔡芳王治伟张立志高强尹卫东
Owner SINOVAC RES & DEV
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