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Filamentous hemagglutinin detection kit and its application

A filamentous hemagglutinin and kit technology, applied in applications, biological tests, measurement devices, etc., can solve problems such as differences in registered quality standards and no FHA toxins, and achieve effective immune effects, high and high neutralization titers.

Active Publication Date: 2021-09-03
SINOVAC RES & DEV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although there is currently an ELISA kit for PT toxin, which can facilitate the rapid detection and content detection of PT antigen, but there is no kit for pertussis disease diagnosis for FHA toxin
[0006] In addition, in the actual production process, it was found that the acellular-adsorbed DTP vaccine or the DPT-based combination vaccine used for the prevention of pertussis was different among different manufacturers or between different combination vaccines of the same manufacturer. There are differences in the registration quality standards of products in different countries

Method used

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  • Filamentous hemagglutinin detection kit and its application
  • Filamentous hemagglutinin detection kit and its application
  • Filamentous hemagglutinin detection kit and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Example 1 Preparation of hybridoma cells

[0061] (1) After the cyclopathy seeds were recovered, and the shake bottle was cultured in fermentation medium, 35.0 ± 2.0 degreulation was cultured for 44-46 hours, and the index growth period was gains.

[0062] (2) After harvest, it is sterilized, clarified, crude, and pure.

[0063] (3) Get purified, the FHA stock solution is obtained after detoxification of formaldehyde.

[0064] (4) After mixing emulsification after mixing emulsification of FHA stock solution and Frego adjuvant (0 day immunization is complete adjuvant, 14 days, 28 days immunized into Fernant Adjuvant), etc., 14 days, 28 days Multi-point immunized BALB / C mice in the back is 0.2 ml / only.

[0065] (5) Last immunization first blood test antibody titer, serum dilution 10 4 The indirect ELISA of the immunized mouse serum was 1.311 with FHA stock solution. After two weeks of last immunization, the selected mice were shocked by the harvest solution, and the splee...

Embodiment 2

[0066] Example 2 Cell fusion and strain

[0067] (1) Cell fusion recovery culture SP2 / 0 cell strain was expanded 3 days before the fusion, and the RPMI1640 cell culture medium was removed 1 day before fusion, and the culture solution was re-added, and SP2 / 0 cells were prepared.

[0068] (2) Immunization mice were sacrificed, and the spleen cell suspension of mice was prepared according to conventional methods.

[0069] (3) According to the results of splenocytes and SP2 / 0 cell counts, the amount of incomplete IMDM culture solution (GIBCO), SP2 / 0 cells are shaken, and the splenocytes are blown well with a pipette. The spleen cells were then mixed with SP2 / 0 cells from 1: 2 to 10: 1 in 50 ml of centrifuge tubes.

[0070] (4) Add incomplete IMDM culture solution to 50ml, centrifuge for 5-10 minutes, and pour a clean. Tap the fusion tube, so that the precipitated cell is loose, and the centrifugal can be placed 37 ° C water bath, ready to fusion.

[0071] (5) 1 ml of 50% of ...

Embodiment 3

[0078] Example 3 Sequence and Stability Detection of Hybridoma Cell Series Secrecy

[0079] Remove the frozen FHA mouse monoclonal hybridoma cell line from liquid nitrogen to expand cultivation, 10 6 The total nucleic acid is extracted at the above number, entrusted Beijing Liuhehua Great Technology Co., Ltd. via the PCR amplification of the heavy chains and light chains and sequencing, and then determines the corresponding amino acid sequence by nucleotide sequence, determined polynucleotides measured. Sequences and amino acid sequences for sequence lists.

[0080] After the completion of hybridoma cells, the freezed FHA mice monoclonal hybridoma cell line was removed from liquid nitrogen in the preparation of hybridoma cells. After expanding the culture, the antibody effectiveness of indirect ELISA was detected. Price, the ascites prepared in the previous period were tested simultaneously. The results showed that the monoclonal antibody of the hybridoma cell line was reached in ...

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Abstract

The invention relates to a kit for detecting filamentous hemagglutinin and its application. The present invention first obtains an antibody titer up to 10 7 , the affinity constant is 3.42×10 11 m ‑1 The filamentous hemagglutinin (FHA) monoclonal antibody of the present invention, the monoclonal antibody of the present invention is coated with ELISA kit, can be used for enzyme-linked immunoassay detection pertussis, detect the content of filamentous hemagglutinin in the vaccine production process and finished vaccine, In order to carry out quality control of vaccines, it has broad application prospects and market value.

Description

Technical field [0001] The present invention relates to the preparation of immunological and kits, and specific relates to a filamentous hemagglutinoid kit and its applications thereof. Background technique [0002] The Hundred Alternative cough is a acute respiratory infectious disease caused by the 100th Cthiobacterium, which is the highest-life infectious disease before the cyclic cough vaccine. By implementing the vaccination of a hundred-day cough vaccine, in particular, after the global implementation of immunization planning (EPI), the combined vaccine immunization coverage increases, and the global cyclopcia has been effectively controlled, and the incidence and mortality have declined significantly. According to the World Health Organization (WHO), the vaccination has been reduced after vaccination, which has been reduced by millions of diseases and millions of death. After the implementation of the immunization plan, China has been implemented in 1978, Bai Ba is incorpo...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/12C12N15/13G01N33/68G01N33/577G01N33/569A61K39/40A61P31/04
CPCA61P31/04C07K16/1225C07K2317/565C07K2317/76C07K2317/92C07K2317/94G01N33/56911G01N33/577G01N33/68G01N2333/235G01N2469/10
Inventor 程会欣张立志王治伟王巍巍刘波马国涛
Owner SINOVAC RES & DEV
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