A pertussis toxin detection kit and its application

A detection kit, a technique for pertussis, which is applied in measurement devices, immunoassays, instruments, etc., can solve the problems of long test period, high cost, and inability to quantify, and achieve the effect of effective immune effect.

Active Publication Date: 2020-10-30
SINOVAC RES & DEV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, animal experiments have disadvantages such as long test period, high cost, and large fluctuations in test results, and can only be used for identification and cannot be quantified.
In the actual production process, it was found that the acellular-adsorbed DPT vaccine used for the prevention of pertussis or the combined vaccine prepared based on DPT was not effective among different manufacturers, between different combined vaccines of the same manufacturer, even if the same product of the same manufacturer There are also differences in the registration quality standards in different countries

Method used

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  • A pertussis toxin detection kit and its application
  • A pertussis toxin detection kit and its application
  • A pertussis toxin detection kit and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] The preparation of embodiment 1 hybridoma cells

[0033] (1) Inoculate pertussis working seeds into fermentation medium after resuscitation and shaking flask culture, culture at 35.0±2.0°C for 44-46 hours, and harvest at the late stage of exponential growth phase.

[0034] (2) After harvesting, it is sterilized, clarified, rough and pure.

[0035] (3) The purified solution is obtained, and the PT stock solution is obtained after glutaraldehyde detoxification.

[0036] (4) Mix and emulsify equal volumes of the vaccine stock solution and Freund's adjuvant (Freund's complete adjuvant for immunization on day 0, incomplete Freund's adjuvant for immunization on day 14 and 28) BALB / c mice were immunized subcutaneously on the back with 0.2ml / mouse.

[0037] (5) One week after the last immunization, blood was collected to detect the antibody titer, and the serum was diluted 10 4The OD value of the indirect ELISA of the PT-detox solution immunized mouse serum was 1.126. Two w...

Embodiment 2

[0038] Example 2 Cell Fusion and Strain Construction

[0039] (1) Resuscitate and culture the SP2 / 0 cell line before cell fusion, expand the culture 3 days before fusion, remove RPMI1640 cell culture medium (Gibco) 1 day before fusion, and add culture medium again to prepare SP2 / 0 cells.

[0040] (2) The immunized mice were sacrificed, and the mouse splenocyte suspension was prepared according to conventional methods.

[0041] (3) Add an appropriate amount of incomplete IMDM culture medium (Gibco) according to the counting results of splenocytes and SP2 / 0 cells, shake and mix the SP2 / 0 cells, and pipette the splenocytes evenly. Then the splenocytes and SP2 / 0 cells were mixed in a 50ml centrifuge tube at a ratio of 1:2 to 10:1, and mixed well.

[0042] (4) Add incomplete IMDM culture medium to 50ml, centrifuge for 5-10 minutes, and pour out the supernatant. Lightly tap the bottom of the fusion tube to loosen and evenly precipitate the cells, and place the centrifuge tube in a...

Embodiment 3

[0051] Example 3 Stability Detection of Monoclonal Antibody Secreted by Hybridoma Cell Lines

[0052] After the 3rd month and the 9th month after the preparation of the hybridoma cells, the frozen PT mouse monoclonal antibody hybridoma cell line with the preservation number CGMCC No. 15288 was taken out from liquid nitrogen for recovery and expanded culture. The ascites was prepared, and the antibody titer was detected by indirect ELISA, and the ascites prepared in the previous stage was used as a control for detection at the same time. As a result, the monoclonal antibody ascites titer prepared by the hybridoma cell line of the present invention reaches 10 7 Above, there is no difference with the titer of ascites in the previous stage, indicating that the titer of ascites prepared after cell preservation has not decreased. Therefore, the antibody secretion activity of the monoclonal cell line is not reduced, and the stability is good. The monoclonal antibody ascitic fluid p...

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Abstract

The invention relates to a pertussis toxin detection reagent box and application thereof. Firstly, a pertussis toxin monoclonal antibody with the antibody valence being as high as 10<7>, and the affinity constant being 3.85*10<11>M<-1>, the monoclonal antibody is obtained through secretion of a hybridoma cell strain with the preservation number being CGMCC No. 15288, the monoclonal antibody is coated with the ELISA reagent box and can be used for detecting pertussis vaccine production process and the content of pertussis toxin in finished product vaccines by using a enzyme linked immunosorbentassay, therefore quality control over the vaccines can be conducted conveniently, and the reagent box has a wide application prospect and market value.

Description

technical field [0001] The invention relates to the technical field of immunology and kit preparation, in particular to a pertussis toxin detection kit and its application. Background technique [0002] Pertussis is a bacterial infectious disease that spreads through the respiratory tract. Before the pertussis vaccine was widely vaccinated, it was the infectious disease with the highest mortality rate in children. Through the implementation of pertussis vaccination, especially after the global implementation of the expanded immunization program, the immunization coverage of DPT combined vaccine has been improved, the global pertussis has been effectively controlled, and the morbidity and mortality have been significantly reduced. According to World Health Organization (WHO) estimates, the implementation of vaccination has reduced tens of millions of cases and millions of deaths. Since EPI was implemented in China in 1978, DPT was included in the children's immunization plan...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/569C12N5/20C07K16/12A61K39/40A61P31/04C12R1/91
CPCA61K2039/505C07K16/1225C07K2317/33C07K2317/35C07K2317/76C07K2317/92C07K2317/94G01N33/56911G01N2469/10
Inventor 程会欣吴君兰张立志王治伟王巍巍刘波马国涛
Owner SINOVAC RES & DEV
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