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Sabin strain poliovirus type III monoclonal antibody and application thereof

A technology for polio and monoclonal antibodies, applied in the field of immunology, can solve the problems of lack of vaccines and test results that cannot truly reflect the type III immunogenicity of type III antigen vaccines, and achieve good virus-specific effects

Active Publication Date: 2015-02-25
SINOVAC BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Due to the multi-antibody coating-multi-antibody sandwich detection system commonly used in the detection of type III virus antigens, this system has a large cross-reaction for types I and II, especially for the type III mixed vaccine Sabin strain IPV. When detecting type 3 antigens, the test results cannot truly reflect the content of type Ⅲ antigens and the immunogenicity of type Ⅲ vaccines due to cross-reaction. Type antigen content; at present, there is still a lack of standardized reagents for the detection of Sabin strain IPV D antigen in the world, and the preparation of monoclonal antibodies with good specificity and neutralizing activity can lay the foundation for the standardization of Sabin strain IPV in vitro potency detection methods

Method used

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  • Sabin strain poliovirus type III monoclonal antibody and application thereof
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  • Sabin strain poliovirus type III monoclonal antibody and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0030] Example 1 Sabin strain poliovirus type III immunogen preparation and animal immunization

[0031](1) Take the Vero cell working cell bank and culture it at 36.5±0.5°C after recovery until the cell concentration is 0.1-10×10 6 cells / ml, the virus was inoculated.

[0032] (2) Inoculate Vero cells with working seeds prepared from sabin strain polio type III virus derived from Intravacc at MOI=10-0.05, and culture at 32.5±0.5°C.

[0033] (3) The virus was cultured for 2-4 days, and the cell supernatant was harvested, which was the poliovirus Sabin strain type III virus harvest liquid.

[0034] (4) Type III harvest liquid is clarified and concentrated more than 5 times with ultrafiltration membrane bag.

[0035] (5) Then carry out molecular sieve chromatography and ion exchange chromatography, the monitoring wavelength is 280nm, collect the eluate and flow-through respectively to obtain the purified solution, and obtain the type III vaccine stock solution after formaldehyd...

Embodiment 2

[0037] Example 2 Cell Fusion and Strain Construction

[0038] (1) Recover and culture the SP2 / 0 cell line before cell fusion, expand the culture 3 days before fusion, remove RPMI 1640 cell culture medium (Gibco) 1 day before fusion, and add culture medium again to prepare SP2 / 0 cells.

[0039] (2) The immunized mice were sacrificed, and the mouse splenocyte suspension was prepared according to conventional methods.

[0040] (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.

[0041] (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 37...

Embodiment 3

[0049] Example 3 Monoclonal Antibody Cell Line Ascites Preparation and Antibody Titer Detection

[0050] Resuscitate the frozen hybridoma cells obtained in Example 2 according to conventional methods, and cultivate them. When the cells cover more than 50% of the bottom of the 25ml cell culture bottle, BALB / c mice can be inoculated intraperitoneally according to conventional methods, and the ascites can be collected regularly. SIPV-Ⅲ.

[0051] Poliomyelitis type III vaccine stock solution was diluted with 0.01M PBS1:20, 100μl / well was coated with an enzyme-titer plate, and left overnight at 2-8°C to detect the antibody titer of poliomyelitis type III ascites SIPV-Ⅲ, and the antibody titer was 10 6 , higher potency. The results are shown in Table 1.

[0052] Table 1 Antibody Titer Test Results

[0053]

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Abstract

The invention provides a Sabin strain poliovirus type III monoclonal antibody and an application thereof, and belongs to the field of immunology. After a mouse is immunized and inoculated with Sabin strain poliovirus type III, mouse spleen cells are fused with mouse myeloma cells, a hybridoma cell strain producing the anti-Sabin strain poliovirus type III specific monoclonal antibody is screened and has the preservation number of CGMCC No.9233, and the secreted monoclonal antibody has high titer, has strong neutralizing activity, and can effectively block infection of the poliovirus type III; at the same time, the antibody can specifically distinguish the poliovirus type III from poliovirus type I, poliovirus type II and other various enteroviruses, has good specificity, and can be used for preparing poliovirus type III antigen content detection kits and antibody detection kits, also can be used for identification detection of the poliovirus type III, and has the broad application prospect.

Description

technical field [0001] The invention relates to the field of immunology and vaccinology, in particular to a neutralizing monoclonal antibody against Sabin strain poliovirus type III, a hybridoma cell line producing the antibody and the application of the antibody. Background technique [0002] Poliomyelitis disease existed in human society as early as 1580-1350 BC. The earliest evidence comes from a figure in a standing posture in ancient Egyptian stone murals, with obvious symptoms of poliomyelitis disease: bent and atrophied legs, Deformity of the top of the foot. However, it was not until 1789 that Englishman Michael Underwood first described the clinical symptoms of polio disease, which he called "weakness of the lower extremities". Half a century later, research by the Germans showed that polio disease could be contagious. In 1894, the largest catastrophe of paralyzing infants on record occurred in the United States, which was later confirmed to be a disease of polio....

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/20C07K16/10G01N33/577G01N33/569A61K39/42A61P31/14C12R1/91
CPCY02A50/30
Inventor 江志华童钦金亚明王欣程会欣王一丁姜德玉蔡芳高强尹卫东
Owner SINOVAC BIOTECH
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