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Sabin strain poliomyelitis type I virus monoclonal antibody and application thereof

A poliomyelitis and monoclonal antibody technology, applied in the field of immunology, can solve the problem that the test results cannot truly reflect the content of type I antigens and the immunogenicity, etc., and achieve the effect of good virus specificity

Active Publication Date: 2015-02-18
SINOVAC BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

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

Method used

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  • Sabin strain poliomyelitis type I virus monoclonal antibody and application thereof
  • Sabin strain poliomyelitis type I virus monoclonal antibody and application thereof
  • Sabin strain poliomyelitis type I virus monoclonal antibody and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0030] Example 1 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) The working seeds prepared from Intravacc-derived sabin polio type I virus were inoculated into Vero cells at MOI=10-0.05, and cultured 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 I virus harvest liquid.

[0034] (4) Type I 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 I vaccine stock solution after formaldehyde inactivation.

[0036] (6) Mix and emul...

Embodiment 2

[0038] Example 2 Cell fusion and strain establishment

[0039] (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.

[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 3...

Embodiment 3

[0050] Example 3 Preparation of monoclonal antibody cell line ascites and detection of antibody titer

[0051] 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-I.

[0052] Dilute the stock solution of polio type Ⅰ vaccine with 0.01M PBS 1:20, coat 100 μl / well on a microtiter plate, overnight at 2-8°C, and detect the antibody of polio type Ⅰ ascites SIPV-I secreted by the hybridoma cells of the present invention Titer, antibody titer up to 105, higher titer. The results are shown in Table 1.

[0053] Table 1 Antibody titer test results

[0054]

[0055]

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Abstract

The invention provides a Sabin strain poliomyelitis type I virus monoclonal antibody and application thereof, and belongs to the field of immunology. The preparation method of the Sabin strain poliomyelitis type I virus monoclonal antibody comprises the following steps: carrying out immunization on mice with the Sabin strain poliomyelitis type I virus, mixing the mouse spleen cells with the mouse myeloma cells, screening and generating specific monoclonal antibody hybridoma cell strain resistaing against the Sabin strain poliomyelitis type I virus, and preserving the hybridoma cell strain with a preservation No. of CGMCC No.9231. The monoclonal antibody secreted by the hybridoma cell strain is high in valence, and has a large application prospect in diagnosis and treatment of poliomyelitis type I virus; meanwhile, the antibody can be used for specifically distinguishing poliomyelitis virus type I, type II and type III as well as other multiple enteroviruses to achieve good specificity; the Sabin strain poliomyelitis type I virus monoclonal antibody can be used for preparing an antigen content check kit for the poliomyelitis type I virus, and further used for identification and testing of the poliomyelitis type I virus, so that the Sabin strain poliomyelitis type I virus monoclonal antibody is wide in application prospect.

Description

technical field [0001] The present invention relates to the field of immunology and vaccinology, in particular to a monoclonal antibody against Sabin strain poliomyelitis type I virus, 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 poli...

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 程会欣金亚明王欣童钦江志华姚志东蔡芳高强尹卫东
Owner SINOVAC BIOTECH
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