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Sabin strain poliovirus type II monoclonal antibody and application thereof

A polio, monoclonal antibody technology, applied in the field of immunology, can solve the problem that the test results cannot truly reflect the immunogenicity of the content of type II antigen, and achieve the effect of good virus specificity

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 polyantibody coating-polyantibody sandwich detection system commonly used in the detection of poliovirus type Ⅱ virus antigens, this system has a large cross-reaction for types Ⅰ and Ⅲ, especially when it is used for the type 3 mixed vaccine Sabin strain During the detection of IPV type II antigen, due to cross-reaction, the test results cannot truly reflect the content of type II antigen and the immunogenicity of vaccine type II. At the same time, WHO also recommends the matching method of polyclonal antibody and monoclonal antibody to detect trivalent polio vaccine The content of various antigens in the test; 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 the detection method of Sabin strain IPV in vitro efficacy

Method used

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  • Sabin strain poliovirus type II monoclonal antibody and application thereof
  • Sabin strain poliovirus type II monoclonal antibody and application thereof
  • Sabin strain poliovirus type II 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) Inoculate Vero cells with working seeds prepared from sabin polio type Ⅱ virus derived from Intravacc at an MOI of 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 II virus harvest solution.

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

[0036] (6) Mix and...

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-II.

[0052] Dilute the polio type Ⅱ vaccine stock solution with 0.01M PBS 1:20, coat 100 μl / well on an enzyme-titer plate, and overnight at 2-8°C to detect the antibody titer of polio type Ⅱ ascites SIPV-Ⅱ, and the antibody titer is the highest up to 10 6 , the results are shown in Table 1.

[0053] Table 1 Antibody titer test results

[0054]

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Abstract

The invention provides a Sabin strain poliovirus type II monoclonal antibody and an application thereof, and belongs to the immunology field. After a mouse is immunized and inoculated with Sabin strain poliovirus type II, mouse spleen cells are fused with mouse myeloma cells, a hybridoma cell strain producing the anti-Sabin strain poliovirus type II specific monoclonal antibody is screened and has the preservation number of CGMCC No.9232, and the secreted monoclonal antibody has high titer, has strong neutralizing activity, and can effectively block infection of the poliovirus type II; at the same time, the antibody can specifically distinguish the poliovirus type II from poliovirus type I, poliovirus type III and other various enteroviruses, and can be used for preparing type II antigen content detection kits and antibody detection kits of the poliovirus type II and a poliomyelitis inactivated vaccine, also can be used for identification detection of the poliovirus type II, and has the broad application prospect.

Description

technical field [0001] The invention relates to the fields of immunology and vaccinology, in particular to a neutralizing monoclonal antibody against Sabin strain poliovirus type II, 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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IPC IPC(8): C12N5/20C07K16/10G01N33/577G01N33/569A61K39/42A61P31/14C12R1/91
CPCY02A50/30
Inventor 王欣江志华程会欣童钦金亚明阴彦辉蔡芳高强尹卫东
Owner SINOVAC BIOTECH
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