Poliovirus type II monoclonal antibody and application thereof

A monoclonal antibody and polio technology, applied in the field of immunology, can solve the problems of good preparation specificity, inability to distinguish D antigen and C antigen well, and inability to truly reflect type II antigen vaccine type II immunogenicity, etc. , to achieve good virus-specific effect

Active Publication Date: 2016-08-31
NAT INST FOR FOOD & DRUG CONTROL
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

[0008] Due to the polyclonal antibody coating-polyclonal antibody sandwich detection system commonly used in the detection of poliovirus type II virus antigens, this system not only has a large cross-reaction for types I and III, but also cannot distinguish D well. Antigen and C antigen, especially when used in the detection of 3-type mixed vaccine Sabin IPV type Ⅱ antigen, the test results cannot truly reflect the content of type Ⅱ antigen and the immunogenicity of vaccine type Ⅱ due to cross-reaction, and WHO also recommends using The polyclonal antibody and monoclonal antibody are matched to detect the content of various antigens in the trivalent polio vaccine; 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 Antibodies that may form the basis for standardization of SabinIPV in vitro potency assays

Method used

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  • Poliovirus type II monoclonal antibody and application thereof
  • Poliovirus type II monoclonal antibody and application thereof
  • Poliovirus type II monoclonal antibody and application thereof

Examples

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Embodiment 1

[0035] Example 1 Immunogen preparation and animal immunization

[0036] (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.

[0037] (2) Inoculate Vero cells with working seed batch prepared by Sabin strain poliovirus type II virus at MOI=5-0.1, and culture at 32.5±0.5°C.

[0038] (3) The virus was cultured for 2-4 days, and the cell supernatant was harvested, which was the Sabin strain poliovirus type II virus harvesting liquid.

[0039] (4) Type II virus harvest liquid is clarified and concentrated by ultrafiltration membrane bag for more than 10 times.

[0040] (5) Then carry out molecular sieve chromatography and ion exchange chromatography, the monitoring wavelength is 280nm, respectively collect eluent and flow-through liquid to obtain purified liquid, and obtain purified and inactivated Sabin strain poliomyelitis II after formaldehyde inactivation Type vir...

Embodiment 2

[0043] Example 2 Cell fusion and strain establishment

[0044] (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.

[0045] (2) The immunized mice were sacrificed, and the mouse splenocyte suspension was prepared according to conventional methods.

[0046] (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.

[0047] (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

[0055] Example 3 Monoclonal Antibody Cell Line Ascites Preparation and Antibody ELISA Titer Detection

[0056] 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. JII-175.

[0057] Dilute the purified and inactivated Sabin strain poliomyelitis type Ⅱ virus with 0.01M PBS 1:200, coat 100 μl / well on the microtiter plate, overnight at 2-8°C, then add 100 μl / well 1:10 2 Initially 10-fold serially diluted negative control and JII-175 monoclonal antibody ascites, react at 37°C for 1 hour, add 1:4000 diluted HRP-labeled goat anti-mouse secondary antibody at 100 μl / well, react at 37°C for 1 hour, wash Plate, color development, termination, read OD450nm, thus detect the indirect ELISA antibody titer of the poliotype ...

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Abstract

The invention relates to a poliovirus type II monoclonal antibody and an application thereof, belonging to the field of immunology and the field of vaccines. In particular, the invention relates to a hybridoma cell strain generating the poliovirus type II monoclonal antibody, the monoclonal antibody produced by the hybridoma cell strain and applications of the hybridoma cell strain and the monoclonal antibody, wherein the collection number of the hybridoma cell strain is CGMCC No.12291.

Description

technical field [0001] The present invention relates to the field of immunology and vaccinology, in particular to an anti-poliomyelitis type II virus monoclonal antibody, a hybridoma cell line producing the antibody and the application of the antibody. Background technique [0002] Poliomyelitis is an acute infectious disease caused by poliovirus that seriously endangers children's health. The virus is a neurotropic virus that mainly invades the motor nerve cells of the central nervous system, and mainly damages the motor neurons in the anterior horn of the spinal cord. Most of the patients are children aged 1 to 6. The main symptoms are fever, general discomfort, severe limb pain, and flaccid paralysis with irregular distribution and varying severity, commonly known as polio. The clinical manifestations of poliomyelitis are varied and include very mild nonspecific lesions, aseptic meningitis (nonparalytic poliomyelitis), and flaccid weakness of various muscle groups (paraly...

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
CPCA61K39/00C07K16/1009G01N33/56983G01N33/577G01N2469/10G01N2469/20Y02A50/30
Inventor 李长贵徐康维英志芳王剑锋江征
Owner NAT INST FOR FOOD & DRUG CONTROL
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