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A kind of poliovirus type Ⅱ monoclonal antibody and its application

A monoclonal antibody, poliomyelitis 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 the type II immunogenicity of type II antigen vaccines. , to achieve a good virus-specific effect

Active Publication Date: 2019-06-11
NAT INST FOR FOOD & DRUG CONTROL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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 Antigen and C antigen, especially when used in the detection of the 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 Polyclonal antibodies and monoclonal antibodies are matched to detect the content of various antigens in 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 Antibody that may form the basis for standardization of Sabin IPV in vitro potency assays

Method used

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  • A kind of poliovirus type Ⅱ monoclonal antibody and its application
  • A kind of poliovirus type Ⅱ monoclonal antibody and its application
  • A kind of poliovirus type Ⅱ monoclonal antibody and its application

Examples

Experimental program
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Effect test

Embodiment 1

[0035] Example 1 Preparation of immunogen and animal immunization

[0036] (1) Resuscitate the working cell bank of Vero cells and incubate at 36.5±0.5℃ until the cell concentration is 0.1-10×10 6 When cells / ml, inoculate virus.

[0037] (2) The working seed batch prepared by Sabin strain poliomyelitis type II virus was used to inoculate Vero cells at MOI=5~0.1 and cultured at 32.5±0.5℃.

[0038] (3) The virus is cultured for 2 to 4 days, and the cell supernatant is harvested, which is the harvest liquid of Sabin strain poliovirus type II virus.

[0039] (4) After clarification of the type II virus harvest liquid, it is concentrated more than 10 times with an ultrafiltration membrane package.

[0040] (5) Then perform molecular sieve chromatography and ion exchange chromatography, monitor the wavelength at 280nm, collect the eluate and flow-through respectively, and then obtain the purified solution. After the formaldehyde inactivation, the purified and inactivated Sabin strain poliomye...

Embodiment 2

[0043] Example 2 Cell fusion and establishment

[0044] (1) Resuscitate and culture SP2 / 0 cell line before cell fusion, expand the culture 3 days before fusion, remove RPMI1640 cell culture medium (Gibco) one day before fusion, add culture medium again, and prepare SP2 / 0 cells.

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

[0046] (3) Add an appropriate amount of incomplete IMDM culture medium (Gibco) according to the count results of splenocytes and SP2 / 0 cells, shake and mix the SP2 / 0 cells, and pipette the splenocytes evenly. Then mix the spleen cells and SP2 / 0 cells 1:2-10:1 in a 50ml centrifuge tube and mix well.

[0047] (4) Add incomplete IMDM culture medium to 50 ml, centrifuge for 5-10 minutes, and pour out the supernatant. Tap the bottom of the fusion tube to loosen the pelleted cells evenly, and place the centrifuge tube in a 37°C water bath to prepare for fusion.

[0048] (5) Use a ...

Embodiment 3

[0055] Example 3 Preparation of ascites of monoclonal antibody cell line and detection of antibody titer by ELISA

[0056] Resuscitate and culture the frozen hybridoma cells obtained in Example 2 according to conventional methods. When the cells cover more than 50% of the bottom of the 25ml cell culture flask, BALB / c mice can be inoculated intraperitoneally according to conventional methods, and ascites are collected regularly. JⅡ-175.

[0057] The purified and inactivated Sabin strain poliomyelitis type II virus was diluted 1:200 with 0.01M PBS, 100μl / well was coated on the ELISA plate, 2-8℃ overnight, and then 100μl / well was added 1:10 2 Start a 10-fold dilution of the negative control and JII-175 monoclonal antibody ascites, react at 37°C for 1 hour, add 100μl / well of 1:4000 diluted HRP-labeled goat anti-mouse secondary antibody, and wash at 37°C for 1 hour Plate, develop, stop, and read OD450nm, thereby detecting the antibody titer of the indirect ELISA method of the poliovirus...

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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 a monoclonal antibody against polio II virus, a hybridoma cell strain producing the antibody, and the application of the antibody. Background technique [0002] Poliomyelitis is an acute infectious disease caused by polio virus that seriously endangers children's health. The virus is a neurotropic virus, which mainly invades the motor nerve cells of the central nervous system, and mainly damages the motor neurons of the anterior horn of the spinal cord. The patients are mostly children aged 1 to 6 years. The main symptoms are fever, general malaise, severe limb pain, irregularly distributed and delayed paralysis of varying severity, commonly known as polio. The clinical manifestations of polio are diverse, including mild non-specific lesions, aseptic meningitis (non-paralytic poliomyelitis), and flaccid weakness of various muscle groups (paralytic poliomyelitis). In...

Claims

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Application Information

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Patent Type & Authority Patents(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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