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PRRSV broad-spectrum neutralizing monoclonal antibody 5d9 and its application

A monoclonal antibody, broad-spectrum technology, applied in the direction of antibodies, applications, antiviral agents, etc., can solve the problems of unreported monoclonal antibody application, lack of neutralizing activity, etc., to prevent virus invasion and protect from infection. Effect

Active Publication Date: 2022-03-25
NORTHWEST A & F UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Monoclonal antibodies with high neutralizing activity have also appeared in the prior art. For example, the MAb LM26 prepared by Liu Mengying et al. and activity, the highest neutralization titer is 1:50; B. Pirzadeh et al. ( Journal of General Virology (1997), 78, 1867–1873) the monoclonal antibody against GP5 had neutralizing activity against American type VR-2332, and the neutralizing potency was 1:32-1:128, but it had neutralizing activity against European type (Lelystad prototype) does not have neutralizing activity
However, monoclonal antibodies with broad-spectrum neutralization and their applications have not been reported in existing studies

Method used

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  • PRRSV broad-spectrum neutralizing monoclonal antibody 5d9 and its application
  • PRRSV broad-spectrum neutralizing monoclonal antibody 5d9 and its application
  • PRRSV broad-spectrum neutralizing monoclonal antibody 5d9 and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1: Preparation and identification of monoclonal antibody 5D9

[0026] 1.1 Establishment of hybridoma cell lines

[0027] The PRRSV virus SD16 (provided by the Laboratory of Immunobiology, Northwest Agriculture and Forestry College of Veterinary Medicine) was used as the immunogen, and emulsified with Freund's complete adjuvant (Sigma) 1:1 to immunize 6-week-old female Balb / c mice (provided by Provided by Xi'an Jiaotong University), subcutaneous injection in the abdomen, the dose is (3 × 10^7 PFU / only), booster immunization once every 14 days. Seven days after the third immunization, blood was collected from the tail vein and IFA was used to detect the antibody titer against the immunogen in the serum of the mice. The mice with the best titer were immunized by tail vein injection, and the immunogen was mixed with normal saline 1:1. , the dose is (3×10^7 PFU / only).

[0028] cell fusion

[0029] (1) Aseptically obtain the spleen cells of the immunized mice to ...

Embodiment 2

[0053] Example 2: Determination of neutralizing activity of monoclonal antibody 5D9

[0054] 2.1 Neutralizing activity assay

[0055] Virus neutralization experiments were performed using the monoclonal antibody 5D9 to detect its neutralization activity. PAM cells were plated into 24-well plates, and 0.01 MOI of different types of PRRSV SD16 virus were added to each virus. Each virus was added with 100 μg / ml, 200 μg / ml, 300 μg / ml, and 400 μg / ml of antibodies. After incubation at 37°C for 1 h, the cells were replaced. After 36 hours, Western blot and qPCR were performed to confirm that it had neutralizing activity. For specific results, see figure 1 .

[0056] based on figure 1 The results showed that the natural target cells of PRRSV in the host alveolar macrophages (PAMs) were cultured in vitro, and the purified monoclonal antibody 5D9 was used according to the concentration of 0.05, 0.1, 0.2, 0.4 μM / mL (micromoles per milliliter). 0.01M PRRSV-SD16 virus was incubated ...

Embodiment 3

[0062] Example 3: Preparation of monoclonal antibody 5D9 ascites and detection of neutralizing activity

[0063] 3.1 Preparation of ascites

[0064] Mice were sensitized by injection of paraffin oil one week in advance, and the selected hybridoma cells in the logarithmic growth phase were intraperitoneally injected into mice one week later, with 5×10^5 cells per mouse. The ascites was collected 7 days after the injection of hybridoma cells. The removed ascites was centrifuged at 5000 g × 10 min at 4°C, and the supernatant ascites was aspirated and collected in an EP tube, and stored at -20°C.

[0065] Detection of Neutralizing Activity of Monoclonal Antibody 5D9 in Ascites

[0066] Using the ascites fluid containing the monoclonal antibody, a virus neutralization experiment was performed by a 4-fold dilution method to detect its neutralization activity. PAM cells were plated to a 24-well plate, and 0.01MOI PRRSV-SD16 virus (PRRSV-II type) or PRRSV-GZ11 (PRRSV-I type virus) w...

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Abstract

The present invention uses PRRSV virus fluid-SD16 as an immunogen to immunize Balb / c mice. After cell fusion and virus infection, Marc145 cells were screened for clones to obtain a positive hybridoma cell line that efficiently secreted monoclonal antibodies, and the mouse monoclonal antibody 5D9 was obtained. The subtype of the monoclonal antibody 5D9 was determined as an IgM monoclonal antibody by ELISA technique. PRRSV-I and PRRSV-II viruses infected Marc145 cells, and the monoclonal antibody was detected by IFA technology, which proved that the monoclonal antibody 5D9 had broad-spectrum reactivity to PRRSV-I and PRRSV-II viruses. It was then used for virus neutralization experiments, and Western blot technology and qPCR technology were used to prove that the monoclonal antibody has neutralizing activity against both PRRSV-I and PRRSV-II viruses, which can prevent virus invasion and protect the body from infection.

Description

technical field [0001] The invention belongs to the biological field, in particular to a PRRSV monoclonal antibody with broad-spectrum neutralizing activity, which can neutralize type I and type II viruses in a broad spectrum, and can be used for the development of a therapeutic drug for porcine reproductive and respiratory syndrome. Background technique [0002] Porcine reproductive and respiratory syndrome (PRRS) is a viral infection caused by porcine reproductive and respiratory syndrome virus (PRRSV), which is mainly characterized by abortion of sows and respiratory disorders of piglets. disease and can cause severe immunosuppression. The virus has spread widely in pig herds around the world, causing huge economic losses to the world pig industry. It has become one of the major epidemics in large-scale pig farms around the world and a major problem in the control of global pig diseases. PRRSV is an enveloped single-stranded positive-stranded RNA virus belonging to the f...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/10C12N15/13G01N33/577G01N33/569A61K39/42A61P31/14
CPCC07K16/10G01N33/577G01N33/56983A61P31/14C07K2317/56A61K2039/505
Inventor 南雨辰周恩民武春燕
Owner NORTHWEST A & F UNIV
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