Hybridoma cell strain and secretion monoclonal antibody and application thereof

A technology of hybridoma cells and monoclonal antibodies, applied in the direction of antibodies, analytical materials, antiviral agents, etc., can solve the problems of high cost, re-emergence of piglets, single preventive effect of vaccines, etc., and achieve good neutralizing activity and high binding strength Effect

Active Publication Date: 2015-06-10
LUOYANG PULIKE WANTAI BIOTECH
View PDF6 Cites 26 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the existing commercial vaccines at home and abroad are all classic vaccine strains. Although they have a certain effect on controlling the disease, they still cannot provide good passive protection for piglets.
At the same time, the level of maternal antibody obtained by piglets is not uniform, which can easily cause some piglets to get sick again and cause the pathogen to spread rapidly
Although yolk antibodies have been used to prevent or treat porcine epidemic diarrhea virus, yolk antibodies ar

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Hybridoma cell strain and secretion monoclonal antibody and application thereof
  • Hybridoma cell strain and secretion monoclonal antibody and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Example Embodiment

[0056] Example 1 Acquisition of hybridoma cell line

[0057] 1.1 Propagation and purification of PEDV virus

[0058] 1.1.1 Preparation of antigens

[0059] The Vero cells that have grown into a good monolayer (Vero cells are from Shanghai Institute of Biochemistry) were washed 2-3 times with PBS, and then inoculated with the inoculation dose of MOI=0.001 to PEDV HN1301 strain. The total amount of virus solution and maintenance solution during inoculation Incubate at 37°C for 60 minutes, mix gently every 20 minutes, discard the liquid in the bottle after adsorption, add 5mL of fresh maintenance solution, and place at 37°C CO 2 In an incubator, the virus solution was harvested when the cytopathic effect reached 90%, and was frozen at -20°C for later use.

[0060] 1.1.2 Purification of antigen

[0061] Take 200mL of virus solution, add 20mL of TritonX-100, shake at room temperature for 10min, centrifuge at 5000rpm for 30min in a high-speed refrigerated centrifuge, take the cen...

Example Embodiment

[0110] Example 2 Preparation and identification of monoclonal antibodies

[0111] 2.1 Preparation of monoclonal antibody ascites BALB / C female mice were first sensitized with sterilized liquid paraffin, 0.5 mL / mice was injected into the abdominal cavity, and 0.5 mL (about 2.0 × 10) was injected into the abdominal cavity after 7-10 days. 6 cells / mL) of hybridoma cells. It was observed that ascites was withdrawn when the abdomen of the mouse was significantly distended. The ascites was placed at 37°C, 1 h later at 4°C overnight. The next day, the supernatant was collected by centrifugation, aliquoted, and stored at -70°C.

[0112] 2.2 Identification of monoclonal antibody Ig types

[0113] Sigma monoclonal antibody typing reagent was used to identify the Ig type of the monoclonal antibody according to the instructions. The heavy chain of 8A3A10 monoclonal antibody was IgG2a subtype, and the light chain was Kappa chain.

[0114] 2.3 Determination of monoclonal antibody titer ...

Example Embodiment

[0134] Example 3 Evaluation of the effect of monoclonal antibody on prevention and treatment of viral infection

[0135] 3.1 Evaluation of the efficacy of monoclonal antibodies in preventing viral infection

[0136] Screening 15 2-3 day-old piglets with double-negative PEDV, RV, TGEV antigens and antibodies, and randomly divided them into 3 groups. The first group and the second group were orally administered 2 mL of the monoclonal antibody prepared in Example 2, and the third group was orally administered 2mL of PBS buffer solution, 24 hours after oral administration, the first and third groups were orally administered with 2mL of PEDV epidemic strain HN1301 enterovirus (including 1000 minimum incidence doses), and the second group was orally administered with CV777 enterovirus (including 1000 minimum incidence doses), The clinical symptoms of piglets were observed daily for 14 consecutive days, and the efficacy of the monoclonal antibody in preventing diarrhea virus infectio...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

Abstract

The invention discloses a hybridoma cell strain and an anti-porcine-epidemic-diarrhea-virus monoclonal antibody produced by the hybridoma cell strain. The anti-porcine-epidemic-diarrhea-virus monoclonal antibody has the preventing and/or treating function for porcine epidemic diarrhea viruses, solves the problem of piglet epidemic diarrhea virus infection caused when existing vaccines and maternal antibodies are insufficient, overcomes the defect that an existing vaccine is single in prevention function and can also be used for developing diagnostic reagent products for the porcine epidemic diarrhea viruses.

Description

technical field [0001] The invention relates to a hybridoma cell strain, a monoclonal antibody secreted by it, a pharmaceutical composition prepared by using the secreted monoclonal antibody, a test kit and an application, and belongs to the field of biomedicine. Background technique [0002] Porcine epidemic diarrhea (porcine epidemic diarrhea, PED) is an acute, contact and highly contagious digestive tract disease caused by porcine epidemic diarrhea virus (porcine epidemic diarrhea virus, PEDV), the main symptoms are vomiting, watery diarrhea and dehydration , The mortality rate of piglets within 7 days of infection is as high as 80%-100%. Cause huge loss to my country's pig industry. Since 2010, there have been large-scale outbreaks of diarrheal diseases in autumn and winter in my country. At the same time, large-scale outbreaks of diarrheal diseases have also occurred in South Korea, Japan, Taiwan, the United States and other places. Molecular biology studies have show...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
IPC IPC(8): C12N5/20C07K16/10A61K39/42A61P31/14G01N33/577G01N33/569
Inventor 田克恭王延辉张云静
Owner LUOYANG PULIKE WANTAI BIOTECH
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products