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Hybridoma cell strain, monoclonal antibody generated by hybridoma cell strain and application

A hybridoma cell line and monoclonal antibody technology, applied in biochemical equipment and methods, microorganisms, biomaterial analysis, etc., can solve the problems of false positives, false negatives, inability to distinguish specific types of antibodies, etc. Efficient secretion effect

Active Publication Date: 2018-07-27
SHANDONG AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The PCR method is easily interfered by many factors, and false positive and false negative results may occur; the ELISA method currently has no finished kits for specific detection of ALV-A on the market, and the existing kits are produced by IDEXX in the United States ALV-A / B antibody detection kit, but cannot distinguish the specific type of antibody in the sample; immunohistochemistry and indirect immunofluorescence methods also require highly specific monoclonal antibodies

Method used

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  • Hybridoma cell strain, monoclonal antibody generated by hybridoma cell strain and application
  • Hybridoma cell strain, monoclonal antibody generated by hybridoma cell strain and application
  • Hybridoma cell strain, monoclonal antibody generated by hybridoma cell strain and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Embodiment 1: the preparation of hybridoma cell line

[0050] (1) Prepare and purify subgroup A gp85 recombinant protein by conventional methods;

[0051] (2) Dilute the purified gp85 recombinant protein of subgroup A to 1.0 mg / mL with sterilized PBS, and immunize 6-week-old female Blab / C mice by intraperitoneal injection, 0.2 mL / mouse, four times.

[0052] Mix the above recombinant protein with complete / incomplete Freund's adjuvant in equal volume. The 3rd and 5th w booster immunization after the first immunization, the protein dose used was the same as the first immunization. The adjuvant used for the first immunization was complete Freund's adjuvant, and the adjuvant was changed to incomplete Freund's adjuvant for booster immunization. One week after the third immunization, blood was collected from the tail vein of the mice, the serum was separated, and the antibody level of the mice was detected. Four weeks after the third immunization, a booster immunization was...

Embodiment 2

[0077] Example 2: Preparation of ascites antibody using hybridoma cells

[0078] (1) Select healthy non-immunized 10-week-old female Balb / c mice, and inject liquid paraffin intraperitoneally 1-2 weeks before hybridoma cell inoculation, 0.5 mL / mouse.

[0079] (2) Pipette the adherent hybridoma cells (preservation number: CGMCC NO.14290) into a single-cell suspension, wash the tumor cells with DMEM basal medium 2-3 times, and count and dilute the tumor cells after low-speed centrifugation up to 5×10 6 individual / mL. Slowly inject 0.2 mL of cell suspension intraperitoneally into the treated mice in multiple directions.

[0080] (3) After injecting hybridoma cells for 10 days, observe the production of ascites in mice (such as figure 2 shown). If the abdomen is obviously swollen and the skin feels tense when you touch it with your fingers, you can use a syringe with a 16-gauge needle to slowly draw out the ascites. Generally, it can be collected 2-3 times continuously, with ...

Embodiment 3

[0082] Example 3: Specific detection of monoclonal antibodies

[0083] Using indirect immunofluorescence (IFA) to detect the specificity of the ascites antibody prepared in Example 2, the steps are as follows:

[0084] Chicken embryo fibroblasts (CEF) were grown in a 96-well cell culture plate. After the cells grew into a monolayer, the CEF cells were infected with ALV-A, ALV-B and ALV-J respectively. After 5-7 days of infection, they were treated with cold acetone -fixed with ethanol (6:4) for 5 minutes, washed once with PBS, dried in the air, and stored at -20°C as the antigen for detection. When performing IFA, properly dilute the ascites antibody to be detected and drop it on the virus-infected 96-well cell culture plate, incubate at 37°C for 30 minutes, wash 5 times with PBS, and then add goat anti-mouse IgG fluorescent labeling antibody, continue to incubate for 30 min, wash 5 times with PBS, and finally observe under a fluorescent microscope, the results are as follows...

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Abstract

The invention relates to the technical field of biology, and discloses a hybridoma cell strain, a monoclonal antibody generated by the hybridoma cell strain and application. The hybridoma cell strainhas the preservation number being CGMCC NO.14290. The monoclonal antibody generated by the hybridoma cell strain can recognize the subgroup A avian leukosis virus gp85 envelope protein and can be usedfor developing subgroup A avian leukosis diagnosis and treatment reagents or medicine; a material basis is provided for subgroup A avian leukosis clinical differential diagnosis and laboratory research.

Description

technical field [0001] The invention relates to the technical field of biotechnology, in particular to a hybridoma cell line and the monoclonal antibody it produces and its application. Background technique [0002] Avian Leukosis Virus (ALV) belongs to the family Retroviridae, the subfamily Oncoviridae, and the genus Avian Alpharetrovirus, and can cause various positive or malignant tumors in poultry. According to the characteristics of viral envelope glycoprotein, virus interference experiment, host range and other molecular biological characteristics, ALV is classified into 11 subgroups, of which A, B, C, D, E, J, K subgroups are from chicken Separated to get. Subgroups A and B are the most common exogenous viruses in commercial layers. [0003] Avian leukemia (Avian leukemia, AL) is an infectious tumor disease caused by subgroup A avian leukemia virus (ALV-A), which mainly infects laying hens, resulting in decreased immunity and production performance of laying hens, e...

Claims

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Application Information

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IPC IPC(8): C12N5/20C07K16/10G01N33/577G01N33/569
CPCC07K16/1036G01N33/56983G01N33/577G01N2333/15
Inventor 郭慧君郭晗璞李宏梅张丹丹胡卫国闫泽一
Owner SHANDONG AGRICULTURAL UNIVERSITY
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