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Hybridoma cell strain 8D3, monoclonal antibody and application thereof

A technology of hybridoma cell line and monoclonal antibody, applied in the direction of antiviral immunoglobulin, instrument, biochemical equipment and method, etc., can solve the problem of not being able to distinguish between mad cow disease and sheep scrapie

Pending Publication Date: 2022-07-15
中国海关科学技术研究中心
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the current TSE diagnostic method evaluated by the European Union is the same method, which is to detect PrPsc in brain tissue, which can be used to detect both cattle and sheep. If the bovine brain is positive, it is judged as mad cow disease. If the brain test is positive, it is judged as scrapie, but mad cow disease and scrapie cannot be distinguished

Method used

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  • Hybridoma cell strain 8D3, monoclonal antibody and application thereof
  • Hybridoma cell strain 8D3, monoclonal antibody and application thereof
  • Hybridoma cell strain 8D3, monoclonal antibody and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Establishment of hybridoma cell line 8D3

[0040] 1. Synthesis of immunogens

[0041] Sheep PrP polypeptide (ovPrP 89-104) was synthesized by Shenggong Bioengineering (Shanghai) Co., Ltd., the synthetic peptide sequence: GGGGWGQGGSHSQWNK (see the sequence listing for details), and the carrier protein keyhole limpethemocyanin (hemocyanin, KLH) was coupled with glutaraldehyde ), the purity is more than 95%, and the OvPrP(89-104aa)-KLH polypeptide conjugate is obtained.

[0042] 2. Immunization of mice

[0043] Three 6-7 week old Balb / c mice (purchased from the Institute of Genetics, Chinese Academy of Sciences) were immunized with the synthetic OvPrP(89-104aa)-KLH polypeptide conjugate as the immunizing antigen. The route, dosage and adjuvant of each immunization are shown in the table 1. The first three immunizations were separated by 4 weeks each time, and a small amount of blood was collected from the tail 12-15 days after the fourth immunization to determine the se...

Embodiment 2

[0093] Preparation of monoclonal antibodies

[0094] 1. Recovery of hybridoma cells

[0095] Take out the cryovial from the liquid nitrogen tank, quickly put it into a 37°C water bath, and shake it to thaw as soon as possible. Centrifuge at 1000 rpm for 10 min and discard the supernatant. Suspend the pellet with 20% serum DMEM, add it to the cell flask, and set CO 2 Cultivated in an incubator. The next day, the medium was exchanged with DMEM containing 20% ​​serum.

[0096] 2. Monoclonal antibodies can be prepared by in vitro culture method and / or in vivo induced ascites method

[0097] in vitro culture

[0098]The established hybridoma cell line 8D3 was expanded and cultured until all cells died. The cell culture supernatant was collected, centrifuged at 1000 rpm for 10 min, and the supernatant was stored at -20°C for later use.

[0099] In vivo induced ascites method

[0100] The multiparous Balb / c mice were selected and injected intraperitoneally with sterilized par...

Embodiment 3

[0102] Identification of monoclonal antibodies

[0103] 1. Identification of immunoglobulin subclasses and light chain species of monoclonal antibodies

[0104] Identified with SouthernBiotech kit. The specific method is as follows:

[0105] (1) Coating: Dilute the antibody provided in the kit with a phosphate buffer solution with a pH value of 9.6 to a concentration of 5-10ug / ml, 100ul / well, overnight at 4°C.

[0106] (2) Washing: washed three times with PBS containing 0.05% Tween-20.

[0107] (3) Blocking: block with PBST containing 1% bovine serum albumin, 200ul / well, and incubate at 37°C for 1h.

[0108] (4) Washing: same as above.

[0109] (5) Sample loading: add hybridoma cell culture supernatant, 100ul / well, and incubate at 37°C for 1h.

[0110] (6) Washing: same as above.

[0111] (7) Enzyme addition: Dilute alkaline phosphatase-labeled immunoglobulins, subclasses, light chains and heavy chains with PBST containing 1% bovine serum albumin to appropriate concentra...

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Abstract

The invention provides a hybridoma cell strain 8D3, a monoclonal antibody and application of the monoclonal antibody, and belongs to the technical field of Ruan virus detection. The hybridoma cell strain 8D3 is preserved in the China General Microbiological Culture Collection Center, and the preservation number is CGMCC (China General Microbiological Culture Collection Center) No.45105. The hybridoma cell strain 8D3 can efficiently and stably secrete the monoclonal antibody, the monoclonal antibody obtained through secretion is good in specificity, prion protein can be specifically recognized, and mad cattle disease and sheep itch disease pathogens can be distinguished.

Description

technical field [0001] The invention belongs to the technical field of Nguyen virus detection, in particular to a hybridoma cell line 8D3, a monoclonal antibody and applications thereof. Background technique [0002] Prions are the causative agent of transmissible spongiform encephalopathy in animals and humans. Since normal PrP (PrP c ) into pathogenic PrP (PrPSc), thereby causing a series of neurodegenerative diseases, named "transmissible spongiform encephalopathy" based on their contagious nature and common neuropathological changes, also called prion disease. The common feature of prion diseases is a chronic degenerative disease that can cause fatal central nervous system, and its typical pathological features include neuronal death, vacuolar degeneration, glial and astrocytic hyperplasia, PrPSc (prion) Abnormal form) accumulation and amyloid plaque formation, many cavities in the brain and central nervous system, spongy changes, usually bilaterally symmetrical lesio...

Claims

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Application Information

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IPC IPC(8): C12N5/20C07K16/08G01N33/577G01N33/569C12R1/91
CPCC07K16/08G01N33/577G01N33/56983C07K2317/94G01N2333/005G01N2469/10Y02A40/70
Inventor 李炎鑫刘全国史喜菊徐立伟侯颖
Owner 中国海关科学技术研究中心
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