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Preparation method and application of monoclonal antibody for resisting echinococcus multilocularis adult pellicle antigen

A technology of Echinococcus multilocularis and monoclonal antibodies, applied in biochemical equipment and methods, anti-animal/human immunoglobulins, instruments, etc., can solve the problem of echinococcosis transmission, pollute the environment, increase the number of host animals and humans Dissemination and other issues, to achieve the effect of wide application value

Pending Publication Date: 2022-06-24
VETERINARY INST XINJINAG ACADEMY OF ANIMAL SCI CLINIC MEDICAL SCI RES CENT XINJIANG ACADEMY OF ANIMAL HUSBANDRY SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the wide distribution of wild foxes, herding dogs and rodents in the western pastoral areas of my country, the life cycle of Echinococcus multilocularis is complicated, which increases the risk of transmission and infection of host animals and humans, and can cause serious socioeconomic problems
[0003] Diagnosis and detection techniques for terminal host infection with Echinococcus multilocularis include arecoline hydrobromide diarrhea method, autopsy method, etc. These methods may infect operators and pollute the environment, resulting in the spread of hydatid disease and serious public health problems health and safety

Method used

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  • Preparation method and application of monoclonal antibody for resisting echinococcus multilocularis adult pellicle antigen
  • Preparation method and application of monoclonal antibody for resisting echinococcus multilocularis adult pellicle antigen
  • Preparation method and application of monoclonal antibody for resisting echinococcus multilocularis adult pellicle antigen

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Create a cell line:

[0063] Antigen preparation:

[0064] Materials: rats infected with Echinococcus multilocularis cysts, healthy Beagle dogs aged 10-12 months and weighing 8-10 kg (purchased from Changzhou Beile Experimental Animal Breeding Co., Ltd.), 50mL centrifuge tubes, 1.5mL centrifuge tubes, Phosphate buffer saline (PBS: 8g sodium chloride, 0.2g potassium chloride, 1.44g disodium hydrogen phosphate, 0.24g potassium dihydrogen phosphate, add distilled water to 800mL, adjust pH to 7.2 with 1M HCL, add water to 1000mL), 1% Triton X-100 (Triton X-100, prepared in PBS), benchtop centrifuge (Eppendorf).

[0065] Steps: Euthanize the mouse infected with Echinococcus multilocularis cyst, sterilize with 75% alcohol for 3 minutes, move it into a biological safety cabinet, fix it on the dissection board with its abdomen facing upward. Cut the abdominal skin and peritoneum with sterile scissors and tweezers, open the abdominal cavity, take out the cyst, remove the surro...

Embodiment 2

[0111] Determination of antibody subtypes of hybridoma cell lines:

[0112] Material: Pierce Rapid Isotyping Kit with Kappa and Lambda-Mouse kit (Sigma Company), 1.5mL centrifuge tube;

[0113] Steps: Take 450μL of the dilution solution and add it to a 1.5mL autoclaved centrifuge tube, then take 50μL of the cell supernatant, mix well, take out the test plate in the Pierce Rapid Isotyping Kit with Kappa and Lambda-Mouse kit, drop on each plate Add 150 μL diluted cell supernatant and let it stand for about 10 minutes. The antibody subtype of the obtained hybridoma cell line Anti-XJEmSfAg-4D6 is IgG1, and the light chain is Kappa chain.

Embodiment 3

[0115] Karyotype analysis of hybridoma cell lines:

[0116] Materials: colchicine, methanol, glacial acetic acid (Tianjin Yongsheng Fine Chemical Co., Ltd.), Giemsa dye solution, neutral gum (Beijing Dingguo Changsheng Biotechnology Co., Ltd.), slide glass, cover glass, microscope and imaging system (Qianxin Instrument Co., Ltd.);

[0117] Steps: add colchicine to the cell culture flask during the logarithmic growth phase of the cells, so that the final concentration is 0.04 μg / mL, continue to cultivate for 14 hours, centrifuge at 1000 rpm for 10 minutes, collect the cells, add 0.075 mol / L KCL at a temperature of 37 ° C Suspend cells in 5 mL of the solution, place in a water bath at 37°C for 30 min, add 1 mL of freshly prepared fixative (formaldehyde: glacial acetic acid at a volume ratio of 3:1), mix well, centrifuge at 1000 rpm for 10 min, discard the supernatant; add 5 mL of fixative, Resuspend the cells, let stand at room temperature for 30 minutes, centrifuge at 1000rpm ...

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Abstract

The invention discloses a preparation method and application of a monoclonal antibody for resisting echinococcus multilocularis adult pellicle antigen. According to the method, a hybridoma cell strain Anti-XJEmSfAg-4D6 is established by fusing splenocytes of a Balb / C mouse with SP2 / 0 cells through a hybridoma technology. A specific monoclonal antibody is obtained from the cell strain through an in-vitro culture method and an animal in-vivo ascites inducing method, and the echinococcus multilocularis adult immunohistochemical kit is prepared. The specific monoclonal antibody for resisting echinococcus multilocularis adult pellicle antigen is provided for clinical and scientific research institutions, can be used for detecting clinical samples in an ELISA (enzyme-linked immuno sorbent assay) experiment, can also be used for detecting pathological specimens in an immunohistochemical experiment, can also provide a convenient research tool for an echinococcosis research department, and has a wide application prospect. The method has a wide application value in the detection of the echinococcus multilocularis infected by a final host, and provides a technical support for baseline investigation and prevention and control effect evaluation of the alveolar echinococcosis.

Description

technical field [0001] The invention belongs to the technical field of biology and cell engineering, and relates to a preparation method and application of a monoclonal antibody against the surface membrane antigen of Echinococcus multilocularis adults. Background technique [0002] Alveolar echinococcosis (AE, Alveolar Echinococcosis) is one of the deadliest helminth diseases in humans caused by the larval stage of Echinococcus multilocularis (Em), mainly in the natural circulation of wild animals, and is prevalent In the northern hemisphere, it is locally endemic. In my country, it is mainly prevalent in western pastoral and semi-agricultural pastoral areas such as Tibet, western Sichuan, Gansu, southern plateau of Qinghai, northern Xinjiang, and Hulun Buir Grassland in eastern Inner Mongolia. Due to the wide distribution of wild foxes, herding dogs and rodents in western pastoral areas of my country, the life history of Echinococcus multilocularis is complicated, which inc...

Claims

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Application Information

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IPC IPC(8): C07K16/18C12N5/20G01N33/569G01N33/577
CPCC07K16/18G01N33/56966G01N33/577G01N2333/43543Y02A50/30
Inventor 赵莉班万里王冰洁吴小霞段兰利郭宝平徐宁张佳睿徐晶汪保穆尼拉·特列吾汗阿热艾·阿哈太俞进喻昌盛郭凯张壮志
Owner VETERINARY INST XINJINAG ACADEMY OF ANIMAL SCI CLINIC MEDICAL SCI RES CENT XINJIANG ACADEMY OF ANIMAL HUSBANDRY SCI
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