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Enzyme linked immunosorbent assay kit for detecting rabies virus glycoprotein antigen and application of kit

An enzyme-linked immunosorbent reagent and glycoprotein antigen technology, which can be used in enzyme-linked immunosorbent assay kits, rabies virus glycoprotein antigen specific, accurate detection, and rapid field, which can solve problems such as time-consuming and inability to meet the needs of rapid diagnosis.

Active Publication Date: 2020-11-27
CHINA ANIMAL HUSBANDRY IND
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, the methods for determining rabies virus glycoprotein antigen are mainly immunofluorescence method, RT-PCR nucleic acid detection method, and virus isolation methods mainly include cell culture separation and suckling mouse inoculation method separation, because the above detection methods must be in P2 or P3 laboratories and the above method must be completed by professional experimenters and equipment, and it takes a long time to meet the needs of a wide range of rapid diagnosis. Therefore, it is necessary to develop a convenient, specific, sensitive, accurate and reliable Rabies virus glycoprotein antigen detection method

Method used

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  • Enzyme linked immunosorbent assay kit for detecting rabies virus glycoprotein antigen and application of kit
  • Enzyme linked immunosorbent assay kit for detecting rabies virus glycoprotein antigen and application of kit
  • Enzyme linked immunosorbent assay kit for detecting rabies virus glycoprotein antigen and application of kit

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Experimental program
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Effect test

Embodiment 1

[0046] Embodiment 1, the screening of the specific hybridoma cell line of rabies virus monoclonal antibody

[0047] Include the following steps:

[0048] 1) Obtain rabies virus inactivated antigen (provided by China Animal Husbandry Co., Ltd. Jiangxi Biopharmaceutical Factory) by hollow fiber method, with a purity ≥ 80%, and use it as an immunogen;

[0049] 2) The immunized animals were BALB / c mice (purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.), which were immunized four times in a row with an interval of 14 days. Immunization by intraperitoneal injection, each mouse was injected with 1 mg of antigen;

[0050] 3) Seven days after the last immunization, the tail blood of the mice was taken to separate the serum, and the serum was detected by indirect ELISA. After the titer was >1:50000, the splenocytes of the immunized animals were separated and combined with the splenocytes of the well-growing myeloma cells SP2 / 0 for fusion, and hybridoma ce...

Embodiment 2

[0053] Embodiment 2, pairing test of rabies virus monoclonal purified antibody

[0054] 1) Prepare coated plates with specific monoclonal antibodies against rabies virus

[0055] Dilute the purified specific monoclonal antibody with pH 9.6 carbonate solution into 0.5μg / ml, 1μg / ml, 2μg / ml, 4μg / ml coating working solution, then add to 96-well polystyrene enzyme Linked reaction plate, 100μl / well, placed at 2-8°C for 8-12 hours to fully combine the specific monoclonal antibody with the enzyme-linked reaction plate, and then add 10mg / ml bovine serum albumin pH7.4 at 300μl / well PBS buffer solution, 37°C blocking treatment for 2-3 hours, after drying, the enzyme-linked reaction plate was dried and stored in a sealed manner at 2-8°C.

[0056] 2) Preparation of horseradish peroxidase-labeled rabies virus-specific monoclonal antibody

[0057] The specific monoclonal antibody of rabies virus is coupled with horseradish peroxidase (HRP) by the glutaraldehyde oxidation method, fully dial...

Embodiment 3

[0071] Embodiment 3, subculture and preservation of rabies virus monoclonal antibody

[0072] Subculture of hybridoma cells includes the following steps:

[0073] 1) The hybridoma cells RV1 screened above were cultured and passaged in DMEM medium (containing 100 U / ml penicillin and 100 μg / ml streptomycin) containing 10% fetal bovine serum;

[0074] 2) After cultured to the 10th passage, the hybridoma cell line can still grow well and be passed down stably, and the titer in the supernatant can still reach above 1:50. Hybridoma cell lines against toxic monoclonal antibodies.

[0075] The preservation of hybridoma cells includes the following steps: in the subculture of hybridoma cells, due to the possibility of mutations during the continuous subculture process and the loss of the ability to secrete monoclonal antibodies, and in order to prevent possible contamination during the culture process, therefore, A portion of hybridoma cells must be preserved;

[0076] 1) Remove the...

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Abstract

The invention discloses an enzyme linked immunosorbent assay kit for specific quantitative detection of rabies virus glycoprotein antigen and application of the kit. The kit comprises an enzyme-linkedreaction plate coated with a capture antibody and a monoclonal antibody of an enzyme-labeled antibody. A heavy chain variable region of the monoclonal antibody is characterized in that CDR1 is shownas amino acids from the 31st site to the 36th site of a sequence 1; CDR2 is shown as the 51st to 66th amino acids of the sequence 1; CDR3 is as shown in amino acids from the 9th site to the 105th siteof the sequence 1; and in a light chain variable region: CDR1 is as shown in the 24th-40th amino acid of the sequence 2; CDR2 is shown as the 56th to 62nd amino acids of the sequence 2; CDR3 is shownas the 95th-103rd amino acid of the sequence 2. The enzyme-linked immunosorbent assay quantitative detection kit prepared from the rabies virus specific monoclonal antibody has high sensitivity and strong specificity, and can effectively distinguish and detect the content of rabies virus glycoprotein antigen in a sample.

Description

technical field [0001] The invention belongs to the technical field of biological detection. More specifically, the invention relates to an enzyme-linked immunoassay kit for specific quantitative detection of rabies virus glycoprotein antigen, which is suitable for specific, rapid and accurate detection of rabies virus glycoprotein antigen. Background technique [0002] Rabies virus is a member of the genus Lyssavirus in the family Rhabdoviridae. According to the analysis and identification of rabies virus N protein monoclonal antibody and G protein series, rabies viruses around the world are mainly divided into 4 serotypes. Among them, serotype 1 is the well-known rabies virus with the widest geographical distribution and the greatest threat to the health of people in the world, including wild viruses and fixed laboratory strains (such as SAD). With the research progress of modern molecular biology, referring to the similarity rate of the N-terminal 500 nucleotides of the ...

Claims

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Application Information

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IPC IPC(8): G01N33/569G01N33/543G01N33/535G01N33/577C07K16/10
CPCG01N33/56994G01N33/543G01N33/535G01N33/577C07K16/10C07K2317/56C07K2317/565G01N2333/145Y02A50/30
Inventor 董春娜张蕾宋芳肖进齐鹏李静李鹏宇刘新月
Owner CHINA ANIMAL HUSBANDRY IND
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