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Ribavirin monoclonal antibody hybridoma cell strain and application thereof

A hybridoma cell line and monoclonal antibody technology, applied in biochemical equipment and methods, instruments, microorganisms, etc., can solve the problems of expensive instruments, cumbersome handling, and complicated operations, and achieve good detection sensitivity and affinity

Inactive Publication Date: 2015-11-25
JIANGNAN UNIV
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Problems solved by technology

Although the instrument detection method is accurate, the instrument is expensive, the operation is complicated, and the sample pretreatment is cumbersome

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  • Ribavirin monoclonal antibody hybridoma cell strain and application thereof
  • Ribavirin monoclonal antibody hybridoma cell strain and application thereof

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Embodiment 1

[0019] Embodiment 1: Preparation of hybridoma cell line 1D4

[0020] 1. Synthesis of complete antigen

[0021] Ribavirin 100mg, dissolved in 5mL of anhydrous DMF and 5mL of anhydrous pyridine mixed solution, added 50mg of succinic anhydride, reflux reaction at 60°C for 12h, and recrystallized at pH about 3, standing at 0°C to obtain white The precipitate was centrifuged and dried to obtain the ribavirin hapten. Take 10mg ribavirin hapten, add 2mL ultrapure water to dissolve, then add NHS (N-hydroxysuccinimide), EDC (carbodiimide) respectively, the molar ratio of hapten:NHS:EDC is 1: 2︰2, at room temperature, mix well, stir for 6h, then react for 12h at room temperature 25°C for activation. Take 25mg bovine serum albumin (BSA), the molar ratio of hapten and bovine serum albumin is 80:1, add 5mL0.1MpH9.6 carbonate buffer. The activated ribavirin hapten solution was slowly added dropwise to the protein solution, and reacted at room temperature for 24 hours. Dialyze with PBS b...

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Abstract

The invention relates to a ribavirin monoclonal antibody hybridoma cell strain and application thereof, and belongs to the field of food safety immunology detection. According to the method, ribavirin complete antigens and an equal amount of a Freund's immunologic adjuvant are uniformly mixed, and the mixture is injected to the backs of BALB / c mice in a multipoint manner for immunization. Splenocytes of the immunized mice are fused with myeloma cells of the mice through a PEG method, and the monoclonal antibody hybridoma cell strain, named 1D4, is obtained through indirect ELISA, indirect competition ELISA screening and three times of subcloning. The monoclonal antibody hybridoma cell strain is preserved in the general microorganism center of the China committee for culture collection of microorganisms, and the collection number is CGMCC No.10864. Monoclonal antibodies excreted from the cell strain 1D4 have better affinity (the affinity constant is 8.04*10<9> L / mol) and detection sensitivity (the IC50 value is 1 ng / mL) on ribavirin, and can be used for ribavirin content detection in food safety.

Description

technical field [0001] A ribavirin monoclonal antibody hybridoma cell line and its application relate to the hybridoma cell line 1D4 and the ribavirin monoclonal antibody produced therefrom. The invention belongs to the field of food safety immunology detection. Background technique [0002] Ribavirin is an antiviral drug, which is a broad-spectrum and powerful antiviral drug. Due to its strong toxic and side effects, it is easy to cause immune system damage and teratogenicity. As early as 2005, the Ministry of Agriculture clearly prohibited the sale and use of ribavirin and other antiviral veterinary drugs. [0003] At present, the main methods for detecting ribavirin are instrumental detection method and immunological detection method. Although the instrumental detection method is accurate, the instrument is expensive, the operation is complicated, and the sample pretreatment is cumbersome. Compared with instrumental detection methods, immunoassay methods have the chara...

Claims

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Application Information

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IPC IPC(8): C12N5/20C07K16/44G01N33/577C12R1/91
Inventor 匡华彭双胥传来徐丽广马伟刘丽强宋珊珊胡拥明
Owner JIANGNAN UNIV
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