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Hybridoma cell strain secreting isoprothiolane monoclonal antibody and application thereof

A hybridoma cell line, monoclonal antibody technology, applied in analytical materials, biochemical equipment and methods, material testing products, etc., can solve the problems of high solvent, consumption, expensive equipment, etc., and achieve the effect of good detection sensitivity

Active Publication Date: 2022-04-05
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Despite the high sensitivity and specificity of these chromatography-based methods, there are some disadvantages such as the need for thorough sample cleanup, high solvent consumption, expensive equipment, and skilled technicians

Method used

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  • Hybridoma cell strain secreting isoprothiolane monoclonal antibody and application thereof

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Experimental program
Comparison scheme
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Embodiment 1

[0044] Embodiment 1: the synthesis of rice blastin hapten

[0045] Since the small molecule of rice blastin is not immunogenic and cannot stimulate the immune response of mice to produce antibodies, it is necessary to couple rice blastin to the protein through protein linkage technology to obtain immunogenicity; protein coupling Active groups commonly used in technology include amino, carboxyl, hydroxyl, mercapto, etc. Since the molecular structure of Daobenling does not contain these active groups, Daobenling was derivatized.

[0046] Dissolve Blastiline (2g, 6.89mmol, 1.00eq) in absolute ethanol (15mL) and cool to -15°C. Fresh filtered KOH (405mg, 1.05eq) was added slowly at 25°C. The resulting mixture was stirred at 25 °C for 17 h, then concentrated to give some white sticky material. treated with water and extracted with ethyl acetate, followed by Na 2 SO 4 Drying, concentration on a rotary evaporator, and purification on a silica gel column afforded a white solid (500...

Embodiment 2

[0047] Embodiment 2: the synthesis of complete antigen of rice blastling

[0048] Weigh 6 mg of blasticillin hapten, 4.4 mg of N-hydroxysuccinimide (NHS), dissolve in 200 μL of N,N-dimethylformamide (DMF), stir at room temperature for 10 min; then weigh 7.5 mg of 1 -(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), added to the rice blasten hapten solution, stirred at room temperature for 6-8 hours, and activated. Take 6mg of keyhole limpet hemocyanin (KLH), add it to 3mL 0.01M carbonate buffer solution (CBS), fully dissolve, slowly add the activated hapten to the diluent dissolved in KLH, and stir overnight at room temperature. Then it was dialyzed with 0.01M PBS solution to remove unreacted small molecular substances to obtain relatively pure complete antigen, which was identified by ultraviolet absorption scanning method.

Embodiment 3

[0049] Embodiment 3: the synthesis of rice blastling coating former

[0050] Dissolve 3.2mg rice blastin hapten and 2.4mg N-hydroxysuccinimide (NHS) in 200μL anhydrous N,N-dimethylformamide (DMF), stir at room temperature for 10min; dissolve 4.2mg 1- (3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) was dissolved in the above solution, stirred at room temperature and reacted for 6-8h to obtain a hapten activation solution; 6mg chicken ovalbumin ( OVA) was dissolved in carbonate buffer solution (CBS); the hapten activation solution was slowly added to the protein diluent, and stirred overnight at room temperature. Then, the reaction solution was dialyzed with 0.01M PBS solution to remove unreacted small molecular substances to obtain the coating original.

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Abstract

The invention provides a hybridoma cell strain secreting an isoprothiolane monoclonal antibody and application of the hybridoma cell strain, and belongs to the technical field of immunochemistry. A complete antigen of isoprothiolane and an equal amount of a Freund's adjuvant are mixed and emulsified, and BALB / c mice are immunized through neck and back subcutaneous multipoint injection. A complete Freund's adjuvant is used for emulsification for first immunization (100 micrograms per duck), an incomplete Freund's adjuvant is used for multiple booster immunization, and an isoprothiolane complete antigen is used for sprint immunization (intraperitoneal injection) for the last time. The method comprises the following steps: fusing splenocytes of a high-titer and high-sensitivity mouse with myeloma cells of the mouse, and screening out hybrid cells after fusing the two cells by adopting a selective culture medium; and screening cells through indirect competitive enzyme-linked immunosorbent assay, and carrying out multiple subcloning to finally obtain the monoclonal antibody hybridoma cell strain. The monoclonal antibody secreted by the cell strain has good detection sensitivity to isoprothiolane, and can be used for residue detection of isoprothiolane.

Description

technical field [0001] The invention belongs to the technical field of food safety immunoassay, in particular to a hybridoma cell strain secreting a rice blastilin monoclonal antibody and an application thereof. Background technique [0002] The English common name of Daobunling is: Isoprothiolane, chemical name: 1,3-dithiopentane-2-ylidene malonate diisopropyl ester; mechanism of action: After absorbed by rice plants, it inhibits phospholipid N-methyltransferase , thus hindering the formation of fatty acids, triglycerides, and phospholipids. Main purposes: This product is a systemic fungicide, which has special effects on rice blast, and is also effective on rice seedling blast and Sclerotinia sclerotiorum. It can also treat rice planthopper and white-backed planthopper when used in a large area. It is mainly used to prevent and control rice blast, and it is effective for ear blast and leaf blast, and it is more effective for ear blast. [0003] At present, the detection ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/20C07K16/44G01N33/577G01N33/53
CPCY02A50/30
Inventor 胥传来刘洋匡华徐丽广孙茂忠吴晓玲刘丽强马伟朱建平郝昌龙宋珊珊胡拥明吴爱红郭玲玲胥欣欣
Owner JIANGNAN UNIV
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