Hybridoma cell strain secreting meloxicam monoclonal antibody and application of hybridoma cell strain

A hybridoma cell line and monoclonal antibody technology, which is applied to cells modified by introducing foreign genetic material, fusion cells, hybrid peptides, etc., can solve the problems of time-consuming, expensive, and inability to realize rapid detection of a large number of samples

Active Publication Date: 2018-12-07
JIANGNAN UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, the methods for detecting meloxicam are mainly liquid chromatography (HPLC), liquid chromatography tandem mass spectrometry (LC-MS / MS), enzyme-linked immunosorbent assay, immunoaffinity chromatographic column and electrochemical sensor, etc. However, these methods have disadvantages such as cumbersome operation, time-consuming, and expensive costs, and cannot realize rapid detection of a large number of samples.

Method used

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  • Hybridoma cell strain secreting meloxicam monoclonal antibody and application of hybridoma cell strain
  • Hybridoma cell strain secreting meloxicam monoclonal antibody and application of hybridoma cell strain
  • Hybridoma cell strain secreting meloxicam monoclonal antibody and application of hybridoma cell strain

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Experimental program
Comparison scheme
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Embodiment 1

[0091] Embodiment 1: the synthesis of meloxicam hapten

[0092] Synthetic routes such as figure 1 .

[0093]1g of compound 1 was dissolved in 50mL of methanol solution, and 157mg of MeONa was added and stirred overnight at 65°C to obtain mixture 1; the obtained mixture was concentrated to obtain compound 2; 1.20g of compound 2 was dissolved in 30mL of DMF, and 1.20mg of 4- Ethyl bromobutyrate was stirred overnight at 80°C to obtain a mixture 2; the step 4 was to concentrate the obtained mixture 2 and then purify it with a silica gel column to obtain compound 3; 800 mg of compound 3 was dissolved in 3 mL of tetrahydrofuran and 1 mL of water 180 mg of lithium hydroxide monohydrate was added to the mixed solution to adjust the pH to 4-6, and stirred at room temperature overnight to obtain a mixture 4; the aqueous layer of the mixture 4 was extracted with ethylamine, and the organic layers were combined, washed with brine, and dried over sodium sulfate. Concentrate to obtain mel...

Embodiment 2

[0096] Embodiment 2: the synthesis of complete antigen of meloxicam

[0097] Dissolve 1.7 mg of meloxicam hapten (Melo), 2.2 mg of 1-ethylcarbodiimide hydrochloride, 1.4 mg of N-hydroxysuccinimide in 400 μL of anhydrous N,N-dimethylformamide , stirred at room temperature for 6-8 hours to obtain A1 solution; take 10 mg of keyhole limpet hemocyanin (KLH) (the molar ratio of Melo to KLH is 1500:1) and dilute it with an appropriate amount of boric acid buffer solution to obtain B1 solution; at room temperature, dropwise Add solution A1 to solution B1, react overnight at room temperature to obtain a mixed solution; separate the complete antigen and uncoupled small molecule hapten (Melo) from the mixed solution by dialysis to obtain the complete antigen (Melo-KLH).

[0098] Dissolve 1.7 mg of meloxicam hapten (Melo), 2.2 mg of 1-ethylcarbodiimide hydrochloride, 1.4 mg of N-hydroxysuccinimide in 400 μL of anhydrous N,N-dimethylformamide , and stirred at room temperature for 6-8h to ...

Embodiment 3

[0100] Embodiment 3: the synthesis of meloxicam coating former

[0101] Dissolve 1.8 mg of meloxicam hapten (Melo), 2.4 mg of 1-ethylcarbodiimide hydrochloride, and 1.45 mg of N-hydroxysuccinimide in 300 μL of anhydrous N,N-dimethylformamide, Stir at room temperature for 6-8 hours to obtain liquid A2; dissolve 5 mg of chicken ovalbumin (OVA) in 2 mL of boric acid buffer solution to obtain liquid B2; at room temperature, add liquid A2 dropwise to liquid B2, and react overnight at room temperature to obtain Mixed solution; the mixed solution was separated by dialysis from the complete antigen and the uncoupled small molecule hapten (Melo), and the coated original (Melo-OVA).

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Abstract

The invention relates to a hybridoma cell strain secreting meloxicam monoclonal antibody and a preparation method of the hybridoma cell strain and belongs to the field of food safety immunodetection.The hybridoma cell strain has a collection number of CGMCC No. 14700. The preparation method comprises the following steps: performing primary immunization on BALB / c mice with a complete Freund's adjuvant, performing booster immunization for three times by using an incomplete Freund's adjuvant, performing impact immunization once on an adjuvant-free meloxicam complete antigen, and enabling the BALB / c mice to be immunized; fusing high titer low-IC50 immunized mice spleen cells with mice myeloma cells by a PEG method, and performing indirect competitive ELISA (Enzyme-Linked Immuno Sorbent Assay)screening and three-times subcloning, thereby obtaining the cell strain. The monoclonal antibody secreted by the cell strain has excellent specificity and detection sensitivity (IC50 value of 0.1ng / mL) on meloxicam, and can be used for detecting residual meloxicam in foods.

Description

technical field [0001] The invention relates to a hybridoma cell line secreting meloxicam monoclonal antibody and an application thereof, belonging to the field of food safety immunoassay. Background technique [0002] Meloxicam is an enol nonsteroidal anti-inflammatory drug with anti-inflammatory, analgesic and antipyretic effects, mainly used to relieve osteoarthritis, painful osteoarthritis, rheumatoid arthritis and symptoms of ankylosing spondylitis. In the veterinary clinical field, meloxicam has been used in dairy cows and pets abroad. And other animal research reports, domestic scholars have also reported that meloxicam is used to treat mastitis in dairy cows, postoperative pain in sows, and auxiliary treatment of mastitis in sows and agalactia syndrome. On July 26, 2016, the Australian Pesticides and Veterinary Medicines Administration (APVMA) issued Bulletin No. 15, revising the maximum residue limits (MRLs) of meloxicam in food. The maximum residue limits of mel...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/20C07K16/44C07K14/795C07K14/77C07D417/12G01N33/577C12R1/91
CPCG01N33/577C07D417/12C07K14/77C07K14/795C07K16/44C07K19/00C12N5/12G01N33/02G01N33/48714G01N33/94
Inventor 匡华林璐胥传来徐丽广马伟刘丽强吴晓玲宋珊珊胡拥明
Owner JIANGNAN UNIV
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