Hybridoma cell line of secreting meloxicam monoclonal antibodies and application thereof

a technology of meloxicam and monoclonal antibodies, which is applied in the field of hybridoma cell lines of secreting meloxicam monoclonal antibodies and application thereof, can solve the problems of cumbersome methods, time-consuming and expensive, and the inability to achieve rapid detection of a large number of samples, and achieve good specificity and detection sensitivity

Active Publication Date: 2020-02-06
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The patent describes a new monoclonal antibody cell line that can detect meloxicam with high sensitivity and specificity. The invention also provides a new method for synthesizing the meloxicam hapten, complete antigen, and coating antigen. The monoclonal antibody cell line can be used for immunoassay detection. The patent also describes a hybrid tumor cell line that produces monoclonal antibodies to meloxicam. This cell line has been deposited with the General Microbial Center of China under the Accession No. CGMCC No. 14686 at Dec. 5, 2017.

Problems solved by technology

However, these methods are cumbersome, time-consuming and expensive, and cannot achieve rapid detection of a large number of samples.

Method used

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  • Hybridoma cell line of secreting meloxicam monoclonal antibodies and application thereof
  • Hybridoma cell line of secreting meloxicam monoclonal antibodies and application thereof
  • Hybridoma cell line of secreting meloxicam monoclonal antibodies and application thereof

Examples

Experimental program
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Effect test

example 1

[0084]Synthesis of Meroxicam Hapten

[0085]The synthesis path is shown in FIG. 1.

[0086]1 g of compound 1 was dissolved in 50 ml of methanol solution, and stirred at 157° C. for the night after adding 157 mg MeONa, and then the compound 1 was gotten. 1.20 g of compound 2 was dissolved 30 ml DMF, and added 1.20 mg Ethyl 4-bromobutyrate stir at 80° C. for the night, and then the mixture 2 was gotten. The step 4 is to concentrate the obtained mixture 2 and then purify it with a silica gel column to obtain compound 3. The 800 mg compound 3 was dissolved into 3 mL tetrahydrofuran and 1 mL water, adding 180 mg 1-hydrated lithium hydroxide, adjusting pH to 4-6, and stirring at room temperature overnight to get mixture 4. The aqueous solution layer with ethylamine from mixture 4 was extracted, and the organic layer was combined, washing with salt water, drying with anhydrous sodium sulfate and being concentrated to obtain meloxicam hapten.

[0087]The products of compound 3 and haptens are calcul...

example 2

[0089]Complete Antigen Synthesis of Meloxicam

[0090]1.7 mg Melo, 2.2 mg 1-ethyl carbodiimine hydrochloride and 1.4 mg N-hydroxysuccinylimide was dissolved into 400 phenyl-anhydrous N, N-dimethylamine, stirring 6-8 h at room temperature to obtain A1 solution. 10 mg keyhole blood blue protein (KLH) (Melo to KLH Moore ratio of 1500:1) was diluted with an appropriate amount of boric acid buffer solution, and B1 solution was obtained. At room temperature, A1 solution was added to B1 solution one drop at a time. Complete antigen (Melo-KLH) is obtained by separating complete antigen and uncoupled small molecule hapten (Melo) by dialysis.

[0091]1.7 mg Melo, 2.2 mg 1-ethyl carbodiimine hydrochloride and 1.4 mg N-hydroxysuccinylimide was dissolved into 400 phenyl-anhydrous N, N-dimethylamine, stirring 6-8 h at room temperature to obtain A1 solution. 10 mg keyhole blood blue protein (KLH) (Melo to KLH Moore ratio of 3000:1) was diluted with an appropriate amount of boric acid buffer solution, an...

example 3

[0093]Synthesis of the Meroxicam Coating Antigen

[0094]1.8 mg Melo 2.4 mg 1-ethylenediamine hydrochloride salt 1.45 mg N-hydroxysuccinylimide was dissolved into 300 phenyl-anhydrous N, N-dimethylamine, and stir it 6-8 h at room temperature to obtain A2 solution. 5 mg of chicken egg albumin (OVA) was dissolved in 2 mL boric acid buffer solution to obtain B2 solution. At room temperature, A2 solution was added to B2 solution, and mixed solution was obtained after overnight reaction at room temperature. Complete antigens, uncoupled small molecule haptens (Melo) and coating antigen (Melo-OVA) were separated by dialysis.

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Abstract

A hybridoma cell line of secreting meloxicam monoclonal antibodies with a preservation number of hybridoma cell line of CGMCC No. 14700 belongs to the field of food safety immunological detection. The. BALB / c mice are immunized through one time immunization with complete freund's adjuvant, three times of booster immunization with incomplete freund's adjuvant and one time of rush immunization with meroxicam complete antigen without adjuvant; the spleen cells from BALB / C mice immunized with high potency and low value of IC50 are fused with murine myeloma cells; and then the hybridoma cell line is obtained through indirect competitive ELISA screening and three sub-clones. The monoclonal antibody secreted by this cell line has good specificity and detection sensitivity to meloxicam (value of IC50 is 0.1 ng / ml), being suitable for detection of meroxicam in food.

Description

CROSS-REFERENCES TO RELATED APPLICATIONS[0001]This application claims priority from China Patent Application Serial Number 2018108758612, which was filed on Aug. 3, 2018, the entire content of which is incorporated herein as reference.BACKGROUND OF THE INVENTION1. Technical Field[0002]The invention relates to the hybridoma cell line of secreting meloxicam monoclonal antibodies and application thereof, belongs to the technical field of food safety and immunological detection.2. Background Art[0003]Meloxicam is an enol-type non-steroidal anti-inflammatory drug that has anti-inflammatory, analgesic and antipyretic effects, and is mainly used to relieve symptoms of osteoarthritis, painful osteoarthritis, rheumatoid arthritis, and strong and rigid spondylitis. In the field of veterinary clinic, meloxicam has been used in cows and pets and other animals. It was reported that meloxicam was used for the treatment of dairy cow mastitis, postoperative pain in gilts, adjuvant treatment of gilt...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C07K16/44C12N5/12G01N33/02C07D417/12G01N33/487G01N33/94
CPCC07D417/12G01N33/48714G01N33/02G01N33/94C12N5/12C07K16/44G01N33/577C07K14/77C07K14/795C07K19/00
Inventor KUANG, HUAXU, CHUANLAILIN, LUXU, LIGUANGMA, WEILIU, LIQIANGWU, XIAOLINGSONG, SHANSHANHU, YONGMING
Owner JIANGNAN UNIV
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