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Diclazuril-resistant specific monoclonal antibody hybridoma cell strain and applications thereof

A hybridoma cell line and monoclonal antibody technology, which is applied to specific peptides, biochemical equipment and methods, and material inspection products, can solve problems such as complex operations, expensive, and restrictive detection methods, and achieve simplified synthesis steps and good results. Effect of Assay Sensitivity and Affinity

Inactive Publication Date: 2019-01-29
JIANGNAN UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The above detection methods can perform quantitative analysis and have a low detection limit, but usually require expensive instruments and complicated operations, and the pretreatment and detection time is long, which seriously restricts the promotion of these detection methods, and cannot achieve the rapid detection of large quantities on site. Require

Method used

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  • Diclazuril-resistant specific monoclonal antibody hybridoma cell strain and applications thereof
  • Diclazuril-resistant specific monoclonal antibody hybridoma cell strain and applications thereof
  • Diclazuril-resistant specific monoclonal antibody hybridoma cell strain and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Embodiment 1: the preparation of hybridoma cell line

[0029] (1) Synthesis of complete antigen:

[0030] Synthesis of hapten: A solution of Dic (1 g, 2.5 mmol) was added to a mixture solution of concentrated sulfuric acid (50 mL) and deionized water (10 mL). The mixture was reacted at 110°C for 24h. After the reaction, the reaction mixture was diluted with 1 L of deionized water to terminate the reaction, and then extracted three times with 20 mL of ethyl acetate. The organic layer was washed with NaCl and washed with MgSO 4 Drying and evaporation in vacuo yielded the hapten Dic-hapten-1. Concrete synthetic route is as follows:

[0031]

[0032] Synthesis of complete antigen: Take 5mg of the above hapten Dic-hapten-1, then add 5.0mg of EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) and 3.0mg of NHS (N-hydroxysuccinimide), dissolved in DMF (N,N-dimethylformamide), stirred at room temperature, activated for 6h; another 10mg KLH (keyhole limpet ...

Embodiment 2

[0041] Example 2 Establishment of Indirect Competitive ELISA

[0042] The monoclonal antibody prepared by the hybridoma cell line through the in vivo ascites was applied to the establishment of the indirect competitive ELISA method, and the specific steps were as follows:

[0043] 1. Coat 96-well ELISA plate with 0.1 μg / mL diluted with carbonate buffer solution (CBS) as the original coating, 100 μL per well, after coating at 37°C for 2 hours, wash the plate three times with PBST washing solution, each time 200 μL per well, 3 min each time, pat dry;

[0044] 2. Block with CBS containing 0.2% gelatin, 200 μL per well, block for 2 hours at 37°C, wash the plate three times with PBST washing solution, 200 μL per well for 3 minutes each time, and pat dry;

[0045] 3. Prepare diclazuril standard solutions of 0, 2, 5, 10, 20, 50, 100, and 200 μg / L in phosphate buffered saline (PBS). Add the standard solution to the already-blocked ELISA plate, 50 μL per well, repeat 3 wells for each...

Embodiment 3

[0050] Example 3 Application of Monoclonal Antibody

[0051] Sample pretreatment and addition recovery experiment: the addition and recovery experiment of actual samples, taking chicken as an example for testing. First, weigh three parts of 1.0g chicken, add 100μL, 200μL and 500μL diclazuril standard solution (100ng / mL) respectively, mix well, add 10mL acetonitrile, homogenize at high speed for 2min, ultrasonically extract for 10min, and let stand for 5min Finally, 200 μL of the supernatant was diluted 4 times with PBS, and used as the ELISA sample extract, and the indirect competition ELISA was used for the addition recovery test, and the recovery rates were 91%, 97%, and 95%, respectively.

[0052] Table 1. Isotype Identification of Monoclonal Antibodies

[0053]

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Abstract

The invention provides a diclazuril-resistant specific monoclonal antibody hybridoma cell strain and applications of the diclazuril-resistant specific monoclonal antibody hybridoma cell strain, and belongs to the technical field of food safety immunological detection. The diclazuril-resistant specific monoclonal antibody hybridoma cell strain SS0711 is preserved in the China General Microbiological Culture Collection Center (CGMCC), and is assigned with the accession number of CGMCC No.14689. The diclazuril-resistant specific monoclonal antibody is secreted by the diclazuril-resistant specificmonoclonal antibody hybridoma cell strain SS0711 with the accession number of CGMCC No.14689. The diclazuril-resistant specific monoclonal antibody hybridoma cell strain is applied to the analysis and detection for diclazuril residue in food safety detection. The obtained diclazuril-resistant monoclonal antibody cell strain has the good detection sensitivity and affinity for diclazuril, for the monoclonal antibody provided by the invention, the IC50=0.49ng / mL, the detection linear range is 0.16-1.55 ng / mL, and the lowest detection limit value (LOD) is 0.08ng / mL.

Description

technical field [0001] The invention relates to an anti-diclazuril specific monoclonal antibody hybridoma cell line and application thereof, belonging to the technical field of food safety immunology detection. Background technique [0002] Diclazuril is a polyether drug and an ideal anticoccidial drug with high efficiency and low toxicity. Diclazuril was researched and developed by the Belgian Janssen Company in 1986, and it was listed in the UK, Germany and other 43 countries with the use of fungicide. Diclazuril belongs to triazine broad-spectrum anticoccidial drugs, which can effectively prevent and kill various protozoa including coccidia. Among them, the anticoccidial index for the 6 most important Eimeria coccidia such as tender, heaped, Brucellian, giant, and mild all exceeds 180, which is a highly effective anticoccidial drug and is widely used for chicken coccidiosis. [0003] At present, high-performance liquid chromatography (HPLC), liquid chromatography-mass s...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/20C07K16/44G01N33/577
CPCC07K16/44G01N33/577G01N33/94
Inventor 胥传来王忠兴匡华徐丽广刘丽强马伟吴晓玲宋珊珊胡拥明
Owner JIANGNAN UNIV
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