Hybridoma WXX-1 capable of secreting anti-lomefloxacin monoclonal antibody and application thereof

A monoclonal antibody, hybridoma cell line technology, applied in the field of immunochemistry, can solve the problems of relying on instruments and equipment, time-consuming, etc.

Inactive Publication Date: 2017-07-21
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these methods all rely on expensive equipment and require professionals to operate
In addition, these methods require complex sample preprocessing, which is time-consuming

Method used

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  • Hybridoma WXX-1 capable of secreting anti-lomefloxacin monoclonal antibody and application thereof
  • Hybridoma WXX-1 capable of secreting anti-lomefloxacin monoclonal antibody and application thereof
  • Hybridoma WXX-1 capable of secreting anti-lomefloxacin monoclonal antibody and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0016] Example 1: Synthesis of Immunogen and Coating Gen

[0017] Synthesis of immunogen: First, bovine serum albumin (Bovine Serum Albumin, BSA) was cationized with EDEA. Dissolve 200mg BSA and 14mg EDEA in 10 mL PBS, adjust its pH to 6.5; dissolve 18.2mg carbodiimide (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, EDC) in 200 μL PBS , slowly drop it into the BSA solution, adjust the pH to 7.5, and stir at room temperature for 2h. Dialysis removes EDEA not bound to BSA, yielding BSA-EDEA.

[0018] BSA-EDEA is also coupled with the small molecule lomefloxacin by the carbodiimide method. Dissolve 10 mg of lomefloxacin, 15 mg of EDC, and 9 mg of N-hydroxysuccinimide (NHS) in 5 mL of 0.05M MES at pH 4.7, and stir at room temperature for 6 h. Then the activated solution was slowly added dropwise to the EDEA-BSA solution (0.3 μmol EDEA-BSA dissolved in 10 mL, 0.05 M, pH 9.6 carbonate buffer solution), and reacted at room temperature for 3 h. The reaction liquid after the react...

Embodiment 2

[0020] Example 2: Mouse immunization

[0021] The immunogen was diluted to an appropriate concentration with physiological saline, emulsified with an equal volume of Freund's adjuvant, and injected at multiple points on the back of 6-8 week-old BALB / c mice. For the first immunization, complete Freund's adjuvant was used at a dose of 100 μg. The following four booster immunizations used incomplete Freund's adjuvant, the immunization dose was 50 μg, and the immunization interval was 21 days. After the third immunization, blood was collected from the tail vein of the mice, and the sera were tested by indirect ELISA. After the fifth immunization, the mice with high titer and inhibition were screened for intraperitoneal immunization, and the spleens of the mice were taken out three days later for cell fusion.

Embodiment 3

[0022] Example 3: Cell Fusion and Screening

[0023] Mouse splenocytes and tumor cells were fused under the action of PEG 1500 to form hybridoma cells. Detection was carried out on the seventh day after fusion, and the cell lines with high titer and good recognition of lomefloxacin were screened for subcloning, and so on, 5 times of subcloning were performed to obtain pure cell lines.

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Abstract

The invention discloses hybridoma WXX-1 capable of secreting an anti-lomefloxacin monoclonal antibody and an application thereof, and belongs to the technical field of immunochemistry. A murine monoclonal cell strain WXX-1 is prepared by a conventional cell fusion technique, and has reserved in China General Microbiological Culture Collection Center, with a preservation number of CGMCC No.13088. The monoclonal antibody secreted by the cell strain is measured by using an indirect competitive-euzymelinked immunosorbent assay method; IC50 of the antibody to lomefloxacin, norfloxacin and enoxacin are 0.19microgram/L, 0.17microgram/L and 0.23microgram/L, respectively. Recovery ranges of lomefloxacin, norfloxacin and enoxacin in milk are 98.02-107.40%, 82.175%-105.55% and 92.47%-105.52%, respectively. The hybridoma provided by the invention provides materials for immunodetection of residual quinolone antibiotics in food, and has an actual application value.

Description

technical field [0001] The present invention relates to a mouse-derived hybridoma cell strain WXX-1 and the monoclonal antibody secreted therefrom which can recognize lomefloxacin, norfloxacin and enoxacin, which can be used for the detection of quinolone antibiotic residues in food, It belongs to the technical field of immunochemistry. Background technique [0002] Lomefloxacin (LOM), norfloxacin (NOR) and enoxacin (ENO) are all quinolone antibiotics. Lomefloxacin is a third-generation quinolone antibacterial drug with a broad antibacterial spectrum. Its antibacterial activity against Gram-positive bacteria is the same as that of norfloxacin, but stronger than that of enoxacin; its effect on Gram-negative bacteria is similar to that of Enoxa The same as Floxacin, weaker than Norfloxacin. Lomefloxacin is widely used in animal husbandry and aquaculture. However, due to the abuse of quinolone antibiotics in the aquaculture industry, the residues of quinolone antibiotics in ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/20C07K16/44G01N33/577C12R1/91
CPCC07K16/44G01N33/577G01N33/9446
Inventor 胥传来陈燕妮匡华徐丽广刘丽强宋珊珊吴晓玲胡拥明
Owner JIANGNAN UNIV
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