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Specific cefquinome-resistant monoclonal antibody hybridoma cell strain 2D4 and application thereof

A hybridoma cell line, monoclonal antibody technology, applied in specific peptides, biochemical equipment and methods, instruments, etc., can solve the problems of human microbial environment balance disorder, bacterial drug resistance, dysregulation, etc., and achieve good specific monoclonal antibody. The effect of cloned antibody cell line, low cross-reaction rate, good detection sensitivity and affinity

Active Publication Date: 2016-05-11
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

As far as the human body is concerned, long-term consumption of animal foods with cefquinome residues can cause bacterial resistance, resulting in disordered and imbalanced microbial environment balance in the human body
Since cefquinome is widely used to treat bovine mastitis and other dairy cow diseases, the residue of the drug in milk will also seriously affect the quality of dairy products

Method used

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  • Specific cefquinome-resistant monoclonal antibody hybridoma cell strain 2D4 and application thereof
  • Specific cefquinome-resistant monoclonal antibody hybridoma cell strain 2D4 and application thereof
  • Specific cefquinome-resistant monoclonal antibody hybridoma cell strain 2D4 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Embodiment 1: the preparation of hybridoma cell line 2D4

[0022] (1) Synthesis of complete antigen: take 4.8 mg of CEF, add 2.5 mg of EDC and 2.0 mg of NHS, dissolve in DMF, stir at room temperature, and activate for 4 hours; another 5 mg of BSA is dissolved in 2 mL of 0.05 M, pH 9.6 CB solution, and the above activation The final cefquinome solution was slowly added dropwise to the CB solution of BSA, stirred and reacted at room temperature overnight, and then dialyzed at 4°C for three days to obtain the immunogen, which was subpackaged and stored at -20°C.

[0023] (2) Animal immunization: healthy BALB / c mice aged 6-8 weeks were selected for immunization. After the cefquinome complete antigen (1 mg / mL) was emulsified with the same amount of Freund's adjuvant, BALB / c mice were immunized by subcutaneous multi-point injection, each 100 μL. Freund's complete adjuvant was used for the first immunization, and Freund's incomplete adjuvant was used for the booster immunizat...

Embodiment 2

[0026] Example 2 Preparation and Identification of Monoclonal Antibody

[0027] Take 8-10 week-old BALB / c mice, and inject 1 mL of paraffin oil into each mouse; 7 days later, each mouse is injected with 1×10 6 Hybridoma 2D4, ascites fluid was collected from the seventh day, the ascites fluid was purified by octanoic acid-saturated ammonium sulfate method, and the obtained monoclonal antibody was stored at -20°C.

[0028] The mouse monoclonal antibody Ig class / subclass identification was used to determine the subtype of the monoclonal antibody. The specific results are shown in Table 1, and the antibody subtype was IgG2b.

[0029] Table 1

[0030]

[0031] Determination of IC of monoclonal antibodies against CEF, CEX and EFT using indirect competition ELISA and indirect ELISA 50 They were 1.2ng / mL, 56.34ng / mL and 19.02ng / mL respectively, and the cross-reactivity rates were all less than 10%. It can be used for specific rapid detection of cefquinome.

[0032] IC of hybridom...

Embodiment 3

[0035] Example 3 Antibody Application

[0036] The monoclonal antibody prepared by hybridoma cell line 2D4 through in vivo ascites was applied to the ELISA addition and recovery test of cefquinome, and the specific steps were as follows:

[0037] a. 0.5 μg / mL CEF-EDC-OVA diluted with carbonate buffer (CBS) was used as the original coating to coat the 96-well microtiter plate, 100 μL per well, after coating at 37°C for 2 hours, wash with PBST washing solution Plate three times, each time with 250 μL per well, each time for 3 minutes, and pat dry;

[0038] b. Block with CBS containing 0.2% gelatin, 200 μL per well, block at 37°C for 2 hours, wash the plate three times with PBST washing solution, 250 μL per well for 3 minutes each time, and pat dry;

[0039] c. Prepare 0, 0.1, 0.2, 0.5, 1, 2, 5, and 10 ng / mL cefquinome standard solutions with phosphate buffered saline (PBS). Add the standard solution and the extract of the sample to be tested to the sealed microtiter plate, 50 ...

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Abstract

The invention relates to a specific cefquinome-resistant monoclonal antibody hybridoma cell strain 2D4 and application thereof, and belongs to the field of food safety immunodetection. The monoclonal cell strain 2D4 is preserved in China General Microbiological Culture Collection Center, CGMCC for short, and the preservation number is CGMCC No.10872. Coupling is conducted on drug cefquinome and BSA to prepare immunogen through an active ester method, and coupling is conducted on the cefquinome and OVA to prepare a coating antigen. The specific cefquinome-resistant monoclonal antibody hybridoma cell strain 2D4 is obtained after immunization is conducted. A monoclonal antibody secreted by the cell strain only specifically aims at the cefquinome, the crossing-over rates of the monoclonal antibody and other cephalosporin antibiotics of cefalexin and ceftiofur are 2.13% and 6.31% respectively, the crossing-over rates of the monoclonal antibody and other cephalosporin antibiotics are all smaller than 0.1%, and therefore high specificity is achieved. According to the obtained cefquinome-resistant monoclonal antibody hybridoma cell strain, good detection sensitivity and affinity are achieved, the cross-reaction rate to other cephalosporin antibiotics is low, and the purpose of specifically detecting the cefquinome can be achieved.

Description

technical field [0001] The invention relates to a hybridoma cell line 2D4 with a specific anti-cefoquine monoclonal antibody and an application thereof, belonging to the field of food safety immune detection. Background technique [0002] Cefquinome (CEF), English name is Cefquinome, molecular formula is C 23 h 24 N 6 o 5 S 2 ·H 2 SO 4 , the molecular weight is 626.69, and the chemical name is [[(6R,7R)-7-[[(2Z)-(2-amino-4-thiazolyl)(methoxyimino)acetyl]amino]-2-carboxy- 8-Oxo-5-thio-1-azabicyclo[4.2.0]oct-2-en-3-yl]methyl]-5,6,7,8-tetrahydroquinoline sulfate monohydrate, The CAS accession number is 84957-30-2. [0003] Cefquinome is currently the only fourth-generation cephalosporin antibiotic dedicated to animals. It has the characteristics of broad antibacterial spectrum and strong antibacterial activity, and is suitable for parenteral administration. Cefquinome is highly stable to β-lactamase, and its intrinsic antibacterial activity is stronger than that of the...

Claims

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Application Information

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IPC IPC(8): C12N5/20C07K16/44G01N33/577C12R1/91
CPCC07K16/44C07K2317/92
Inventor 匡华朱雨婷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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