Enrofloxacin monoclonal antibody as well as preparation method and application thereof

A monoclonal antibody, enrofloxacin technology, applied in the field of immunochemistry, can solve the problems of lack of antibody affinity for hapten, restriction of use and promotion, amplification of signal strength, etc., to achieve high affinity, reduce difficulty and cost, and increase selection sexual effect

Active Publication Date: 2015-03-04
SUZHOU UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, this method only amplifies the signal intensity, but does not fundamentally improve the affinity of the corresponding antibody to the hapten. At the same time, it expands the effective detection concentration span and greatly reduces the detection accuracy; w

Method used

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  • Enrofloxacin monoclonal antibody as well as preparation method and application thereof
  • Enrofloxacin monoclonal antibody as well as preparation method and application thereof
  • Enrofloxacin monoclonal antibody as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1: Preparation and detection of enrofloxacin immunogen.

[0039] The specific experimental steps of immunogen preparation are as follows:

[0040] 1. Blocking of carrier protein: Dissolve ethylenediamine (EDA) (20 μl), bovine serum albumin (BSA) (23.6 mg), dicyclohexylcarbodiimide (EDC) (25.6 mg) in sequence at pH=7.0 In 0.01M PBS solution (1mL), under the action of the coupling agent EDC, block the carboxyl residues in the BSA molecule, shake overnight (20°C, 110rpm), dialyze with PBS for 24h, remove excess EDA and EDC, and obtain cations BSA back up.

[0041] 2. Coupling of enrofloxacin and carrier protein: dissolve enrofloxacin standard (ENR) (15.3mg), N-hydroxyoxalimide (NHS) (5.2mg), EDC (9.3mg) In N,N-dimethylformamide (DMF) (500μl), shake overnight at room temperature in the dark (20°C, 110r / min) to form an activated ester; centrifuge for 5min (3000r / min), take the supernatant (100μl ) into cationized BSA solution (2ml, which contains 5mg cationized BS...

Embodiment 2

[0043] Embodiment 2: Animal immunity and antiserum detection.

[0044] 1. Animal immunization: BALB / c mice were used as immunized animals, and immunized according to the procedures in Table 1.

[0045] Table 1. Mouse immunization program schedule

[0046]

[0047] Among them: E-B represents ENR and cBSA conjugate; E represents ENR;

[0048] CFA stands for complete Freund's adjuvant; IFA stands for incomplete Freund's adjuvant.

[0049] The injection dose of each immunization was set to be 100 μg / mouse, and the time interval between two immunizations was 4 weeks; the first immunization was performed by intraperitoneal injection after the immunogen was emulsified with an equal volume of Freund's complete adjuvant; The method and dose of the third immunization are the same as the first one, in which the adjuvant is Freund's incomplete adjuvant, and its volume ratio to the immunogen is 1:3; the method and dose of the fifth immunization are the same as the first time , wherei...

Embodiment 3

[0090] Example 3: cell fusion, screening of hybridoma cells and cloning culture.

[0091] Three days after the seventh booster immunization, splenocytes were collected for cell fusion, positive clones were selected for subcloning, and hybridoma cell lines capable of stably secreting monoclonal antibodies were obtained. The specific experimental process is as follows:

[0092] 1. Cell fusion and culture:

[0093] (1) Preparation of myeloma cells: On the day of fusion, take 4 to 5 bottles (50ml standard cell bottles) of Sp2 / 0Ag14 mouse myeloma cells in the logarithmic growth phase, gently tap the wall of the bottle to make the cells fall off and collect them in a 50ml free In a bacterial centrifuge tube, centrifuge at 1000rpm for 5min to remove the culture medium, add serum-free RPMI-1640 culture medium to wash, centrifuge once, then resuspend with 20ml serum-free RPMI-1640 culture medium, and count the cells for later use;

[0094] (2) Preparation of feeder cells: the day befo...

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Abstract

The invention provides an enrofloxacin monoclonal antibody as well as a preparation method and an application thereof. The preparation method of the enrofloxacin monoclonal antibody comprises the following steps: simultaneously carrying out immunogen and envelope antigen on enrofloxacin and a conjugate of the enrofloxacin and cationic bovine serum albumin, building an indirect checkerboard ELISA method so as to calibrate appropriate envelope antigen concentration and antiserum working concentration, analyzing affinity of to-be-detected enrofloxacin monoclonal antibody on a standard sample by indirect competition ELISA, selecting candidate rats based on the fact that the antiserum working concentration is greater than 1/50000 and 50% inhibiting concentration IC50 is not greater than 5ng/ml as indexes, strictly screening by a cell fusion technique to prepare hybridoma cell strains 14H4A1 and 14F8B1, inducing two strains of cells to generate ascitic fluid so as to obtain a target monoclonal antibody. By virtue of the enrofloxacin monoclonal antibody with a high titer and a high affinity, the actual detection of an extremely small amount of enrofloxacin residue is implemented; the technical support and material basis are provided for implementing efficient and fast field detection.

Description

technical field [0001] The invention belongs to the technical field of immunochemistry, and specifically relates to an enrofloxacin monoclonal antibody with high titer, high affinity and high specificity and a preparation method thereof, the application of the monoclonal antibody to detect enrofloxacin residues, and the secretion of enrofloxacin A hybridoma cell line producing the monoclonal antibody. Background technique [0002] Enrofloxacin (Enrofloxacin), also known as ethyl ciprofloxacin, is the first special fluoroquinolone drug for livestock and poultry, because of its advantages of broad antibacterial spectrum, strong antibacterial activity, easy absorption and wide distribution in the body. It is widely used in veterinary clinical prevention and treatment. Due to the discovery of the drug resistance of human pathogenic bacteria induced by residues of the drug in animal foods, the Ministry of Agriculture of my country stipulates that the maximum residue limit (Maxim...

Claims

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Application Information

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IPC IPC(8): C12N5/20C07K16/44C07K16/06G01N33/577C12R1/91
Inventor 吴康宋学宏杨彩根徐逍
Owner SUZHOU UNIV
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