Preparation method of anti-fluoroquinolone rabbit monoclonal antibody and application thereof

A monoclonal antibody, anti-fluoroquinolone technology, applied in the field of immunochemistry, can solve problems such as harm to human health, unfavorable development of livestock and poultry products, drug residues, etc., and achieve high-broad-spectrum effects

Inactive Publication Date: 2011-03-09
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, due to the long-term and extensive application of such drugs, a large number of drug residues in livestock products have caused a series of negative effects, whi

Method used

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  • Preparation method of anti-fluoroquinolone rabbit monoclonal antibody and application thereof
  • Preparation method of anti-fluoroquinolone rabbit monoclonal antibody and application thereof
  • Preparation method of anti-fluoroquinolone rabbit monoclonal antibody and application thereof

Examples

Experimental program
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Embodiment 1

[0038] Example 1 Synthesis of Immunization Antigen and Coating Antigen

[0039] The following six fluoroquinolone drugs were selected for immunoantigen synthesis to be coupled to the carrier protein bovine serum albumin: ofloxacin, dafloxacin, ciprofloxacin, norfloxacin, enrofloxacin, flumequine . The six drug structures are as follows:

[0040]

[0041] 1) Dissolve 30 mg of ciprofloxacin, 45 mg of N-hydroxysuccinimide and 150 mg of carbodiimide hydrochloride in 1.5 mL of dimethylformamide, and stir for 24 hours in the dark to obtain a ciprofloxacin reaction solution; 100 mg of bovine serum albumin was dissolved in 15 mL of phosphate buffer to obtain a bovine serum albumin solution, and the ciprofloxacin reaction liquid was added dropwise to the bovine serum albumin solution, stirred for 3 hours, and dialyzed with phosphate buffer. Obtain ciprofloxacin immune antigen, and store at -20°C; ofloxacin immune antigen, enrofloxacin immune antigen and ciprofloxacin immune antige...

Embodiment 2

[0047] Example 2 Fluoroquinolone rabbit monoclonal antibodies produced

[0048] 2.1 Immunization of rabbits

[0049] The six immune antigens synthesized above were mixed and immunized into rabbits by subcutaneous injection on the back, and 2ml of blood was collected before immunization. For the first immunization, 0.5 mg of each of the six immune antigens was mixed with an equal amount of Freund's complete adjuvant for immunization, and the second was immunized. From the second to the fifth immunization, 0.25 mg of each of the six immunization antigens was mixed with an equal amount of Freund’s incomplete adjuvant for immunization, and the immunization interval was two weeks, and the booster immunization was arranged within 4-8 weeks after the fifth immunization. Mix 0.5 mg of each of the six immune antigens and inject intravenously, kill the rabbit 4 days after the injection, and take the spleen for later use;

[0050] 2.2 Cell Fusion

[0051] Spleen is aseptically taken, a...

Embodiment 3

[0056] Indirect ELISA and direct ELISA operating steps in the screening process of embodiment 3

[0057] 3.1 The steps of indirect ELISA method are as follows:

[0058] 1. Coating plate: Dilute the six coating antigens to 0.5 μg / mL with coating buffer (CBS) and mix the coating plate, 50 μl / well, coat overnight at 4°C or coat at 37°C for 2 hours;

[0059]2. Plate washing: wash once with TBST, 200μl / well;

[0060] 3. Blocking: 0.2% gelatin / TBS, 100 μl / well, 37°C for 1 hour;

[0061] 4. Plate washing: wash three times with TBST, 200 μl / well;

[0062] 5. Adding samples: 50 μl / well of the supernatant stock solution or diluted solution of each cell culture well, reacting on a shaker at 30°C for 1 hour;

[0063] 6. Plate washing: wash the plate three times with TBST, 200 μl / well;

[0064] 7. Secondary antibody: add 2500-fold diluted alkaline phosphatase-labeled goat anti-rabbit, 50 μl / well, and place on a constant temperature shaker at 30°C for 40 minutes;

[0065] 8. Plate wash...

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Abstract

The invention discloses a preparation method of anti-fluoroquinolone rabbit monoclonal antibodies and application thereof. Six fluoroquinolone medicaments are chosen in the invention and coupled with Bovine serum albumin (BSA) to synthesize immunoantigens respectively. The above six immunoantigens are injected subcutaneously in the backs of immune rabbits after being mixed together. The anti-fluoroquinolone rabbit monoclonal antibodies are obtained by hybridoma technique. Rabbit monoclonal antibodies, which are superior to rat monoclonal antibodies, are prepared in the invention. More antigenic determinants are identified when rabbits are chosen as the experimental animal instead of rats. In addition, more integrated experiments can be carried out in rabbit spleens, since the spleens of rabbits are larger than that of rats. The invention provides the possibility of high throughput screening for fusion cells. The anti-fluoroquinolone antibodies of wide-adaptability in the invention has good detection effect on fluoroquinolones, such as enrofloxacin, ofloxacin, Difloxacin, Pefloxacin, Fleroxacin, Danofloxacin, etc., thus making it applicable to fluoroquinolone residue detection on food, forage and environmental samples.

Description

technical field [0001] The invention relates to the technical field of immunochemistry, in particular to a preparation method and application of an anti-fluoroquinolone rabbit monoclonal antibody. Background technique [0002] Fluoroquinolones (fluoroquinolones, FQNS or FQS) drugs belong to the third generation of quinolone antibacterial drugs. They were invented in the 1980s. A fluorine atom was introduced into the 6-position of the quinolone naphthyridine ring, and piperazine was connected to the 7-position. Ring, so collectively referred to as fluoroquinolones, is a series of new fluorine-substituted quinolone derivatives. [0003] FQNS has the advantages of good absorption, long half-life, and broad antibacterial spectrum, and has become a commonly used drug in veterinary clinics. However, due to the long-term and extensive application of such drugs, a large number of drug residues in animal products have been caused, causing a series of negative effects, which not only...

Claims

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Application Information

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IPC IPC(8): C07K16/44G01N33/577
Inventor 郑晓冬刘娜陆蕾倪庚
Owner ZHEJIANG UNIV
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