Anti-fluoroquinolone drug cluster-specific monoclonal antibody hybridoma cell line and the monoclonal antibody produced by it and its application

A hybridoma cell line and fluoroquinolone technology, applied in the biological field, can solve the problems of false negative detection accuracy, false positive detection specificity, low detection limit, etc., and achieve strong enrichment, strong versatility, and high sensitivity Effect

Active Publication Date: 2019-05-17
SUZHOU UNIV
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Problems solved by technology

[0005] 1. The detection limit is low: the single-component detection limit (the T line completely disappears) of the commercialized gold standard quinolone drug is about 10ppb, while the Ministry of Agriculture’s minimum residue standard for the above-mentioned 8 kinds of drugs is: 30-100ppb, based on this standard Use 5-10× volume buffer solution to prepare the homogenate supernatant of the sample to be tested. Under the premise of not enriching the target substance, its content is very likely to be lower than the detection limit, and the sample to be tested cannot be effectively screened qualitatively. Use less than The preparation of the homogenate supernatant of the sample to be tested with 5× volume buffer is not conducive to the full release of the target substance in the sample to be tested, which affects the detection accuracy, and false negatives lead to missed detection. Identification affects detection specificity, and false positives lead to false detections. Therefore, the single-component detection limit of the immune gold standard card should be less than or equal to 3ppb to fully meet the actual needs of users;
[0006] 2. The versatility is not strong: Zhao Yinli and others reported in the literature that using self-prepared strong specific monoclonal antibodies against enrofloxacin as labeled antibodies to develop gold-labeled khakis, the detection limit of enrofloxacin (T line disappears completely) is less than or equal to 3ppb , and using chicken muscle as the model matrix enrofloxacin external standard incorporation recovery test obtained good recovery results, but because of no cross-reaction with other quinolones, it can only be used for strong specificity analysis of single-component residues of enrofloxacin What is more unfavorable is that enrofloxacin is partially converted into ciprofloxacin through in vivo metabolism, and the mixed residues of enrofloxacin and ciprofloxacin in the body after enrofloxacin administration cannot be accurately determined by using the immune gold standard card. There are also some sporadic reports on immunoassay products for targets of other fluoroquinolones such as norfloxacin, ofloxacin, and pefloxacin, but most of them are more suitable for single-component assays due to their strong specificity for their targets
However, the residues of fluoroquinolones in food samples tend to be multi-component mixed residues, so it is impossible to use a single antibody immunoassay product for qualitative or semi-quantitative analysis of multiple component residues at the same time

Method used

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  • Anti-fluoroquinolone drug cluster-specific monoclonal antibody hybridoma cell line and the monoclonal antibody produced by it and its application
  • Anti-fluoroquinolone drug cluster-specific monoclonal antibody hybridoma cell line and the monoclonal antibody produced by it and its application
  • Anti-fluoroquinolone drug cluster-specific monoclonal antibody hybridoma cell line and the monoclonal antibody produced by it and its application

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preparation example Construction

[0056] In the preparation of the immunogold standard card in the present invention, the enrofloxacin standard sample is preferably coupled with high-purity cationized bovine serum albumin (cBSA) by carbodiimide coupling method or mixed anhydride method. The conjugate is prepared as a test line (T line) for the coated antigen. The two methods are as follows:

[0057] 1. Carbodiimide coupling method

[0058] First, ethylenediamine (EDA) and bovine serum albumin were mixed and dissolved in 0.01M PBS (pH7.0) solution, and under the action of chemical coupling agent-dicycloethylcarbodiimide (EDC), the bovine serum was blocked. The -COOH residue in the albumin molecule is removed by dialysis to remove excess EDA and EDC, and cationized bovine serum albumin (cBSA) is obtained as a protein carrier.

[0059]Dissolve the standard sample of enrofloxacin (EF) in N,N-dimethylformamide (DMF), add N-hydroxysuccinimide (NHS), and react overnight in EDC in the dark at room temperature to form...

Embodiment 1

[0069] Example 1: In vivo induction of monoclonal antibody samples of the present invention

[0070] 1. Recovery and proliferation of hybridoma cell line -11F10

[0071] Take the hybridoma cell line-11F10 cryopreservation tube from the liquid nitrogen tank, melt it rapidly in a water bath at 37°C, centrifuge at 600r / min for 5min, discard the supernatant, add fresh 15% FBS / RPMI-1640 culture medium to suspend the cells, and supplement each After adding the above-mentioned culture solution to 5ml, plant it in a 50ml cell culture bottle and culture it in a carbon dioxide incubator. After the cells grow to a density of 30%, the medium is replaced halfway. Passage once every 2-3 days according to 1:3-4.

[0072] 2. In vivo induction of monoclonal antibody protein and ascites acquisition

[0073] 7-10 days before planting hybridoma cells in vivo, 12 8-week-old male BALB / c mice were injected intraperitoneally with pristane at 0.5ml / mouse, and carefully reared for later use.

[0074...

Embodiment 2

[0077] Embodiment 2: Purification of the monoclonal antibody protein of the present invention

[0078] Pre-cool 0.01M PBS (pH 8.0) 4× diluted ascitic fluid in an ice-water bath as a preparation solution. According to 50mg of total protein: 1ml Protein G Resin, fill the gel in a small column for chromatography (10ml), equilibrate the column with 50× pre-cooled 0.01M PBS (pH 8.0) of the column bed volume, and prepare the solution at room temperature Filter five times at the natural flow rate of the Protein G Resin column, so that the monoclonal antibody protein in the ascitic fluid can fully specifically bind to protein G, and then fully rinse with pre-cooled 0.01M PBS (pH8.0) solution 50 times the volume of the column bed Chromatographic column to remove foreign proteins adhering to the chromatographic gel.

[0079] Pre-add 100μl 1M Tris-Cl (pH 9.6) to each 1.5ml EP tube, add ice-cold 0.1M Glycine-HCl (pH 2.7) to the above-mentioned treatment column to elute, and collect the f...

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Abstract

The invention discloses a fluoroquinolones-resistant specific monoclonal antibody hybridoma cell strain, a monoclonal antibody generated based on the cell strain, and the application thereof. The preservation number of the cell strain is CCTCC No.C2015221. The cell strain can stably secrete a monoclonal antibody specific to fluoroquinolones. The result of an indirect ELISA test shows that, the monoclonal antibody is high in sensitivity and strong in universality for the drug detection of ciprofloxacin, enrofloxacin, norfloxacin, pefloxacin, enoxacin and the like. The monoclonal antibody is applied to other immunoassay products, such as the preparation of immunogold labeling cards, immune affinity chromatography gels and the like. Obtained products are still high in sensitivity, high in selectivity, strong in enrichment and strong in universality for target detection. Therefore, the monoclonal antibody can be widely applied to the trace residual qualitative and semi-quantitative analysis of mainly fluoroquinolones in the fields of agriculture, food, environment and the like.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to anti-fluoroquinolone drug cluster-specific monoclonal antibody hybridoma cells and the monoclonal antibody produced therefrom and applications thereof. Background technique [0002] Fluoroquinolones, as artificially synthesized antibiotics, are widely used in medicine (as first-line antibacterial drugs in clinical practice), agricultural and sideline medicines, etc. In the production of aquatic products (an important antibacterial drug used for the prevention and treatment of bacterial diseases in livestock, poultry and aquatic animals), the current trend of fluoroquinolone antibacterial drugs in food samples is mixed with multiple components and high residues. What is more worrying is that related pharmaceuticals The diffusion of the above-mentioned antibiotics in the environment in enterprises, hospitals, and agricultural and sideline aquatic products production enterprises causes ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/20C07K16/44G01N33/577G01N33/531C12R1/90
CPCC07K16/44G01N33/531G01N33/577G01N2430/00
Inventor 吴康宋学宏杨彩根
Owner SUZHOU UNIV
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