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An anti-beta-receptor agonist cluster-specific monoclonal antibody hybridoma cell line and its secreted monoclonal antibody and application

A hybridoma cell line, receptor agonist technology, applied in the biological field, can solve the problems of limited application, difficult, time-consuming and so on

Active Publication Date: 2021-10-22
SUZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] First, there are many operation steps, a large amount of toxic and harmful organic solvents, many instruments and equipment (such as rotary evaporators, nitrogen blowing instruments, etc.), many conversions of liquid phase dissolution systems, and long time-consuming
[0005] Second, the loss rate of the target is high: the loss of the target may be caused in multiple operations such as the removal of biological macromolecules, liquid-liquid extraction / solid phase extraction, purification of the target, reconstitution of the injection buffer, etc.
[0006] Third, although clenbuterol hydrochloride, ractopamine, and salbutamol are all phenethylamines in chemical structure, the physical and chemical properties of the three are quite different, and it is difficult to use a unified extraction solution and extraction solution or a single solid-phase extraction column and ensure that each drug can obtain satisfactory extraction-purification effect
[0007] Fourth, liquid-liquid extraction / solid-phase extraction has only a certain selectivity. While purifying the target, it is inevitable to mix in matrix interferences with similar physical and chemical properties to the target (especially samples with complex matrices such as animal liver and kidney). If the interference peak appears in the retention time region of the target, it will seriously affect the interpretation of the machine-tested data
[0014] Because of its strong selectivity, its application in the pretreatment of mixed residual samples of multi-component targets is greatly limited; therefore, the preparation of multi-component cluster-specific monoclonal antibodies against β-agonists expands its target coverage to replace SPE column made possible

Method used

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  • An anti-beta-receptor agonist cluster-specific monoclonal antibody hybridoma cell line and its secreted monoclonal antibody and application
  • An anti-beta-receptor agonist cluster-specific monoclonal antibody hybridoma cell line and its secreted monoclonal antibody and application
  • An anti-beta-receptor agonist cluster-specific monoclonal antibody hybridoma cell line and its secreted monoclonal antibody and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0070] Example 1: Acquisition of ascites samples containing monoclonal antibodies according to the present invention

[0071] 1. Recovery and proliferation of hybridoma cell line 1C3A and patent CN104311438A hybridoma cells

[0072] Take the cryopreservation tube of the hybridoma cell line from the liquid nitrogen tank, thaw it quickly in a water bath at 37°C, centrifuge at 600r / min for 5min, discard the supernatant, add 15% FBS / RPMI-1640 culture medium to suspend the cells, and add the above After the culture solution reaches 5ml, plant it in a 50ml cell culture bottle and culture it in a carbon dioxide incubator. After the cells grow to 30% density, change the medium halfway. - Passage once every 3 days according to 1:3-4.

[0073] 2. In vivo induction of monoclonal antibody protein and ascites acquisition

[0074] 7-10 days before planting hybridoma cells in vivo, 12 male BALB / c mice aged 8-10 weeks were injected intraperitoneally with 0.5ml / mouse of pristane, and careful...

Embodiment 2

[0077] Example 2: Purification of monoclonal antibody protein of the present invention and ractopamine monoclonal antibody of patent CN104311438A hybridoma cells

[0078] Aspirate 2mL protein G resin suspension, transfer it to a disposable PE column with a column capacity of 12mL, then add 20mL binding buffer (3.3768g Na 2 HPO 4 12H 2 O, 0.0888g NaH 2 PO 4 2H 2 O, 8.5g NaCl, 1LH 2 O preparation), the flow rate is 1mL min -1 , to remove impurities in the suspension and to precipitate the suspension. Then slowly add 4mL of monoclonal antibody ascites to make it slowly flow through the gel layer. Protein G is the cell wall protein of group G streptococci, which can capture the Fc region of IgG antibodies, while other subtypes cannot be captured, so as to achieve the purpose of purification . After the ascites has completely flowed through the gel layer, add 100 mL of binding buffer to wash to remove non-specific adsorption, and then add 15 mL of glycine buffer (0.1 mol L ...

Embodiment 3

[0080] Example 3: Analysis of Immune Crossover

[0081] The present invention selects clenbuterol hydrochloride, terbutaline, bromobuterol, sibuterol, and these four beta-adrenoceptor agonists to detect the cross-reaction rate. Albuterol concentration range 0-30ng mL -1 , the concentration range of cross-products is 0.1-10000ng mL -1 . Make standard curves for albuterol and other 4 cross-products, and calculate their respective ICs 50 , so as to obtain their respective CR values, and the results are shown in Table 1.

[0082] Table 1

[0083]

[0084] As can be seen from Table 1, when the CR value of albuterol was determined to be 100%, it was found that the cross-reaction rates of clenbuterol hydrochloride, terbutaline, bromobuterol and sibuterol were very large, and the CR values ​​were respectively 66.8%, 57.3%, 42.1%, 121.5%. These four substances are very similar to salbutamol in structure, and all have a tert-butyl group connected to a nitrogen atom, indicating ...

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Abstract

The invention relates to the field of biotechnology, and discloses an anti-β-receptor agonist cluster-specific monoclonal antibody hybridoma cell line and the monoclonal antibody secreted therefrom. The hybridoma cell line of the present invention is prepared from salbutamol artificial antigen, and the monoclonal antibody secreted by it can simultaneously recognize salbutamol, clenbuterol hydrochloride, terbutaline, bromobuterol and sibuterol and other β-receptor agonists, Has cluster specificity; the immunoaffinity chromatography gel prepared from this is mixed with other β‑receptor agonists, such as ractopamine, as a purification reagent for machine test pretreatment and HPLC‑FLD, LC‑MS / The combination of MS detection technology can efficiently detect various clenbuterol components in pork, pig liver and pig urine samples, with high accuracy and sensitivity.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to an anti-beta-receptor agonist cluster-specific monoclonal antibody hybridoma cell line, the monoclonal antibody secreted thereto and applications thereof. Background technique [0002] Represented by clenbuterol hydrochloride, ractopamine, and albuterol, the three main types of clenbuterol residues in meat and animal offal have repeatedly been banned and have become the primary targets of veterinary drug residues monitored and supervised by food safety regulators. Immunoassay technologies represented by ELISA kits and immunocolloidal gold test strips have been widely used in high-throughput rapid qualitative screening of residual samples of the above three targets. However, in order to confirm and accurately quantify the above target residues, it must be determined by high performance liquid chromatography with fluorescence detector (HPLC-FLD) or even liquid-mass tandem spectrometry ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/20C07K16/44G01N33/577G01N33/559G01N33/531C12R1/91
CPCC07K16/44G01N33/531G01N33/559G01N33/577
Inventor 吴康黄芳胡仁莉原茵
Owner SUZHOU UNIV
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