Formoterol resistant monoclonal antibody and application thereof

A monoclonal antibody, formoterol technology, applied in the fields of immunology and veterinary drug residue analysis, can solve the problems of high experimental equipment and technical requirements, cumbersome operation, not suitable for rapid detection of large quantities of samples, etc., to achieve strong controllability and repeatable effects

Active Publication Date: 2017-05-31
BIOLOGY INST OF HEBEI ACAD OF SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Currently, the detection techniques for formoterol residues mainly include high-performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC-MS), liquid chromatography-tandem mass spectrometry (LC-MS / MS) and immunoassay. technology, the first three methods are sensitive and accurate, but the operation is cumbersome and requires high experimental equipment and technology, which is not suitable for rapid detection of large batches of samples

Method used

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  • Formoterol resistant monoclonal antibody and application thereof
  • Formoterol resistant monoclonal antibody and application thereof
  • Formoterol resistant monoclonal antibody and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Embodiment 1 Preparation of formoterol antigen of the present invention

[0042] (1) Preparation of activated p-aminobenzoic acid: take 6 mg of sodium nitrite, dissolve it in 0.35 mL of distilled water, then weigh 10 mg of p-aminobenzoic acid, dissolve it in 1.1 mL of 1mol / L hydrochloric acid, stir in an ice bath, and The above-mentioned sodium nitrite solution was added dropwise to the p-aminobenzoic acid solution, and reacted in the dark for 1 hour to obtain activated p-aminobenzoic acid;

[0043] (2) Preparation of formoterol active intermediate with carboxyl group: Weigh 30 mg of formoterol and dissolve it in 5 mL, 0.05 mol / L ice-cold borax buffer solution (pH=9, containing 0.15 mol / L chlorine sodium chloride), stirred in an ice bath, this solution was added dropwise to the activated p-aminobenzoic acid solution, and reacted in the dark at 4°C for 3 hours to obtain a carboxyl-containing formoterol active intermediate solution;

[0044] (3) Synthesis of immunogen: A...

Embodiment 2

[0046] Example 2 Preparation of formoterol monoclonal antibody of the present invention

[0047] (1) Animal immunization: immunize 6-8 week-old female Balb / c mice with bovine serum albumin as the carrier protein, and immunize once every 2 weeks. The immunization process is shown in Table 1. After the third immunization, the tail was docked to collect blood Determine the titer and inhibition rate, and select the mouse with the best result to prepare for fusion;

[0048] Table 1 Immunization flow chart

[0049]

[0050] (2) Cell fusion: the fused mice were bleed from the eyeballs, and the serum was used as a positive control. After the neck was killed, the spleen was taken out under aseptic conditions to prepare spleen cells, which were fused with SP2 / 0 cells by PEG at a ratio of 5:1. The fused cell suspension was added to a 96-well plate with feeder cells, and placed in a 37°C, 5% CO 2 cultivated in an incubator;

[0051] (3) Screening of positive hybridoma cell lines: th...

Embodiment 3

[0053] Example 3 Characterization of the Formoterol Monoclonal Antibody of the Present Invention

[0054] (1) Potency determination

[0055] The indirect ELISA method is used, and the specific steps are as follows: Coating: Dilute the coating material with carbonate buffer solution to a concentration of 2 μg / mL, 100 μL / well in a 96-well microtiter plate, overnight at 4°C; washing: return the coated plate to room temperature , pour off the coating solution, add 300 μL of washing solution to each well, let stand for 1 min each time, wash 3 times, and pat dry for the last time; sealing: add 200 μL of washing solution containing 10% calf serum to each well, 37°C for 1 hour; Pour off the blocking solution, wash 3 times, and pat dry; add primary antibody: use the washing solution to dilute the monoclonal antibody at a ratio of 1:2000, add 100 μL to each well, and set a blank control well (PBS) and a negative control (negative control) Serum), placed at 37°C for 45min; poured off th...

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Abstract

The invention relates to a formoterol resistant monoclonal antibody and application thereof, which belong to the technical field of immunology and the technical field of veterinary drug residue analysis. The resistant monoclonal antibody is generated by virtue of the secretion of a hybridoma cell strain with a preservation number of CCTCC No. C2016182, a titer of the antibody is 1: 512000, a subtype is IgG1, an affinity constant Ka is equal to 7.24*10<8>L / mol, a suppression rate IC50 for formoterol is 1.1 microgram / L, and the antibody has no cross reaction with other formoterol similar substance. The monoclonal antibody can be used for preparing an enzyme linked immunosorbent assay kit and colloidal gold immunochromatographic strip for detecting formoterol so as to achieve a purpose of rapidly and sensitively detecting the formoterol in animal tissues, urine and feed.

Description

technical field [0001] The invention relates to an anti-formoterol monoclonal antibody, a hybridoma cell line producing the monoclonal antibody, and the application of the antibody in detecting formoterol, belonging to the technical fields of immunology and veterinary drug residue analysis. Background technique [0002] Formoterol (Formoterol) is the third-generation β-beta drug developed by the Central Research Institute of Yamanouchi Pharmaceutical Co., Ltd. 2 - Adrenergic receptor agonist anti-asthmatic drugs, which have bronchodilator effect, anti-allergic and edema-inhibiting effects, and can be used to treat chronic bronchial asthma, nocturnal asthma, exercise-induced asthma, chronic obstructive pulmonary disease and non-asthmatic airway in children disease. In recent years, with the strengthening of food safety supervision in my country, the supervision on the use of prohibited veterinary drugs has gradually increased, and the phenomenon of abuse of prohibited veteri...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/20C07K16/44G01N33/532G01N33/535G01N33/543G01N33/577G01N33/94
CPCC07K16/44C07K2317/33C07K2317/35C07K2317/92G01N33/532G01N33/535G01N33/543G01N33/577G01N33/9433
Inventor 李春生刘静静李玉静吴萌程华邸禄芹张小兵李亚璞陈英珠
Owner BIOLOGY INST OF HEBEI ACAD OF SCI
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