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ELISA (Enzyme Linked Immunosorbent Assay) kit for detecting formoterol as well as preparation method and application of ELISA kit

An enzyme-linked immunosorbent reagent, formoterol technology, applied in the fields of immunology and veterinary drug residue analysis, can solve the problems of cumbersome operation, unsuitable for rapid detection of large quantities of samples, and high requirements for experimental equipment and technology

Active Publication Date: 2017-05-24
石家庄市博生环境科技有限公司
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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

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  • ELISA (Enzyme Linked Immunosorbent Assay) kit for detecting formoterol as well as preparation method and application of ELISA kit
  • ELISA (Enzyme Linked Immunosorbent Assay) kit for detecting formoterol as well as preparation method and application of ELISA kit
  • ELISA (Enzyme Linked Immunosorbent Assay) kit for detecting formoterol as well as preparation method and application of ELISA kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Embodiment 1 Preparation of formoterol antigen described in the present invention

[0061] (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;

[0062] (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;

[0063] (3) Synthesis of im...

Embodiment 2

[0065] Embodiment 2 The preparation of the microtiter plate coated with formoterol antigen according to the present invention

[0066] Dilute the formoterol antigen to 0.25 mg / ml with pH9.6 carbonate buffer, add 100 μL / well to the wells of the microtiter plate, where the formoterol antigen includes the immunogen, the detection agent, and the immunogen Serum albumin was used as carrier, and proovalbumin was detected as carrier; incubate overnight at 4°C or at 37°C for 2 hours, wash 4-5 times, and pat dry; add blocking buffer containing 10% calf serum, 200 μL / well, 37°C Incubate for 2 hours; drain in a vacuum oven at room temperature for 5 hours, and vacuum seal with an aluminum foil bag.

Embodiment 3

[0067] Example 3 Preparation of Formoterol Monoclonal Antibody According to the Present Invention

[0068] (1) Animal immunization: immunize female Balb / c mice aged 6-8 weeks with the immunogen whose carrier protein is bovine serum albumin, and immunize once every 2 weeks. , select the mouse with the best result to prepare for fusion;

[0069] (2) Cell fusion: Take mouse splenocytes and mouse myeloma SP2 / 0 cells for fusion, select the wells with high positive positives for the indirect ELISA method, and subclone the positive wells by the limiting dilution method until the cells are established. A hybridoma cell line producing a single monoclonal antibody against formoterol;

[0070] (3) Large-scale preparation of monoclonal antibodies: select large female Balb / c mice, use the method of in vivo induction of ascites, prepare a large amount of ascites, and purify the ascites by octanoic acid-ammonium sulfate precipitation, divide into small tubes, and store at -20 °C , Deformot...

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Abstract

The invention relates to an ELISA (Enzyme Linked Immunosorbent Assay) kit for detecting formoterol as well as a preparation method and application of the ELISA kit, and belongs to the technical field of immunology and the technical field of veterinary drug residue analysis. The ELISA kit for detecting formoterol contains an ELISA plate coated with a formoterol antigen, a formoterol monoclonal antibody, an enzyme marker, a formoterol standard solution, a substrate developing solution, a stopping solution, a concentrated washing solution and a sample compound solution. The ELISA kit has advantages of high sensitivity, high precision, high accuracy, high cross-reactivity, good stability, long storage time, simple operation, high speed and suitability for primary screening of bulk samples.

Description

technical field [0001] The invention relates to an ELISA kit for detecting formoterol, a preparation method and application thereof, which is particularly suitable for detecting formoterol residues in animal tissues, and belongs to the technical field of immunology and the technical field of veterinary drug residue analysis. Background technique [0002] Formoterol is a third-generation β2-adrenoceptor agonist anti-asthma drug developed by the Central Research Institute of Yamanouchi Pharmaceutical Co., Ltd., Japan. It has bronchiectasis, anti-allergic and anti-edema effects. For the treatment of chronic bronchial asthma, nocturnal asthma, exercise-induced asthma, chronic obstructive pulmonary disease and non-asthmatic respiratory diseases in children. 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 veterinary drugs ha...

Claims

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Application Information

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IPC IPC(8): G01N33/94G01N33/531G01N33/543G01N1/34G01N1/38
CPCG01N1/34G01N1/38G01N33/531G01N33/54306G01N33/9433
Inventor 李春生刘静静吴萌李玉静杜顺丰武孝利曹秀梅闫玉杰
Owner 石家庄市博生环境科技有限公司
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