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ELISA (Enzyme-linked Immunosorbent Assay) kit used for detecting Cimaterol medicine residue, as well as preparation method and application of ELISA kit

An enzyme-linked immunosorbent reagent, the technology of cimaterol, applied in the biological field, can solve the problems of high cost, cumbersome and time-consuming sample processing, and inability to apply on-site detection.

Active Publication Date: 2014-03-12
河北冠卓检测科技股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The instrument detection method is sensitive and accurate, but the sample processing is cumbersome, time-consuming and costly, and requires specially trained professionals to operate complex instruments and equipment, which cannot be applied to on-site detection, thus limiting its wide application

Method used

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  • ELISA (Enzyme-linked Immunosorbent Assay) kit used for detecting Cimaterol medicine residue, as well as preparation method and application of ELISA kit
  • ELISA (Enzyme-linked Immunosorbent Assay) kit used for detecting Cimaterol medicine residue, as well as preparation method and application of ELISA kit
  • ELISA (Enzyme-linked Immunosorbent Assay) kit used for detecting Cimaterol medicine residue, as well as preparation method and application of ELISA kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Embodiment one The preparation of cimaterol medicine antigen of the present invention

[0059] (1) Diazotization of cimaterol: Weigh 3.5 mg of CIM into a 10 ml screw bottle, dissolve with 1 ml of 0.1 mol / L HCL, cool in an ice bath, keep away from light, add sterilized bismuth drop by drop while stirring Distill water until dissolved and add an appropriate amount of pre-cooled 1mol / L NaNO2 solution (starch potassium iodide test paper is blue-black), and react at 4°C for 6 hours to obtain diazotized CIM;

[0060] (2) Synthesis of immunogen: Weigh 10 mg of BSA and dissolve it in 1 mL of 0.1 mol / L pre-cooled PBS (pH 7.4), then add the solution obtained in (1) dropwise, stirring while adding. Adjust the pH value to about 8.5 with 1mol / L NaOH, react at 4°C for 12 hours, dialyze the reactant with 0.01mol / L PBS for 3 days while stirring at 4°C, and change the liquid 3 times a day to obtain CIM-BSA;

[0061] (3) Synthesis of the detection agent: Weigh 8 mg of OVA and dissolve i...

Embodiment 2

[0062] Embodiment 2 The preparation of the enzyme plate plate coated with cimaterol drug antigen according to the present invention

[0063] Dilute the cimaterol drug antigen to 0.25mg / ml with PH9.6 carbonate buffer, add 100ul / well to the well of the microtiter plate, wherein the cimaterol drug antigen includes immunogen, detection agent, immunogen Original bovine serum albumin was used as carrier, and original ovalbumin was detected as carrier; incubate overnight at 4°C or 37°C for 2 hours, wash 4-5 times, and pat dry; add blocking buffer containing 10% calf serum, 200ul / well, Incubate at 37°C for 2 hours; drain in a vacuum oven at room temperature for 5 hours, and vacuum seal with an aluminum foil bag.

Embodiment 3

[0064] Embodiment three The preparation of cimaterol drug monoclonal antibody of the present invention

[0065] (1) Animal immunization: immunize 6-8 week-old female Blab / c mice with the immunogen whose carrier protein is bovine serum albumin, and immunize once every two weeks. , select the mouse with the best result to prepare for fusion;

[0066] (2) Cell fusion: Take mouse splenocytes and SP2 / O cells preserved in our laboratory for fusion, and select the wells with high positive positive levels for the indirect ELISA method, and subclone the positive wells by limiting dilution method until Establishment of a hybridoma cell line producing a single monoclonal antibody against cimaterol;

[0067] (3) Large-scale preparation of monoclonal antibodies: select large female Blab / 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°...

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Abstract

The invention relates to an ELISA (Enzyme-linked Immunosorbent Assay) kit used for detecting Cimaterol medicine residue, as well as a preparation method and application of the ELISA kit, and belongs to the field of biotechnology. The ELISA kit comprises an ELISA plate coated with a Cimaterol medicine antigen, a Cimaterol medicine monoclonal antibody, an enzyme marker, a Cimaterol standard sample solution, a substrate color developing solution, a stop solution, a concentrated cleaning solution and a sample diluent. The application of the ELISA kit mainly comprises the steps of sample pretreatment, operation and result analysis. The ELISA kit can be used for detecting Cimaterol medicine residue in animal tissues (muscles and livers), urine and fodders; no special instrument is required during pretreatment; the operation is simple and fast; the precision, accuracy, sensitivity, specificity and the like are high; the ELISA kit is suitable for screening and on-site monitoring of a great amount of samples.

Description

technical field [0001] The invention relates to an ELISA kit for detecting drug residues, a preparation method and application thereof, in particular to an ELISA kit for detecting cimaterol drug residues, a preparation method and application thereof, and belongs to the field of biotechnology. Background technique [0002] Cimaterol (CIM), also known as cimaterol, cimaterol, belongs to a kind of phenethylamine drugs, is a potent selective B 2 - Receptor agonists, clinically mainly used to dilate trachea and increase pulmonary ventilation, and to treat diseases such as asthma, obstructive pneumonia, smooth muscle spasm and shock. Therefore, the drug can increase the lean meat rate, reduce fat deposition and promote animal growth, so it is often illegally used as a feed additive in the production of animal-derived food. Long-term use may cause CIM to accumulate and remain in edible animal tissues. After entering the human body, it can cause clinical symptoms such as palpitatio...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/577G01N33/531G01N1/28
CPCG01N1/28G01N33/535G01N33/577
Inventor 李春生李君华吴萌程华李亚璞陈英珠张小兵董超
Owner 河北冠卓检测科技股份有限公司
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