Zopiclone hapten, antigen, antibody, colloidal gold chromatography detection device and application thereof

A technology for zopiclone and hapten, applied in the field of zopiclone hapten and colloidal gold chromatography detection device, can solve the problems of expensive equipment, false positive, complicated operation, etc., and achieve short detection time, strong specificity and easy operation simple effect

Active Publication Date: 2020-07-31
SHENZHEN BIOEASY BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

LC and LC-MS / MS detection have the advantages of high precision, high separation efficiency, good selectivity, low false positive rate, good reproducibility, etc., but the operation is complicated, the equipment is expensive, the operator has high technical requirements, and it cannot be displayed immediately Results and other issues, not suitable for rapid online detection and monitoring
TLC, NIR and RS are easy to operate and fast in analysis, but their sensitivity is poor, there is often interference between multiple components, the specificity is not good, and Chinese patent medicines and complex food matrices are prone to false positives, generally need to use dedicated Further validation with more robust methods

Method used

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  • Zopiclone hapten, antigen, antibody, colloidal gold chromatography detection device and application thereof
  • Zopiclone hapten, antigen, antibody, colloidal gold chromatography detection device and application thereof
  • Zopiclone hapten, antigen, antibody, colloidal gold chromatography detection device and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Example 1. Preparation of zopiclone hapten

[0062] The synthetic route to obtain the zopiclone hapten molecule by modifying the zopiclone intermediate (CAS: 43200-81-3) with succinic anhydride is as follows:

[0063]

[0064] Specifically, weigh 1.31 g of zopiclone intermediate (CAS: 43200-81-3) and 1.0 g of succinic anhydride, dissolve it completely with anhydrous pyridine, stir and react at 80°C overnight, and detect the reaction by thin layer chromatography (TLC). Process, after the reaction is complete, evaporate the solvent to dryness, add water to dissolve, extract 2-3 times with ethyl acetate, combine the organic phases, evaporate to dryness and pass column purification to obtain white solid H1, which is identified as the target product by MS, see Figure 4 .

Embodiment 2

[0065] Example 2. Preparation and Identification of Zopiclone Antigen

[0066] Preparation: Dissolve 0.1 mmol of the zopiclone hapten prepared in Example 1 in 2 mL of dimethylformamide (DMF), stir and add 0.2 mmol of dicyclohexylcarbodiimide (DCC) and 0.15 mmol of N-hydroxysuccinyl imine (NHS). Magnetic stirring was carried out overnight at 4°C. After centrifugation, the supernatant was liquid A, and 140 mg of BSA was weighed and dissolved in 10 mL of 0.1 mol / L phosphate buffered saline (PBS) (pH=8.0). Add DMF 1mL, stir and dissolve to prepare solution B, under magnetic stirring, gradually drop solution A into solution B, and react at 4°C for 12h. After centrifugation, the supernatant was taken, dialyzed with normal saline at 4°C for 3 days, and the dialysate was changed 3 times a day. The obtained whole antigen was dispensed into 0.5 mL centrifuge tubes at a concentration of 1 mg / mL. Store frozen in a -20°C freezer. At the same time, the zopiclone antigen with OVA as the ...

Embodiment 3

[0068] Example 3. Preparation of Zopiclone Antibody

[0069] 3.1 Animal immunity

[0070] Using the zopiclone immunization antigen obtained in Example 2, immunized four 6-week-old BALB / C mice with an immunization dose of 200 μg / mouse, and after three times of booster immunization, it produced antiserum.

[0071] 3.2 Cell fusion and cloning

[0072] Under sterile conditions, spleen cells were prepared from the spleen of immunized BALB / C mice, and the quantity ratio was adjusted according to the spleen cell: myeloma cell (SP2 / 0) = 9:1 for fusion. After more than 3 times of cloning culture and detection, A hybridoma cell line stably secreting anti-zopiclone monoclonal antibody was screened.

[0073] 3.3 Cell cryopreservation and recovery

[0074] The hybridoma cells were made into 5×10 6cells / mL for long-term storage in liquid nitrogen. When resuscitating, take out the cryopreservation tube from the liquid nitrogen irrigation, quickly put it into a warm water bath at 37°C, a...

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Abstract

The invention provides a zopiclone hapten, a zopiclone antigen obtained by coupling the hapten with carrier protein, a zopiclone antibody obtained by immunizing an animal with the zopiclone antigen, azopiclone colloidal gold chromatography detection device prepared therefrom and application thereof. By virtue of the principle of chromatographic immunocolloidal gold, the content of zopiclone illegally added in a Chinese patent medicine or health food is semi-quantitatively detected through colorimetric assay between a detection line and a quality control line in a test strip. The detection device can quickly and accurately detect the zopiclone illegally added in the Chinese patent medicine or the health food and can meet on-site supervision and enforcement needs of a supervision departmentand an inspection organization. Compared with the prior art, the detection device provided by the invention has the advantages of high sensitivity, strong specificity, low cost, simple operation, short detection time and a long shelf life. The detection device can be applied to quick detection of the zopiclone illegally added in the Chinese patent medicine or the health food or the like.

Description

technical field [0001] The invention relates to the field of biological detection. More specifically, the present invention relates to a zopiclone hapten, a zopiclone antigen, a zopiclone antibody, a colloidal gold chromatography detection device comprising the above-mentioned antigen and antibody, and uses thereof. Background technique [0002] With the increasingly fierce competition in modern society, the pressure from study, life, and employment has also increased. Anxiety and insomnia have become common problems that plague people. Many people have to rely on sedative and tranquilizing psychotropic drugs to maintain normal sleep and life, but because sedative and hypnotic drugs, such as zopiclone, estazolam, barbiturate, diazepam, etc., all belong to the second category of psychotropic drugs. The use of drugs is strictly restricted, so traditional Chinese medicine preparations and health foods with sedative and hypnotic effects may become the first choice of patients. ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07D487/04C07K14/765C07K14/77C07K14/795C07K16/44G01N33/558
CPCC07D487/04C07K14/765C07K14/77C07K14/795C07K16/44C07K19/00G01N33/558
Inventor 王炳志杨星星李细清许稳健金虹张鑫高荣健谢迪琴万秋玲
Owner SHENZHEN BIOEASY BIOTECHNOLOGY CO LTD
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