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Plate for detecting immunity of cannabis and tetrahydrocannabinol monoclonal antibody through collaurum tag

A tetrahydrocannabinol and detection plate technology, applied in the fields of molecular immunology and toxicology, can solve the problems of slow instrument testing, low monoclonal antibody titer, and long analysis operation period.

Active Publication Date: 2009-11-18
SHANGHAI CRIMINAL SCI TECH RES INST +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The routine method of testing drugs in the laboratory is instrumental analysis, but the speed of instrumental testing is slow, the cycle of analysis is long, the purchase of instruments is expensive, special training is required, and the high cost of instrumental testing (more than 400 yuan for a sample) is difficult for grassroots units. to promote
[0006] Although foreign countries (such as the United States, etc.) have developed a marijuana monoclonal antibody immunoassay panel, there are two problems: (1) domestic and foreign marijuana identification is mainly based on whether the sample contains tetrahydrocannabinol (THC). The developed cannabis monoclonal antibody can only recognize the metabolite 11-nor-Δ8-tetrahydrocannabinol-9 carboxylic acid, which is the least abundant in urine samples, but cannot detect tetrahydrocannabinol (THC); (2) current cannabis monoclonal antibody immunity The sensitivity of the detection board is above 100.0ng. To accurately detect 11-nor-Δ8-tetrahydrocannabinol-9 carboxylic acid, the sensitivity of the cannabis monoclonal antibody immunoassay board must be below 10.0ng
Since the molecular weight of BSA is only 400,000-600,000 Da, the source of the animal is close to the human species, so the prepared monoclonal antibody has a low titer and poor specificity

Method used

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  • Plate for detecting immunity of cannabis and tetrahydrocannabinol monoclonal antibody through collaurum tag
  • Plate for detecting immunity of cannabis and tetrahydrocannabinol monoclonal antibody through collaurum tag
  • Plate for detecting immunity of cannabis and tetrahydrocannabinol monoclonal antibody through collaurum tag

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preparation example Construction

[0066] The principle and steps of the preparation of the complete antigen are shown in the following process: (1) the hydroxyl-containing compound is first reacted with succinic anhydride to generate a carboxyl-containing intermediate, (2) and then reacted with N-hydroxysuccinimide (NHS) In the reaction, the produced ester bond reacts with the amino group of the protein to form an amide bond under the condition of fat-soluble dicyclohexylcarbodiimide (DCC). A succinyl group is inserted between two cross-linked compounds, a small molecule hapten and a protein.

[0067]

[0068] Preparation Reaction Formula of THC Complete Antigen

[0069] The esterification and condensation reactions can be carried out by any method known to those skilled in the art under any suitable conditions. For example, the half-esterification reaction of the present invention can be carried out under the following conditions: the reaction temperature is 0-100°C, preferably room temperature-80°C, more...

Embodiment 1 4

[0122] The preparation of embodiment 1 tetrahydrocannabinol-KLH complete antigen

[0123] Weigh 100.0 mg of THC, dissolve it in 10.0 mL of pyridine solution, add 300.0 mg of succinic anhydride to the solution, react at 60°C for 4 h, and distill off pyridine. Wash with ethanol to obtain solid powder. Weigh 20.0mg of the above solid powder and dissolve it in 1.0mL DMF (N,N-dimethylformamide), add 10.0mg N-hydroxysuccinimide (NHS) and 65.0mg DCC to it, and react at room temperature for 3h , centrifuge at 10000.0rpm to remove insoluble matter, drop the supernatant into 10.0mL, 10.0mg / mL KLH protein solution drop by drop, react at room temperature for 2h, and dialyze the reaction product at 4°C with 0.01M, pH 7.4 phosphate buffer , during which the buffer was changed 3 to 4 times to remove unreacted small molecular substances, and finally identified by ultraviolet spectroscopy to obtain the complete antigen THC-KLH.

Embodiment 2 4

[0124] Example 2 Preparation of tetrahydrocannabinol monoclonal antibody (CCTCC NO.C200915)

[0125] Balb / C mouse immune procedure

[0126] First emulsify the antigen with Freund's adjuvant, prepare the complete antigen THC-KLH with PBS to make a 1.0 mg / mL solution, then mix the complete antigen solution and Freund's adjuvant in equal volumes, and shake with a high-speed shaker A uniform emulsion is formed, and this emulsion is used for immunization of animals.

[0127] Select 8-week-old mice for injection immunization. Twelve 8-week-old healthy Balb / C mice were taken, and each mouse was intraperitoneally injected with complete Freund's adjuvant emulsified antigen 100.0uL on day 0. On the 14th day, each mouse was intraperitoneally injected with 100.0uL of emulsified antigen in incomplete Freund's adjuvant. On the 21st day, the blood was collected by picking the mouse eyeball, and the antibody titer (titer) in the serum was detected by the ELISA method. On the 28th day, 100...

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Abstract

The invention discloses a complete antigen for detecting tetrahydrocannabinol and preparing an antibody. The invention also discloses an anti-tetrahydrocannabinol monoclonal antibody prepared by the complete antigen and a tetrahydrocannabinol monoclonal antibody immunity detection plate through a collaurum tag for detecting the tetrahydrocannabinol in medicine, a urine specimen or other human specimens. Compared with HPLC and other methods, the detection plate is simple, portable and easy to carry, can carry out field detection and does not need expensive equipment. When the detection plate is used to detect the tetrahydrocannabinol, the whole test can be completed within 1.5 minutes; the detecting sensitivity can reach 0.5ng; and the detection plate has no crossed reaction with more than 60 kinds of common medicaments, drugs and internal metabolins of the tetrahydrocannabinol.

Description

technical field [0001] The present invention relates to the fields of molecular immunology and toxicology. Specifically, the present invention relates to the detection of illegal drugs, especially to the enzyme-linked immunoassay detection of tetrahydrocannabinol. Background technique [0002] At present, the situation of drug crime is very serious. There are many kinds of drugs. According to the statistics of the United Nations in 2004, there are more than 200 million drug addicts in the world, of which at least 140 million people smoke marijuana, more than 9 million people smoke heroin, and more than 13 million people smoke cocaine, amphetamine and other drugs. Synthetic drugs are even more difficult to count. At present, the number of drug addicts in our country continues to rise. The number of registered drug addicts in our country exceeds one million, and the number of young drug addicts has increased sharply. The scope of drug abuse continues to expand, and counties...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/435C07K16/44C12N5/18G01N33/53G01N33/543
Inventor 曾立波
Owner SHANGHAI CRIMINAL SCI TECH RES INST
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