Amantadine hapten, antigen, chemiluminiscence enzyme-linked immunoassay kit and application thereof

Abstract

The invention discloses an amantadine hapten, an antigen, a chemiluminiscence enzyme-linked immunosorbent assay kit thereof and an application thereof. The invention discloses the amantadine hapten, and also discloses the corresponding artificial antigen and the chemiluminiscence enzyme-linked immunoassay kit and the application thereof. The amantadine hapten provided by the invention is coupled with a carrier protein to obtain an amantadine antigen. The amantadine antigen can be applied to preparation of an amantadine specific antibody. An amantadine chemiluminiscence enzyme-linked immunoassay method is established on the basis of antibody preparation. The detection range is 0 to 16.2 [mu]g / L; the half inhibition concentration is 0.45 [mu]g / L; the detection limit is 0.20 [mu] g / L, the addition recovery rate is 90.00%-104.22%, the whole detection process can be completed only in about 30 min, the detection limit is lower, the sensitivity is higher, and a more convenient, faster and simpler method is provided for detection of residues of amantadine in tissue samples.

Description

technical field

[0001] The invention belongs to the technical field of biological detection. More specifically, it relates to an amantadine hapten, an antigen and a chemiluminescence enzyme-linked immunoassay kit and application thereof. Background technique

[0002] Amantadine (AMA), molecular formula C 10 h 17 N, is an antiviral and anti-Parkinson's disease drug for humans. It is often used for the prevention and treatment of upper respiratory diseases caused by influenza A virus. Overdose or allergic constitution will be caused by stimulation or poisoning of the central nervous system. Adverse reactions to hallucinations, confusion, mood or other mental changes. There is a lack of scientific, standardized, safe and effective test data for human amantadine antiviral drugs transplanted into animals. The use of amantadine in the treatment of animal viral diseases not only brings adverse consequences to animal disease control, but also affects the implementation of nationa...

Examples

Embodiment 1

[0070] The preparation of embodiment 1 amantadine hapten

[0071] 1. Preparation of amantadine hapten

[0072] (1) Add 15.0g of 2-adamantanone and 6.3g of 80% hydrazine hydrate to 30mL of methanol solution, react at -10°C for 30min, evaporate the methanol solvent to obtain a transparent oily product, which is directly used without purification react in the next step.

[0073] (2) The above product was dissolved in 30 mL of chloroform, 10.12 g of triethylamine and 12.01 g of succinic anhydride, and after reflux and stirring for 1 h, the solvent was evaporated, and the residue was purified by column chromatography to obtain a white solid product, which was amantadine hapten. It was identified and successfully synthesized by mass spectrometry and nuclear magnetic resonance spectroscopy, and its spectral data are as follows: ESI-MS(+):265.155[M+H] + .

[0074] Its structural formula is shown in formula (I):

[0075]

[0076] The synthetic route of this amantadine hapten is...

Embodiment 2

[0077] Embodiment 2 Preparation of amantadine antigen, coating agent and specific antibody

[0078] 1. Synthesis of amantadine antigen

[0079] (1) Weigh 6.12 mg of the amantadine hapten prepared in Example 1, dissolve it in 1 mL of N,N-dimethylformamide (DMF), add 4.3 mg of 1-(3-dimethylaminopropyl) -3-Ethylcarbodiimide hydrochloride and 5.2 mg of N-hydroxysuccinimide were reacted with stirring at room temperature for 2 hours to obtain solution C;

[0080] (2) 50 mg of BSA was dissolved in 5 mL of 0.1 mol / L sodium bicarbonate buffer to obtain a protein solution;

[0081] (3) Add the above solution C to the protein solution drop by drop, stir overnight at room temperature, dialyze with PBS solution at 4°C for 3 days, and change the dialysate 6 times during the period; Filter, aliquot into ampoules, and store at -20°C.

[0082] This amantadine antigen was used as the amantadine immunogen.

[0083] 2. Synthesis of Amantadine Coating Source

[0084] The method is the same as...

Embodiment 3

[0104] The preparation of the chemiluminescent ELISA kit of embodiment 3 amantadine

[0105] 1. Prepare reagents:

[0106] (1) ELISA plate coated with amantadine antigen: 96-well detachable ELISA plate, coated with amantadine antigen and blocking solution, the coating concentration is 65.5 μg / L. The amantadine antigen is a conjugate of amantadine hapten and BSA.

[0107] Dilute the coated antigen to 65.5 μg / L with coating solution, add 150 μL of coating solution to each well, incubate overnight at 37°C, pour off the liquid in the well, wash twice with washing solution, and pat dry. Then add 170 μL of blocking solution to each well, incubate at 37°C for 2 hours, pour off the liquid in the well, dry in an oven at 37°C, and store in a vacuum-sealed aluminum foil bag at 4°C.

[0108] (2) Preparation of amantadine standard solution: Accurately weigh amantadine standard solution, dilute to 1.00mg / mL with chromatographic grade methanol, and then use 0.04mol / L PB buffer solution (fo...

technical field

[0001] The invention belongs to the technical field of biological detection. More specifically, it relates to an amantadine hapten, an antigen and a chemiluminescence enzyme-linked immunoassay kit and application thereof. Background technique

[0002] Amantadine (AMA), molecular formula C 10 h 17 N, is an antiviral and anti-Parkinson's disease drug for humans. It is often used for the prevention and treatment of upper respiratory diseases caused by influenza A virus. Overdose or allergic constitution will be caused by stimulation or poisoning of the central nervous system. Adverse reactions to hallucinations, confusion, mood or other mental changes. There is a lack of scientific, standardized, safe and effective test data for human amantadine antiviral drugs transplanted into animals. The use of amantadine in the treatment of animal viral diseases not only brings adverse consequences to animal disease control, but also affects the implementation of nationa...

Examples

Embodiment 1

[0070] The preparation of embodiment 1 amantadine hapten

[0071] 1. Preparation of amantadine hapten

[0072] (1) Add 15.0g of 2-adamantanone and 6.3g of 80% hydrazine hydrate to 30mL of methanol solution, react at -10°C for 30min, evaporate the methanol solvent to obtain a transparent oily product, which is directly used without purification react in the next step.

[0073] (2) The above product was dissolved in 30 mL of chloroform, 10.12 g of triethylamine and 12.01 g of succinic anhydride, and after reflux and stirring for 1 h, the solvent was evaporated, and the residue was purified by column chromatography to obtain a white solid product, which was amantadine hapten. It was identified and successfully synthesized by mass spectrometry and nuclear magnetic resonance spectroscopy, and its spectral data are as follows: ESI-MS(+):265.155[M+H] + .

[0074] Its structural formula is shown in formula (I):

[0075]

[0076] The synthetic route of this amantadine hapten is...

Embodiment 2

[0077] Embodiment 2 Preparation of amantadine antigen, coating agent and specific antibody

[0078] 1. Synthesis of amantadine antigen

[0079] (1) Weigh 6.12 mg of the amantadine hapten prepared in Example 1, dissolve it in 1 mL of N,N-dimethylformamide (DMF), add 4.3 mg of 1-(3-dimethylaminopropyl) -3-Ethylcarbodiimide hydrochloride and 5.2 mg of N-hydroxysuccinimide were reacted with stirring at room temperature for 2 hours to obtain solution C;

[0080] (2) 50 mg of BSA was dissolved in 5 mL of 0.1 mol / L sodium bicarbonate buffer to obtain a protein solution;

[0081] (3) Add the above solution C to the protein solution drop by drop, stir overnight at room temperature, dialyze with PBS solution at 4°C for 3 days, and change the dialysate 6 times during the period; Filter, aliquot into ampoules, and store at -20°C.

[0082] This amantadine antigen was used as the amantadine immunogen.

[0083] 2. Synthesis of Amantadine Coating Source

[0084] The method is the same as...

Embodiment 3

[0104] The preparation of the chemiluminescent ELISA kit of embodiment 3 amantadine

[0105] 1. Prepare reagents:

[0106] (1) ELISA plate coated with amantadine antigen: 96-well detachable ELISA plate, coated with amantadine antigen and blocking solution, the coating concentration is 65.5 μg / L. The amantadine antigen is a conjugate of amantadine hapten and BSA.

[0107] Dilute the coated antigen to 65.5 μg / L with coating solution, add 150 μL of coating solution to each well, incubate overnight at 37°C, pour off the liquid in the well, wash twice with washing solution, and pat dry. Then add 170 μL of blocking solution to each well, incubate at 37°C for 2 hours, pour off the liquid in the well, dry in an oven at 37°C, and store in a vacuum-sealed aluminum foil bag at 4°C.

[0108] (2) Preparation of amantadine standard solution: Accurately weigh amantadine standard solution, dilute to 1.00mg / mL with chromatographic grade methanol, and then use 0.04mol / L PB buffer solution (fo...

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