Antigen, antibody and enzyme-linked immunosorbent assay (ELISA) kit of amantadine

An enzyme-linked immunoassay detection and amantadine technology, which is applied in the direction of measuring devices, color/spectral characteristic measurement, instruments, etc., can solve the problem that the accuracy, sensitivity, and specificity cannot fully meet the detection requirements, the process is complicated, and the adamantine does not meet the requirements. Alkylamine detection requirements and other issues

Active Publication Date: 2018-06-12
广东标允生物科技有限公司 +1
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Enzyme-linked immunosorbent assay kits capable of detecting amantadine have been developed abroad, but domestically produced kits cannot fully meet the detection requirem...

Method used

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  • Antigen, antibody and enzyme-linked immunosorbent assay (ELISA) kit of amantadine
  • Antigen, antibody and enzyme-linked immunosorbent assay (ELISA) kit of amantadine
  • Antigen, antibody and enzyme-linked immunosorbent assay (ELISA) kit of amantadine

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Experimental program
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Effect test

Embodiment 1

[0068] Preparation and identification of embodiment 1 amantadine hapten

[0069] 1. Preparation of amantadine hapten

[0070] (1) Accurately weigh 10 mg of N-(1-adamantane)hydrazine carboxamide and dissolve it in 125 μL of methanol, stir and dissolve to obtain liquid A;

[0071] (2) Dissolve 4.0 mg of glyoxylic acid in 125 μL of methanol to obtain liquid B;

[0072] (3) Mix A and B liquids and put them into a round-bottomed flask with a magnetic stirrer, stir and react at room temperature for 48 hours, centrifuge the reacted mixed solution at 4500r / min for 15min, then discard the supernatant solution, and The precipitate was vacuum-dried at 50°C, and finally a white solid powder was obtained, which was the amantadine hapten.

[0073] 2. Identification of amantadine hapten

[0074] The resulting hapten was identified by mass spectrometry, -MS: M-1 = 264.6, such as figure 2 shown. The results show that the chemical structural formula of the amantadine hapten is as shown in...

Embodiment 2

[0076] The preparation of embodiment 2 amantadine artificial antigen

[0077] 1. Synthesis of amantadine immune antigen

[0078] (1) Weigh 5.93 mg of hapten and dissolve it in 1 mL of DMF, add 4.3 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 5.2 mg of N-hydroxysuccinate imide, stirred and reacted at room temperature for 2h to obtain solution C;

[0079] (2) 50 mg of BSA was dissolved in 5 mL of 0.1 mol / L sodium bicarbonate buffer to obtain a protein solution;

[0080] (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.

[0081] 2. Synthesis of Amantadine Coating Source

[0082] The amantadine coating agent was prepared in the same synthesis method as the above-mentioned amantadine immune antigen.

Embodiment 3

[0083] Example 3 Preparation and Specific Identification of Amantadine Monoclonal Antibody

[0084] 1. Preparation of amantadine monoclonal antibody

[0085] (1) Animal immunization: 150 μg / mouse of the immunogen prepared above was dissolved in normal saline, mixed with Freund's complete adjuvant in an equal volume, and subcutaneously injected into the back of the neck to immunize 6- and 8-week-old Balb / c female mice On the 7th, 14th, and 28th day after the initial immunization, the immunogen and Freund's incomplete adjuvant were mixed in equal volumes, and each additional immunization was given once. The immune complex was 150 μg per mouse 3 days before fusion, and no Freund's adjuvant was added. One booster immunization;

[0086] (2) Cell fusion: Take splenocytes from immunized mice and mix them with mouse myeloma cells (SP2 / 0) in the logarithmic growth phase, and then slowly add preheated fusion agent (PEG4000) for fusion within 45s. Suspend the HAT medium evenly, then ad...

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Abstract

The invention discloses an antigen, an antibody and an enzyme-linked immunosorbent assay (ELISA) kit of amantadine. Firstly, an amantadine hapten is prepared, wherein the molecular structural formulaof the amantadine hapten is as shown in formula (I) (The formula is shown in the description.); then, the amantadine hapten is coupled with carrier protein to obtain an artificial antigen; and then, amonoclonal antibody of amantadine is obtained through a hybridoma technique, thereby establishing a competitive ELISA method and kit according to the monoclonal antibody of amantadine. The ELISA kitof amantadine, disclosed by the invention, can be used for detecting the content of amantadine in an animal tissue sample, the detection limit is 0.10mu g/kg, IC50 is 0.79mu g/L, and the linear detection range is 0-16.2mu g/L; and the method is simple, convenient and quick, is low in cost, high in sensitivity and highstrong in specificity, and is suitable for on-site quick detection.

Description

technical field [0001] The invention belongs to the technical field of food safety detection. More specifically, it relates to an amantadine antigen, antibody and ELISA kit. Background technique [0002] Amantadine (Amantadine) belongs to the antiviral drug that inhibits influenza virus. It was once used in poultry to resist avian influenza, so it is favored by the majority of farmers. However, due to the lack of safety and efficacy data, amantadine is not available in the United States. It is recommended to treat influenza again, and it has certain toxic and side effects on the nervous system and cardiovascular system. Therefore, the Ministry of Agriculture of my country issued the "Urgent Notice on Checking Amantadine and Other Antiviral Drugs" in 2005 to prohibit the production, operation and sale of antiviral drugs. Use antiviral drugs such as amantadine. The U.S. FDA banned adamantanes and neuraminidase inhibitors anti-influenza drugs from being used in poultry in 2006....

Claims

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Application Information

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IPC IPC(8): G01N33/577G01N33/535G01N21/31
CPCG01N21/31G01N33/535G01N33/577
Inventor 杨金易沈玉栋曾道平谭庶孙远明徐振林陈丽韦田
Owner 广东标允生物科技有限公司
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