Atropine alkaloid hapten and artificial antigen as well as preparation methods and application thereof

An artificial antigen and atropine technology, applied in biological testing, chemical instruments and methods, material inspection products, etc., can solve the problems of not being able to adapt to multiple types of preliminary screening, rapid screening, high professional requirements, and complicated pretreatment procedures , to achieve the effect of good application prospect, high sensitivity and simple preparation method

Active Publication Date: 2022-01-25
CHINA AGRI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The biggest advantage of the instrument method is that it has sufficient sensitivity and accuracy, but the instrument is expensive, has high requirements for the detection environment and the professionalism of the operator, and requires complex pre-processing procedures, so the instrument method cannot adapt to various types, Preliminary screening of a large number of samples or rapid screening of residues in multiple links of the animal-derived food industry chain, and immunological methods with antigen-antibody specific recognition reactions as the core can better meet this demand

Method used

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  • Atropine alkaloid hapten and artificial antigen as well as preparation methods and application thereof
  • Atropine alkaloid hapten and artificial antigen as well as preparation methods and application thereof
  • Atropine alkaloid hapten and artificial antigen as well as preparation methods and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Embodiment 1, preparation and characterization of atropine alkaloid hapten

[0044] One, the preparation of atropine alkaloid hapten

[0045] The preparation of atropine alkaloid hapten shown in formula I, reaction equation is as figure 1 shown.

[0046] The first step: the reaction uses pyridine as a solvent, mixes 1eq tropinol standard product with 1.1eq succinic anhydride, dissolves in a 5mL round bottom flask, installs a reflux device, places in a 70°C oil bath, and stirs magnetically for 24h.

[0047] Step 2: Add 0.1eq succinic anhydride to the reaction solution, stir at 600r / min for 6h, remove most of the pyridine by nitrogen blowing in the fume hood, let it stand for natural volatilization, and form brown crystals without purification.

[0048] 2. Characterization of atropine alkaloid hapten

[0049] Mass spectrometry identification: mass spectrometry identification result of atropine alkaloid hapten shown in formula I: MS m / z[M+H] + Theoretical value: 242.28; ...

Embodiment 2

[0050] Example 2, Preparation and Characterization of Atropine Alkaloid Artificial Antigen

[0051] In the preparation methods of the immunogen and the coating source, the difference lies in the type of carrier protein used. The carrier protein of the immunogen is mainly BSA and KLH, the carrier protein of the coating source is mainly OVA, and the coupling method used is the active ester method.

[0052] 1. Synthesis and identification of atropine alkaloid artificial antigen

[0053] 1. Preparation of atropine alkaloid artificial antigen

[0054] (1) Calculate based on the hapten and BSA feeding ratio of 1:100, then accurately weigh 7.3mg of hapten, 8.6mg of EDC, and 5.2mg of NHS according to the molar mass ratio of hapten:EDC:NHS=2:3:3 and dissolve them respectively In 1mL DMF, put it on a magnetic stirrer and activate it for 16h, which is the solution I.

[0055] (2) Dissolve 20 mg of KLH, BSA, and OVA in 10 mL of PBS respectively, pre-cool in a -20°C refrigerator, and the...

Embodiment 3

[0061] Embodiment 3, preparation of atropine alkaloid monoclonal antibody

[0062] The TRO-HS-BSA and TRO-HS-KLH artificial antigens prepared in Example 2 were used to immunize 8 BALB / c female mice aged 6-8 weeks respectively. Each immunogen was diluted to 1 mg / mL with 0.01 M PBS, and emulsified with an equal amount of Freund's adjuvant to form a water-in-oil structure. Except Freund's complete adjuvant was used for the first immunization, Freund's incomplete adjuvant was used for the rest of the booster immunization, and the immunization dose was 100 μg per mouse, and multi-point intradermal injection was used on the back of the neck. After 4 weeks, booster immunization was carried out and immunization was given once every 3 weeks, for a total of 2 additional immunizations, which were changed to multi-point subcutaneous injections on the back of the neck. 7-10 days after the two immunizations, the orbital blood of each mouse was centrifuged to take the supernatant for determ...

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Abstract

The invention discloses an atropine alkaloid hapten and artificial antigen as well as preparation methods and application thereof The structural formula of the atropine alkaloid hapten is shown as a formula I. The atropine alkaloid hapten is prepared according to the following method: 1) taking pyridine as a solvent, and carrying out reaction on tropine and succinic anhydride; and 2) adding the succinic anhydride into the reaction liquid after the reaction in the step 1) again, and stirring to obtain the atropine alkaloid hapten. A carrier protein can be coupled to carboxyl carbon of the hapten by adopting an active ester method to obtain the atropine alkaloid artificial antigen. The invention also provides a monoclonal antibody of the atropine alkaloid, which is obtained by immunizing an experimental animal (female BALB / c mouse) through the atropine alkaloid artificial antigen and carrying out cell fusion, screening and in-vitro induction. The monoclonal antibody of the atropine alkaloid can be used for preparing an atropine alkaloid detection reagent or kit. Thehapten and artificial antigen have a good application prospect in veterinary drug residue detection.

Description

technical field [0001] The invention relates to a hapten of atropine alkaloids, an artificial antigen, a preparation method and application thereof, and belongs to the field of food safety. Background technique [0002] Atropine alkaloids are mainly secondary metabolites produced by Solanaceae plants such as datura and hyacinth. They are compounds formed based on tropane ring and various esterifications, mainly including atropine, scopolamine, and anisodamine Alkali etc. Atropine alkaloids are essentially M-choline receptor blockers, they can antagonize the muscarinic effect of acetylcholine or its analogues, and are widely used clinically. However, its toxicological effects are closely related to pharmacological effects. Normal doses of medication will not cause adverse reactions in animals, and due to rapid metabolism, it will hardly remain in animal tissues. However, if it involves eating contaminated feed or Failure to strictly abide by the drug dosage regulations may ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07D451/10C07K14/765C07K14/77C07K14/795C07K16/44G01N33/53G01N33/543G01N33/577
CPCC07D451/10C07K14/765C07K14/77C07K14/795C07K16/44G01N33/5308G01N33/577G01N33/54346
Inventor 江海洋沈建忠丁双阳王梓乐郑丕苗温凯
Owner CHINA AGRI UNIV
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