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10-hydroxymesaconitine hapten, artificial antigen, antibody, and preparation methods and application thereof

A new aconitine and artificial antigen technology, applied in the field of food safety, can solve problems such as insufficient sensitivity of antibodies and uncertain quality of artificial antigens

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

AI Technical Summary

Problems solved by technology

During the synthesis of haptens into immunized animals, the quality of artificial antigens is uncertain, which may completely or partially change the epitopes of aconitum alkaloids, resulting in insufficient sensitivity of the prepared antibodies, or even inability to stimulate the body to produce anti-aconitum organisms Alkaline antibody

Method used

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  • 10-hydroxymesaconitine hapten, artificial antigen, antibody, and preparation methods and application thereof
  • 10-hydroxymesaconitine hapten, artificial antigen, antibody, and preparation methods and application thereof
  • 10-hydroxymesaconitine hapten, artificial antigen, antibody, and preparation methods and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1、10

[0036] Example 1, 10-hydroxyaconitine artificial antigen

[0037] 1. Preparation of 10-hydroxynaconitine artificial antigen

[0038] Flowchart such as figure 2 shown.

[0039] (1) Weigh 19 mg of 10-hydroxyaconitine and dissolve it in 2 mL of anhydrous pyridine, then add 29.3 mg of SA, wherein the molar ratio of 10-hydroxyaconitine to SA is 1:100, and stir at room temperature Reaction 22h. Then, 2 mL of ethyl acetate and water were added respectively, and extracted three times with ethyl acetate. The aqueous phase was discarded, and the ethyl acetate phase was retained, and dried by rotary evaporation to obtain the hapten HMA-SA.

[0040] Mass Spectrometry Identification: Mass Spectrometry Identification Result of Hapten HMA-SA: MS m / z[M+H] + Theoretical value: 748.7; measured value: 747.9, consistent with the molecular weight of the target product, mass spectrum as figure 1 shown.

[0041] (2) Dissolve HMA-SA with 1mL DMF, stir magnetically, add 2mg EDC and 1.2mgNHS wh...

Embodiment 2

[0050] Embodiment 2, monoclonal antibody

[0051] 1. Animal immunity and immune effect determination

[0052] 1.1. Animal immunity

[0053] Select 8 healthy SPF-grade 6-week-old female BALB / c mice, and use HMA-SA-BSA for immunization, and the immunization dose is 100 μg protein (HMA-SA-BSA) / mouse; Inject at 8 points, and immunize four times in total, with an interval of 2 weeks between immunizations. For the first immunization, dilute HMA-SA-BSA with PBS to 100 μg protein / 100 μL, then add an equal amount of FCA, and fully emulsify (take a drop of the emulsified emulsion, put it on the water surface, and the milk drop will float on the water surface, and no diffusion occurs, which proves that the emulsification is complete), inject 200 μL subcutaneously into BALB / c mice. For the subsequent two booster immunizations, FIA can be used instead of FCA for emulsification, and other reagents and procedures remain unchanged.

[0054] 1.2. Determination of immune effect

[0055] (1...

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Abstract

The invention discloses a 10-hydroxyl mesaconitine hapten, artificial antigen, antibody and preparation methods and application thereof. The structural formula of the 10-hydroxyl mesaconitine hapten is shown as a formula I, and the 10-hydroxyl mesaconitine hapten can be prepared according to the following method: taking pyridine as a solvent, and reacting 10-hydroxyl mesaconitine with succinic anhydride. The invention also provides the monoclonal antibody of the 10-hydroxyl mesaconitine, an experimental animal is immunized by the artificial antigen of the 10-hydroxyl mesaconitine to generate antiserum, hybridoma cells secreting the monoclonal antibody resisting the 10-hydroxyl mesaconitine are screened out by ELISA (enzyme-linked immunosorbent assay), the monoclonal antibody of the 10-hydroxyl mesaconitine is prepared, and the antibody has the advantages of high sensitivity and broad spectrum, and can crossly recognize aconite alkaloids. The broad-spectrum specific antibody provided by the invention can be used for establishing immunological detection methods such as enzyme-linked immunosorbent assay, chemiluminescence, time resolution, fluorescence polarization and the like of the aconite alkaloid, can be used for preparing a kit detection product of the aconite alkaloid, and is suitable for detection of a large number of samples and on-site rapid detection.

Description

technical field [0001] The invention relates to a 10-hydroxyaconitine hapten, an artificial antigen, an antibody and a preparation method and application thereof, belonging to the field of food safety. Background technique [0002] 10-Hydroxyaconitine is one of the in vivo metabolites of neoaconitine, which has the common parent ring structure of aconitine alkaloids and is also highly toxic. Aconitum alkaloids are widely found in traditional Chinese medicinal materials of the genus Aconitum, such as Chuanwu, Aconitum, and Aconite, and are widely used in China, Japan, Korea and other countries to treat various pains caused by exogenous wind-cold-damp pathogens. However, these compounds are extremely toxic, can damage the nervous system and cardiovascular system, and can cause death in severe cases. According to the 2015 edition of "Chinese Pharmacopoeia", calculated as dry products, the total amount of neoaconitine, hypoaconitine, and aconitine in Chinese herbal medicines of...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07D221/22C07K14/765C07K14/77C07K1/107C07K16/44G01N33/577
CPCC07D221/22C07K14/765C07K14/77C07K16/44G01N33/577G01N2333/415
Inventor 江海洋王战辉史为民任祯慧温凯郑丕苗
Owner CHINA AGRI UNIV