CN-119192016-B - Amantadine hydrophobic hapten as well as synthesis method and application thereof

Abstract

The invention relates to the technical field of biochemistry, in particular to an amantadine hydrophobic hapten, a synthesis method and application thereof. Based on the structure of the amantadine target, different hydrophobic spacer arms are introduced, so that the hydrophobicity of hapten is improved. Subsequently, hapten is coupled to carrier protein and the immunogenicity of artificial antigen is evaluated. The invention explores the influence of hapten hydrophobicity increase on immunogenicity, provides theoretical support for reasonable design of hapten on one hand, and provides a material basis for preparing high-affinity antibody of amantadine on the other hand. The invention provides a new idea for reasonably designing small molecular compound hapten and preparing high-affinity antibody.

Inventors

• WANG ZHANHUI
• WEN KAI
• SHEN JIANZHONG
• YU XUEZHI
• LIU RUI
• JIANG HAIYANG
• ZHANG YINGJIE
• Pan Yantong

Assignees

• 中国农业大学

Dates

Publication Date

20260508

Application Date

20240905

Claims (7)

1. 1. The amantadine hapten is characterized by comprising a hydrophobic spacer, wherein the structural formula of the amantadine hapten is any one of the following structural formulas: I; VI。
2. 2. An amantadine artificial antigen comprising the amantadine hapten of claim 1 and a carrier protein coupled to the amantadine hapten.
3. 3. The amantadine artificial antigen of claim 2, wherein the amantadine hapten is coupled to the carrier protein by an amide bond.
4. 4. An amantadine artificial antigen according to claim 2 or 3, wherein the carrier protein is selected from bovine serum albumin, ovalbumin, keyhole limpet hemocyanin, thyroxine and human serum albumin.
5. 5. The method for preparing the amantadine artificial antigen according to any one of claims 2 to 4, wherein the carrier protein is coupled to the carboxyl group of the amantadine hapten by an active ester method.
6. 6. The preparation method of claim 5, wherein the coupling molar ratio of the amantadine hapten to the carrier protein is 10-15:1.
7. 7. An amantadine hapten as claimed in claim 1 or an amantadine artificial antigen as claimed in any of claims 2 to 4 for any of the following applications: For evaluating the effect of hapten hydrophobicity on immunogenicity; Use in the preparation of an amantadine-specific antibody; use in the detection of an amantadine-specific antibody; the use is for non-disease diagnostic purposes.

Description

Amantadine hydrophobic hapten as well as synthesis method and application thereof Technical Field The invention relates to the technical field of biochemistry, in particular to an amantadine hydrophobic hapten, a synthesis method and application thereof. Background Amantadine (AMANTADINE, AMD), which is a chemical risk factor, is the first antiviral agent approved by the U.S. Food and Drug Administration (FDA), and is used clinically for many years. AMD is low in price and good in antiviral effect, and domestic breeding enterprises illegally use AMD for preventing and treating diseases of animals caused by virus infection. With the wide application of amantadine, the side effects caused by the amantadine are gradually developed, including drug resistance problems, promotion of strain variation, neurotoxicity and the like, and serious potential risks are caused to the physical health of people. Therefore, the method for detecting the forbidden drugs, which has high sensitivity, strong specificity, simplicity, convenience and practicability, is an important technical support for guaranteeing the food safety and has important significance for guaranteeing the health of people. The rapid detection technology of the immunoassay based on antigen and antibody specific recognition can effectively make up for the defects of instrument analysis and detection due to the advantages of low cost and batch screening. Antibodies are the core reagents for immunoassay methods. Because amantadine belongs to a small molecular compound with smaller molecular weight, the monoclonal antibody has the problems of poor specificity and sensitivity, difficult screening and the like in the aspect of preparation. In view of this, the present invention has been made. Disclosure of Invention In order to solve the technical problems, the invention provides an amantadine hydrophobic hapten, and a synthesis method and application thereof. Specifically, the technical scheme of the invention is as follows: in a first aspect, the present invention provides an amantadine hapten comprising a hydrophobic spacer arm having any one of the following structural formulas: I; II; III; IV; V; VI; VII; VIII。 The invention is based on the structure of an amantadine target, and aims to increase the hydrophobicity of hapten by introducing a spacer containing a saturated fatty chain, an unsaturated fatty chain, a benzene ring, ethylene glycol and other structures. The invention finally screens 8 hapten structures with better performance in hydrophobicity and enhancing immune response reaction. In a second aspect, the present invention provides an amantadine artificial antigen comprising the amantadine hapten and a carrier protein coupled to the amantadine hapten. Preferably, the amantadine hapten is coupled to the carrier protein via an amide bond. Preferably, the carrier protein is selected from bovine serum albumin, ovalbumin, keyhole limpet hemocyanin, thyroxine and human serum albumin. Preferably, the carrier protein is coupled to the carboxyl group of the amantadine hapten using an active ester method. Preferably, the coupling molar ratio of the amantadine hapten to the carrier protein is 10-15:1. The invention couples hapten and carrier protein to evaluate the immunogenicity of artificial antigen. On one hand, the influence of the increase of hapten hydrophobicity on immunogenicity is explored, and theoretical support is provided for reasonable design of hapten. On the other hand, a material basis is provided for the preparation of high affinity antibodies to amantadine. In a third aspect, the invention provides any one of the following uses of the amantadine hapten or the amantadine artificial antigen: For evaluating the effect of hapten hydrophobicity on immunogenicity; Use in the preparation of an amantadine-specific antibody; use in the detection of an amantadine-specific antibody; the use is for non-disease diagnostic purposes. The 8 haptens provided by the invention are specially prepared aiming at the influence of hapten hydrophobicity on monoclonal antibody preparation in an immunoassay technology, and a new thought and a new method are provided for reasonable design of small molecular compound haptens and preparation of high-affinity antibodies. In a fourth aspect, the present invention provides specific antibodies prepared from the artificial antigen, wherein the specific antibodies are polyclonal antibodies and/or monoclonal antibodies. The specific antibody provided by the invention has higher affinity. In a fifth aspect, the invention provides an amantadine drug detection reagent or kit prepared from the specific antibody. In a sixth aspect, the invention provides any one of the following uses of the specific antibody: the application in the fluorescence polarization detection kit for detecting the amantadine drug; the application of the ELISA kit for preparing amantadine drug; application in preparing a lateral flow chromatography detectio