CN-119192017-B - Paranitroaniline hydrophobic hapten as well as synthetic method and application thereof

Abstract

The invention relates to the technical field of biochemical engineering, in particular to a paranitroaniline hydrophobic hapten, a preparation method and application thereof. The invention is specifically based on p-nitroaniline, the hydrophobicity of the target being regulated by introducing polar atoms (F, N or O) from the spacer. Subsequently, hapten is coupled to carrier protein and the immunogenicity of artificial antigen is evaluated. The invention explores the influence of hapten hydrophobicity change on immunogenicity, provides theoretical support for reasonable design of hapten on one hand, and provides a material basis for preparing high-affinity antibody of paranitroaniline 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 paranitroaniline hapten is characterized by comprising a hydrophobic spacer, wherein the structural formula of the paranitroaniline hapten is any one of the following structural formulas: II; III; IV。
2. 2. A paranitroaniline artificial antigen comprising the paranitroaniline hapten of claim 1 and a carrier protein coupled to the paranitroaniline hapten.
3. 3. The paranitroaniline artificial antigen of claim 2, wherein the paranitroaniline hapten is coupled to the carrier protein via an amide bond.
4. 4. A para-nitroaniline 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 paranitroaniline artificial antigen according to any one of claims 2 to 4, wherein the carrier protein is coupled to the carboxyl group of the paranitroaniline hapten by an active ester method.
6. 6. The preparation method of claim 5, wherein the coupling molar ratio of paranitroaniline hapten to carrier protein is 4.75-13.38:1.
7. 7. Use of the paranitroaniline hapten of claim 1 or the paranitroaniline artificial antigen of any one of claims 2-4 for any one of the following: For evaluating the effect of hapten hydrophobicity on immunogenicity; the application of the specific antibody in preparing paranitroaniline; the application of the kit is used for detecting the paranitroaniline specific antibody; the use is for non-disease diagnostic purposes.

Description

Paranitroaniline hydrophobic hapten as well as synthetic method and application thereof Technical Field The invention relates to the technical field of biochemical engineering, in particular to a paranitroaniline hydrophobic hapten, a synthesis method and application thereof. Background P-nitroaniline (p-Nitroaniline) belongs to aniline compounds and is widely applied to the production process of chemicals such as pesticides, veterinary drugs, rubber, dyes, medicines and the like. The excessive use and discharge of such compounds has resulted in a great degree of pollution to the environment (particularly the water body). Paranitroaniline has high chemical toxicity, and can produce toxic action through various modes such as inhalation, oral administration, skin contact and the like, and induce diseases such as hypoxia, jaundice, anemia and the like. Paranitroaniline is used as a chemical for high incidence and characteristic pollutants of sudden environmental events, and is an important point for environmental risk prevention and control. Antibodies are the core reagents for immunoassay methods. One of the key steps in the preparation of small molecule antibodies is the rational design and synthesis of haptens. The nature of the hapten has an important impact on its immunogenicity, directly determining the specificity and potency of the antibody produced. In conventional hapten designs, hydrophobicity is often an important consideration as it can significantly affect the stability and affinity of antigen-antibody interactions. In the related fields of proteins and nano vaccines, researches show that enhancing the hydrophobicity of an immunogen can induce stronger immune response, but in the field of preparation of small molecular compound antibodies, the influence of hapten hydrophobicity on the immune response is unknown. Because paranitroaniline 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 a paranitroaniline 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 a paranitroaniline hapten, where the paranitroaniline hapten contains a hydrophobic spacer arm, and the structural formula of the paranitroaniline hapten is any one of the following structural formulas: I; II; III; IV。 The invention is based on the structure of the p-nitroaniline target, and adjusts the hydrophobicity of the target by introducing polar atoms (F, N or O) from a spacer arm, so that the 4 hapten structures with excellent performances in hydrophobicity and immune response enhancement reaction are finally screened. In a second aspect, the present invention provides a paranitroaniline artificial antigen comprising the paranitroaniline hapten and a carrier protein coupled to the paranitroaniline hapten. Preferably, the paranitroaniline 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 paranitroaniline hapten by an active ester method. Preferably, the coupling molar ratio of the paranitroaniline hapten to the carrier protein is 4.75-13.38:1. 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 preparing high affinity antibodies to nitroaniline. In a third aspect, the invention provides any one of the following uses of the paranitroaniline hapten or the paranitroaniline artificial antigen: For evaluating the effect of hapten hydrophobicity on immunogenicity; the application of the specific antibody in preparing paranitroaniline; the application of the kit is used for detecting the paranitroaniline specific antibody; the use is for non-disease diagnostic purposes. The 4 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 inventio