The Construction Of Nonylphenol Immunoassay

Nonylphenol（NP）is a typical endocrine disruptor（EDs）.Due to high yield,high utilization rate,and often seen in the aquatic environment,and causing serious pollution to the environment,it is urgent for researchers to establish a nonylphenol.Complete set of inspection systems.However,traditional detection methods are time consuming and expensive,so this article will focus on how to establish a simple and easy to operate and inexpensive immunochemical technique for detecting NP.The mechanism of action of immunobiochemical detection is antibody/antigen specific binding,which can achieve the purpose of immediate and rapid detection and has strong sensitivity.In this thesis,based on the study of the synthesis of nonylphenol immunoantigen,the preparation of polyclonal antibody and its immunoassay technology,a rapid and simple method for the detection of nonylphenol immunoassay（ELISA）was established.The specific work is as follows:（1）The use of organic synthesis to modify nonylphenol to obtain four nonylphenol haptens（NPH）,Abbreviated as NPH1、NPH2-1、NPH2-2 and NPH3,preliminary determination of the chemical structure of several haptens by nuclear magnetic and mass spectrometry,through The carbodiimide method uses two nonylphenol haptens to couple with two carriers such as bovine serum albumin（BSA）and ovalbumin（OVA）,and synthesizes four antigens NPH-BSA（1）.NPH-OVA（1）,NPH-BSA（2）and NPH-OVA（2）.（2）Injecting and immunizing New Zealand white rabbits and Balb/c mice with（1）mesophenol-based immune antigen NPH-BSA（1）,using NPH-BSA（2）and NPH-OVA（2）The mice were immunized to obtain four kinds of nonylphenol polyclonal antibodies,and the prepared serum antibody has strong ability to bind antigen,and the antibody titer was64000,the immunizing antigen was nonylphenol hapten-coupled BSA carrier protein and immunized.The species obtained good serum titers for New Zealand white rabbits.（3）Optimized the indirect competition ELISA conditions,using NPH-OVA（2）as the coating antigen,and the coating concentration was determined to be 5μg/mL.The mice were immunized with NPH-BSA（2）to obtain antibodies,and the dilution of the antibody serum was 1:8000.The dilution of Ig goat anti-mouse antibody is 1:5000.The blocking agent used in the experiment is skim milk powder.The optimum antigen-antibody reaction pH is 7.4,and the constant temperature reaction time is 1 h.The indirect competition of nonylphenol is established by the previous experimental conditions.The ELISA standard curve,the final NP sensitivity IC₅₀ is 158μg/L,the minimum detection limit is 1.4695μg/L.