Study On Rapid Detection Technology Of 1-indole Butyric Acid Immunology

1-Pyrenebutyric acid is the most common derivative of polycyclic aromatic hydrocarbons,mainly found in water,air and soil.It has the properties of estrogen,which can covalently bind to DNA molecules in the human body,causing errors in its transcription and replication,and also causing damage to the body's immune system,nervous system and blood circulation.At present,the detection methods of polycyclic aromatic hydrocarbons such as 1-anthracene acid are mainly gas phase,liquid phase,fluorescence and other instrument detection methods.These methods are costly,require strict operators and are time-consuming,in contrast,immunology.Detection technology detection is currently used for the detection of polycyclic aromatic hydrocarbons in foods because of its high speed,good sensitivity,high specificity and rapid detection for large samples.In this paper,1-indole acid artificial antigen was prepared by carbodiimide method(EDC),and the monoclonal antibody with high sensitivity and specificity against 1-indoleic acid was screened by hybridoma cell technology.The indirect competitive ELISA kit for 1-butyric acid with high sensitivity,good specificity and high accuracy was assembled.1.Preparation and identification of 1-Pyrenebutyric acid artificial antigenThe complete artificial antigens PBA-BSA and PBA-OVA were synthesized by coupling 1-Pyrenebutyric acid with carrier protein bovine serum albumin(BSA)and chicken ovalbumin(OVA)by EDC.The properties were identified by UV scanning,polypropylene gel electrophoresis and animal immunization.The results showed that the maximum absorption peaks of PBA-BSA and PBA-OVA were different from those of BSA and OVA,respectively.The surging speed of PBA-BSA was slower than that of BSA,and the tailing phenomenon appeared.The antibody serum was determined to have a titer of up to 1:1.28×104 and IC50=5.94 ng/mL.It can be seen that the complete artificial antigen preparation of 1-indolebutyric acid is successful.2.Preparation and identification of 1-Pyrenebutyric acid monoclonal antibodyThe mice with good sensitivity after immunization with complete artificial antigen of indolebutyric acid were selected for cell fusion assay.A positive hybridoma with stable secretion of monoclonal antibody to 1-Pyrenebutyric acid was prepared by positive hybridoma cell screening technique.The cell line,designated 10C2A6,was identified for its potency,sensitivity,affinity,subtype and specificity.The results showed that the titer was as high as 1:2.56×105,the subtype was IgG2b,the IC50 was 0.86 ng/mL,the cross-reactivity with other analogs was less than 1.7%,and the affinity was 1.96×109 L/mol.A monoclonal antibody with high specificity and sensitivity is well established,which lays a good foundation for the establishment of immunological rapid detection method for 1-Pyrenebutyric acid3.Preparation and identification of 1-Pyrenebutyric acid indirect competitive ELISA kitThe optimal workflow of the ELISA kit was determined by the checkerboard method and orthogonal test.The 1-Pyrenebutyric acid indirect competitive ELISA kit was assembled and its sensitivity,sensitivity,accuracy and stability were identified.The results showed that:1-Pyrenebutyric acid ndirect competition ELISA kit standard curve R2=0.9939,good regression;sensitivity was 0.010ng/mL,the recovery rate was 77.2%-95.8%,the coefficient of variation was 3.01%-6.46%,less than 15%,indicating higher accuracy;cross-reaction can be seen that the cross-reaction rate with 1-oxime methanol,1-Pyrenebutyric acid and hydrazine and other analogs is 0.33%or less,with phenanthrene,naphthalene,benzopyrene,BSA and The cross-reaction rate of OVA is less than 0.05%,and its specificity is good.The kit is stable and can be stored for at least 180 days in the dark.