Study On Rapid Immunoassay And Detection Techniques For The Adulteration Marker Of Gutter Cooking Oil

It is difficult to find gutter cooking oil after being micromixed into edible oil through refining and processing.Routine identification methods need to combine multiple indicators of different sample pretreatment as the basis for judgment.In order to realize the rapid detection of gutter cooking oil,it is urgent to find new detection indicators and synchronous detection means.A large number of studies have shown the feasibility of capsaicinoids as a marker of gutter cooking oil adulteration.In addition,ginger is widely used in food cooking.The main components of gingerols have interconversion in the process of gutter cooking oil processing.Therefore,it can also be used as an important marker of kitchen waste oil adulteration.Broad-spectrum antibodies in immunoassays that identify a set of compounds with similar chemical structures can meet our needs.Considering the problems of low efficiency and heavy workload of broad-spectrum antibody screening,this study proposed a strategy to design broad-spectrum haptens of capsaicinoids and gingerols based on molecular docking,and verified the feasibility of the method from both theoretical and practical levels.Finally,broad-spectrum antibodies of capsaicinoids and gingerols was realized the effective screening,and it was initially applied.At the same time,the Fluorescence Polarization Immunoassay（FPIA）detection method realized the rapid field detection of gutter cooking oil adulteration markers.The main research results of this topic were as follows:In order to solve the problems of single detection index,large workload,long time consuming and great difficulty of broad-spectrum antibody preparation,this study proposed to use capsaicinoids and gingerols as adulteration markers.The CPC Ab-D8 of capsaicinoids was used for homology modeling,and molecular docking was cunducted with gingerols,and the hapten was designed in reverse on the basis of identifying important functional groups.The mice were immunized with complete antigen and finally obtained the broad-spectrum monoclonal antibody cell lines producing antibodies against capsaicinoids and gingerols.Through molecular docking,it was found that the benzene ring,phenol hydroxyl group,methoxy and the carbonyl in long chain alkyl in gingerols are the main functional groups that could form hydrogen bonds with CPC Ab-D8.Based on this,Hapten 1 and Hapten 7 of capsaicinoids and gingerols were successfully designed and synthesized,and the corresponding complete antigens Hapten 1-BSA/OVA and Hapten 7-BSA/OVA were prepared.Finally,three broad-spectrum monoclonal antibodies with good recognition against capsaicinoids and gingerols were obtained,1-F12,1-G12 and 1-D6,with titer of 128000,64000 and 126000,respectively.The IC₅₀ of broad-spectrum monoclonal antibody 1-F12 of capsaicinoids and gingerols to six analytes（Capsaicin,Dihydrocapsaicin,N-Vanillylnonanamide,6-Shogaol,6-Gingerol and Vanillylacetone）ranged between 251.58 and 974.98 ng/m L.The reversed strategy of designing broad-spectrum hapten based on molecular docking provides an important guarantee for broad-spectrum prepared.The availability of broad-spectrum monoclonal antibodies significantly improves the reliability of the detection discrimination results,and also verified the feasibility of designing a broad-spectrum hapten strategy based on molecular docking from the perspective of practical detection.To solve the problem that the recognition mechanism of antigen-antibody for capsaicinoids and gingerols was not clear.This study demonstrated the feasibility of the reversed strategy of designing broad-spectrum hapten through the establishment and validation of electrostatic potential model of the hapten and hapten-polypeptide.At the same time,the cross-reactivity experiment can well reflect the important role of different functional groups,the position and length of the connecting arms on the recognition mechanism.Electrostatic potential（ESP）analysis of haptens（Hapten X,Hapten A,Vanillic acid,N-vanillylnonanamide,Hapten 2,Hapten 3,Hapten 4,Hapten 5,Hapten 6）and universal hapten with lysine tripeptide conjugates（Hap-X-3T-A,Hap-4-3T and Hap-5-3T）was conducted.Combined with the existing data,this proved the important potential role of ESP in the efficient screening of haptens.Five reported antigens and antibodies were collected for cross-combination,and finally the best combination（Hapten 5-OVA/1-F12）and key functional groups were identified.With the optimal combination,the IC₅₀ of all analytes was intermediate between 88.13～499.16 ng/m L.These studies provided a theoretical basis for the subsequent broad-spectrum antigen design.At the same time,the preliminary test of lateral flow immunoassay（LFIA）verified the possibility of broad-spectrum monoclonal antibodies of capsaicinoids and gingerols applied in practical monitoring.These studies provided theoretical basis and technical support for simultaneous immunoanalysis of various small molecule exogenous markers in edible oil.In view of the complexity of ingredient of gutter cooking oil and the special requirements of rapid field detection,the FPIA detection method was established by using CPC Ab-D8 to realize the rapid field detection of gutter cooking oil.The tracer was obtained by coupling the prepared fluoresceinthiocarbamyl ethylenediamine with the universal Hapten 5 to capsaicinoids.The tracer purified by TLC showed good binding to CPC Ab-D8.Through the optimization of the concentration of tracer and recognition element,the type and p H of buffer and incubation time,the linear range(IC₂₀～IC₈₀)of established FPIA was 0.00397～0.09799μg/m L,and the IC₅₀ was 0.01973μg/m L.The recovery rate of corn germ oil,soybean oil and peanut blend oil were between 90 and 135%.Compared with other methods for the detection of capsaicinoids,FPIA showed the comprehensive advantages of high sensitivity,high specificity,simple operation,small equipment volume and short incubation time,realized the simple and rapid on-site detection of gutter cooking oils.