Rapid Immunoassay For The Detection Of Four Fishery Drug Residues In Fish

As we all know,the issue of food safety is a matter of close global concern,especially the residue of veterinary drugs in aquatic products,such as fluoroquinolones and chloramphenicols,etc.Our country regulated the amount of drug residues in aquatic products by issuing departmental announcements or limit standards.However,some newly abused compounds,such as fish narcotics,were used in aquaculture,transportation or sales in large quantities and led to drug residues,which were potentially harmful for humans.Therefore,it is of great significance to develop and establish detection methods for drugs that are easily abused in aquatic products.In this project,four fishery medicines of praziquantel,rifampicin,eugenol and tricaine were used as research objects to carry out the preparation of monoclonal antibodies and the development of analytical methods for indirect competitive enzyme-linked immunosorbent assay?ic-ELISA?and colloidal gold test strips.By analyzing the determinant characteristics of praziquantel,rifampicin,eugenol and tricaine four small molecule compounds,designed the hapten coupling site,respectively,and the carrier protein bovine serum albumin?BSA?,keyhole limpet hemocyanin?KLH?,chicken egg ovalbumin?OVA?,etc.were coupled to prepare complete antigens,immunized BALB/c mice,and monoclonal antibodies were screened using hybridoma cell technology.Finally,four hybridoma cells were screened for anti-praziquantel antibody 2A11,anti-rifampicin antibody 4F1,anti-eugenol antibody 2C9 and anti-tricaine antibody 4B5.The cross-reaction experiment showed that antibody 2C9 had different degrees of cross-linking to eugenol,isoeugenol,methyl eugenol and methyl isoeugenol,which belonged to group-selective antibody;the cross-over rates of antibodies 2A11,4F1 and 4B5 to similar drugs were less than 5.0%,which were highly selective antibodies.The affinity of the antibodies was characterized.The affinity constants K_a were 3.143×10⁹ L/mol,2.533×10⁹ L/mol,1.321×10⁹L/mol and 7.527×10⁸ L/mol,respectively.The corresponding antibody subtypes were IgG1,IgG2a,IgG2a and IgG2b.The ic-ELISA detection method was established based on the prepared monoclonal antibodies.First,the antibody or antigen concentration was optimized,and secondly,the organic solvent content,ionic strength and pH in the measurement solution were optimized.Half of the monoclonal antibodies to the four fishery drugs of praziquantel,rifampin,eugenol and tricaine The inhibitory concentrations(IC₅₀)were 1.517 ng/m L,5.312 ng/mL,42.884ng/m L and 43.399 ng/m L;the linear range of the standard curves(IC₂₀?IC₈₀)were 0.541ng/m L?4.251 ng/m L,1.698 ng/mL?16.620 ng/mL,11.711 ng/mL?157.041 ng/m L and11.824 ng/m L?159.292 ng/m L.Selected the common fish sample?Qingzhan fish?as the matrix sample,and evaluated the ic-ELISA method by adding a recovery experiment.Among them,when the concentration of praziquantel was 0.5,2.0,4.0 ng/g,the recovery rate was91%?97%,the coefficient of variation was 3.871%?5.137%;the concentration of rifampicin was 2.0,8.0,16 ng/g,the recovery rate was 89%?96%,the coefficient of variation was2.524%?4.466%;when the concentration of eugenol was 25,50,150 ng/g,recovery rate was between 89%and 93%,the coefficient of variation was 1.879%?2.181%;the concentration of tricaine was 25,50,150 ng/g,the recovery rate was between 87%and 95%,and the coefficient of variation was 2.660%?3.246%.In addition,four kinds of monoclonal antibodies were labeled with colloidal gold,and a detection method based on colloidal gold test strips was developed.The conditions of colloidal gold labeling and detection solutions were optimized,and the test performance of test strips was evaluated through fish samples.Praziquantel in fish samples had a line of extinction value of less than 5 ng/g;rifampicin had a line of elimination value of less than 100ng/g;tricaine had a line of elimination value of less than 500 ng/g;eugenol had a line of elimination value of less than 1000 ng/g,the structural analogues isoeugenol,methyl eugenol and methyl isoeugenol had a naked-line extinction value below 5000 ng/g.