Preparation Of Monoclonal Antibody For Pyrene And Benzo[a]pyrene And Establishment Of An Enzyme-linked Immunosorbent Assay

Cyclic aromatic hydrocarbons（PAHs）are organic compounds composed of multiple benzene rings in different permutations,which widely exist in the environment.Human and animal exposure to PAHs can cause a variety of adverse effects,including carcinogenicity,teratogenicity,mutagenicity,genotoxicity.At present,the residues of PAHs in aquatic products are common in the world.The accumulation of PAHs in aquatic products will not only affect the quality of aquatic products,but also cause extensive and lasting serious harm to human health through the accumulation of organisms in vivo.Therefore,it is necessary to strengthen the monitoring of PAHs residues in aquatic products.At present,the detection of PAHs is mostly instrumental analysis.Although this method has high sensitivity,the pretreatment process is complex,time-consuming,and cannot be detected on site.Compared with instrumental analysis,immunoassay,which is the mainstream technology of rapid detection methods nowadays.It is very suitable for screening analysis and on-site detection of PAHs residues.However,the immunoassay of PAHs mainly focuses on air,soil and water samples,and there are few studies on the immunoassay of PAHs in aquatic products.Therefore,the establishment of immunoassay method for PAHs residues in aquatic products has important application value.In this study,pyrene butyric acid（PBA）was used as hapten to synthesize complete antigens with different coupling ratios,and finally a monoclonal antibody that can recognize pyrene（PYR）and benzo[a]pyrene（BaP）was obtained.Based on this antibody,an enzyme-linked immunoassay was established and applied to detect PYR and BaP residue in fish,shrimp and crab.1.Using PBA as a hapten,the complete antigen with different coupling ratio was successfully prepared by coupling with BSA and OVA according to carbon diimine method（EDC）.The coupling ratios of PBA-BSA and PBA-OVA were 8.1（A₁）,12.8（A₂）,18.3（A₃）,9.4（B₁）,11.7（B₂）and 19.2（B₃）,respectively.2.The different coupling ratio of immunogens and different doses were used to immunize mice.The titer and inhibition of mice serum were monitored with coating antigen with different coupling ratios.The results showed that when mice were immunized with PBA-BSA-A₃ as immunogen at a dose of 50μg,and PBA-OVA-B₃as coating antigen to monitor the serum of mice,the serum titer was higher and inhibition rate was better.3.Through multiple cell fusion and screening,a hybrioma cell line was finally obtained,named 4D6,which could recognize both PYR and BaP.The antibody subtype was Ig G₁.The ic-ELISA method was established and optimized.The results showed that when PBA-OVA-B₃ was used as the coating antigen and the concentration was 2μg/m L,the dilution of 4D6 was 1:20000,the working concentration of the secondary antibody was 1:6000,and 20%DMF was used as the dilution solution of the competing drug,ELISA method has the highest sensitivity.Using PYR as a competitive drug,an ic-ELISA method was established.The equation was y=-54.756 x+79.143 ranged from 1 to 16 ng/m L,R²=0.9956,the IC₅₀ was 3.73±0.43 ng/m L（n=5）.The antibody primarily recognizes PYR（100%）and BaP（38%）.4.In the application of ELISA,the limits of detection（LOD）of PYR and BaP were 0.43-0.54μg/kg and 0.92-0.98μg/kg,and the limits of quantification（LOQ）were 0.56-0.71μg/kg and 1.12-1.22μg/kg,respectively.5.Standard solutions of PYR and BaP were added to fish,shrimp and crab blank samples according to 1×LOQ,2×LOQ and 4×LOQ.The results showed that the recoveries of PYR and BaP in fish,shrimp and crab were 81.5%-101.9%and84.9%-94.0%,respectively.Compared with HPLC-FLD,the results of the two methods were consistent,which indicated that the ic-ELISA method can be used as a reliable tool for the detection of PYR and BaP residues in actual samples.6.For stability studies,PYR standard solution could be stored at 4℃for at least one year.Under the accelerated condition of 37℃,the PBA-OVA coating antigen could be stored for 8 d,which was equivalent to 12 months at 4℃.4D6 antibody could be stably stored for more than 6 d,which was equivalent to 9 months stored at4℃.In this study,through the selection of hapten,the optimization of complete antigen synthesis,multiple cell fusion and clonal screening,a monoclonal antibody with high sensitivity and simultaneous recognition of PYR and BaP was obtained.Based on this antibody,an ic-ELISA method for the detection of PYR and BaP residue in fish,shrimp and crab was established.This method filled the gap in the immunoassay of PYR and BaP in aquatic products,laid a foundation for the development and application of PYR and BaP residue detection kits in aquatic products,and had high application value in guaranteeing quality.