Preparation Of Histamine Monoclonal Antibody And Establishment Of Immunological Detection Method

Histamine is a biogenic amine associated with allergies and food poisoning.It is an important indicator of food freshness and quality.It has also been reported in recent years that antibiotics produced by fermentation cause a series of medical accidents due to histamine contamination.In order to efficiently and rapidly detect histamine,developed two immunoassays and compared with conventional high performance liquid chromatography(HPLC)analysis.In this study,histamine standard was used as a hapten,and histamine was coupled to carrier protein(BSA,OVA)by MBS,according to the UV spectrophotometer,the results of UV scanning were obtained and the artificial antigen was successfully synthesized.After immunizing BALB/c mice with histamine-BSA,successful screening of two specific anti-histamine hybridoma cell lines:1G3 and 2H1.The resulting histamine antibody of 2H1 was IgG1 subtype of the κ light chain.Two immunoassay methods were established.The linear regression equation of ELISA was Y=1.59-354X,R2=0.987,the lowest detection limit(IC10)was 88.84 ng/mL,and the detection range was 170.2 ng/mL-8.4 μg/mL.The linear regression equation of CLEIA was Y=1.525-335X,R2=0.991,IC10 was 73.24 ng/mL,and the detection range was 145.6 ng/mL-9.0 μg/mL.In the cross-reaction assay,compounds with similar structures to histamine(L-tryptophan,phenethylamine,L-histidine,tryptamine)did not cross-react,indicating that the prepared anti-histamine monoclonal antibody has good specificity for histamine.Histamine was added to foods(fish,shrimp,soy sauce and fish balls)and medicines(gentamicin,amikacin,penicillin injection,fish peptone and fermentation broth).The results showed that the histamine recovery rates of ELISA,CLEIA,HPLC were 73.39%-131.3%,77.06%-119.54%,73.2%-129.44%,respectively.Among them,the recovery rate of CLEIA is better,indicating that the accuracy of CLEIA detection method is higher.The samples(fish,shrimp,scallops,cooking wine)were used to monitor histamine changes at room temperature for 36 h by three detection methods(ELISA,CLELA and HPLC).The results showed that compared with HPLC,ELISA and CLEIA have lower detection limits,lower cost,easier operation,and HPLC needs to derivatize the sample,which is not suitable for large-scale sample detection.