Preparation Of Anti-cronobacter Monoclonal Antibody And Large-volume Magnetic Separation For Detection Of Listeria Monocytogenes

Traditional methods for the detection of foodborne pathogen usually takes 6~7d.Such a long detection period cannot meet the requirements of enterprises and relevant departments in routine monitoring of product quality.Therefore,the development of a rapid,sensitive and simple detection method is of great significance for the prevention of foodborne diseases.In this study,Cronobacter spp.and Listeria monocytogenes have been chosen as target bacteria,and the detailed research contents are as follows:First,anti-Cronobacter monoclonal antibodies(mAbs)have been prepared.The cell debris of Cronobacter spp.was used as immune-antigen.After four rounds of immunization,two rounds of subcloning,and screening process,one hybridoma cell line(5-H6)which can produce anti-Cronobacter spp.mAbs was obtained successfully.The results of dot-blot indicated that anti-Cronobacter spp.mAbs can bind with three Cronobacter species named Cronobacter muytjensii,Cronobacter turicensis,and Cronobacter dublinensis,respectively.The mAbs also show the certain cross-reaction with Staphylococcus aureus,and the slight cross-reaction with Micrococcus luteus and Bacillus subtilis.The titer of mAbs(1 mg/mL)against C.muytjensii ATCC51329,C.muytjensii SR1074B,C.turicensis CICC24178 and C.dublinensis CICC24179 was 1:32000,1:32000,1:16000,and 1:16000,respectively.The result of western-blot showed that antigenic substance recognized by mAbs is lipopolysaccharide of Cronobacter spp..Secondly,a two-step large-volume(10 mL)magnetic separation system was developed for Listeria monocytogenes detection,in which the mAb consumption is14-folds lower than that of traditional immune-magnetic separation(IMS)method.Under optimal conditions,the amounts of biotin-mAbs and magnetic beads(MBs)were 5?g and 700?g,respectively.The immunoreation time between biotin labeled mAbs and Listeria monocytogenes was 60 min,the capture time between streptavidin labeled MBs and biotin-mAbs bound Listeria monocytogenes was 30 min,while the magnetic separation time was 3 min.Combined with a PCR assay,the detection limit of the proposed method for Listeria monocytogenes was 8×10~0 CFU/mL in pure culture and 8×10~1 CFU/mL in pasteurized milk.The proposed method also showed a high specificity for Listeria monocytogenes with a negligible cross-reaction with other bacteria.The overall detection time including two-step magnetic separation and PCR assay took less than 7 h.In 50 m L magnetic separation system,the various parameters that could influence the separation efficiency were also optimized.The results showed that the amounts of SA-MB were highly achieved at 3.5 mg per test.Therefore,how to further reduce SA-MB consumption in 50 mL magnetic separation system is still explored in the future research work.