Development Of Rapid Immunoassays For Main Foodborne Pathogens In Raw Milk And Dairy Products

Pollution of foodborne pathogens in raw milk and during the process of dairy products has been one of the main threat to the safety of dairy products. The traditional culture based detection method was accurate but time-consuming, labor-intensive and inefficient. Therefore, an accurate, simple, fast analytical method with low cost was urged in the food industry and basic inspection organization to control the foodborne pathogens in the material, progress, and final products. In this study, we reviewed the popular pathogens in the raw milk and dairy products, and selected the Escherichia coli(E. coli O157: H7), five main enterotoxins of Staphylococcal aureus(S. aureus), Salmonella, and Listeria monocytogenes(L. monocytogenes) as our objects of study. Subsequently, we prepared effective immunogens against different pathogens according to their characteristic. After cell screening, the monoclonal antibodies(mAbs) with homogeneous cross reactivity and excellent specificity were obtained. Based on the paired antibodies, reliable, simple and fast immunoassays with low cost were developed for rapid detection of the main foodborne pathogens in raw milk samples.First, to establish the specific immunoassay against E. coli O157: H7, we prepared the boiled bacteria cells of E. coli O157: H7 as immunogen, and produced specific mAbs against the E. coli O157: H7 after cell fusion and selection. After pairwise study, paired mAbs were obtained. Based on the paired mAbs, sandwich ELISA and lateral flow assay of E. coli O157: H7 were established, the detection limit(P/N?2.1) was 1.1×105 CFU/mL and 3×105 CFU/m L, respectively. No cross reactivity with other tested bacteria was observed. The study with western blot revealed that the target antigen of the two paired mAbs was the membrane protein with molecular weight of 37 k Da.Secondly, to establish the specific immunoassay against five main enterotoxins of Staphylococcal aureus, we used the five recombinant Staphylococcal Enterotoxins(SEs) as immunogen and produced the specific mAbs. The optimal paired mAbs against the five SEs were obtained after pairwise study and further comparison. On this basis, specific ELISA and lateral flow assay were respectively developed against the five main SEs. The detection limit of the ELISA method against SEA, SEB, SEC, SED, SEE was 0.15, 0.08, 0.14, 0.12, and 0.25 ?g/kg, respectively. We further developed a multiplexed lateral flow assay that can simultaneously detect the five main SEs. These methods were applied to the detection of SEs in the milk sample.Thirdly, to develop specific immunoassay against the genus of salmonella, we first prepared mAbs against the flagella and lipopolysaccharide(LPS) of the Salmonella. typhimurium(S. typhimurium), and found that paired mAbs against LPS enable a detection of S. typhimurium with higher sensitivity. The detection limit of established ELISA and lateral flow assay was 104 CFU/m L and 1.3×105 CFU/mL, respectively. Cross reactivity with S. paratyphi B which shared the same O antigen was observed but not with the other salmonella strains and other tested bacteria. Meanwhile, we synthetized LPS and BSA conjugates as immunogen with Na IO4 and EDC method, and prepared mAbs against conserved structure of the LPS outer core. Based on the paired mAb that specific to the salmonella spp., ELISA method against salmonella was developed, the detection limit of twelve salmonella strains with different O antigen was 1.3×105- 1.2×106 CFU/m L. Based on the genus specific LPS mAb 5H12 and a heterogenous LPS-BSA conjugate synthetized by Na IO4 method, we developed a competitive lateral flow assay for Salmonella spp., the twelve tested salmonella strains were detected with a detection limit of 106 CFU/mL and without any cross reactivity against the other tested strains.Fourthly, to establish L. monocytogenes specific immunoassay, we first use recombinant P60 protein of L. monocytogenes as immunogen and found the prepared mAbs were homogeneously specific to the listeria spp. Based on the paired mAbs, ELISA and lateral flow assay of listeria spp. were developed, the detection limit against eight L. monocytogenes strains and four Listeria strains were 105-106 CFU/mL, and no cross reaction was observed with other tested strains. At the same time, we synthetized the peptide pepD of L. monocytogenes and conjugated it to BSA with a bifunctional coupling linker SMCC, to prepare the immunogen of L. monocytogenes. As a result, mAbs specific to L. monocytogenes were obtained and was found can be paired to the listeria specific mAbs against the P60 protein. The established ELISA and lateral flow assay successfully identified the eight L. monocytogenes strains from four listeria strains without any cross reactivity with other tested strains.Fifthly, to enhance the sensitivity of the classical sandwich ELISA method against SEB, we reduced Ag layer with certain thickness(6.6 nm) on the Au nano particles modified with 4-NTP, and synthetized the Au@Ag core-shell structure with embedded Raman label, and excellent SERS intensity. After labeled with SEB mAb, this material was successfully applied to the Raman sensing of SEB on the microplate, the detection limit of this method was 1.3 ng/kg, which was 25 times lower than the classical sandwich ELISA, and can be used for the detection of low level of SEB in milk sample.