Preparation Of POMs-based Fluorescent Biological Probe And Its Application In LM Immunoassay

Malignant events of global food safety occurred frequently in recent years,thus the problem of food safety has become one of the public health issues of common concern around the world.The unqualified food is not only a threat to the consumer's health and safety,also seriously affected and restricted the economic development of the region.According to the world health organization?WHO?report,pathogenic microorganisms contamination is one of the most important food safety risk factors that cause foodborne disease.Establishment and improvement of the detection system of foodborne pathogens and diseases,preventing and controlling the spread of the foodborne disease has attached great importance by the department of health and food safety throughout the world.Listeria monocytogenes?L.monocytogenes,LM?is an important foodborne and zoonotic pathogen which could widely found in in meat,eggs,poultry,seafood,dairy products,vegetables and refrigerated Food medium.The infectious diseases caused by LM can lead to human and animal encephalitis,meningitis,sepsis and pregnant women abortion premature,abortion,and stillbirth with high the mortality rate.Since the disease poses a serious threat to animal husbandry and human health,LM has been listed as one of the main surveillance bacteria for foodborne pathogens according to WHO standard.Therefore,there is urgent need to establish a rapid and accurate LM detection technology for food safety control and people's health and social stability improvement.In our strategy,a novel POM-based microsphere was designed and synthesized.Then monoclonal antibody?mAbs?against LM was coupled with the fluorescent microspheres to form the fluorescent bioprobe.Based on the principle of double antibody sandwich fluorescence immunoassay,a rapid,accurate,sensitive and specific method for detection LM had been established via flow cytometry.This study includes the following three parts:Part?.Preparation and identification of polyclonal antibody and monoclonal antibody against LM.?1?Preparation and identification of polyclonal antibodies: First,vaccine against the whole bacteria of LM was prepared by Freund's complete and incomplete adjuvant.New Zealand white rabbits were immunized with multi-subcultaneous injections of vaccine.After the immunization,serum was collected for antibody purification by octanoic acid-saturated ammonium sulfate method and Protein G affinity chromatography.The content and purity of antibody was determined by BCA protein quantitative kit and SDS-PAGE gel electrophoresis.The indirect ELISA method was used to evaluate the titer and specificity of the antibody.The purified polyclonal antibody IgG of rabbit anti-LM was biotinylated using biotin labeling kit for further use.The results showed the prepared polyclonal IgG with high purity and specificity exhibited two clear bands on SDS-PAGE gel.Protein content was 10.65 mg · L^-1 and the titer were up to 1: 25600.The labeled ratio of IgG to biotin was 3.63.?2?Preparation and identification of monoclonal antibody: BALB/c mice were immunized with whole LM bacteria.The spleen cells from immured mouse were fused with SP2/0 cells by using hybridoma cell technology.Positive hybridoma cells were screened by indirect ELISA.The hybridoma cells which secreted stable monoclonal antibodies against LM antigen have been obtained by limited dilution method.The screened hybridoma cells were expended cultured to prepare mouse monoclonal antibody ascites.The titer and specificity of monoclonal antibody in ascites were determined by indirect ELISA.Antibody subtypes were identified using the monoclonal antibody subtype identification kit.Protein was purified by HiTrap IgM HP affinity chromatography combined ammonium sulfate precipitation method and characterized by SDS-PAGE gel electrophoresis.The results showed that hybridoma cell line 5-B3 which could secrete LM monoclonal antibody was screened out by subcloning.The titer of the ascites antibody was higher than 1: 12800 as an identified subtype IgM.After protein concentration,the purity of the antibody with good specificity was up to 1.748 mg · L^-1.Part ?.Synthesis of polyoxometalate?POM?-decorated fluorescent microspheres and bioprobe for Listeria monocytogenes.?1?Preparation and characterization of polyoxometalate-decorated fluorescent microspheres: the polystyrene?PS?microspheres were used as the matrix,which contain rich negatively charged on the surface.PEI and PAA were used alternately coated onto the PS surface to form six positive and/or negative functional layers through layer-by-layer electrostatic self-assembly techniques.On the other hand,the fluorescent dye([N?C₄H₉?₄]₂[V₆O₁₃{?OCH₂?₃CNH-CH₂-C₁₆H₉}₂],POM-FD)was synthesized by ourselves from polyoxometalate anions,which successful applied to coating on the PS surface.Additional,the surface of microspheres was further fabricated with carboxyl groups by polyelectrolyte method.The as-prepared fluorescent microspheres were characterized by Zeta potential,fluorescence spectroscopy,laser scanning confocal microscopy,and flow cytometry?FCM?.As shown in the results,the surface of PS was decorated by POM-FD successfully.And the POM-FD@PS possess superior properties of high fluorescence intensity and good photo-stability.?2?Preparation and characterization of bioprobe based on POM: the fluorescent microspheres POM-FD@PS and the as-prepared mAbs against Listeria monocytogenes were coupled via the amide reaction between the carboxyl groups of POM-FD@PS and amino groups of m Abs,which using EDC/NHS crosslinking.The fluorescent bioprobe based on POM for Listeria monocytogenes was well-prepared through optimized mAbs amount.Based on the flow cytometry assays,the as-prepared fluorescent bioprobe was used initially to detection of Listeria monocytogenes.The results showed that the fluorescent bioprobe was excellent prepared,and the best value of coupling ratio of microspheres to mAbs was 105 : 56 ?g.Furthermore,the bioprobe exhibited high-performance to detection of Listeria monocytogenes by using liquid-array system.Part ?.Establishment and evaluation of the method for detection of Listeria monocytogenes based on liquid-array system.?1?Establishment and evaluation of fluorescent microsphere-based suspension immunoassay: Establishment of suspension immunoassay for detection of Listeria monocytogenes using the as-prepared fluorescent bioprobe,which based on the double antibody sandwich fluorescence immunoassay.The experimental conditions of the assay were further optimized,which the data were collected by flow cytometry.The results of the fluorescent microsphere-based suspension immunoassay showed that the optimum concentration of coupling mAbs was 28 ?g/m L,the best working concentration of biotin-labeling pAbs and SAPE were 250 ?g/mL and 15 ?g/mL,respectively.?2?Evaluation of fluorescent microsphere-based suspension immunoassay: the validity of the assay was also investigated by determining the limit of detection?LOD?,specificity and reproducibility.To comprehensively evaluate the validity of the assay,the assay was further compared to the Luminex-based suspension immunoassay based on the commercial PS fluorescent microspheres and conventional culture method.The results the PS fluorescent microsphere-based suspension immunoassay showed that the optimum concentration of coupling m Abs was 15 ?g/m L,the best working concentration of biotin-labeling pAbs and SAPE were 250 ?g/m L and 8 ?g/m L,respectively.The LOD of the method was 1.0 * 106 CFU/mL with high specificity and reproducibility.However,the LOD of the fluorescent microsphere-based suspension immunoassay was lower than that of commercial PS fluorescent microspheres-based suspension immunoassay with more stability.To compare the test result difference between the above two methods,48 model samples were selected to pre-enrichment 72 h.Each sample was tested in triplicate by fluorescent microsphere-based suspension immunoassay,Luminex-based suspension immunoassay and conventional culture method.As exhibited in the results,the LOD of the both suspension immunoassay was 6 CFU/g with a coincidence rate of 100%.Based on these results,both accuracy of suspension immunoassay were pretty good.Nevertheless,suspension immunoassay consumed much less time of detection process,and had the advantage of operation without bacteria,security and liability.