Developing an immunogold nanoparticle-based lateral flow assay for the rapid detection of Bacillus cereus in milk

Since foodborne diseases and outbreaks increase considerably, food safety is always a widely held concern with respect to human health in the world. According to the suevey results in 2007 to 2012 from the Center for Disease Cotrol (CDC) of the Department of Health in Taiwan, Bacillus cereus was rated as a common pathoge in food poisoning, with approximately 17%patients were infected. Instead of conventional detecting methods for which require time, skilled technicians and special equipment, lateral flow assay (LFA) utilizing the specific binding between antigen and antibody is currently as an on-site detection tool applied in the realm of environmental, food and clinical analyses. Therefore, in this study, a rapid, simple, snesitive and specific LFA strip using polyclonal antibody we raised against 28.5-kDa cell surface protein of B. cereus and 30-nm immunogold nanoparticles had been successfully deveoped for the detection of B. cereus in milk. Whole assay could be completed within 20min. The visual detection limit (VDL) was determined as 10^3 CFU/mL with the dynamic range from 10^4 to 10^8 CFU/mL in both phosphate buffer (PB) system and milk samples, showing a high sensitivity to B.cereus. The specificity of LFA was also good, despite a little reactivity with Vibrio parahaemolyticus. The semi-quantitative results of the strip assay were in a good agreement with those of conventional spread plate method. In the precision analysis, the coefficients of variation (CV) of intra- and inter-assay were 9.6% and 12.8%, indicating hogh result reproducibility. Hence, the polyclonal antibody-based LFA platform we developed has a great potential as a user-friendly, rapid and sensitive on-site tool for detecting B. cereus in milk. within 20min. The visual detection limit (VDL) was determined as 10^3 CFU/mL with the dynamic range from 10^4 to 10^8 CFU/mL in both phosphate buffer (PB) system and milk samples, showing a high sensitivity to B.cereus. The specificity of LFA was also good, despite a little reactivity with Vibrio parahaemolyticus. The semi-quantitative results of the strip assay were in a good agreement with those of conventional spread plate method. In the precision analysis, the coefficients of variation (CV) of intra- and inter-assay were 9.6% and 12.8%, indicating hogh result reproducibility. Hence, the polyclonal antibody-based LFA platform we developed has a great potential as a user-friendly, rapid and sensitive on-site tool for detecting B. cereus in milk.CHAPTER ONE: LITERATURE REVIEW……………………………1 1. Foodborne diseases and reported outbreaks in Taiwan…………… 1 2. Bacillus cereus …………………………………………………… 2 2.1 Bacterial morphology and colony morphology………………… 2 2.2 Pathogenesis and characterization of two types of B. cereus foodborne diseases…… 3 2.2.1 Diarrheal illness………………………………………………… 4 2.2.2 Emetic illness…………………………………………………… 5 2.3 Presence of B. cereus in different types of foods…………………… 6 2.4 Current food legislations and commercial kits for Bacillus cereus… 8 3. Cell surface proteins and their applications as target antigens for immunoassays………12 3.1 Function of cell surface proteins……………………………………… 13 3.2 Applications of using cell surface proteins as target antigens for immunoassays…………… 13 4. Lateral flow assay (LFA)…………………………………………… 14 4.1 Theory of LFA……………………………………………………15 4.2 The components of a LFA and their functions…………………… 15 4.3 Different formats of LFA………………………………………… 17 4.3.1 Antibody-based LFA…………………………………………… 17 4.3.2 DNA-based LFA………………………………………………… 18 5. Gold nanoparticles (AuNPs)………………………………………… 19 5.1 Characteristics of AuNPs………………………………………… 20 5.2 Production of AuNPs………………………………………………20 5.2.1 Preparation methods…………………………………………… 21 5.2.2 Characterization methods……………………………………… 22 5.2.3 Various attachment methods for ligand molecules to the surface of AuNPs…………………23 5.3 Applications of AuNPs in antibody-based LFAs…………………23 CHAPTER TWO: MATERIALS AND METHODS………………… 28 1. The objective of this study………………………………………… 28 2. Research design…………………………………………………… 29 3. Materials……………………………………………………………30 4. Instruments…………………………………………………………31 5. Methods……………………………………………………………32 5.1 Production of monoclonal (mAb) and polyclonal antibody (pAb)……32 5.1.1 Preparation of the immunogen……………………………………32 5.1.2 Characterization of the immunogen using SDS-PAGE……………32 5.1.3 Immunization……………………………………………………33 5.1.4 Determine the titer of antiserum using ELISA……………………33 5.1.5 Production of mAb………………………………………………34 5.1.5.1 Hybridoma production for mAb………………………………34 5.1.5.2 Antibody screening……………………………………………34 5.1.6 Production of pAb………………………………………………35 5.1.6.1 Purification of pAb…………………………………………35 5.1.7 Determine Ab reactivity using western blot assay………………35 5.1.8 Determine cross-reactivity of Ab using ELISA…………………36 5.2 Preparation of immunogold nanoparticles…………………………36 5.2.1 Preparation of AuNPs……………………………………………36 5.2.2 Conjugation of AuNPs with Ab…………………………………36 5.2.2.1 Optimization of K2CO3 concentration…………………………36 5.2.2.2 Optimization of Ab concentration………………………………37 5.2.2.3 Preparation of immunogold nanoparticles…………………… 37 5.2.3 Characterization of immunogold nanoparticles…………………… 37 5.3 Preparation of the lateral flow strips………………………………… 37 5.4 Procedures of lateral flow assay (LFA)……………………………… 38 5.5 Optimization of LFA………………………………………………… 38 5.6 Determine the assay sensitivity of the developed LFA………………38 5.7 Determine the specificity of the developed LFA………………………38 5.8 Precision assay………………………………………………………39 5.8.1 Intra-assay……………………………………………………… 39 5.8.2 Inter-assay………………………………………………………… 39 5.9 Matrix effect analyses of milk samples……………………………… 39 CHAPTER THREE: RESULTS AND DISCUSSION…………………… 40 1. Characterization of 26.5- and 28.5-kDa cell surface proteins from B. cereus BCRC 10603……………41 2. Monoclonal antibody (mAb)………………………………………41 2.1 Titer………..……………………………………………………41 2.2 Hybridoma screening…………………………………………… 42 3. Polyclonal antibody (pAb)………………………………………43 3.1 Titer………..……………………………………………………43 3.2 Reactivity………………………………………………………43 3.3 Specificity………..……………………………………………44 4. Different sized AuNPs……………………………………………45 5. Immunogold nanoparticles………………………………………45 5.1 Optimal K2CO3 and antibody concentration……………………45 5.2 Characterization of immunogold nanoparticles………………… 46 6. LFA development…………………………………………………47 6.1 Optimal coating buffer………………………………………47 6.2 Optimal blocking buffer………………………………………48 6.3 Optimal washing buffer…………………………………………48 6.4 Optimal membrane………………………………………………49 6.5 Optimal test-line antibody density………………………………50 6.6 Optimal incubation time………………………………………50 6.7 Optimal sized immunogold nanoparticles……………51 6.7.1 Optimal volume ratio of sample to immunogold nanoparticles…51 6.7.2 Optimal OD520 value of immunogold nanoparticles…….……52 7. Assay performance………………………………………………52 7.1 Sensitivity of LFA………………………………………………52 7.1.1 Dose curve in PB system……………………………………53 7.1.2 Dose curve in skimmed milk…………………………………53 7.2 Specificity of LFA………………………………………………54 7.3 Matrix effects of milk samples………………………………… 55 7.4 Precision…………………………………………………………55 7.5 Cost comparisons with ELISA and PCR………………………56 CHAPTER FOUR: CONCLUSION…………………………………57 CHAPTER FIVE: FUTURE WORK…………………………………57 CHAPTER SIX: REFERENCES…………………..…………………8