Immunomicroarray for multipathogens

Transcription

1 Funding period: Jan 3-Feb ultipathogen screening and /or confirmation via microarray detection Arun Bhunia, ark organ, Shu-I Tu Center for Food Safety and Engineering, Purdue University ERRC, Wyndmoor, PA Primary objective to develop microarray based platforms to detect three major pathogens (Listeria,, E. coli) simultaneously Focus. Nucleic acid based platform Applegate (PI). - based platform Bhunia (PI) Team members: BK Hahm (PD), Priya Banada (PD), Viswaprakash Nanduri (PD), Tao Geng (PD), Amanda Lathrop (GS), Hyochin Kim (GS), Andrew Gehring (USDA- ARS, ERRC) CFSE Annual Review Oct -3, Immunomicroarray for multipathogens Essentials Specific antibodies and immune reaction (Objective & b) Platforms: Fiber optic and 9-well/slide (Objective ) Sample preparation strategy- (Objective 3) Selective growth media (Objective ) Objective : and antibody reaction a. Development: Genomic and proteomic approaches in antibody development for Listeria, E. coli and. b. Antigen Expression: Effect of stress responses (media and environments) on antigen expression and immunoassays 3 Objective a: Genomic and proteomic approaches in antibody development for Listeria, E. coli and Search for species-specific surface exposed amino acid sequence Determine hydropathy profiles, surface localization, and antigenic properties Synthesize candidate peptide (5- amino acid) antigen Immunize rabbits and test for antibody production Strategies 5 -TCC-CGT-ACT-GAC-ATT-CTC-3 N - S - R - T - D - I - L -C N C 5 E. coli-specific antibodies Anti-E. coli O57:H7 F Ab ST39 ST33 ST33 ST33 Anti- Anti- CSA- Target antigen Anti-E. coli O57:H7 LPS Anti-E. coli Whole cells Intimin -specific antibodies LPS 3 kda 3 kda 5 kda 5 kda ultiple proteins ultiple proteins List specificity E. coli O57:H7 E. coli E. coli O57:H7 Sal. Enteritidis Sal. Enteritidis Listeria specific antibodies CE9 Ab E-7G Ab E-E Ab P Ab-E Anti-Listeria Lm Lm7 LmC39 Target antigen kda kda 97 and 3 kda kda kda,, 5, 5, 3 and 3 kda 3 kda InlB 5 kda 9 kda ActA specificity, L. innocua

2 Objective b: Effect of stress responses on antigen expression and immunoassays otivation: As biosensors are becoming increasingly sensitive, a slight reductions in immune reactions due to cellular or environmental factors will result in false results Effect of selective enrichment media on InlB and ActA expression for specific antibodies V7 (/a) Scott A (b) urrayb (ab) ATCC 9 (a) SLCC 79 (3c) ATCC 7 (/c) F33 (/b) ATCC 5 (3b) ATCC 97 (d) ATCC 9 (e) ATCC 9 (c) SLCC (7) SLCC 373 (3a) V7 (/a) Scott A (b) urrayb (ab) ATCC 9 (a) SLCC 79 (3c) ATCC 7 (/c) F33 (/b) ATCC 5 (3b) ATCC 97 (d) ATCC 9 (e) ATCC 9 (c) SLCC (7) SLCC 373 (3a) Effect of selective enrichment broths on InlB, ActA and several unknown antigens in L. Effect of environmental stresses on antibody based detection of E. coli O57:H7, S. enterica and L. 7 No expression observed anti-inlb (Lm) Lathrop et al (Submitted) anti-acta (LmC39) RT-PCR ELISA OD9 OD9 F (b) F-ActA.5.5 F-InlB.5.5 acta inlb S No temp ctrl ~9 bp OD9 OD9 F33 (/b).5 F33-ActA.5 F33-InlB.5.5 acta inlb S 9 Transmission electron microscopic (TE) analysis of InlB and ActA expression in different media Lm LmC39 (a) InlB (c) ActA (b) InlB (d) ActA InlB ActA Effect of enrichment broth on antigen expression for a Listeria specific Protein Identity Calculated Gene name molecular mass Putative LPXTG-motif cell wall 5 kda anchor domain protein Lmo,. kda 5 kda Flavocytochrome C Fumarate Reductase chain A homolog Lmo 355 5,. kda eno 5 kda Enolase,7. kda (Lmo 55) 5 kda Glyceraldehyde 3-phosphate gap 3,. kda dehydrogenase (Lmo 59) 35 kda Putative phosphosugar binding protein Hypothetical regulator Lmo 3,3.7 kda Effect of environmental stresses on antibody based detection of E. coli O57:H7, S. enterica and L. Stress conditions investigated Heat 5 o C; Cold ( o C); Acid (ph 5.); Salt (NaCl- 5.5%), oxidative stress (H O ;5 m, ethanol (5%) Duration Short-term (3h) Long term (> h) Geng et al.. J. Food Prot. 9(): 79- Hahm and Bhunia.. J. Appl. icrobiol. :7-7 Lm

3 Western blot analysis of antigen expression with long-term combined stress kda 3 kda 3 kda 5 kda Sal. Enteritidis (our lab) 37 C, no stress C, ph 5.5, C, ph 5.5, h kda kda 5 kda 5 kda 3 kda 3 kda L. (our lab) 37 C, no stress C, ph 5.5, C, ph 5.5, h ELISA9+SD Examination of antibody reactions with bacteria grown in hotdogs in the presence of stress E.coliO 57:H7 ELISA analysis Sal.C SA- 37`C,no stress Sal.Enteritidis (ourlab) `C,pH 5.5,NaCl3.5% L. (ourlab) kda 3 kda 3 kda 5 kda Western blot analysis E. coli O57:H 7 Sal. Sal. CSA- Enteriti dis (our lab) kda kda 5 kda 5 kda 3 kda 3 kda L. (our lab) 3 Summary: ELISA response to environmental stress E. coli O57:H7 Sal. CSA- Sal. Enteritidis L. ELISA response Stress (Compared to control -37C) C % 5 C % 5.5% NaCl 5% C up to h 3% (ph 5.5, ) C up to h 33% C 3% (ph 5.5, ) (ph 5.5, ) (ph 5.5, ) C or C up to h 3-% C % 5 C 37% 5.5% NaCl 3% 5 m HO % C up to h 3% ph 5.5 % 5.5% NaCl % 5% ethanol 53% 5 C 53% C up to h % C or C up to h -3% 5 Objective : Fiber optic sensor for food pathogens Listeria E. coli O57:H7 Enteritidis Fiber optic sensor for pathogen detection Detector FO based detection of L. from inoculated meat samples after h-enrichment F Fluorescent molecules Laser detector Fiber laser Evanescent wave Signal (pa 5 5 Bologna Hotdog () () (3) 5 top middle bottom ^ inoculum ^ inoculum ^3 inoculum neg control pos control 7 Geng et al. Appl. Environ. icrobiol. 7:3-3

4 L. detection using RAPTOR Detection of cells with fiber optic sensor grown in or EC at h intervals Continous detection of E. coli O57:H7 grown in, EC or GB at h interval Signal,pA L (.mg/ml) L (.mg/ml) L P (.mg/ml) L P (.mg/ml) coupon RAPTOR SIGNAL,pA L. devoid 35 E. fecalis 35 L. rhamnosus E+ - - Time (hours) Listeria,cfu/ml CONCENTRATION OF BACTERIA, cfu/ml PBS Hotdog Geng et al. Sensors : Nanduri et al. Sensors :- Signals (pa) Signals from E. coli grown in.e+ Signals from E. coli grown in EC.E+9 35 Signals from E. coli grown in GB w/ EC Concentration of E. coli grown in.e+ 3 Concentration of E. coli grown in EC Concentration of E. coli grown in GB w/ EC.E+7 5.E+.E+5.E+ 5.E+3.E+ 5.E+ E. coli concentration (CFU/ml) Summary: Fiber optic sensor for food pathogens Objective 3. Sample preparation strategy for use with microarray platforms Listeria E. coli O57: H7 SLT Detection limit Detection time 3 cfu/ml 3 h - 5 cfu/ml meat 3 cfu/ml meat.5 ug/ml cfu/ml - h?? Publications Geng et al.. AE. 7: 3 Nanduri et al. Sensors, : Kim et al.. Key Eng. aterials Geng et al.. Sensors, :79 Tu et al.. SPIE organ et al.. Key Eng. aterials enrichment detection device () Self-contained, easyto-use device to deliver particle free and ph adjusted bacterial suspension Performance evaluation of with food sample Growth of pathogens spiked onto food sample in Food sample edia Growth Detection E. coli O57:H7 L. E. coli O57:H7 Ground beef mec + novobiocin CT-SAC Typhimurium Egg RV XLD Listeria Hotdog OX CFU/ml CFU/ml CFU/ml Bag CFU/ml Bag CFU/ml Bag CFU/ml 3

5 Lateral flow immunoassay of L. retrieved from CFU/g E. coli O57:H7 CFU/g Typhimurium CFU/g h incubation h incubation 5 h incubation After Before PCR tube PCR analysis E. coli O57:H7 culture before or after passing through the cartridge h h h after before after before after before : bp DNA ladder,,, : CFU/ml of inoculation PCR mix 7 eaea stx stx Objective : Formulation of selective universal enrichment broth for, E. coli and Listeria (SEL) edia is called SEL, E. coli and Listeria Base Buffered Listeria Enrichment Broth () with acriflavin, cyclohexamide, fosfomycin, nalidixic acid) edia performance will be addressed in our next project. Hyochin Kim ultipathogen Screening Fiber optic Sensor Test sample Enrichment in using universal selective enrichment broth (eg., SEL media) icroarray ELISA 9 Need work! Conclusions Specific antibodies are available for immunomicroarray assay Stress and media affect antibody reaction either by affecting transcription, translation or translocation Data with physiologically stressed bacterial cells would be helpful in formulating assay procedure for optimum signal Fiber optic sensor data are promising and multiplex assay could be developed using the automated system- RAPTOR system removes particles and inhibitors from food and should be useful for detection by biosensor platforms 3 5