optimized, high-throughput antibody microarray of …microarray detection of heat-killed e. coli...
TRANSCRIPT
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Optimized, High-Throughput Antibody
Microarray of Pathogens
Andrew G. Gehring, Jeffrey D. Brewster,
Yiping He, Peter L. Irwin, George C. Paoli, Tawana Simons, Shu-I Tu, and Joseph Uknalis
United States Department of Agriculture
Agricultural Research Service
Eastern Regional Research Service
600 East Mermaid Lane
Wyndmoor, PA 19038
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OUTLINE
• Microarrays- Historical (Gene expression on glass slides)
• Research Objective- optimize high-throughput multiplexed detection of pathogens with antibody microarray (limit of detection; speed; reagents)
• Results- Fluorescent Sandwich Immunoassay Microarray
• Conclusions- LOD ~2e5 cells/mL [2e6 cell/mL]; ~80 min [~2.5 hr]
• Future- Typing and subtyping of pathogens with microarrays?
2/21
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Microarrays
• Traditionally- Gene Expression (also sequence & gene mutation)
• Substrates (glass, nitrocellulose, plastic, etc.)
• Microarray printers (contact vs. non-contact)
• Printed recognition elements (probes/targets or features); Conjugation chemistry
• Reporters/Labels- fluorescent targets/probes (also precipitating/colorimetric)
• Detection (typically fluorescence-laser scanning)
• Software based microarray analysis
3/21
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Microarray Contact Printer
Tecan LS-400 4 laser
Array scanner
and Laser Scanner
Genomic Solutions OmniGrid Accent Pro
4/21
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Research Objective:
Develop detection microarrays for the high-
throughput screening of pathogens in foods.
• Develop antibody based or protein microarrays for multiplexed detection of live, pathogenic bacteria, toxins, structural protein, metabolites, etc.
• Antibody microarray- fluorescent sandwich immunoassay for E. coli O157 and STX-1
(Specifically)
5/21
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Antibody Microarray Approach
• Passively adsorb capture antibodies on
polystyrene surface
• Reporter (fluorescent) antibodies reacted after
capture of analyte (“sandwich immunoassay”)
• Potential to screen for many (tens - hundreds)
pathogens at once
• Rapid analytical time (< 90 min)
6/21
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Fluorescent Immunoassay Schematic
Reporter antibody
Biotin
Molecular spacer
Capture antibody
Charged Glass or Polystyrene substrate
Streptavidin Bovine serum albumin
7/21
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Fluorescent Immunoassay Schematic
Reporter antibody
Capture antibody
Polystyrene substrate
Bovine serum albumin
8/21
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96, 13x8 Subarray Microarray
9/21
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96, 13x8 Subarray Microarray (inset)
10/21
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11/21
Multiplex detection of E. coli O157:H7
(in the presence of 108 cells/mL Salmonella
typhimurium, and 100 mg/mL Chicken IgG)
-5000
5000
15000
25000
35000
45000
1 2 3 4 5 6 7 8 9
log Escherichia coli O157:H7 concentration (cells/mL)
Re
sp
on
se
(A
FU
s)
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(200 mm diameter spots of biotinylated capture antibody,
exposed to 108 cells/mL heat-killed bacteria)
0
10000
20000
30000
40000
50000
60000
70000
2 10 60
Bacterial capture time (min)
Flu
ore
scen
ce i
nte
nsit
y (
AF
U)
Time Course of Bacterial Capture
12/21
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Microarray Detection of Live E. coli O157:H7
(Variable reaction conditions)
13/21
0
100
200
300
400
500
600
700
800
1 E+04 1 E+05 1 E+06 1 E+07 1 E+08 1 E+09
5'5'
60'5'
5'60'
60'60'
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Microarray Detection of E. coli O157:H7
(Variable conjugate reaction times)
14/21
y = 0.16x0.32
r2 = 0.89
y = 0.052x0.42
r2 = 0.95
y = 0.078x0.39
r2 = 0.94
0
20
40
60
80
100
120
140
1 E+04 1 E+05 1 E+06 1 E+07 1 E+08 1 E+09
Bacterial conc. (cells/mL)
Re
sp
on
se
(A
FU
)
60 min
3 min
30 min
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Microarray Detection of Heat-killed E. coli O157:H7
(Variable reaction conditions)
15/21
0
100
200
300
400
500
1 E+03 1 E+04 1 E+05 1 E+06 1 E+07 1 E+08 1 E+09
Bacterial conc. (cells/mL)
Response (
AF
U)
Static
Aspirated/Dispensed
Centrifuged
Shaken
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Microarray Detection of Live E. coli O157:H7
(Variable reaction conditions)
16/21
0
1,000
2,000
3,000
4,000
5,000
1 E+04 1 E+05 1 E+06 1 E+07 1 E+08 1 E+09
Bacterial cell conc. (cells/mL)
Re
sp
on
se
(A
FU
) 4x centrifuged
static
aspirate/dispensed
shaken
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Microarray Detection of E. coli O157:H7
(Variable centrifugation times)
17/21
0
1,000
2,000
3,000
4,000
1 E+04 1 E+05 1 E+06 1 E+07 1 E+08 1 E+09
Bacterial conc. (cells/mL)
Re
sp
on
se
(A
FU
) 4x
3x
2x
1x
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Microarray Detection of STX-1
(Variable reaction conditions)
18/21
y = 2.29x0.39
r2 = 0.94
0
20
40
60
80
1 E+00 1 E+01 1 E+02 1 E+03 1 E+04
Toxin conc. (ng/mL)
Resp
on
se (
AF
U)
Static
Shaking
Aspirate-Dispense
Centrifugation
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1.0E+00
1.0E+01
1.0E+02
1.0E+03
1.0E+04
1.0E+05
1.0E+06
1.0E+07
1.0E+08
1.0E+09
1.0E+10
E. coli O157:H7 S. typhimurium L. monocytogenes Y. enterocolitica O:8
Lo
g c
ell c
on
ce
ntr
atio
n (
CF
U/m
L).
Mixed Culture in Ground Pork
(30ºC, 24 h enrichment)- BLEB*
Broth + Oxyrase + 2% Casamino acids + Oxyrase & 2% Casamino acids
*very similar results with TSB or UPB 19/21
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Conclusions
• Multiplex detection of E. coli O157:H7
cells and STX-1 toxin in multiwell plate
format
• Total assay time (per 96 samples)-
< 90 min (formerly ~2.5 hr)
• Limit of detection for bacteria- ~2e5
cells/mL (formerly ~2e6 cells/mL)
20/21
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Future Research
• Detection of more pathogens and
associated toxin
• Automation- Plate washers, robotic
manipulation
• Typing and subtyping of pathogens with
microarrays?
21/21