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

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

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

Microarray Contact Printer

Tecan LS-400 4 laser

Array scanner

and Laser Scanner

Genomic Solutions OmniGrid Accent Pro

4/21

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

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

Fluorescent Immunoassay Schematic

Reporter antibody

Biotin

Molecular spacer

Capture antibody

Charged Glass or Polystyrene substrate

Streptavidin Bovine serum albumin

7/21

Fluorescent Immunoassay Schematic

Reporter antibody

Capture antibody

Polystyrene substrate

Bovine serum albumin

8/21

96, 13x8 Subarray Microarray

9/21

96, 13x8 Subarray Microarray (inset)

10/21

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)

(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

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'

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

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

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

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

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

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

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

Future Research

• Detection of more pathogens and

associated toxin

• Automation- Plate washers, robotic

manipulation

• Typing and subtyping of pathogens with

microarrays?

21/21