ECL Seminars: Detecting Environmental Chemicals Using ......Detecting environmental chemicals using...
Transcript of ECL Seminars: Detecting Environmental Chemicals Using ......Detecting environmental chemicals using...
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Detecting environmental chemicals using immunoassay
technology
Shirley J. Gee, Candace Bever, Bruce D. HammockDepartment of Entomology
University of California DavisDavis, CA 95616
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Immunoassay: a complementary technology
ANALYTICAL CHEMIST
Mass spectrometry
Gas chromatography
Capillaryelectrophoresis
Liquid chromatography
Infrared spectrometry
Radioisotope detection
Ultraviolet/visible spectrometry
Immunoassay
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Immunoassay
• Sensitive
• Selective
• Rapid sample preparation
• Cost-effective
• Simple to use
• Adaptable to various formats
• Some targets are difficult
• Cross reactivity/Interferences
• Reagent availability
• New technology
• Difficult to apply to multianalyteanalysis
Advantages Disadvantages
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Sandwich immunoassay for protein analysis
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Antibody binding to a small molecule
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Small molecule immunoassay
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Analyte
Coating antigen
IgG
IgG-HRP
Enzyme tracer
Substrate
Product
B. Indirect Competitive ELISA
A. Direct Competitive ELISA
Immunoassay formats for detection of small molecules
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• Reasonable signal:noise ratio
• High maximum absorbance
• Low background absorbance
• High sensitivity
• low limit of detection
• steep slope (-0.75 to -1)
• Useful range of quantitation
• number of points – well
distributed2,4,5-TCP, nM
Abso
raba
nce
10 -3 10 -2 10 -1 10 0 10 1 10 2 10 30.0
0.2
0.4
0.6
0.8
1.0
1.2
Calibration curve for competitive immunoassay
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Murine Characteristics Camelids
140-150 kDa size 15-20 kDa
tetramer structure monomer
mammalian –doubles every day
culture bacterial –doubles every 20
mins
mgs in weeks yield mgs in a day
chemically labeling chemically or genetically
Antibody differences
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immunization Collect B-cells Isolate mRNA
cDNAlibrary
Digest to produce VHH insertLigation of VHH into pComb3X
amplification
Nanobodylibrary
construction
YY YY
Y
Y
Y Y
Y
Y
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Library screening
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Nanobodies developed or under development to date in the Hammock
Laboratory
• BDE-47
• Tetrabromobisphenol A
• Bisphenol A
• Triclocarban
• Paraquat
• Chlorantraniliprole
• 3-Phenoxybenzoic acid
• Aflatoxins
• Microsomal and soluble epoxide hydrolase
N N CH3H3C+ +
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Nanobody production to a target analyte
O
Br
Br
Br
Br
O OH
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0
20
40
60
80
100
0.001 0.1 10 1000
Res
pons
e (%
)
BDE-47 (ug/L)
0
20
40
60
80
100
0.1 10 1000
Res
pons
e (%
)
BDE-47 (ug/L)
D#8(3)C#16(3)B#11(2)A#7(7)
Nanobody production to BDE-47
Purified nanobody sensitivity
IC50 = 1.4 μg/L
IC50 (pAB) = 1.75 μg/L
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Nanobody characterization
Analyte cross-reactivity
0
20
40
60
80
100
Cro
ss-r
eact
ivit
y (%
)
O
Br
Br
Br
Br
HO
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Antibody characterization
0
20
40
60
80
100
20 40 60 80 100
Act
ivit
y (%
)
Temp (degree C)
VHH 10minsVHH 60minspAb 10minspAb 60mins
Thermal stability
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Nanobody® (sdAb) summary
• Ease of genetic manipulation• Labeled constructs
• Well-expressed products• Easy to scale up/improved costs
• High chemical/temperature stability and solubility• Decreased matrix effects
• Ease of storage, efficient refolding
Employs phage-display technology…
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Non competitive assays provide clear difference in signals at low concentrations of analyte from signals at zero concentrations
0
50
100
Sign
al (%
)
Analyte concentration
competitive
Non competitive
+ 20%
– 20%
Increase in analyte concentration
Competitive vs noncompetitive assays
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Phage displayed peptide selection
Analyte Analyte
Antibody
Trivalent complex
Eludisp
Phagemid containing phage particle
Peptide
Phage displayed peptides effectively result in sandwich type assays for small molecules.
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PHAIAs improve assay sensitivity
AnalyteCompetitive ELISA IC50
(ng/ml)
Noncompetitive PHAIA SC50
(ng/ml)
Sensitivity Enhancement
Molinate 69.2 5.0 14x
Atrazine 0.64 0.051 12x
Digoxin 2.1 0.22 10x
3-Phenoxybenzoic acid 1.65 0.31 5x
BDE-47 1.7 0.7 2x
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Phage peptide-based dipstick assay
50 25 12.5 6.3 3.1 1.6 0.8 0
BDE 47 (ng/mL)
0.6
0
.8
1.5
3
1309
PA
b(μ
g/sp
ot)
50 25 12.5 6.3 3.1 1.6 0.8 0
BDE 47 (ng/mL)
0.6
0
.8
1.5
3
1309
PA
b(μ
g/sp
ot)
3 1 0.5 0.1 0
BDE 47 (%, w/w)
1309
PA
b(μ
g/sp
ot)
0.3
0.5
1
2
3 1 0.5 0.1 0
BDE 47 (%, w/w)
1309
PA
b(μ
g/sp
ot)
0.3
0.5
1
2
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HRP
AnalyteAnalyte
HRPHRP
AnalyteAnalyte
3-PBA (ng/mL)10-2 10-1 100 101A
bsor
banc
e at
450
nm
0.00.51.01.52.02.53.03.5
2 10,000 0.15 2 20,000 0.2 2 40,000 0.3 1 10,000 1.0
Incubation time (h)
SC50 (ng/ml)
Dilution of 2nd
antibody
Bead-based PHAIA
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6 Kb ssDNA
DNA release
Target sequence (peptide encoding sequence)
Real time qPCR
PCR product
Phage peptide mediated real time quantitative PCR
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3-PBA, ng/ml0.001 0.01 0.1 1 10 100
Relative C
t
73
74
75
76
77
78
79
80
81 Absorbance at 450 nm
0.0
0.5
1.0
1.5
2.0
PHAIA-PCRPHAIA
PCR cycles15 20 25 30 35
Fluorescence
0
1
2
3
4
5
2566416410.250.060.020.004
3-PBA, ng/ml
PHAIA-qPCR is more sensitive than PHAIA
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PCR cycles15 20 25 30 35
Fluo
resc
ence
0.0
0.5
1.0
1.5
2.0
3-PBA, ng/ml0.001 0.01 0.1 1 10
Rel
ativ
e C
t
70
72
74
76
78
80
PCR cycles0 10 20 30
Fluo
resc
ence
0.0
0.5
1.0
1.5
2.0
PHAIA-qPCR can be selective
3-PBA
molinate
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Spiking (ng/ml)
aExpecteddetection
after dilution
Run 1 Run 2CV (%)Detection Recovery
(%) Detection Recovery (%)
Recovery of 3-PBA
50 5 5.0 ± 0.2 100 5.1 ± 0.1 102 3.120 2 2.2 ± 0.1 110 2.3 ± 0.06 115 4.35 0.5 0.4 ± 0.05 80 0.5 ± 0.04 100 102 0.2 0.18 ± 0.02 90 0.2 ± 0.01 100 10
adetection level by the PHAIA-qPCR from diluted samples
Assay validation
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Amount of beads (ul)2 5 10
Ct
16
18
20
22
24
26
20.250.030
3-PBA, ng/ml
3-PBA, ng/ml0.1 1 10
Rel
ativ
e C
t
79
80
81
82
83
84
85
Magnetic bead-based PHAIA-qPCRBead-based qPCR PHAIA
Ab coating Not necessary 1 h at 37 oC
Blocking Not necessary when beads are stored in PBS containing 1% BSA 1 h at 37 oC
Interaction 1 h at RT 1 h at RTSecondary Ab Not necessary 1 h at RTDetection 30 min 15 minAssay time 1h and 30 min 4 h and 15 min
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Phage displayed peptide assays
• All the advantages of ‘sandwich-type’ assays
• Often improves sensitivity compared to competitive assay
• Easy to produce, but requires an antibody
• Multiple assay formats possible
AnlyteAnlyteAnlyte
AnalyteAnalyteAnalyte
AnalyteAnalyte
HRPHRP
AnalyteAnalyte
Alkaline Phosphatase
Analyte
Alkaline Phosphatase
Alkaline Phosphatase
AnalyteAnalyte
TbTb
TbTbAnalyteAnalyte
Analyte
Tb
AnalyteAnalyte
TbTb
Dipstick assay
PHAIA
Magnetic bead PHAIA
Enzyme-peptide fusion
Antibody FRET
Phage FRET
RT-PCR
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Acknowledgements
Bruce Hammock
Candace Spier
Zuzana Majkova
Mark McCoy
Hee Joo Kim
Yanru Wang
Ki Chang Ahn
Julie Dechant
Gualberto Gonzalez-Sapienza
Martin Rossotti
Supported in part by NIEHS Superfund P42ES004699, NIOSH Western Center for Agricultural Health and Safety PHS OH07550NIH Fogarty International Center TW05718
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Resources
• Bever, C.R.S, Z. Majkova, R. Radhakrishnan, I. Suni, M. McCoy, Y. Wang, J. Dechant,
S.J. Gee and B.D. Hammock. 2014. Development and utilization of camelid VHH
antibodies from alpaca for BDE-47 detection. Anal Chem. In press
• Kim, H-J., M. McCoy, S.J. Gee, G.G. Gonzalez-Sapienza and B.D. Hammock. 2011.
Noncompetitive phage anti-immunocomplex real-time polymerase chain reaction for
sensitive detection of small molecules. Anal. Chem. 83(1):246-253.
• Kim, H-J., M.A. Rossotti, K.C. Ahn, G.G. González-Sapienza, S.J. Gee, R. Musker and
B.D. Hammock. 2010. Development of a noncompetitive phage anti-immunocomplex
assay for brominated diphenyl ether 47. Anal. Biochem. 401(1):38-46. PMID: 20152791.
PMCID: PMC2861364.
• Additional publications at www.biopestlab.ucdavis.edu/immunoassay/
• Bruce Hammock ([email protected]), Shirley Gee ([email protected]), Candace
Spier-Bever ([email protected])