ECL Seminars: Detecting Environmental Chemicals Using ......Detecting environmental chemicals using...
Transcript of ECL Seminars: Detecting Environmental Chemicals Using ......Detecting environmental chemicals using...
Detecting environmental chemicals using immunoassay
technology
Shirley J. Gee, Candace Bever, Bruce D. HammockDepartment of Entomology
University of California DavisDavis, CA 95616
Immunoassay: a complementary technology
ANALYTICAL CHEMIST
Mass spectrometry
Gas chromatography
Capillaryelectrophoresis
Liquid chromatography
Infrared spectrometry
Radioisotope detection
Ultraviolet/visible spectrometry
Immunoassay
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
Sandwich immunoassay for protein analysis
Antibody binding to a small molecule
Small molecule immunoassay
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
• 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
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
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
Library screening
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+ +
Nanobody production to a target analyte
O
Br
Br
Br
Br
O OH
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
Nanobody characterization
Analyte cross-reactivity
0
20
40
60
80
100
Cro
ss-r
eact
ivit
y (%
)
O
Br
Br
Br
Br
HO
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
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…
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
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.
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
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
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
6 Kb ssDNA
DNA release
Target sequence (peptide encoding sequence)
Real time qPCR
PCR product
Phage peptide mediated real time quantitative PCR
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
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
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
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
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
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
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])