Immunoassay for Monitoring Human and Environmental Exposure … · 2018. 12. 4. · Br atoms in the...
Transcript of Immunoassay for Monitoring Human and Environmental Exposure … · 2018. 12. 4. · Br atoms in the...
Immunoassay for Monitoring Human and Environmental Exposure to
Personal Care Products and Brominated Flame Retardants
Ki Chang Ahn, Ph.D.Immunochemist/Agricultural Chemist
Shirley J. Gee, and Bruce D. Hammock
Department of Entomology,University of California, Davis, Davis, CA 95616
Overview1. Introduction of analytical targets for immunoassay2. Development of immunoassay3. Improvement of the immunoassay4. Application for environmental/human monitoring
Current targets of interest1. Pyrethroid insecticides2. High-production-volume (HPV) chemicals:
- Industrial contaminants PBDEs and TBBPA in brominated flame retardants
- Antimicrobials triclocarban and triclosan in personal care products
Brominated Flame Retardants: delay ignition and save lives- How can? Br atoms in the molecule act as an oxygen
scavenger.
O
BrX BrYPolybrominated diphenylethers (PBDEs)
Tetrabromobisphenol A (TBBPA)
Br Br
HO
Br
OH
Br
Why BDE-47 was considered as the target analyte for development of an immunoassay?
- BDE-47 is predominant along with BDE-99, BDE-100, and BDE-153, and can be used as a maker of exposure of all the PBDEs.
Antimicrobials Triclocarban and Triclosan in Personal Care Products
NH
NH
OCl Cl
Cl
O
Cl
Cl
OH
Cl
Chemical Structures of Test Compounds
Androgen-/Estrogen-Based Bioassays:TCC and analogs amplify the activity of sex steroid
hormones, being potential EDCs.In ER-based
recombinant cellsIn AR-based
recombinant cells
Ahn et al. EHP, 2007
Enzyme-Based Assays:TCC and its analogs strongly inhibit sEH
AUDA
NSPA
I
II
III (TCC)
2’-OH TCC
2’-OH TCC (sulfate conjugate)
IV
V
VI (DCC)
VII (TFC)
VIII (TCS)
>100000
390 ± 10
>100000
>100000
>100000
>100000
>100000
>100000
>100000
> 20000
> 10000
>100000
3 ± 0.02
-
390 ± 3
59 ± 4
13 ± 1
73 ± 5
101 ± 4
28 ± 2
18 ± 1
52 ± 3
16 ± 1
4764 ± 123
CompoundIC50 (nM)
mEH sEH
Ahn et al. EHP, submitted
Therapeutic applications:TCC and its analogs reduce high blood pressure, are
strongly anti-inflammatory, synergize NSAIDs and aspirin, and have analgesic properties
COOH
COOH
Arachidonate
P450
OEpoxyarachidonate (EET)
COOH
HO OH
Diol of arachidonate (DHET)
sEH inhibition by TCCIκK-NFκB cascade
Inflammation
X
PainHypertensionImflammation
Ahn et al. EHP, submitted
Ryanodine Receptor-Mediated Bioassay: noncoplanrPCBs (PCB-95) is highly active in the assay
TCS significantly increases [3H]ryanodine
binding in skeletal muscle SR vesicles.
TCS treatment increases cytosolic Ca2+ concentration in
resting myotubes in a dose-dependent manner.
Ahn et al. EHP, 2007Being potential EDC
Enzyme-Based Assays:TCS inhibits human carboxylesterase 1 (hCE1)
With an IC50 = 260 nM
Benzil
Ethacrynic acid
I
II
III (TCC)
IV
V
VI (DCC)
VII (TFC)
VIII (TCS)
94.7 ± 2.5
-
12.3 ± 6.3
22.0 ± 5.8
13.4 ± 6.0
13.4 ± 3.5
3.0 ± 6.6
4.0 ± 8.1
24.9 ± 8.8
83.4 ± 3.5
77.5 ± 1.3
-
22.4 ± 1.3
10.7 ± 4.7
11.5 ± 3.5
8.0 ± 1.3
11.5 ± 0.8
6.9 ± 3.7
13.9 ± 6.6
40.0 ± 5.2
44.7 ± 1.7
-
<1
<1
<1
<1
<1
1.8 ± 2.3
8.2 ± 1.2
56.2 ± 5.0
Compound(1 uM)
% InhibitionhCE1 hCE2 hCE3
Ahn et al. EHP, submitted
Cl
ClO
O
O
HO
O
Cl
Cl
O
HO
ESTERASE
Permethrin ester
meta-Phenoxybenzyl alcohol Dichlorovinyl chrysanthemic acidpermethrin esterase.cdx
TCS Inhibits Esterases that Degrade Pesticides and Drugs
Biosensors and Immunoassay: a complementary tool
MS GC HPLC
IR UV/VIS
CE
Analytical Chemist
Immunoassay/Biosensor
Not a replacement for GC or HPLC, but cost-effective alternative• Direct immunochemical analysis• Prioritization of samples for other methods• Immunoaffinity cleanup for other methods
• Analytical cost high
Atrazine $50 - $150/Sample
Paraquat $200 - $500/Sample
TCDD $1,500 - $5,000/Sample
• Analysis time relatively long
• Analysis not field portable
• Analysis not in environmentalist
and hygienists’ hands
• Instrument >$150,000
(purchase)
• Sensitive (low detection limits)• High analyte selectivity• Rapid sample prep. (reduced sample
preparation)• Relative cost effective for large
number of samplesPCB $35/Sample
• Adaptable to field use• Rapid results from abiotic and biotic
samples of interest such as water, soil, biosolid, consumer products, blood and urine
• Instrument: >$1,000 (purchase)
Instrumental analysis using classical methods
Immunoassays as analytical methods - Advantages
Analytical methods: PBDEs
• GC-ECD or GC-MS: Sensitive,expensivesimultaneous multi-detection for all PBDE congeners in any sample.
• X-ray fluorescence: Nondestructive and rapid detection for total bromine in solid consumer products.
• Immunoassay: Sensitive, rapid detection specific to BDE-47 in any sample.
Development of Immunoassay
• BDE-47• Triclocarban
• Synthesis of derivatives of target analyte (Hapten chemistry)
• Production of antibodies
• Assay optimization
Development of BDE-47-Specific Immunoassay
- For production of a specific antibody and an assay competitor, hapten should imitate the target compound BDE-47
OBr
Br
Br
O
NH KLH
OBr
Br
Br
Br
Immunizing Hapten B2RMS: 0.08
Competitive coating hapten D3
For Ab production For competitive assay
N
BrBr
O
O
NH BSA
Ahn et al. EST, submitted
Optimized conditions for BDE-47 immunoassay
50% DMSOin 1×PBS (pH 7.5)
PolyclonalAb #1309(1:4000)
produced against B2-KLH
0.5% BSA 1 μg/mL of
D3-BSA
Assay buffer
Antiserum (dilution)Blocking agent
Coating antigen
Ahn et al. EST, submitted
Competitive indirect ELISA as standard immunoassay format: - Sensitive assay for BDE-47- Detection range: 0.35-8.50 ng/mL- Limit of detection: 0.20 ng/mL
OBr
Br
Br
Br
1 0 -3 1 0 -2 1 0 -1 1 0 0 1 0 1 1 0 2 1 0 3 1 0 4 1 0 5
0 .0 0 0
0 .1 0 0
0 .2 0 0
0 .3 0 0
0 .4 0 0
0 .5 0 0
0 .6 0 0
0 .7 0 0
0 .8 0 0 C o m p e tive in d ire c t E L IS A
F o u r p a ra m e te r s ig m o id a l cu rveA m a x= 0 .7 7 4A m in = 0 .0 2 4IC 5 0= 1 .7 5 u g /LS lo p e = 0 .9 2
IC 2 0 -8 0= 0 .3 5 -8 .5 0 u g /L
Aso
rban
ce 4
50-6
50 n
m
B D E -47 (μg /L )BDE-47
BDE-15BDE-99
BDE-100BDE-153
BDE-154BDE-209
PCB-77TCDD
3-OH-BDE-47
3-CH3O-BDE-47
5-OH-BDE-47
5-CH3O-BDE-47
Triclosan
0
20
40
60
80
100
Cro
ss-r
eact
ivity
(%)
Correlation between BDE-47 data in house dust measured by GC-MS and ELISA (n=17) - Highly positive correlation- The immunoassay over-evaluated the occurrence of BDE-47 than that measured by GC-MS.
GC/MS vs. ELISA
y = 2.7453xR2 = 0.778
1
10
100
1000
10000
100000
10 100 1000 10000
BDE-47 (ng/g) in dustmeasured by GC/MS
BD
E-47
(ng/
g) in
dus
tm
easu
red
by E
LIS
A
Ahn et al. EST, submitted
Correlation between BDE-47 data in furniture foam measured by ELISA and total Br by XRF (n=11).
- Low positive correlationship- The ELISA can selectively detect BDE-47 among the brominatedcompounds in polyurethane foam.- XRF is hard to distinguish the banned PBDEs .
ELISA vs. XRF
y = 0.8293x + 2.7118R2 = 0.3807
0.01.02.03.04.05.06.07.0
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0
BDE-47 (%, w/w) in furniture foam measured by ELISA
Tota
l Br
(%, w
/w) i
n fu
rinitu
re fo
am
mea
sure
d by
XRF
Ahn et al. EST, submitted
Development of TCC Immunoassay
- For production of a specific antibody and an assay competitor, hapten should imitate the target compound
TCC
NH
NH
OS
Cl
ClCOOH
N
NH
NH
SF3CO
O COOH
O
Immunizing Hapten Competitive coating hapten
NH
NH
OCl
Cl
Cl
For Ab production For competitive assay
Optimized conditions for TCC immunoassay
40% DMSO in 1×PBS (pH 7.5)
PolyclonalAb #1648(1:5000)
Produced against BDH-
215-Thy
0.5% BSA 1 μg/mL of BDH 291-9-
BSA
Assay buffer
Antiserum (dilution)Blocking agent
Coating antigen
TCC-Specific immunoassay
10-4 10-3 10-2 10-1 100 101 102 103 1040.000
0.200
0.400
0.600
0.800
1.000
1.200
1.400 NH
NH
OCl
Cl
Cl
Sigmoidal fit (quadruplicate x n, n=5)Chi^2 0.337R^2 0.998
Absmax=1.162Absmin=0.014IC50=0.69 ng/mLSlope=0.84
Linear detection range (IC20-80)= 0.13-3.60 ng/mLLow detection limit (IC10)=0.03 ng/mLHigh detection limit (IC90)=10 ng/mLAb
s 45
0-65
0 nm
TCC concentration (ng/mL)
TCCTCC-O
H
Sulfate
of TCC-O
H
TFC
TCSDinu
ronsE
Hi, #15
55sE
Hi, #17
09TCC1TCC2TCC4
0
20
40
60
80
100
Cro
ss-r
eact
ivity
(%)
Comparison of TCC data measured by LC/MS/MS and ELISA
10945±8177040±50C9827±1368142±178B11991±673 ng/g8654±1481 ng/gBiosolid A103.50±0.71143.16±0.469D24.97±1.9816.38±0.298C45.1±7.7186.42±0.493BLOQ (2 ng/mL)5.87±0.159 ng/mLMouse serum A
ImmunoassayLC/MS/MS
TCC concentration measured(ng/mL serum or ng/g dried biosolid)Sample
Comparison of the immunoassay with LC/MS/MS method for TCC in whole blood
General laboratory analystSpectrophotometricspecialist
Operator
99 data/2.5hrs25 data/2.5hrsHigh through-put assay
Relatively less expensiveexpensiveCost
0.03 ng/mL~5 ng/mL in whole blood
0.56 ng/mL~5 ng/mL in whole blood
Sensitivity- LOD- LOQ
Not requiredRequired(13C-TCC)
Surrogate
Not requiredRequired (CUDA)Internal standard
LLELLESample preparation
10 uL10 uLSample size
ImmunoassayLC/MS/MS
Improvement of immunoassay- In terms of sensitivity, and speed
• How can I increase sensitivity? In place of enzyme reporter,1. Fluorescent dye or inorganic particles2. Chemiluminescent dye
• How can I increase speedy?1. Automatic detection system
Improvement I:Fluorescent inorganic particles Eu2O3 as
reportersAdvantages
• Large Stokes shift• Sharp emission spectrum
increases spectral sensitivity• Long lifetime emission
permits gated detection (increases signal to
noise ratio)• Resistant to photobleaching• Cost effective
67
Europium oxide (Eu2O3) spectra
80x103
70
60
50
40
30
20
10
Inte
nsity
(arb
itrar
y un
its)
650600550500450400350Wavelength (nm)
Excitation spectrumobserved at 610 nm Emission spectrum
excited at 466 nm
4x106
3
2
1
Inte
nsity
(arb
itrar
y un
its)
600580560540520500Wavelength (nm)
Excitation spectrum observed at 548 nm
Emission spectrum excited at 524 nm
Europium Oxide
Rhodamine
Putative structure of europium particle-labeled hapten
O
Si
NH2
OOSi Si
O O
NH2
H2N
O
SiO
SiO
OH2N
HN
O
OEu2O3
NH2-Fuctionalized Eu2O3 particle as fluorescent label Hapten
Ahn et al. ACS Symposium book, 2008
Europium oxide in a pyrethroid metabolite assay
1E-9 1E-8 1E-7 1E-6 1E-5 1E-4 1E-3 0.01 0.1 1 10 100
100000
150000
200000
250000
300000
350000
400000
Amin=1.1377E5Amax=3.1735E5Sope=0.96IC50=1.90E-4 ng/mL
Fluo
resc
ence
inte
nsity
3-PBAcid (ng/mL)
• Competitive assay for 3-phenoxybenzoic acid (PBA) with magnetic separation
•• Europium oxideEuropium oxideparticle conjugated toparticle conjugated tocompetitive competitive haptenhapten
• IC502 x 10 –4 ng mL-1
(10,000-fold sensitive)Ahn et al. ACS
Symposium book, 2008
Improvement II:Automated Magnetic Particle-Based
Chemiluminescent Immunoassay using ACS-180 Autoanalyzer:
- Competitive indirect10-4 10-3 10-2 10-1 100 101 102 103
4000
6000
8000
10000
12000
14000
16000
Rel
ativ
e lig
ht u
nit (
RLU
)
Concentration of inhibitor
Ahn et al. Analytical Chemisty, 2007
Analytical comparison between the conventional ELISA and automated luminescent magnetic particle-based for 3-PBA, a biomarker of human
exposure to pyrethroid insecticides
AutomaticHand-operatedAnalytical procedure
130 results in 1 h96 results in a microplate in approximately 2 h
Analytical time after a competition step
0.100.025-0.060.005
1.650.20-5.00.1
Analytical sensitivity:- IC50 (µg/L)- IC20-80 (µg/L)- LOD (µg/L)
150 µL in the cuvette(competitive hapten, sample, and
antibody)
100 µL in the well (sample and antibody)
Total volume (µL) of the mixture of immunoreagents
1:4 (0.1 mg IgG/mg magnetic particles/mL of antibody coated-
paramagnetic particles)
1:3000 (from 1.1 mg/mL of IgG-HRP conjugate)
- Secondary antibody (dilution)
1:72,000 (from 5 mg/mL of 3-PBA-BSA-A+ conjugate,
in the cuvette)
1:5,600 (from 2.8 mg/mL of 3-PBA-BSA
conjugate)
- Competitive hapten-BSA conjugate (dilution)
1:48,000 (in the cuvette)1:10,000 (in the well)-Polyclonal antibody (dilution)
Immunoreagents:
Automatedimmunoassay
Indirect coating antigen-immoblized ELISA
Ahn et al. Analytical Chemistry, 2007
Immunoassay Application I
- Human exposure study of pyrethroidsusing 3-PBA Immunoassay
Human exposure study• Included 73 pine cone
harvest workers who were not previously exposed to pyrethroid insecticides.
• The study was to determine if exposure occurred under normal pyrethroid use and harvesting practices.
• 300 urine samples were collected in successive days for seven days.
• and were analyzed for 3-PBA using ELISA
Cl
ClO
O
O
Esterase
Cl
ClO
OH
Free acidsGlycine conjugatesGlucuronides
Other conjugates
General metabolism in mammals of pyrethroidscontaining a phenoxy benzyl moiety
- Pemethrin, cypermethrin, deltamethrin, cyfluthrin, esfenvalerate
O
O
OH
OH
O
Dehydrogenase
3-PBA, common metabolite
Sample preparation of urine for 3-PBA immunoassay
• Acid hydrolysis of urine to form free metabolite• Dual mode SPE (C8 and anionic exchange) to reduce matrix effect
• Acidic eluate (1% AA in ethyl acetate/hexane=30:70, v/v)• Evaporation• 5-Fold dilution with 10% MeOH-PBS
Si
OO
O O
Si
Si
Si
O
Hydrophobic binding
Si
O
O OH
Si
Si
H3+N
ionic binding
Mixed-mode SPE (C8 and anion exchange) O
O
-O
Pyrethroid metabolite (3-PBA) Immunoassay
O COOHOOHC
O COOH
Immunizing Antigen Coating AntigenO COOH
2
LOQ(ng/mL)
2-40
Detection range (ng/mL)
Human urine
BIOMARKER OF EXPOSURE = 3-PBA
Comparison of 3-PBA concentrations determined with a LC/MS/MS and ELISA, urine extract being prepared with a
mixed mode SPE. - The ability of the immunoassay throughout the whole
concentration range was highly positive correlation (R = 0.99) with slope = 0.95.
0 20 40 60 80 100 120 140 160
0
20
40
60
80
100
120
140
160Y=0.95X+1.04R=0.985
3-P
BA
det
erm
ined
by
SP
E-L
C/M
S/M
S (u
g/L)
3-PBA determined by SPE-ELISA (ug/L)
0 2 4 6 8 10 12 14 160
2
4
6
8
10
12
14
16
18
20Y=1.14X+0.24R=0.964
Ahn et al. Prepared.
Application II
• Analysis of total type II pyrethroid insecticides in orange oil
• Hydrolysis of type II pyrethroids
• Formation of 3-PBA using an oxidizing reagent
• Immunoassay for 3-PBA
OOR1
O CN
R1
O
OH+ O
H
O
OHO
O
Acid
HOH
3-Phenoxybenzyl aldehyde
Oxidant
3-PBA
Type II pyrethroids
Immunoassay under developing
• Total PBDEs immunoassay• TBBPA immunoassay• TCS immunoassay
Summary
Immunoassay
• In parallel with the instrument assay, an immunological method currently under development will significantly improve expensive and labor intensive process by making the tests cheaper and assay time faster.
• Advance to a high throughput format and a biosensor format based on antibody- Rapid and sensitive automated immunoassay- On site assay with an biosensor format
Development
Improve to high-throughput format
Improve to on site assay
AcknowledgementsNIEHS Superfund Basic Research ProgramNIEHS Center for Environmental Health
SciencesNIOSH Center for Agricultural Disease and
Research, Education and PreventionSynthia-LLC
Thank you!!!!• Dr. Bruce Hammock• Ms. Shirley Gee• Dr. Chrisophe Morrisseau• Ms. Hsing-Ju Tsai• Dr. Huazhang Huang• Dr. Bill Lasely• Dr. Jiangang Chen• Dr. Michael Denison• Dr. Bin Zhao• Dr. Isaac Pessah• Dr. Gennady Cherednichenko• Dr. Daniel P. Y. Chang• Dr. Hee Joo Kim• Dr. Takeo Kasagami• Dr. Pavel Aronov• Dr. Arlene Blum• Dr. Deborah Bennett