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