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 (bdhammock@ucdavis.edu), Shirley Gee (sjgee@ucdavis.edu), Candace

Spier-Bever (crspier@ucdavis.edu)