Rapid Assessment Tools for Monitoring Food and Forage

RAPID ASSESSMENT TOOLS FOR MONITORING FOOD AND FORAGE

Sponsoring Institution

National Institute of Food and Agriculture

Project Status

TERMINATED

Funding Source

HATCH

Reporting Frequency

Annual

Accession No.

0210216

Grant No.

(N/A)

Project No.

CA-D-ENM-7654-H

Proposal No.

(N/A)

Multistate No.

(N/A)

Program Code

(N/A)

Project Start Date

Oct 1, 2012

Project End Date

Sep 30, 2017

Grant Year

(N/A)

Project Director
Hammock, B.

Recipient Organization
UNIVERSITY OF CALIFORNIA, DAVIS
410 MRAK HALL
DAVIS,CA 95616-8671

Performing Department
Entomology and Nematology

Non Technical Summary
To make rational decisions about animal, human and environmental health - whether those decisions be about pesticides, fungal toxins, drugs or other intentional or inadvertent contaminants it is important to have information about concentrations that are present. Immunoassays have been used in human clinical medicine for more than 30 years and over the last 20 years, become an important tool in monitoring feed and forage for a variety of materials ranging from genetic engineering products through food adulterants and pesticides. Immunoassays use an antibody to bind to the chemical or toxin of interest and then an enzyme or other label to detect the binding. The technique termed ELISA or enzyme linked immunosorbent assay has dominated the immunoassay field. We propose to increase the throughput of the technology, develop new labels to detect binding and ultimately to develop field portable formats to replace the classical ELISA. At the same time, we will use classical immunoassay methods to develop assays for pesticides or other compounds of immediate interest to the agricultural sector.

Animal Health Component

100%

Research Effort Categories

Basic

(N/A)

Applied

(N/A)

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
133	0110	1150	10%
711	0210	2000	10%
723	5010	2000	10%
723	5220	2000	45%
723	6010	1170	15%
723	6020	1170	10%

Knowledge Area
133 - Pollution Prevention and Mitigation; 711 - Ensure Food Products Free of Harmful Chemicals, Including Residues from Agricultural and Other Sources; 723 - Hazards to Human Health and Safety;

Subject Of Investigation
0110 - Soil; 0210 - Water resources; 6010 - Individuals; 6020 - The family and its members; 5010 - Food; 5220 - Pesticides;

Field Of Science
2000 - Chemistry; 1150 - Toxicology; 1170 - Epidemiology;

Keywords

immunoassay

enzyme linked immunosorbent assay

Goals / Objectives
Goals Analytical data is critical for making rational decisions about animal, human and environmental health - whether those decisions be about pesticides, mycotoxins, drugs or other intentional or inadvertent contaminants. Immunoassays have been used in human clinical medicine for more than 30 years and over the last 20 years, become an important tool in monitoring feed and forage for a variety of materials ranging from genetic engineering products through food adulterants and pesticides. The technique termed ELISA or enzyme linked immunosorbent assay has dominated the immunoassay field. Our objectives strive to increase the throughput of the technology, develop new reporter molecules and more robust reagents and ultimately to develop field portable methods to replace the classical ELISA. Objectives Develop rapid methods for the detection of pesticides, mycotoxins, bioterrorism agents and other foreign compounds in feed and forage. A. Develop and validate new immunoassays for compounds of interest to agricultural industry. B. Develop more robust and sensitive binding molecules and labels for compounds in Objective A. C. Use the above systems in field portable formats to monitor exposure. Expected Outputs Activities. The project is primarily developmental and application, thus a number of experiments are conducted and the data analyzed. This process results in peer-reviewed publications. In the most recent 5 year period at least 30 publications were a result of this project. This body of work will be presented at local and national conferences each year. We will continue to lecture each year in the UC Davis Environmental Toxicology graduate class in the analysis of toxicants. At least one graduate student and 2-3 postdoctoral researchers are mentored in this research area. Events. A senior researcher has given a one-week workshop on immunoassay technology in each of the last 5 years, most recently this workshop was at the invitation of a collaborator in China. Services. We will continue to host graduate students and postdoctoral researchers from several foreign programs for periods from 2 weeks to 18 months to provide instruction in immunoassay technology.Products. We anticipate filing patent disclosures on work resulting from the phage peptide and single domain antibody work. Reagents and protocols developed in the project are available for licensing to the commercial sector and will be provided gratis to universities and non-profit institutions. Our website describes the basic technology and provides links to relevant publications and a link to contact us with questions. Dissemination. We will continue to give lectures in the technology to state agencies such as the Office of Environmental Health Hazard Assessment (OEHHA) and the California Department of Pesticide Regulation. Dissemination of information to peer scientists will occur through conferences and personal meetings.

Project Methods
The enzyme linked immunosorbent assay (ELISA) has been used in the clinical field for more than 30 years. In agriculture it has been applied to monitoring products of genetic engineering, mycotoxins and pesticides in food, forage and the environment. In this project we will develop new immunoassays for compounds of interest to the agricultural industry. These include primarily pesticides and other contaminants that reach the food chain, such as brominated flame retardants and antimicrobial compounds. In a classical dipstick assay in a competitive format it is difficult to clearly distinguish the intensity of colors between zero and near-zero concentrations. To overcome this drawback, we developed an innovative technology termed the phage anti-immunocomplex assay (PHAIA) in which a phage-displayed short peptide loop forms a trivalent complex binding to the small hapten-antibody complex, generating positive signals that are directly proportional to analyte concentrations. This feature makes the PHAIA an excellent tool to develop a dipstick or lateral flow system, particularly when the presence of the analyte needs to be visualized in a yes or no fashion. The assays will be developed using methods developed in the previous project period. We will replace the trapping antibody used in the dipstick with the more robust single domain antibodies to improve field portability of these assays. Camelids have the normal IgG antibody and antibodies that are formed from only heavy chains. These antibodies offer similar affinities to their two chain counterparts and they are extraordinarily heat and chemically stable. Since sdAbs are relatively small and encoded on a single polypeptide chain they are easily expressed recombinantly and cDNA can be stored indefinitely and used to produce industrial quantities of reagents cheaply. sdAbs will be produced to our targets by methods recently developed in our laboratory. We plan to develop a novel lateral flow device for application to field studies. The reagents to be used will be antibodies, phage displayed peptides or sdAbs depending upon the outcome of studies in earlier aims and the needed sensitivity. We are using conventional commercially available gold colloids and have published a powerful dipstick assay for the pyrethroid metabolite 3-PBA using this technology. However, we also plan to utilize a new class of nanoparticle that consists of a gold-coated up-converting lanthanide nanophosphor developed in the Kennedy laboratory at UCD as either platforms for assays or as labels with improved detectability. An advantage is that the particles do not quench like organic dyes, fluoresce with a long t1/2 in the red region with little or no background, and can be interrogated by the naked eye in visible light or using very inexpensive red laser diodes with or without detection with a photomultiplier. Ultimately these portable assays will be utilized to monitor human exposure to pesticides in studies involving agricultural, and landscape workers in collaboration with Debbie Bennett of the School of Medicine Occupational Health group.

Progress 10/01/12 to 09/30/17

Outputs
Target Audience:The tools we develop specifically are for industry (for their product stewardship), regulators, environmental scientists and public health professionals interested in human exposure to agricultural chemical and environmental and industrial chemicals. We also develop tools for technology companies to offer the immunoassay services more broadly. The tools are applicable to community groups, in this case native Americans to analyze their own environment. Depending on the assay Federal and State Agencies and International Agencies are relevant. These include but are not limited to NIOSH, NIEHS, NINDS, NIDDK, EPA, FDA and others. Some like NIOSH, NIEHS, and related agencies are through grant reporting. The technologies are broader since immunoassays and sensors are used extensively in clinical medicine with both human and veterinary application. These research tools are used to monitor food safety, insure rootstock and seed are free of pathogens and many other applications. We are developing one kit specifically to support the health of cystic fibrosis patients. As mentioned above one example target audience is the Yurok native American tribe in northern California with a concern over pesticides used in illegal growing of medicinal recreational drugs. Academically we reach scientists interested in antibody development, development of biosensors, analysis of pesticides and other environmental chemicals, and nanobody development. The assays are research tools for a variety of projects ranging from the health of the Sacramento Delta and San Francisco Bay to worker exposure in the Central Valley. Target analytes range from pesticides and terror weapons to industrial chemicals and of course include drugs and natural poisons. Changes/Problems:Increasingly we are moving from classical polyclonal and monoclonal antibodies to VHH reagents or nanobodies which are produced recombinantly in bacteria after they are raised in llama and alpaca and screened or panned with a bacteriophage display system. This is far more powerful and cost effective. What opportunities for training and professional development has the project provided?Training and professional development for: 5 Ph.D. students 3 Visiting Ph.D. students 3 Visiting postdoctoral 1 Visiting faculty 6 Postdoctoral trainees How have the results been disseminated to communities of interest?Yes, see above. A project has been submitted to NIH proposing the transfer of this technology to native American nations to facilitate them monitoring their own food and environment to insure safety. This was noted in January in the SF Chronicle regarding rodenticide and herbicide contamination on native American lands associated with cannabis production. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? lmmunoassays, that have been used in clinical medicine for many years, are an important tool for the analytical chemist when approaching problems in analysis of food and feed. Data that the analytical chemist generates are important to regulators and other stakeholders for making decisions about animal, human and environmental health. The technology is being applied for monitoring products of genetic engineering, food adulterants, nutritional components and pesticides, for example. lmmunoassays use an antibody to bind to the chemical or toxin of interest and then an enzyme or other label is used to detect the binding. The technique termed ELISA or enzyme linked immunosorbent assay has dominated the immunoassay field. We propose to make immunoassays to pesticides or other compounds of immediate interest to the agricultural sector while making significant improvements in the technology to increase the speed, lower the limit of detectable chemical, lengthen the stability of reagents and enhance the field portability of the tests. Possibly the most notable use of our technology was to monitor illegal presence of the insecticide fipronil in eggs in Europe. We recently published a study showng fipronil associated with development of Alzheimer's Disease. Specifically we developed assays to support food and forage safety. These include pesticides such as fibronil, neonicatinoids, pyrethroids, and organophosphates and the fungicide malachite green/leucomalachite green used in aquaculture. We have a hydroxyfipronil assay used to monitor exposure in man and in parrie dogs, a TETs assay to monitor bioterror attacks, for organophosphate flame retardants, mycotoxins such as fumonison and aflatoxin, and others We developed new detection methods using PCR for organophosphorus insecticides and ochratoxin A, a novel impededance biosensor for polybrominated diphenyl ether flame retardants and collaborated with and engineer on a nanoparticle array detector. We also have adapted our immunoassays to field assays run on a cell phone platform. A novel platform was developed for rapid detection of Staphylococcal enterotoxin B which is one of the most sensitive assays ever developed for this serious pathogen. We utilized novel recombinant alpaca antibodies or nanobodies in the development of a method to detect the flame retardant tetrabromobisphenol A and the mycotoxin ochratoxin A. We have modified these reagents using recombinant technology to make the assays faster and more sensitive. The development of VHH or nanobody technology is a major accomplishment for this laboratory. We continue to be active on assays for personal care products such as triclosan and triclocarban. As mentioned above we are expanding our work on mycotoxins and food safety exemplified by recent work on ochratoxin and aflatoxins and food flavor chemicals. Not only do we make these assays but we also apply them. For example we have applied our pyrethroid metabolite immunoassay to a study of migrant worker exposure to pyrethroids in the central valley of California. A major goal was driving forward the development of VHH or nanobody technology for immunossays of small molecules and proteins of biological interest. Our productivity to funding ratio remains infinitely large with multiple peer review publications and no resources. Since the new format is increasing cost of reporting generating negative funding for the program, our productivity has increased further. Overall, we have made progress in the utilization of existing assays to monitor chemicals of interest to agriculture and human health, to develop new assays and the explore novel methods in order to reach the goals of the project. This technology hasextended basic science and has the potential to improve food security and safety particularly among diverse and vunerable populations.

Publications

Type: Journal Articles Status: Published Year Published: 2016 Citation: Y.F. Li, Y.M. Sun, R. Beier, H.T. Lei, S.J. Gee, B.D. Hammock, H. Wang, Y.D. Shen and J.Y. Yang. 2016. Immunochemical techniques for multianalyte analysis of chemical residues in food and the environment: A review. Trends Analyt Chem. 88: 25-40.
Type: Journal Articles Status: Published Year Published: 2017 Citation: Chu, S., M.R. Baker, G. Leong, R.J. Letcher, S.J. Gee, B.D. Hammock and Q. Li. 2017. Exploring Adduct Formation between Human Serum Albumin and Eleven Organophosphate Ester Flame Retardants and Plasticizers Using MALDITOF/ TOF and LC-Q/TOF. Chemosphere. 180:169-177. PMID: 28407546.
Type: Journal Articles Status: Published Year Published: 2017 Citation: Vasylieva, Natalia; Barnych, Bogdan; Rand, Amelia ; Inceoglu, Bora; Gee, Shirley; Hammock, Bruce. 2017. Sensitive immunoassay for detection and quantification of the neurotoxin, tetramethylenedisulfotetramine (TETS). Anal. Chem. 89(10):5612-5619. PMID: 28398746
Type: Journal Articles Status: Published Year Published: 2017 Citation: Li, D., Y. Cui, C. Morisseau, S.J. Gee, C.S. Bever, X. Liu, L. Tang, B.D. Hammock and Y. Ting. 2017. Nanobody based Immunoassay for Human Soluble Epoxide Hydrolase Detection using PolyHRP for Signal Enhancementthe Rediscovery of PolyHRP? Anal. Chem. 89(11):6248-6256. PMID: 28460522
Type: Journal Articles Status: Published Year Published: 2017 Citation: B. Barnych, N. Vasylieva, T. Joseph, S. Hulsizer, Hai Nguyen, T. Cajka, I. Pessah, H. Wulff, S.J. Gee, B. D. Hammock. 2017. Development of tetramethylenedisulfotetramine (TETS) hapten library: synthesis, electrophysiological studies and immune response in rabbits. Chemistry A European Journal., PMID: 28411375
Type: Journal Articles Status: Published Year Published: 2017 Citation: Vasylieva, N., B. Barnych, D. Wan, E.A. El-Sheikh, H.M. Nguyen, H. Wulff, R. McMahen, M. Strynar, S.J. Gee and B.D. Hammock. 2017. Hydroxy-fipronil as a new urinary biomarker of exposure to fipronil. Environ Int. 103:91-98. PMID: 28343720.

Progress 10/01/15 to 09/30/16

Outputs
Target Audience:The tools we develop specifically are for industry (for their product stewardship), regulators, environmental scientists and public health professionals interested in human exposure to agricultural chemical and environmental and industrial chemicals. We also develop tools for technology companies to offer the immunoassay services more broadly. The tools are applicable to community groups, in this case native Americans to analyze their own environment. Depending on the assay Federal and State Agencies and International Agencies are relevant. These include but are not limited to NIOSH, NIEHS, NINDS, NIDDK, EPA, FDA and others. Some like NIOSH, NIEHS, and related agencies are through grant reporting. The technologies are broader since immunoassays and sensors are used extensively in clinical medicine with both human and veterinary application. These research tools are used to monitor food safety, insure rootstock and seed are free of pathogens and many other applications. We are developing one kit specifically to support the health of cystic fibrosis patients. As mentioned above one example target audience is the Yurok native American tribe in northern California with a concern over pesticides used in illegal growing of medicinal recreational drugs. Academically we reach scientists interested in antibody development, development of biosensors, analysis of pesticides and other environmental chemicals, and nanobody development. The assays are research tools for a variety of projects ranging from the health of the Sacramento Delta and San Francisco Bay to worker exposure in the Central Valley. Target analytes range from pesticides and terror weapons to industrial chemicals and of course include drugs and natural poisons. Changes/Problems:Increasingly we are moving from classical polyclonal and monoclonal antibodies to VHH reagents or nanobodies which are produced recombinantly in bacteria after they are raised in llama and alpaca and screened or panned with a bacteriophage display system. This is far more powerful and cost effective. What opportunities for training and professional development has the project provided?Training and professional development for the following: 2 Visiting Ph.D. students 3 Visiting postdoctoral trainee 1 Visiting faculty (Ting 4 Postdoctoral trainees How have the results been disseminated to communities of interest?Yes, see above. A project has been submitted to NIH proposing the transfer of this technology to native American nations to facilitate them monitoring their own food and environment to insure safety. What do you plan to do during the next reporting period to accomplish the goals?Generate funding to accomplish research then carry it out.

Impacts
What was accomplished under these goals? lmmunoassays, that have been used in clinical medicine for many years, are an important tool for the analytical chemist when approaching problems in analysis of food and feed. Data that the analytical chemist generates is important to regulators and other stakeholders for making decisions about animal, human and environmental health. The technology is being applied for monitoring products of genetic engineering, food adulterants, nutritional components and pesticides, for example. lmmunoassays use an antibody to bind to the chemical or toxin of interest and then an enzyme or other label is used to detect the binding. The technique termed ELISA or enzyme linked immunosorbent assay has dominated the immunoassay field. We propose to make immunoassays to pesticides or other compounds of immediate interest to the agricultural sector while making significant improvements in the technology to increase the speed, lower the limit of detectable chemical, lengthen the stability of reagents and enhance the field portability of the tests. In this year we utilized assays for antimicrobial agents triclocarban and triclosan. Some mechanistic studies with triclosan indicate that triclosan is a liver tumor promoter. This was selected as one of the 20 best studies supported by NIEHS for the year. An assay for a pyrethroid metabolite was used in three separate studies; one to examine the exposure of farm worker families in the Central Valley of California, second to compare exposure between farmers and consumers in Thailand to these insecticides. Specifically we developed assays to support food and forage safety. These include pesticides such as fibronil, neonicatinoids, pyrthroids, and organophosphates and the fungicide malachite green/leucomalachite green used in aquaculture. We developed new detection methods using PCR for organophosphorus insecticides and ochratoxin A, a novel impededance biosensor for polybrominated diphenyl ether flame retardants and collaborated with and engineer on a nanoparticle array detector. We also have adapted our immunoassays to field assays run on a cell phone platform. A novel platform was developed for rapid detection of Staphylococcal enterotoxin B which is one of the most sensitive assays ever developed for this serious pathogen. We utilized novel recombinant alpaca antibodies or nanobodies in the development of a method to detect the flame retardant tetrabromobisphenol A and the mycotoxin ochratoxin A. We have modified these reagents using recombinant technology to make the assays faster and more sensitive. The development of VHH or nanobody technology is a major accomplishment for this laboratory. We continue to be active on assays for personal care products such as triclosan and triclocarban. As mentioned above we are expanding our work on mycotoxins and food safety exemplified by recent work on ochratoxin and aflatoxins and food flavor chemicals. Not only do we make these assays but we also apply them. For example we have applied our pyrethroid metabolite immunoassay to a study of migrant worker exposure to pyrethroids in the central valley of California. A major goal was driving forward the development of VHH or nanobody technology for immunossays of small molecules and proteins of biological interest. Our productivity to funding ratio remains infinitely large with multiple peer review publications and no resources. Overall, we have made progress in the utilization of existing assays to monitor chemicals of interest to agriculture and human health, to develop new assays and the explore novel methods in order to reach the goals of the project.

Publications

Type: Journal Articles Status: Published Year Published: 2015 Citation: Liu, X., Y. Xu, Y.H. Xiong, D. Wan, .Z.Y. He, X. Wang, S.J. Gee and B.D. Hammock. Development of a nanobody-alkaline phosphatase fusion protein and its application in a highly sensitive direct competitive fluorescence enzyme immunoassay for detection of ochratoxin A in cereal. Anal Chem. 87(2):1387-94. PMID: 25531426. PMCID: PMC4476795 [U50 OH007550, P42 ES004699]
Type: Journal Articles Status: Published Year Published: 2015 Citation: Cui, Y., D. Li, C. Morisseau, J. Dong, J. Yang, D. Wan, M. Rossotti, G. Gonzalez, S.J. Gee and B.D. Hammock. Heavy chain single-domain antibodies to detect native human soluble epoxide hydrolase. Anal Bioanal Chem. 407(24):7275-83. PMID: 26229025. PMCID: PMC4573264. [P42 ES004699, R01 ES002710]
Type: Journal Articles Status: Published Year Published: 2015 Citation: Ranganathan, A., S.J. Gee and B.D. Hammock. An immunoassay for the detection of triclosan-O-glucuronide, a primary human urinary metabolite of triclosan. Anal Bioanal Chem. 407(24):7263-73. PMID: 26255293. PMCID:PMC4580343 [P42 ES004699]
Type: Journal Articles Status: Published Year Published: 2015 Citation: Hua, X., L. Zhou, L. Feng, Y. Ding, H. Shi, L. Wang, S.J. Gee, B.D. Hammock and M. Wang. Competitive and noncompetitive phage immunoassays for the determination of benzothiostrobin. Anal Chim Acta. 890:150-56. PMID: 26347177. PMCID:PMC4791039 [P42 ES004699, U50 OH007550]
Type: Journal Articles Status: Published Year Published: 2015 Citation: Yin, W., X. Hua, X. Liu, H. Shi, S.J. Gee, M. Wang and B.D. Hammock. Development of an enzyme-linked immunosorbent assay for thiacloprid in soil and agro-products with phage-displayed peptide. Anal Biochem. 481:27-32. PMID: 25908560.
Type: Journal Articles Status: Published Year Published: 2015 Citation: Rossotti, M., M. Pirez, A. Gonzalez-Techera, Y. Cui, C. Bever, K.S.S. Lee, C. Morisseau, C. Leizagoyen, S.J. Gee, B.D. Hammock and G. Gonzalez-Sapienza. A method for sorting and pairwise selection of nanobodies for the development of highly sensitive sandwich immunoassays. Anal Chem. 87(23):11907-14. PMID: 26544909. PMCID: PMC4666776. [P42 ES004699, K99 ES024806, P30 ES023513]
Type: Journal Articles Status: Published Year Published: 2016 Citation: Ahn, K.C., A. Ranganathan, S.H. Hwang, E. Fritsch, K. Morisseau, I. Pessah, B.D. Hammock and S.J. Gee. Detection of the antimicrobial triclosan in environmental samples by immunoassay. Environ Sci Technol. 50(7):3754-61. PMID: 26937944. PMCID: PMC4821808. [P42 ES004699, P30 ES023513, U54 OH007550]
Type: Journal Articles Status: Published Year Published: 2015 Citation: Shu, M., Y. Xu, X. Liu, Y. Li, Q. He, Z. Tu, J. Fu, S.J. Gee and B.D. Hammock. Anti-idiotypic nanobodyalkaline phosphatase fusion proteins: Development of a one-step competitive enzyme immunoassay for fumonisin B1 detection in cereal. Anal Chim Acta. 92453-59. PMID: 27181644. [Did not cite any of our grants]
Type: Journal Articles Status: Published Year Published: 2016 Citation: Bever, C.S., J.X. Dong, N. Vasylieva, B. Barnych, Y. Cui, Z.L. Xu, B.D. Hammock and S.J Gee. VHH antibodies: Emerging reagents for the analysis of environmental chemicals. Anal Bioanal Chem. 408(22):5985-6002. PMID: 27209591. PMC: PMCID4983233. [P42 ES004699, U54 OH007550, U54 NS079202, P30 ES023513]
Type: Journal Articles Status: Published Year Published: 2016 Citation: Wagner, S.D, T. Kurobe, B.G. Hammock, C.H. Lam, G. Wu, N. Vasylieva, S.J. Gee, B.D. Hammock and S.J. Teh. Developmental effects of fipronil on Japanese Medaka (Oryzias latipes) embryos. Chemosphere 166:511-20. PMID: 27710886.[P42 ES004699, U50, OH07550]
Type: Journal Articles Status: Published Year Published: 2015 Citation: Wang, J., Z. Majkova, C.R.S. Bever, J. Yang, S.J. Gee, J. Li, T. Xu, B.D. Hammock. One-step immunoassay for tetrabromobisphenol A using a camelid single domain antibody-alkaline phosphatase fusion protein. Anal Chem. 87(9):4741-48. PMID: 25849972. PMCID: PMC4476793 [P42 ES004699, 2U50 OH007550]
Type: Journal Articles Status: Published Year Published: 2015 Citation: Vasylieva, N., K.C. Ahn, B. Barnych, S.J. Gee and B.D. Hammock. Development of an immunoassay for the detection of the phenylpyrazole insecticide fipronil. Environ Sci Technol. 49(16):10038-47. PMID: 26196357. PMCID: PMC4605820 [P42 ES004699, U50 OH07550, U54 NS079202]

Progress 10/01/14 to 09/30/15

Outputs
Target Audience:Target Audience The tools we develop specifically are for industry (for their product stewardship), regulators, environmental scientists and public health professionals interested in human exposure to agricultural chemical and environmental and industrial chemicals. Depending on the assay Federal and State Agencies and International Agencies are relevant. These include but are not limited to NIOSH, NIEHS, NINDS, NIDDK, EPA, FDA and others. Some like NIOSH, NIEHS, and related agencies are through grant reporting. The technologies are broader since immunoassays and sensors are used extensively in clinical medicine with both human and veterinary application. These research tools are used to monitor food safety, insure rootstock and seed are free of pathogens and many other applications. Academically we reach scientists interested in antibody development, development of biosensors, analysis of pesticides and other environmental chemicals, and nanobody development. The assays are research tools for a variety of projects ranging from the health of the Sacramento Delta and San Francisco Bay to worker exposure in the Central Valley. Target analytes range from pesticides and terror weapons to industrial chemicals. Changes/Problems:Increasingly we are moving from classical polyclonal and monoclonal antibodies to VHH reagents or nanobodies which are produced recombinantly in bacteria after they are raised in llama and alpaca and screened or panned with a bacteriophage display system. This is far more powerful and cost effective. What opportunities for training and professional development has the project provided?Training and professional development for: 3 Ph.D. students 1 Visiting Ph.D. students 1 Visiting postdoctoral trainee 1 Visiting faculty 6 Postdoctoral trainees How have the results been disseminated to communities of interest?yes, see above What do you plan to do during the next reporting period to accomplish the goals?yes, see above

Impacts
What was accomplished under these goals? lmmunoassays, that have been used in clinical medicine for many years, are an important tool for the analytical chemist when approaching problems in analysis of food and feed. Data that the analytical chemist generates is important to regulators and other stakeholders for making decisions about animal, human and environmental health. The technology is being applied for monitoring products of genetic engineering, food adulterants, nutritional components and pesticides, for example. lmmunoassays use an antibody to bind to the chemical or toxin of interest and then an enzyme or other label is used to detect the binding. The technique termed ELISA or enzyme linked immunosorbent assay has dominated the immunoassay field. We propose to make immunoassays to pesticides or other compounds of immediate interest to the agricultural sector while making significant improvements in the technology to increase the speed, lower the limit of detectable chemical, lengthen the stability of reagents and enhance the field portability of the tests. In this year we utilized assays for antimicrobial agents triclocarban and triclosan. Some mechanistic studies with triclosan indicate that triclosan is a liver tumor promoter. This was selected as one of the 20 best studies supported by NIEHS for the year. An assay for a pyrethroid metabolite was used in three separate studies; one to examine the exposure of farm worker families in the Central Valley of California, second to compare exposure between farmers and consumers in Thailand to these insecticides. Specifically we developed assays to support food and forage safety. These include pesticides such as fibronil, neonicatinoids, pyrthroids, and organophosphates and the fungicide malachite green/leucomalachite green used in aquaculture. We developed new detection methods using PCR for organophosphorus insecticides and ochratoxin A, a novel impededance biosensor for polybrominated diphenyl ether flame retardants and collaborated with and engineer on a nanoparticle array detector. We also have adapted our immunoassays to field assays run on a cell phone platform. A novel platform was developed for rapid detection of Staphylococcal enterotoxin B which is one of the most sensitive assays ever developed for this serious pathogen. We utilized novel recombinant alpaca antibodies or nanobodies in the development of a method to detect the flame retardant tetrabromobisphenol A and the mycotoxin ochratoxin A. We have modified these reagents using recombinant technology to make the assays faster and more sensitive. The development of VHH or nanobody technology is a major accomplishment for this laboratory. We continue to be active on assays for personal care products such as triclosan and triclocarban. As mentioned above we are expanding our work on mycotoxins and food safety exemplified by recent work on ochratoxin and aflatoxins and food flavor chemicals. Not only do we make these assays but we also apply them. For example we have applied our pyrethroid metabolite immunoassay to a study of migrant worker exposure to pyrethroids in the central valley of California. Our productivity remains infinite with 18 peer review publications and no resources. Overall, we have made progress in the utilization of existing assays to monitor chemicals of interest to agriculture and human health, to develop new assays and the explore novel methods in order to reach the goals of the project.

Publications

Type: Journal Articles Status: Published Year Published: 2013 Citation: Vanrell, L., A. Gonz�lez-Techera, B.D. Hammock and G. Gonzalez-Sapienza. 2013. Nanopeptamers for the Development of Small-Analyte Lateral Flow Tests with a Positive Readout. Anal Chem. 85(2):1177-82. PMID: 23214940. PMCID: PMC3904493. [P42 ES04699, U54 OH007550]
Type: Journal Articles Status: Published Year Published: 2013 Citation: Han, J.H., H.J. Kim, S. Lakshmana, S.J. Gee, B.D. Hammock and I.M. Kennedy. 2013. Photonic crystal lab-on-a-chip for detecting Staphylococcal enterotoxin B at low attomolar concentration. Anal Chem. 85(6):3104-09. PMID: 23418954. PMCID: PMC3608201. [P42 ES004699]
Type: Journal Articles Status: Published Year Published: 2013 Citation: Lee, E.H., Y.A. Kim, Y.T. Lee, B.D. Hammock and H.S. Lee. 2013. Competitive immunochromatographic assay for the detection of the organophosphorus pesticide EPN. Food Agric Immunol. 24(2): 129-38. PMID: 21504817. PMCID: PMC3927651 [/]
Type: Journal Articles Status: Published Year Published: 2013 Citation: Wang, Y., H. Wang, P.W. Li, Q. Zhang, H.J. Kim, S.J. Gee and B.D. Hammock. 2013. Phage-displayed peptide that mimics aflatoxins and its application in immunoassay. J Agric Food Chem. 61(10):2426-33. PMID: 23394544. PMCID: PMC3830680 [Available on 2014/3/13]. [P42 ES004699, U50 OH07550, CounterAct, U54 NS079202]
Type: Journal Articles Status: Published Year Published: 2013 Citation: Ranganathan, A., G. Paradise, C. Hansen, M.R. McCoy, S.J. Gee, P. Zhong, D. Chang, B.D. Hammock. 2013. An indirect homologous competitive enzyme-linked immunosorbent assay for the detection of a class of glycosylated dihydrochalcones. J Agric Food Chem. 61(28):6964-70. NIHMS497122. PMID: 23767873. PMCID: PMC3723731. [P42 ES004699, PHS OH07550]
Type: Journal Articles Status: Published Year Published: 2013 Citation: Wang, Y., P. Li, Z. Majkova, C.R. Spier, H.J. Kim, Q. Zhang, J.E. Dechant, S.J. Gee and B.D. Hammock. 2013 Sept. Isolation of alpaca anti-idiotypic heavy chain single domain antibody for the aflatoxin immunoassay. Anal Chem. 85(17): 8298-303. PMID: 23965250. PMCID: PMC3787825. [P42 ES04699, U50 OH07550, CounterAct Program, U54 NS079202]
Type: Journal Articles Status: Published Year Published: 2014 Citation: Thiphom, S., T. Prapamontol, S. Chantara, A. Mangklabruks, C. Suphavilai, K.C. Ahn, S.J. Gee and B.D. Hammock. 2014. Determination of the pyrethroid insecticide metabolite 3-PBA in plasma and urine samples from farmer and consumer groups in northern Thailand. J Environ Sci Health B. 49(1):15-22. PMID: 24138464. PMCID: PMC4020914. [P42 ES04699, U50 OH07550]
Type: Journal Articles Status: Published Year Published: 2013 Citation: Trunnelle, K.J., D.H. Bennett, D.J. Tancredi, S.J. Gee, M.T. Stoecklin-Marois, T.E. Hennessy-Burt, B.D. Hammock and M.B. Schenker. 2013 Oct. Pyrethroids in house dust from the homes of farm worker families in the MICASA study. Environ Int. 61C:57-63. PMID: 24096042. PMCID: PMC4059492. [U50 OH007550, R01 OH009293, P42 ES004699]

Progress 10/01/13 to 09/30/14

Outputs
Target Audience: The tools we develop specifically are for industry (for their product stewardship), regulators, environmental scientists and public health professionals interested in human exposure to agricultural chemical and environmental and industrial chemicals. Depending on the assay Federal and State Agencies and International Agencies are relevant. These include but are not limited to NIOSH, NIEHS, NINDS, NIDDK, EPA, FDA and others. Some like NIOSH, NIEHS, and related agencies are through grant reporting. The technologies are broader since immunoassays and sensors are used extensively in clinical medicine with both human and veterinary application. These research tools are used to monitor food safety, insure rootstock and seed are free of pathogens and many other applications. Academically we reach scientists interested in antibody development, development of biosensors, analysis of pesticides and other environmental chemicals, and nanobody development. The assays are research tools for a variety of projects ranging from the health of the Sacramento Delta and San Francisco Bay to worker exposure in the Central Valley. Target analytes range from pesticides and terror weapons to industrial chemicals. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Training and professional development for: 5 Ph.D. students 5 Student visitors 6 Faculty visitors 8 Undergraduates 12 Postdoctoral trainees How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Immunoassays, that have been used in clinical medicine for many years, are an important tool for the analytical chemist when approaching problems in analysis of food and feed. Data that the analytical chemist generates is important to regulators and other stakeholders for making decisions about animal, human and environmental health. The technology is being applied for monitoring products of genetic engineering, food adulterants, nutritional components and pesticides, for example. Immunoassays use an antibody to bind to the chemical or toxin of interest and then an enzyme or other label is used to detect the binding. The technique termed ELISA or enzyme linked immunosorbent assay has dominated the immunoassay field. We propose to make immunoassays to pesticides or other compounds of immediate interest to the agricultural sector while making significant improvements in the technology to increase the speed, lower the limit of detectable chemical, lengthen the stability of reagents and enhance the field portability of the tests. In this year we utilized assays for antimicrobial agents triclocarban and triclosan. Some mechanistic studies with triclosan indicate that triclosan is a liver tumor promoter. An assay for a pyrethroid metabolite was used in three separate studies; one to examine the exposure of farm worker families in the Central Valley of California, second to compare exposure between farmers and consumers in Thailand to these insecticides and the third study developed a method for measuring pyrethroids in orange oil. We developed assays for the fungicide malachite green/leucomalachite green used in aquaculture. We developed new detection methods using PCR for organophosphorus insecticides and ochratoxin A, a novel impededance biosensor for polybrominated diphenyl ether flame retardants and collaborated with and engineer on a nanoparticle array detector. We utilized novel antibodies in the development of a method to detect the flame retardant tetrabromobisphenol A and the mycotoxin ochratoxin A. Overall, we have made progress in the utilization of existing assays to monitor chemicals of interest to agriculture and human health, to develop new assays and the explore novel methods in order to reach the goals of the project.

Publications

Type: Journal Articles Status: Published Year Published: 2014 Citation: Ahn, K.C., T. Kasagami, H.J. Tsai, N.H. Schebb, T. Ogunyoku, S.J. Gee and B.D. Hammock. 2012. An immunoassay to evaluate human/environmental exposure to the antimicrobial triclocarban. Environ Sci Technol. 46(1):374-81.
Type: Journal Articles Status: Published Year Published: 2014 Citation: Kim, H.J., M. Mccoy, Z. Majkova, J. Dechant, S.J. Gee, S. Tabares-da Rosa, G.G. Gonzalez-Sapienza and B.D. Hammock. 2012. Isolation of alpaca anti-hapten heavy chain single domain antibodies for development of sensitive immunoassay. Anal Chem. 84(2):1165-71.
Type: Journal Articles Status: Published Year Published: 2014 Citation: Schebb, N.H., K.C. Ahn, H. Dong, S.J. Gee and B.D. Hammock. 2012. Whole blood is the sample matrix of choice for monitoring systemic triclocarban levels. Chemosphere. 87(7):825-27.
Type: Journal Articles Status: Published Year Published: 2014 Citation: McCoy, M., Z. Yang, X. Fu, K.C. Ahn, S.J. Gee, D. Bom, P. Zhong, D. Chang and B.D. Hammock. 2012. Monitoring of total type II pyrethroid pesticides in citrus oils and water by converting to a common product 3-phenoxybenzoic acid. J Agric Food Chem. 60(20):5065-70.
Type: Journal Articles Status: Published Year Published: 2014 Citation: Han, J.H., L. Sudheendra, H.J. Kim, S.J. Gee, B.D. Hammock and I.M. Kennedy. 2012. Ultrasensitive on-chip immunoassays with a nanoparticle-assembled photonic crystal. ACS Nano. 6(10):8570-82.
Type: Journal Articles Status: Published Year Published: 2014 Citation: Thiphom, S., T. Prapamontol, S. Chantara, A. Mangklabruks, C. Suphavilai, K.C. Ahn, S.J. Gee and B.D. Hammock. 2012. An enzyme-linked immunosorbent assay for detecting 3-phenoxybenzoic acid in plasma and its application in farmers and consumers. Anal Methods. 4(11):3772-78.
Type: Journal Articles Status: Published Year Published: 2014 Citation: Han, J.H., H.J. Kim, L. Sudheendra, E.A. Hass, S.J. Gee, B.D. Hammock and I.M. Kennedy. 2012. Electrophoretic build-up of multi nanoparticle array for a highly sensitive immunoassay. Biosens Bioelectron. 41:302-08.
Type: Journal Articles Status: Published Year Published: 2014 Citation: Trunnelle, K.J., D.H. Bennett, K.C. Ahn, M. Schenker, D.J. Tancredi, S.J Gee, M.T. Stoecklin-Marois and B.D. Hammock. 2014. Concentrations of the urinary pyrethroid metabolite 3-phenoxybenzoic acid in farm worker families in the MICASA Study. Environ Res. 131C:153-59.
Type: Journal Articles Status: Published Year Published: 2014 Citation: Radhakrishnan, R., I. Suni, C.S. Bever, and B.D. Hammock. 2014. Impedance biosensors: Applications to sustainability, and remaining technical challenges. ACS Sustain Chem Eng. 2:1649-55. PMID: 25068095.
Type: Journal Articles Status: Published Year Published: 2014 Citation: Liu, X., Y. Xu, Y. Xiong, Z. Tu, Y.P. Li, Q. He, Z. He, Y. Qiu, J. Fu, L. Xiong, S.J. Gee and B.D. Hammock. 2014 Aug. VHH-phage based competitive real-time immuno-PCR for ultrasensitive detection of ochratoxin A in cereal. Anal Chem. 86(15):7471-77.
Type: Journal Articles Status: Published Year Published: 2014 Citation: Bever, C.R.S, Z. Majkova, R. Radhakrishnan, I. Suni, M. McCoy, Y. Wang, J. Dechant, S.J. Gee and B.D. Hammock. 2014 Aug. Development and utilization of camelid VHH antibodies from alpaca for 2,2,4,4-tetrabrominated diphenyl ether detection. Anal Chem. 86(15):7875-82. PMID: 25005746.
Type: Journal Articles Status: Published Year Published: 2014 Citation: Wang, J., C.R.S. Bever, Z. Majkova, J.E. Dechant, J. Yang, S.J. Gee, T. Xu and B.D. Hammock. 2014 Aug. Heterologous antigen selection of camelid heavy chain single domain antibodies against tetrabromobisphenol A. Anal Chem. 86(16):8296-302.
Type: Journal Articles Status: Published Year Published: 2014 Citation: Dong, J., C. Xu, H. Wang, Z. Xiao, S.J. Gee, Z. Li, F. Wang, W. Wu, Y. Shen, J.Y. Yang, Y. Sun, and B.D. Hammock. 2014 Aug. Enhanced sensitive immunoassay: noncompetitive phage anti-immune complex assay for the determination of malachite green and leucomalachite green. J Agric Food Chem. 62(34):8752-58.
Type: Journal Articles Status: Published Year Published: 2014 Citation: Hua, X., W. Yin, H. Wang, Y. Wang, H.J. Kim, S.J. Gee, M. Wang, F. Liu and B.D. Hammock. 2014 Aug. Development of phage immuno-loop-mediated isothermal amplification assays for organophosphorus pesticides in agro-products. Anal Chem. 86(16):8441-47.
Type: Journal Articles Status: Published Year Published: 2014 Citation: Yueh, M.F., K. Taniguchi, S. Chen, R.M. Evans, B.D. Hammock, M. Karin and R.H. Tukey. 2014 Dec. The commonly used antimicrobial additive triclosan is a liver tumor promoter. Proc Natl Acad Sci USA. 111(48):17200-05.

Progress 01/01/13 to 09/30/13

Outputs
Target Audience: Peer scientists through peer-reviewed publications, scientific meetings Peer scientists and the public through laboratory and program websites and social media Federal agencies, NIOSH, NIEHS through grant reporting activities FDA though scientific publications on triclosan Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Training and professional development for 4 Ph.D. students 10 Students visitors 3 Faculty visitors 16 Postdocs How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? During the last year we have developed numerous assays for monitoring food and forage as well as animals and humans for pesticides and other environmental contaminants. We have developed new technologies for this including the use of alpaca - phage generated nanobodies and lanthanide based sensors. We have examined the biology of these materials and based on some of them we are developing assays to treat neuropathic diseases such as equine laminitis, inflammation and diabetes. We have also found deleterious effects for example with the environmental contaminant triclocarban. Our productivity in publications per unit funding continues to increase each year during which we receive no funding. This is reported in peer review publications, national and international meetings, a laboratory and program web site, social media, and the laboratory runs a technology transfer and community engagement core. Our technologies have benefited stake holders through state and federal agencies including NIEHS and NIOSH. Outcome impact There have been numerous outcomes and impacts from this work. One recent impact was the announcement by the US FDA that industry would have to show benefit to continue including triclocarban in personal care products. This was based largely on work from UCD. We have provided reagents from the laboratory for immunoassay and enzyme assay and therapy to over 50 laboratories a year. We also are working on antidotes to chemical threat agents. Thus the work combines to ensure animal and human food is safe from contaminants and that we understand how the contaminants act to cause health effects.

Publications

Type: Journal Articles Status: Published Year Published: 2013 Citation: Wang, Y., P. Li, Z. Majkova, C.R. Spier, H.J. Kim, Q. Zhang, J.E. Dechant, S.J. Gee and B.D. Hammock. 2013 Sept. Isolation of alpaca anti-idiotypic heavy chain single domain antibody for the aflatoxin immunoassay. Anal Chem. 85(17): 8298-303
Type: Journal Articles Status: Published Year Published: 2013 Citation: Ranganathan, A., G. Paradise, C. Hansen, M.R. McCoy, S.J. Gee, P. Zhong, D. Chang, B.D. Hammock. 2013. An indirect homologous competitive enzyme-linked immunosorbent assay for the detection of a class of glycosylated dihydrochalcones. J Agric Food Chem. 61(28):6964-70.
Type: Journal Articles Status: Published Year Published: 2013 Citation: Trunnelle, K.J., D.H. Bennett, D.J. Tancredi, S.J. Gee, M.T. Stoecklin-Marois, T.E. Hennessy-Burt, B.D. Hammock and M.B. Schenker. 2013 Oct. Pyrethroids in house dust from the homes of farm worker families in the MICASA study. Environ Int. 61C:57-63.
Type: Journal Articles Status: Published Year Published: 2013 Citation: Thiphom, S., T. Prapamontol, S. Chantara, A. Mangklabruks, C. Suphavilai, K.C. Ahn, S.J. Gee and B.D. Hammock. 2013. Determination of the pyrethroid insecticide metabolite 3-PBA in plasma and urine samples from farmer and consumer groups in northern Thailand. J Environ Sci Health B. 49:15-22.
Type: Journal Articles Status: Published Year Published: 2013 Citation: Wang, Y., H. Wang, P.W. Li, Q. Zhang, H.J. Kim, S.J. Gee and B.D. Hammock. 2013. Phage-displayed peptides that mimic aflatoxins and its application in immunoassay. J Agric Food Chem. 61(10):2426-33
Type: Journal Articles Status: Published Year Published: 2013 Citation: Lee, E.H., Y.A. kim, Y.T. Lee, B.D. Hammock and H.S. Lee. 2013. Competitive immunochromatographic assay for the detection of the organophosphorus pesticide EPN. Food Agric Immunol. 24(2): 129-38.
Type: Journal Articles Status: Published Year Published: 2013 Citation: Han, J.H., H.J. Kim, S. Lakshmana, S.J. Gee, B.D. Hammock and I.M. Kennedy. 2013. Photonic crystal lab-on-a-chip for detecting Staphylococcal enterotoxin B at low attomolar concentration. Anal Chem. 85(6):3104-09.
Type: Journal Articles Status: Published Year Published: 2012 Citation: Vanrell, L., A. Gonz�lez-Techera, B.D. Hammock and G. Gonzalez-Sapienza. 2013. Nanopeptamers for the Development of Small-Analyte Lateral Flow Tests with a Positive Readout. Anal Chem . 85(2):1177-82.
Type: Journal Articles Status: Published Year Published: 2012 Citation: Lee, J.Y., Y. A. Kim, M. Y. Kim, Y. T. Lee, B.D. Hammock and H.S. Lee. 2012. Importance of membrane selection in the development of immunochromatographic assays for low-molecular weight compounds. Anal Chim Acta. 757:69-74.
Type: Journal Articles Status: Published Year Published: 2012 Citation: Schebb N.H., B.A. Buchholz, B.D. Hammock, and R.H. Rice. Metabolism of the antibacterial triclocarban by human epidermal keratinocytes to yield protein adducts. J Biochem Mol Toxicol.26(6):230-34.
Type: Journal Articles Status: Published Year Published: 2012 Citation: Cherednichenko, G., R. Zhang, R.A. Bannister, V. Timofeyev, N. Li, E.B. Fritsch, W. Feng, G.C. Barrientos, N.H. Schebb, B.D. Hammock, K.G. Beam, N. Chiamvimonvat and I.N. Pessah. 2012. Triclosan impairs excitation-contraction coupling and Ca2+ dynamics in striated muscle. Proc Natl Acad Sci U.S.A. 109(35):14158-63.
Type: Journal Articles Status: Published Year Published: 2012 Citation: Schebb, N.H., B. Franze, R. Maul, A. Ranganathan and B.D. Hammock. 2012. In vitro glucuronidation of the antibacterial triclocarban and its oxidative metabolites. Drug Metab Dispos. 40(1):25-31.
Type: Journal Articles Status: Published Year Published: 2012 Citation: Schebb N.H., B.A. Buchholz, B.D. Hammock, and R.H. Rice. 2012. Metabolism of the antibacterial triclocarban by human epidermal keratinocytes to yield protein adducts. J Biochem Mol Toxicol.26(6):230-34.
Type: Journal Articles Status: Published Year Published: 2012 Citation: Yueh, M.F., T. Li, R.M. Evans, B.D. Hammock and R.H. Tukey. 2012. Triclocarban mediates induction of xenobiotic metabolism through activation of the constitutive androstane receptor and the estrogen receptor alpha. PLoS One. 7(6):e37705.

Progress 01/01/12 to 12/31/12

Outputs
OUTPUTS: Specifically during the last period we have reported on rapid field assays for the antimicrobial agent triclocarban in addition to our continuing work on general pesticides including pyrethroids. Most reports during this period focus on the improving immunoassays through a variety of technologies including use of nanoparticle labels, a phage amplification systems and through development of novel nanobodies. Each of these technologies result in incremental improvements to the immunoassays as well as improving speed, sensitivity and robustness for field applications. In addition to publications this technology has been distributed through NIOSH for worker safety, NIEHS (environmental health research) and numerous state and federal agencies. We have trained scientists from multiple countries in this technology through an ongoing training program supported by the Fulbright Foundation as well as other routes. Of course this work is presented at national and International meetings. PARTICIPANTS: In this reporting period three postdoctoral researchers contributed to the by forming hypotheses, designing and conducting experiments, interacting with colleagues and collaborators and publishing the results. We collaborated with researchers from one University of California campus, one research organization from Thailand, one university in Uruguay and partnered with two companies in the U.S. We have trained two undergraduates from UC Davis, one UC Davis graduate student, 3 foreign graduate students, 6 post-doctoral researchers, and 1 foreign faculty member. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
Assays developed last year and the years immediately prior to have been used to measure exposure of humans to pesticides and antimicrobial compounds demonstrating a practical application. The same assays can now be used to monitor exposure in livestock or feed and food. The application of such rapid assays for environmental contaminants helps to ensure animal and food safety from contamination with fire retardants, industrial chemicals, personal care products, pesticides and other materials. This now has been extended to biological (rift valley fever, ebola, blue tongue) threats. From a more basic standpoint we have continued to develop transduction technologies (predominantly lanthanide nanoparticles), new immunoassay amplification processes (phage amplification PHAIA) and radically new methods of making immunoassays (nanobodies) that are widely applicable from home pregnancy kits, to veterinary surveillance to research.

Publications

Han, J.H., H.J. Kim, L. Sudheendra, E.A. Hass, S.J. Gee, B.D. Hammock and I.M. Kennedy. 2012. Electrophoretic build-up of multi nanoparticle array for a highly sensitive immunoassay. Biosens Bioelectron. 41:302-08. PMCID: PMC 3525773
Thiphom, S., T. Prapamontol, S. Chantara, A. Manhklabruks, C. Suphavilai, K.C. Ahn, S.J. Gee and B.D. Hammock. 2012. An enzyme-linked immunosorbent assay for detecting 3-phenoxybenzoic acid in plasma and its application in farmers and consumers. Anal Methods. 4(11):3772-78. DOI: 10.1039/c2ay25642h
Han, J.H., L. Sudheendra, H.J. Kim, S.J. Gee, B.D. Hammock and I.M. Kennedy. 2012. Ultrasensitive on-chip immunoassays with a nanoparticle-assembled photonic crystal. ACS Nano. 6(10):8570-82. PMID: 22957818. PMCID: PMC3479307
Ahn, K.C., T. Kasagami, H.J. Tsai, N.H. Schebb, S.J. Gee and B.D. Hammock. 2012. An immunoassay to evaluate human/environmental exposure to the antimicrobial triclocarban. Env. Sci. Technol. 46(1):374-81. PMID:22077920. PMCID: PMC 3258304
McCoy, M., Z. Yang, X. Fu, K.C. Ahn, S.J. Gee, D. Bom, P. Zhong, D. Chang and B.D. Hammock. 2012. Monitoring of total type II pyrethroid pesticides in citrus oils and water by converting to a common product 3-phenoxybenzoic acid. J Agric Food Chem. 60(20):5065-70. PMID: 22486225. PMCID: PMC3412423
Schebb, N.H., K.C. Ahn, H. Dong, S.J. Gee and B.D. Hammock. 2012. Whole blood is the sample matrix of choice for monitoring systemic triclocarban levels. Chemosphere. 87(7):825-27. NIHMS348170. PMID: 22273184. PMCID: PMC3538789

Progress 01/01/11 to 12/31/11

Outputs
OUTPUTS: During the last year we have continued to make and use rapid analytical methods for environmental contaminants such as pyrethroid insecticides, herbicides, personal care products and other materials. We have utilized these assays to monitor for these materials in the environment and in human body fluids as indicators of exposure. In addition we also have advanced immunochemical technologies along several fronts. These advances can be applied to immunoassays for human and animal health as well as for environmental analysis. One dramatic advance is the use of heat stable recombinant nanobodies cloned from alpaca. These new binding reagents promise to revolutionize immunoassay development. We immunize alpaca with multiple antigens, collect cDNA from white blood cells, pcr the nanobody single chain genes and place into phage, then pan phage for the desired antibodies. These antibodies can be produced in large amounts in E. coli and used for a variety of immunoassays. During the last year we developed the most sensitive nanobody based assays ever reported for small molecules. In addition we continue to develop phage amplified immunoassays to improve sensitivity and specificity of immunoassays. This technology can be applied to any immunoassay by using phage to bind to the antigen antibody complex. This results in a 5-50x improvement in sensitivity. Finally, we are using novel fluorescent lanthanide nanoparticles to enhance the sensitivity of immunoassays. These materials are particularly suited to biosensor and microfluidic formats. Productivity expressed in terms of publications per unit of funding has reached an exceptionally high level since no funding has been received for many years. From a basic standpoint we are increasing dramatically the sensitivity and specificity of immunoassays in general by integrating the above technological advances. We are applying these technologies to evaluate exposure of farm workers and their families to pesticides and other environmental contaminants. These targets include exposure to pyrethroids in the central valley of California and exposure to the highly toxic and newly reregistered pesticide paraquat to field workers in Hawaii. PARTICIPANTS: The laboratory has been involved in the career development of the following scientists. Assistant Research and Project Scientists (Hee-Joo Kim, Ahmet Bora Inceoglu, Ki Chang Ahn, Christophe Morisseau), postdoctoral researchers (Mark McCoy, Nils Helge Schebb, Anu Ranganathan, Zuzana Majkova) and pre-doctoral students (Pavel Aronov). We have collaborated with Drs. Julie Dechant and Jerry Last at UCDavis. We have partnered with Dr. Robert I. Krieger (University of California Riverside), Dr. A.M. Calafat (CDC), Dr. Gualberto G. Gonzalez-Sapienza (University of Montevideo, Uruguay) and Dr. H.S Lee (Kyungpook National University, Korea). In addition our reagents are available gratis through UC Davis Technology Transfer to academic and non profit organizations and for license to for profit organizations. We have interacted with NIEHS, EPA, NIOSH, and CDC at the national level and with state and local laboratories. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
The above technologies have permitted us to monitor workers for exposure to pesticides as an example of human monitoring, the monitoring of foam in furniture sold in California for the use of illegal fire retardants, the monitoring of biosolids from sewage treatment for the presence of personal care products such as triclosan and triclocarban present in such things as solid soaps, liquid hand cleaners, toothpaste and other products. These results have been presented at meetings and communicated to regulatory agencies including CDFA, US EPA and US NIOSH.

Publications

Ahn, K.C., H-J. Kim, M.R. McCoy, S.J. Gee and B.D. Hammock. 2011. Immunoassays and biosensors for monitoring environmental and human exposure to pyrethroid insecticides. J. Agric. Food Chem. 59(7):2792-2802. PMCID: PMC3070843
Schebb, N.H., B. Inceoglu, K.C. Ahn, C. Morisseau, S.J. Gee and B.D. Hammock. 2011. Investigation of human exposure to triclocarban after showering and preliminary evaluation of its biological effects. Environ. Sci. Technol. 45(7):3109-3115. NIHMS278856.
Kim, Y.A., E.H. Lee, K.O. Kim, Y.T. Lee, B.D. Hammock and H.S. Lee. 2011. Competitive immunochromatographic assay for the detection of the organophosphorus pesticide chlorpyrifos. Anal. Chim. Acta 693(1-2):106-113.
Ye, X., X. Zhou, J. Furr, K.C. Ahn, B. Hammock, E.L. Gray and A.M. Calafat. 2011. Biomarkers of exposure to triclocarban in urine and serum. Toxicology 286(1-30):69-74.
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. PMCID: PMC3031424
Ahn, K.C., S.J. Gee, H.J. Kim, P. Aronov, H. Vega, R.I. Krieger, B.D. Hammock. 2011. Immunochemical analysis of 3-phenoxybenzoic acid, a biomarker of forestry worker exposure to pyrethroid insecticides. Anal. Bioanal. Chem. 401 (4):1285-93.
Tabares da Rosa, S., M. Rossotti, C. Carleiza, F. Carrion, O. Pritsch, K.C. Ahn, J. Last, B. Hammock, G. 2011. Gonzalez-Sapienza. Competitive selection from single domain antibody libraries allows isolation of high-affinity antihapten antibodies that are not favored in the llama immune response. Anal Chem. 83(18)7213-7220. PMCID:PMC 3193053.
Schebb, N.H., B. Franze, R. Maul, A. Ranganathan and B.D. Hammock. In vitro glucuronidation of the antibacterial triclocarban and its oxidative metabolites. 2012. Drug Metab Dispos. 40(1):25-31.
Shim, J.Y., Y.A. Kim, Y.T. Lee, B.D. Hammock and H.S. Lee. 2010. Monoclonal antibody-based enzyme-linked immunosorbent assays for the organophosphorus insecticide O-ethyl O-4-nitrophenyl phenylphosphonothioate (EPN). J Agric Food Chem. 58(9):5241-7.
Gonzalez-Techera, A., L. Vanrell, J.A. Last, B.D. Hammock and G. Gonzalez-Sapienza. 2007. Phage anti-immune complex assay: general strategy for noncompetitive immunodetection of small molecules. Anal Chem. 79(20):7799-806. PMCID: PMC2519879.
Kim, H.J., M. Mccoy, Z. Majkova, J. Dechant, S. Gee, S. Tabares-da Rosa, G. Gonzalez-Sapienza, B.D. Hammock. 2011. Isolation of alpaca anti-hapten heavy chain single domain antibodies for development of sensitive immunoassay. Anal Chem. 84:1165-1171. PMCID: PMC3264785

Progress 01/01/10 to 12/31/10

Outputs
OUTPUTS: Specifically during the last period we have reported on rapid field assays for clomazone, dioxin, triclocarban, and the fire retardant pbde in addition to our continuing work on general pesticides including pyrethroids, triazines, and others. We have increased the speed, reproducibility and sensitivity on of immunoassays with a variety of technologies. The most dramatic improvement is through the implementation of PHIAI. This phage amplification technique increases the absolute sensitivity of all small molecule immunoassays 5-50 X. We have continued our engineering collaboration on the development of optical biosensors which also increase the speed, sensitivity and reproducibility of immunoassays using microfluidic technologies. In addition to publications this technology has been distributed through NIOSH for worker safety, NIEHS (environmental health research) and numerous state and federal agencies. We have trained scientists from multiple countries in this technology through an on going training program supported by the Fulbright Foundation as well as other route. Of course this work is presented at national and International meetings. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
From the standpoint of immediate application rapid assays for environmental contaminants help to insure food safety from contamination with fire retardants, industrial chemicals, personal care products, pesticides and other materials. This now has been extended both to chemical (TETs, parathion) and biological (rift valley fever, ebola, blue tongue) threats. From a more basic standpoint we had developed transduction technologies (predominantly lanthanide nanoparticles), new immunoassay amplification processes (phage amplification PHAIA) and radically new methods of making immunoassays (nanobodies) that are widely applicable from home pregnancy kits, to veterinary surveillance to research.

Publications

752. Kim, H-J., M.A. Rossotti, K.C. Ahn, G.G. Gonzalez-Sapienza, S.J. Gee, R. Musker and B.D. Hammock. 2010. Development of a noncompetitive phage anti-immunocomplex assay for brominated diphenyl ether 47. Analytical Biochemistry 401:38-46. PMCID: PMC2861364 755. Carlomagno, M., C. Matho, G. Cantou, J.R. Sanborn, J.A. Last, B.D. Hammock, A. Roel, D. Gonzalez and G. Gonzalez-Sapienza. 2010. A clomazone immunoassay to study the environmental fate of the herbicide in rice (Oryza sativa) agriculture. J. Agric. Food Chem. 58(7):4367-4371. NIHMS189902 759. Garcia-Nieto, E., M. Nichkova, L. Yanez, R. Costilla-Salazar, A. Torres-Dosal, S.J. Gee, B.D. Hammock, L. Juarez-Santacruz and F. Diaz-Barriga. 2010. Assessment of dioxin-like soil contamination in Mexico by enzyme-linked immunosorbent assay. Arch. Environ. Contam. Toxicol. 58(4):918-926. PMID: 20091164 774. Baumann, A., W. Lohmann, T. Rose, K.C. Ahn, B.D. Hammock, U. Karst and N.H. Schebb. 2010. Electrochemistry-mass spectrometry unveils the formation of reactive triclocarban metabolites. Drug Metab. Dispos. 38(12): 2130-2138. PMCID: PMC2993458 [Available on 2011/12/1] 775. Rossotti, M.A., M. Carlomagno, A. Gonzalez-Techera, B.D. Hammock, J. Last and G. Gonzalez-Sapienza. 2010. Phage anti-immunocomplex assay for clomazone: Two-site recognition increasing assay specificity and facilitating adaptation into an on-site format. Anal. Chem. 82(21):8838-8843. NIHMS242038

Progress 01/01/09 to 12/31/09

Outputs
OUTPUTS: This project develops sensitive laboratory and field based biosensors for pesticides, environmental contaminants, bioterror weapons, and other materials. We have improved the immunoassay technology by developing a novel phage driven amplification system which is generally applicable to all immunoassays from home pregnancy kits through high throughput drug screening. We also have developed the technology to produce recombinant, heat stable single chain antibodies from llama and alpaca. In collaboration with engineers we are developing optical transduction systems including composite fluorescent nanoparticles for microfluidic assays and a novel nanoparticle - nanowell system demonstrating a new concept in highly sensitive analytical chemistry. These technological improvements are generally applicable to biosensors and immunoassays used in medicine, agriculture, and environmental science. We have developed a number of immunoassays and applied many of them to environmental problems. A partial list includes application of insecticide and herbicide assays to orange oil. The movement back to natural citrus oils has resulted in a dramatic increase in pesticide levels in fruit drinks. We have focused on several bioterror products with ricin as a completed example and work on TETs being initiated. Assays for personal care products and fire retardants are needed since these materials are present at far higher concentrations in the environment than pesticides and many of them are environmentally stable. Output continues high considering no input. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
This project has resulted in numerous practical technologies which have been transferred to other academic laboratories, state agencies and the federal government. Of particular note this year we have worked on the brominated fire retardants. These are highly stable materials in the environment with levels in household dust of 1500 ppm (>10 6th more than most pesticides). There may be over 5 pounds of these materials in a couch and 30% of a plastic TV cabinet may be fire retardants. These are high volume chemicals of questionable value in most of their uses but their use is required by antiquated regulations. Among other things we developed a sensitive field assay for PBDE47 and demonstrated that this illegal compound still was commonly detected in furniture stores in California. This assay is being used to illustrate other biosensor technologies developed in the laboratory. As usual no resources were provided for this project.

Publications

Ma, Z., D. Dosev, M. Nichkova, R.K Dumas, S.J. Gee, B.D. Hammock, K. Liu and I.M. Kennedy. 2009. Synthesis and characterization of multifunctional silica core-shell nanocomposites with magnetic and fluorescent functionalities. J. Magnetism & Magnetic Materials 321:1368-1371.
Ma, Z.Y., D. Dosev, M. Nichkova, S.J. Gee, B.D. Hammock and I.M. Kennedy. 2009. Synthesis and bio-functionalization of multifunctional magnetic Fe3O4@Y2O3:Eu nanocomposites. Journal of Materials Chemistry 19:4695-4700.
Nichkova, M., X. Fu, Z. Yang, P. Zhong, J.R. Sanborn, D. Chang, S.J. Gee and B.D. Hammock. 2009. Immunochemical screening of pesticides (simazine and cypermethrin) in orange oil. J. Agric. Food Chem. 57(13):5673-5679.
Shen, S., F. Zhang, S. Zeng, Y. Tian, X. Chai, S. Gee, B.D. Hammock and J. Zheng. 2009. Development of enantioselective polyclonal antibodies to detect styrene oxide protein adducts. Anal. Chem. 81(7):2668-2677.
Ahn, K.C., S.J. Gee, H-J. Tsai, D. Bennett, M.G. Nishioka, A. Blum, E. Fishman and B.D. Hammock. 2009. Immunoassay for monitoring environmental and human exposure to the polybrominated diphenyl ether BDE-47. Environ. Sci. Technol. 43(20):7784-7790

Progress 01/01/08 to 12/31/08

Outputs
OUTPUTS: In this project we develop rapid, field portable, and highly quantitative assays for environmental contaminants in feed, forage, food products and the environment. We also improve immunochemical technology to make all such assays better, faster and cheaper. During the last year the anti microbials triclosan and triclocarban have emerged as the most abundant chlorinated organics found in sludge. This is commonly applied to pasture and agricultural fields. We have developed and are developing new immunoassays for these materials as well as for fire retardants commonly found in milk, meat and meat products. We participated in the largest human exposure study ever done on the highly toxic herbicide paraquat used to produce commercial coffee and bananas. We also have developed the first highly sensitive lc-ms assays for dihydroxy vitamin D3 and its metabolites in milk. We continue to develop immunoassays for a variety of other materials including tetrachlorodioxin, pyrethroid pesticides, and possible biowarefare agents and others. We have developed three technologies which improve immunoassay technology generally. For example we have developed lanthanide nanoparticles as reporters and carriers for microfluidic and dip stick immunoassays. Theses materials increase precision and sensitivity of the resulting assays. The particles have a wide Stokes' shift, no bleaching, long relaxation times and narrow emission spectra allowing assays to be multiplexed. We also developed phase display systems to coat plates with peptidomimetics which accelerate the development of heterologous immunoassays. We finally developed phase display systems to improve the sensitivity and specificity of immunoassays. These later technologies are generally applicable to immunoassays. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
There are numerous outcomes for this project. One that is of particular note is the study integrating job task in agriculture, paraquat exposure, respiratory physiology and general health of farm workers. This exposure study would not have been possible without the development of a sensitive, selective immunoassay for paraquat. There was exposure for all workers, sprayers had the highest levels. Only in one case in case of an accident was there significant exposure. In general the paraquat coffee and banana plantation workers where healthier than age matched non workers and healthier than age matched Americans. There were no resources provided for this project.

Publications

Kim, H.-J.A. Gonzalez-Techera, G.G. Gonzalez-Sapienza, K.C. Ahn, S.J. Gee and B.D. Hammock. 2008. Phage-borne peptidomimetics accelerate the development of polyclonal antibody-based heterologous immunoassays for the detection of pesticide metabolites. Environ. Sci. Technol. 42:2047-2053. doi: 10.1021/es702219a. Gonzalez-Techera, A., H.-J. Kim, S.J. Gee, J.A Last, B.D. Hammock and G. Gonzalez-Sapienza. 2007. Polyclonal antibody-based noncompetitive immunoassay for small analytes developed with short peptide loops isolated from phage libraries. Anal. Chem. 79:9191-9196. doi: 10.1021/ac7016713 Van Emon, J.M., J.C. Chuang, R.A. Lordo, M.E. Schrock, M. Nichkova, S.J. Gee and B.D. Hammock. 2008. An enzyme-linked immunosorbent assay for the determination of dioxins in contaminated sediment and soil samples. Chemosphere 72:95-103. doi:10.1016/j.chemosphere.2008.01.012 Lee, K., E-K. Park, M. Stoecklin-Marois, M.E. Koivunen, S.J. Gee, B.D. Hammock, L.A. Beckett and M.B. Schenker. 2008. Occupational paraquat exposure of agricultural workers in large Costa Rica farms. Int. Arch. Occup. Environ. Health DOI 10.1007/s00420-008-0356-7 Hwang, H-M., E-K. Park, T.M. Young and B.D. Hammock. 2008. Occurrence of endocrine-disrupting chemicals in indoor dust. Sci. Total Environ. 404:26-35. DOI: 10.1016/j.scitotenv.2008.05.031 Gonzalez-Techera, A., M. Umpierrez-Failache, S. Cardozo, G. Obal, O. Pritsch, J.A. Last, S.J. Gee, B.D. Hammock and G. Gonzalez-Sapienza. 2008. High-throughput method for ranking the affinity of peptide ligands selected from phage display libraries. Bioconjugate Chem. 19:993-1000. DOI: 10.1021/bc700279y Ahn, K.C., B. Zhao, J. Chen, G. Cherednichenko, E. Sanmarti, M.S. Denison, B. Lasley, I.N. Pessah, D. Kultz, D.P.Y. Chang, S.J. Gee and B.D. Hammock. 2008. In vitro biologic activities of the antimicrobials triclocarban, its analogs, and triclosan in bioassay screens: receptor-based bioassay screens. Environ. Health. Persp. 16:1203-1210. DOI: 10.1289/ehp.11200 Park, E-K., H.D. Tagles, S.J. Gee, B.D. Hammock, K. Lee and M.B. Schenker. 2008. Recruiting strategy and 24-hour biomonitoring of paraquat in agricultural workers. J. Agromedicine 13(4):207-217. Trindade, M., M. Nording, M. Nichkova, E. Spinnel, P. Haglung, M.S. Last, S. Gee, B. Hammock, J. A. Last, G. Gonzalez-Sapienza and B.M. Brena. 2008. Enzyme-linked immunosorbent assay for screening dioxin soil contamination by uncontrolled combustion during informal recycling in slums. Environmental Toxicology and Chemistry 27(11):2224-2232. Kim, H-J., K. Ahn, A. Gonzalez-Techera, G.G. Gonzales-Sapienza, S.J. Gee and B.D. Hammock. 2009. Magnetic bead-based phage anti-immunocomplex assay (PHAIA) for the detection of the urinary biomarker 3-phenoxybenzoic acid to assess human exposure to pyrethroid insecticides. Anal Biochem. 386(1):45-52. PMID: 19101498

Progress 01/01/07 to 12/31/07

Outputs
As described in the proposal and in review papers published this year it is critical to human and animal health that we know the levels of environmental chemicals to which we are exposed. This laboratory is unique in having the capability to carry out these analyses both by instrumental methods (GLC MS, LC MS) and rapid immunoassay. Pyrethroids are the major insecticides used world wide and their use pattern indicates both human and environmental exposure. We have not only developed methods to detect classes of and specific compounds in the environment but also to detect specific biomarkers of exposure in human body fluids. We have also targeted the personal care products (PCPs) as major materials of concern in publications listed below. We are developing immunoassays for triclosan and triclocarban and have developed LC MS methods to monitor human exposure, which is quite high. These materials are now among the most important halogenated environmental contaminants as well as being present in samples of blood and urine of most Americans. Not only are we developing and employing specific assays but we are advancing the technology of immmunodetection. This is illustrated by our use of nanotechnology to develop fluorescent reporter systems to multiplex assays and miniaturized assays. These fluorescent materials have allowed us to move immunoassays toward microfluidic biosensors, develop internal standard to improve assay precision and to multiplex assays so that multiple assays can be run simultaneously.

Impacts
Our continued work in this area has resulted in rapid, inexpensive analytical methods to monitor the environment, feed and forage and human body fluids for exposure to agricultural chemicals and personal care products. We have advanced the technology of immunochemical detection which extends beyond environmental chemistry to all immunoassays including clinical chemistry.

Publications

Kim, H.-J., K.C. Ahn, S.J. Ma, S.J. Gee and B.D. Hammock. 2007. Development of sensitive immunoassays for the detection of the glucuronide conjugate of 3-phenoxybenzyl alcohol, a putative human urinary biomarker for pyrethroid exposure. J. Agr. Food Chem. 55:3750-3757.
Nichkova, M., D. Dosev, A.E. Davies, S.J. Gee, I.M. Kennedy and B.D. Hammock. 2007. Quantum dots as reporters in multiplexed immunoassays for biomarkers of exposure to agrochemicals. Anal. Lett. 40:1423-1433.
Ahn, K.-C., S.J. Gee, H.-J. Kim, M. Nichkova, N.A. Lee and B.D. Hammock. 2007. Progress in the development of biosensors for environmental and human monitoring of agrochemicals. In: Symposium 225: Rational Environmental Management of Agrochemicals: Risk Assessment, Monitoring and Remedial Action: Proceedings of the American Chemical Society Pacifichem 2005, Honolulu, HI (I.R. Kennedy, K. Solomon, S.J. Gee, A. Crossan, S. Wang, F. Sanchez-Bayo eds.), Vol. 966 pp.138-154.
Nichkova, M., D. Dosev, S.J. Gee, B.D. Hammock and I.M. Kennedy. 2007. Multiplexed immunoassays for proteins using magnetic luminescent nanoparticles for internal calibration. Anal. Biochem. 369:34-40.
Dosev, D., M. Nichkova, R.K. Dumas, S.J. Gee, B.D Hammock, K. Liu and I.M. Kennedy. 2007. Magnetic/luminescent core/shell particles synthesized by spray pyrolysis and their application in immunoassays with internal standard. Nanotechnology. 18:055102 (6pp) doi:10.1088/0957-4484/18/5/055102.
Koivunen, M.E., S.J. Gee, M. Nichkova, K.C. Ahn and B.D. Hammock. 2007. Monitoring human exposure to pesticides using immunoassay. In: ACS Symposium Series, Assessing Exposures and Reducing Risks to People from the Use of Pesticides (R.I. Krieger, J.N. Seiber, N. Ragsdale and J.N. Seiber, eds.), Vol.951, pp. 141-156, American Chemical Society, Washington D.C.
Yuan, W., J. Chung, S.J. Gee, B.D. Hammock and J. Zheng. 2007. Development of polyclonal antibodies for detection of styrene oxide modified proteins. Chem. Res. Toxicol. 20:316-321.
Chen, J., K.C. Ahn, N.A. Gee, S.J. Gee, B.D. Hammock and B.L. Lasley. 2007. Antiandrogenic properties of parabens and other phenolic containing small molecules in personal care products. Toxicol. Appl. Pharm. 221:278-284.