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Candace S. Bever
Foodborne Toxin Detection and Prevention Research Unit, Western Regional Research Center, United States Department of Agriculture, Agricultural Research Service, Albany, California, United States of America

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Research article
Published: 17 April 2020 in PLOS ONE
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The mushroom poison that causes the most deaths is the class of toxins known as amatoxins. Current methods to sensitively and selectively detect these toxins are limited by the need for expensive equipment, or they lack accuracy due to cross-reactivity with other chemicals found in mushrooms. In this work, we report the development of a competition-based lateral flow immunoassay (LFIA) for the rapid, portable, selective, and sensitive detection of amatoxins. Our assay clearly indicates the presence of 10 ng/mL of α-AMA or γ-AMA and the method including extraction and detection can be completed in approximately 10 minutes. The test can be easily read by eye and has a presumed shelf-life of at least 1 year. From testing 110 wild mushrooms, the LFIA identified 6 out of 6 species that were known to contain amatoxins. Other poisonous mushrooms known not to contain amatoxins tested negative by LFIA. This LFIA can be used to quickly identify amatoxin-containing mushrooms.

ACS Style

Candace S. Bever; Catharine A. Adams; Robert M. Hnasko; Luisa W. Cheng; Larry H. Stanker. Lateral flow immunoassay (LFIA) for the detection of lethal amatoxins from mushrooms. PLOS ONE 2020, 15, e0231781 .

AMA Style

Candace S. Bever, Catharine A. Adams, Robert M. Hnasko, Luisa W. Cheng, Larry H. Stanker. Lateral flow immunoassay (LFIA) for the detection of lethal amatoxins from mushrooms. PLOS ONE. 2020; 15 (4):e0231781.

Chicago/Turabian Style

Candace S. Bever; Catharine A. Adams; Robert M. Hnasko; Luisa W. Cheng; Larry H. Stanker. 2020. "Lateral flow immunoassay (LFIA) for the detection of lethal amatoxins from mushrooms." PLOS ONE 15, no. 4: e0231781.

Journal article
Published: 15 February 2020 in Toxins
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Globally, mushroom poisonings cause about 100 human deaths each year, with thousands of people requiring medical assistance. Dogs are also susceptible to mushroom poisonings and require medical assistance. Cyclopeptides, and more specifically amanitins (or amatoxins, here), are the mushroom poison that causes the majority of these deaths. Current methods (predominantly chromatographic, as well as antibody-based) of detecting amatoxins are time-consuming and require expensive equipment. In this work, we demonstrate the utility of the lateral flow immunoassay (LFIA) for the rapid detection of amatoxins in urine samples. The LFIA detects as little as 10 ng/mL of α-amanitin (α-AMA) or γ-AMA, and 100 ng/mL of β-AMA in urine matrices. To demonstrate application of this LFIA for urine analysis, this study examined fortified human urine samples and urine collected from exposed dogs. Urine is sampled directly without the need for any pretreatment, detection from urine is completed in 10 min, and the results are read by eye, without the need for specialized equipment. Analysis of both fortified human urine samples and urine samples collected from intoxicated dogs using the LFIA correlated well with liquid chromatography–mass spectrometry (LC-MS) methods.

ACS Style

Candace S. Bever; Kenneth D. Swanson; Elizabeth I. Hamelin; Michael Filigenzi; Robert H. Poppenga; Jennifer Kaae; Luisa W. Cheng; Larry H. Stanker. Rapid, Sensitive, and Accurate Point-of-Care Detection of Lethal Amatoxins in Urine. Toxins 2020, 12, 123 .

AMA Style

Candace S. Bever, Kenneth D. Swanson, Elizabeth I. Hamelin, Michael Filigenzi, Robert H. Poppenga, Jennifer Kaae, Luisa W. Cheng, Larry H. Stanker. Rapid, Sensitive, and Accurate Point-of-Care Detection of Lethal Amatoxins in Urine. Toxins. 2020; 12 (2):123.

Chicago/Turabian Style

Candace S. Bever; Kenneth D. Swanson; Elizabeth I. Hamelin; Michael Filigenzi; Robert H. Poppenga; Jennifer Kaae; Luisa W. Cheng; Larry H. Stanker. 2020. "Rapid, Sensitive, and Accurate Point-of-Care Detection of Lethal Amatoxins in Urine." Toxins 12, no. 2: 123.

Journal article
Published: 11 December 2019 in Toxins
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Amatoxins (AMAs) are lethal toxins found in a variety of mushroom species. Detection methods are needed to determine the occurrence of AMAs in mushroom species suspected in mushroom poisonings. In this manuscript, we report the generation of novel monoclonal antibodies (mAbs, AMA9G3 and AMA9C12) and the development of a competitive, enzyme-linked immunosorbent assay (cELISA) that is sensitive at 1 ng mL−1 and shows selectivity for α-amanitin (α-AMA) and γ-amanitin (γ-AMA), and less for β-amanitin (β-AMA). In order to decrease the overall time needed for analysis, the extraction procedure for mushrooms was also simplified. A rapid (1 min) extraction procedure of AMAs using solvents as simple as water alone was successfully demonstrated using Amanita mushrooms. Together, the extraction method and the mAb-based ELISA represent a simple and rapid method that readily detects AMAs extracted from mushroom samples.

ACS Style

Candace S. Bever; Robert M. Hnasko; Luisa W. Cheng; Larry H. Stanker. A Rapid Extraction Method Combined with a Monoclonal Antibody-Based Immunoassay for the Detection of Amatoxins. Toxins 2019, 11, 724 .

AMA Style

Candace S. Bever, Robert M. Hnasko, Luisa W. Cheng, Larry H. Stanker. A Rapid Extraction Method Combined with a Monoclonal Antibody-Based Immunoassay for the Detection of Amatoxins. Toxins. 2019; 11 (12):724.

Chicago/Turabian Style

Candace S. Bever; Robert M. Hnasko; Luisa W. Cheng; Larry H. Stanker. 2019. "A Rapid Extraction Method Combined with a Monoclonal Antibody-Based Immunoassay for the Detection of Amatoxins." Toxins 11, no. 12: 724.

Comparative study
Published: 01 September 2019 in Analytical Biochemistry
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Enzyme labeling of an antigen or an antibody helps to visualize and amplify the signal and is an important reagent used in immunoassays for the detection of a target of interest. In this research, soybean peroxidase (SBP), a less commonly used enzyme reporter, was compared in immunoassays with the two most commonly used reagents, horseradish peroxidase (HRP) and alkaline phosphatase (ALP). The enzyme-antibody conjugates were evaluated by their performance in an indirect competitive enzyme-linked immunosorbent assay (icELISA) and in an indirect competitive chemiluminescent enzyme immunoassay (icCLEIA) for ractopamine (RAC). The results revealed that the more affordable SBP offers a long-lasting chemiluminescent signal, which outperformed ALP and HRP. SBP-antibody conjugate (SBP-Ab) based immunoassays produced lower limits of detection (LODs) and better accuracy in the detection of RAC in animal urine samples. Additionally, SBP-Ab has advantages in being more resistant to heat, acid and organic solvents. These results suggest that SBP could be a potentially excellent alternative to HRP and ALP for the development of immunoassay in food safety field.

ACS Style

Huijuan Yang; Candace Bever; Huiyan Zhang; Ghulam Mujtaba Mari; Hongfang Li; Xiya Zhang; Liuchuan Guo; Ziwen Wang; Pengjie Luo; Zhanhui Wang. Comparison of soybean peroxidase with horseradish peroxidase and alkaline phosphatase used in immunoassays. Analytical Biochemistry 2019, 581, 113336 .

AMA Style

Huijuan Yang, Candace Bever, Huiyan Zhang, Ghulam Mujtaba Mari, Hongfang Li, Xiya Zhang, Liuchuan Guo, Ziwen Wang, Pengjie Luo, Zhanhui Wang. Comparison of soybean peroxidase with horseradish peroxidase and alkaline phosphatase used in immunoassays. Analytical Biochemistry. 2019; 581 ():113336.

Chicago/Turabian Style

Huijuan Yang; Candace Bever; Huiyan Zhang; Ghulam Mujtaba Mari; Hongfang Li; Xiya Zhang; Liuchuan Guo; Ziwen Wang; Pengjie Luo; Zhanhui Wang. 2019. "Comparison of soybean peroxidase with horseradish peroxidase and alkaline phosphatase used in immunoassays." Analytical Biochemistry 581, no. : 113336.

Journal article
Published: 13 July 2019 in Toxins
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Botulism is a devastating disease caused by botulinum neurotoxins (BoNTs) secreted primarily by Clostridium botulinum. Mouse bioassays without co-inoculation with antibodies are the standard method for the detection of BoNTs, but are not capable of distinguishing between the different serotypes (A–G). Most foodborne intoxications are caused by serotypes BoNT/A and BoNT/B. BoNT/E outbreaks are most often observed in northern coastal regions and are associated with eating contaminated marine animals and other fishery products. Sandwich enzyme-linked immunosorbent assays (ELISAs) were developed for the detection of BoNT/E3. Monoclonal antibodies (mAbs) were generated against BoNT/E3 by immunizing with recombinant peptide fragments of the light and heavy chains of BoNT/E3. In all, 12 mAbs where characterized for binding to both the recombinant peptides and holotoxin, as well as their performance in Western blots and sandwich ELISAs. The most sensitive sandwich assay, using different mAbs for capture and detection, exhibited a limit of detection of 0.2 ng/ml in standard buffer matrix and 10 ng/mL in fish product matrices. By employing two different mAbs for capture and detection, a more standardized sandwich assay was constructed. Development of sensitive and selective mAbs to BoNT/E would help in the initial screening of potential food contamination, speeding diagnosis and reducing use of laboratory animals.

ACS Style

Candace S. Bever; Miles Scotcher; Luisa W. Cheng; Robert M. Hnasko; Larry H. Stanker. Development and Characterization of Monoclonal Antibodies to Botulinum Neurotoxin Type E. Toxins 2019, 11, 407 .

AMA Style

Candace S. Bever, Miles Scotcher, Luisa W. Cheng, Robert M. Hnasko, Larry H. Stanker. Development and Characterization of Monoclonal Antibodies to Botulinum Neurotoxin Type E. Toxins. 2019; 11 (7):407.

Chicago/Turabian Style

Candace S. Bever; Miles Scotcher; Luisa W. Cheng; Robert M. Hnasko; Larry H. Stanker. 2019. "Development and Characterization of Monoclonal Antibodies to Botulinum Neurotoxin Type E." Toxins 11, no. 7: 407.

Research article
Published: 02 February 2019 in Journal of Consumer Protection and Food Safety
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Organophosphate and pyrethroid insecticides are used for controlling residential and agricultural insect pests and disease-carrying vectors in Thailand especially in the northern region. Chiang Rai and Nan Provinces are well known for agricultural production. The aim of the present study was to assess the level of exposure to organophosphate and pyrethroid insecticides in 51 consumers and 49 farmers living in Chiang Rai and Nan Provinces by monitoring the urinary metabolites 6-dialkylphosphates (DAPs) and 3-phenoxybenzoic acid (3-PBA) as biomarkers of exposure to organophosphates and pyrethroids. The results showed at least one metabolite was present in all subjects indicating that consumers and farmers were exposed to these pesticides. Concentrations of organophosphate and pyrethroid metabolites in consumers and farmers were similar except diethylthiophosphate which was significantly higher in farmers. Diethyldithiophosphate and 3-PBA concentrations were significantly different between the group who had reported using and not using pesticide in their house.

ACS Style

Surat Hongsibsong; Tippawan Prapamontol; Jie-Xian Dong; Candace S. Bever; Zhen-Lin Xu; Shirley J. Gee; Bruce D. Hammock. Exposure of consumers and farmers to organophosphate and synthetic pyrethroid insecticides in Northern Thailand. Journal of Consumer Protection and Food Safety 2019, 14, 17 -23.

AMA Style

Surat Hongsibsong, Tippawan Prapamontol, Jie-Xian Dong, Candace S. Bever, Zhen-Lin Xu, Shirley J. Gee, Bruce D. Hammock. Exposure of consumers and farmers to organophosphate and synthetic pyrethroid insecticides in Northern Thailand. Journal of Consumer Protection and Food Safety. 2019; 14 (1):17-23.

Chicago/Turabian Style

Surat Hongsibsong; Tippawan Prapamontol; Jie-Xian Dong; Candace S. Bever; Zhen-Lin Xu; Shirley J. Gee; Bruce D. Hammock. 2019. "Exposure of consumers and farmers to organophosphate and synthetic pyrethroid insecticides in Northern Thailand." Journal of Consumer Protection and Food Safety 14, no. 1: 17-23.

Research paper
Published: 26 November 2018 in Analytical and Bioanalytical Chemistry
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Phage-displayed peptides have been proven to be powerful reagents for competitive and noncompetitive immunoassays. However, they are unconventional reagents, which greatly limit their analytical commercial applications and require additional reagents for detection. In this work, the peptides that specifically bind with anti-benzothiostrobin monoclonal antibody (mAb) or benzothiostrobin-mAb immunocomplex were synthesized and conjugated with fluorescein isothiocyanate (FITC) as substitutes of the phage-displayed peptides to avoid their shortcomings and extend their applications. Competitive and noncompetitive fluorescence immunoassays (FIAs) for benzothiostrobin were developed by mAb coupling with magnetic nanoparticles as concentration elements and peptides conjugated with FITC as tracers. Compared with enzyme-linked immunosorbent assays, the FIAs reduced the number of steps from 6 to 2 and analysis time from more than 5 to 1.2 h. The competitive FIA showed the half-maximal inhibition concentration (IC50) of 16.8 ng mL−1 and detection range (IC10–IC90) of 1.0–759.9 ng mL−1, while the concentration of analyte producing 50% saturation of the signal (SC50) and detection range (SC10–SC90) of noncompetitive FIA were 93.4 and 5.9–788.2 ng mL−1, respectively. The average spiked recoveries were 68.33–98.50% and 73.33–96.67% for competitive and noncompetitive FIAs, respectively. The FIAs showed good correlation with high-performance liquid chromatography for the detection of benzothiostrobin in authentic samples.

ACS Style

He Chen; Qian Yang; Yuan Ding; Natalia Vasylieva; Candace Bever; Xiude Hua; Minghua Wang; Bruce D. Hammock. Competitive and noncompetitive immunoassays for the detection of benzothiostrobin using magnetic nanoparticles and fluorescein isothiocyanate-labeled peptides. Analytical and Bioanalytical Chemistry 2018, 411, 527 -535.

AMA Style

He Chen, Qian Yang, Yuan Ding, Natalia Vasylieva, Candace Bever, Xiude Hua, Minghua Wang, Bruce D. Hammock. Competitive and noncompetitive immunoassays for the detection of benzothiostrobin using magnetic nanoparticles and fluorescein isothiocyanate-labeled peptides. Analytical and Bioanalytical Chemistry. 2018; 411 (2):527-535.

Chicago/Turabian Style

He Chen; Qian Yang; Yuan Ding; Natalia Vasylieva; Candace Bever; Xiude Hua; Minghua Wang; Bruce D. Hammock. 2018. "Competitive and noncompetitive immunoassays for the detection of benzothiostrobin using magnetic nanoparticles and fluorescein isothiocyanate-labeled peptides." Analytical and Bioanalytical Chemistry 411, no. 2: 527-535.

Full paper
Published: 14 September 2018 in Electroanalysis
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Both direct and non‐competitive two‐site (sandwich) immunoassays are reported for 3‐phenoxybenzoic acid (3‐PBA) utilizing signal transduction by electrochemical impedance spectroscopy (EIS), and the sandwich immunoassay reduces the detection limit for this small molecule analyte by ∼70×. The direct EIS immunoassay utilizes a polyclonal antibody to 3PBA for biomolecular recognition, while the sandwich EIS immunoassay utilizes in addition a previously reported antiimmunocomplex M13 bacteriophage clone. For both immunoassays, the polyclonal antibody film is immobilized atop an Au electrode by amide bond formation. The direct EIS immunoassay exhibits a 3‐PBA sensitivity of 5.4×104 kΩ cm2 M1, and a detection limit of 2.5×10−4 M (54 μg/ml), while the sandwich EIS immunoassay exhibits a 3PBA sensitivity of 4.2×106 kΩ cm2 M−1, and a detection limit of 3.4×10−6 M (0.74 μg/ml). For the sandwich EIS immunoassay, a constant excess (80×109 PFU/ml) of bacteriophage is maintained during all experiments. This is the first report of a bacteriophage‐assisted sandwich EIS assay.

ACS Style

Madhavi Pali; Candace R. S. Bever; Natalia Vasylieva; Bruce D. Hammock; Ian I. Suni. Impedance Detection of 3-Phenoxybenzoic Acid with a Noncompetitive Two-site Phage Anti-immunocomplex Assay. Electroanalysis 2018, 30, 2653 -2659.

AMA Style

Madhavi Pali, Candace R. S. Bever, Natalia Vasylieva, Bruce D. Hammock, Ian I. Suni. Impedance Detection of 3-Phenoxybenzoic Acid with a Noncompetitive Two-site Phage Anti-immunocomplex Assay. Electroanalysis. 2018; 30 (11):2653-2659.

Chicago/Turabian Style

Madhavi Pali; Candace R. S. Bever; Natalia Vasylieva; Bruce D. Hammock; Ian I. Suni. 2018. "Impedance Detection of 3-Phenoxybenzoic Acid with a Noncompetitive Two-site Phage Anti-immunocomplex Assay." Electroanalysis 30, no. 11: 2653-2659.

Journal article
Published: 28 June 2018 in Toxins
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One of the deadliest mushrooms is the death cap mushroom, Amanita phalloides. The most toxic constituent is α-amanitin, a bicyclic octapeptide, which damages the liver and kidneys. To develop a new tool for detecting this toxin, polyclonal antibodies were generated and characterized. Both α- and β-amanitin were coupled to carrier proteins through four different linking chemistries, one of which has never before been described. These conjugates were evaluated for their effectiveness in generating antibodies specific for the free toxin, as well as their utility in formatting heterogeneous assays with high sensitivity. Ultimately, these efforts yielded a newly described conjugation procedure utilizing periodate oxidation followed by reductive amination that successfully resulted in generating sensitive immunoassays (limit of detection (LOD), ~1.0 µg/L). The assays were characterized for their selectivity and were found to equally detect α-, β-, and γ-amanitin, and not cross-react with other toxins tested. Toxin detection in mushrooms was possible using a simple sample preparation method. This enzyme-linked immunosorbent assay (ELISA) is a simple and fast test, and readily detects amatoxins extracted from A. phalloides.

ACS Style

Candace S. Bever; Bogdan Barnych; Robert Hnasko; Luisa W. Cheng; Larry H. Stanker. A New Conjugation Method Used for the Development of an Immunoassay for the Detection of Amanitin, a Deadly Mushroom Toxin. Toxins 2018, 10, 265 .

AMA Style

Candace S. Bever, Bogdan Barnych, Robert Hnasko, Luisa W. Cheng, Larry H. Stanker. A New Conjugation Method Used for the Development of an Immunoassay for the Detection of Amanitin, a Deadly Mushroom Toxin. Toxins. 2018; 10 (7):265.

Chicago/Turabian Style

Candace S. Bever; Bogdan Barnych; Robert Hnasko; Luisa W. Cheng; Larry H. Stanker. 2018. "A New Conjugation Method Used for the Development of an Immunoassay for the Detection of Amanitin, a Deadly Mushroom Toxin." Toxins 10, no. 7: 265.

Journal article
Published: 28 March 2018 in Chemosphere
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Triclosan is frequently used for its antimicrobial properties and has been detected in human serum, urine, and breast milk. Animal and molecular studies have shown that triclosan exerts a wide range of adverse health effects at both high (ppm) and low (ppb) concentrations. Since triclosan is of growing concern to human and environmental health, there is a need to improve extraction procedures and to study additional effects from triclosan exposure. In this study, we have improved triclosan extraction from breast milk by using salt (MgSO4) to reduce emulsion formation and increase water polarity and water (∼80%) to enhance the overall extraction efficiency (∼3.5 fold). This extraction method was applied to breast milk samples collected from donors who i) recorded their use of triclosan-containing personal care products and ii) provided matching infant stool samples. Of the participants who had detectable amounts of triclosan in their breast milk, nine (75%) of them reported daily use of triclosan-containing personal care products. Levels of triclosan in breast milk were compared to the donor's infant's fecal microbiome. We found that the bacterial diversity in the fecal microbiome of the infants exposed to breast milk with detectable triclosan levels differed compared to their peers exposed to milk containing non-detectable amounts. This finding implies that exogenous chemicals are impacting microbiome diversity.

ACS Style

Candace S. Bever; Amy A. Rand; Malin Nording; Diana Taft; Karen M. Kalanetra; David Mills; Melissa A. Breck; Jennifer T. Smilowitz; J. Bruce German; Bruce D. Hammock. Effects of triclosan in breast milk on the infant fecal microbiome. Chemosphere 2018, 203, 467 -473.

AMA Style

Candace S. Bever, Amy A. Rand, Malin Nording, Diana Taft, Karen M. Kalanetra, David Mills, Melissa A. Breck, Jennifer T. Smilowitz, J. Bruce German, Bruce D. Hammock. Effects of triclosan in breast milk on the infant fecal microbiome. Chemosphere. 2018; 203 ():467-473.

Chicago/Turabian Style

Candace S. Bever; Amy A. Rand; Malin Nording; Diana Taft; Karen M. Kalanetra; David Mills; Melissa A. Breck; Jennifer T. Smilowitz; J. Bruce German; Bruce D. Hammock. 2018. "Effects of triclosan in breast milk on the infant fecal microbiome." Chemosphere 203, no. : 467-473.

Research article
Published: 10 May 2017 in Analytical Chemistry
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Soluble epoxide hydrolase (sEH) is a potential pharmacological target for treating hypertension, vascular inflammation, cancer, pain, and multiple cardiovascular related diseases. A variable domain of the heavy chain antibody (termed single domain antibody (sdAb), nanobody, or VHH) possesses the advantages of small size, high stability, ease of genetic manipulation, and ability for continuous manufacture, making such nanobody a superior choice as an immunoreagent. In this work, we developed an ultrasensitive nanobody based immunoassay for human sEH detection using polymeric horseradish peroxidase (PolyHRP) for signal enhancement. Llama nanobodies against human sEH were used as the detection antibody in sandwich enzyme linked immunosorbent assays (ELISA) with polyclonal anti-sEH as the capture antibody. A conventional sandwich ELISA using a horseradish peroxidase (HRP) labeled anti-hemeagglutinin (HA) tag as the tracer showed a marginal sensitivity (0.0015 optical density (OD)·mL/ng) and limit of detection (LOD) of 3.02 ng/mL. However, the introduction of the PolyHRP as the tracer demonstrated a 141-fold increase in the sensitivity (0.21 OD·mL/ng) and 57-fold decrease in LOD (0.05 ng/mL). Systematic comparison of three different tracers in four ELISA formats demonstrated the overwhelming advantage of PolyHRP as a label for nanobody based immunoassay. This enhanced sEH immunoassay was further evaluated in terms of selectivity against other epoxide hydrolases and detection of the target protein in human tissue homogenate samples. Comparison with an enzyme activity based assay and a Western blot for sEH detection reveals good correlation with the immunoassay. This work demonstrates increased competiveness of nanobodies for practical sEH protein detection utilizing PolyHRP. It is worthwhile to rediscover the promising potential of PolyHRP in nanobody and other affinity based methods after its low-profile existence for decades.

ACS Style

Dongyang Li; Yongliang Cui; Christophe Morisseau; Shirley J. Gee; Candace S. Bever; Xiangjiang Liu; Jian Wu; Bruce D. Hammock; Yibin Ying. Nanobody Based Immunoassay for Human Soluble Epoxide Hydrolase Detection Using Polymeric Horseradish Peroxidase (PolyHRP) for Signal Enhancement: The Rediscovery of PolyHRP? Analytical Chemistry 2017, 89, 6248 -6256.

AMA Style

Dongyang Li, Yongliang Cui, Christophe Morisseau, Shirley J. Gee, Candace S. Bever, Xiangjiang Liu, Jian Wu, Bruce D. Hammock, Yibin Ying. Nanobody Based Immunoassay for Human Soluble Epoxide Hydrolase Detection Using Polymeric Horseradish Peroxidase (PolyHRP) for Signal Enhancement: The Rediscovery of PolyHRP? Analytical Chemistry. 2017; 89 (11):6248-6256.

Chicago/Turabian Style

Dongyang Li; Yongliang Cui; Christophe Morisseau; Shirley J. Gee; Candace S. Bever; Xiangjiang Liu; Jian Wu; Bruce D. Hammock; Yibin Ying. 2017. "Nanobody Based Immunoassay for Human Soluble Epoxide Hydrolase Detection Using Polymeric Horseradish Peroxidase (PolyHRP) for Signal Enhancement: The Rediscovery of PolyHRP?" Analytical Chemistry 89, no. 11: 6248-6256.

Review
Published: 21 May 2016 in Analytical and Bioanalytical Chemistry
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A VHH antibody (or nanobody) is the antigen binding fragment of heavy chain only antibodies. Discovered nearly 25 years ago, they have been investigated for their use in clinical therapeutics and immunodiagnostics, and more recently for environmental monitoring applications. A new and valuable immunoreagent for the analysis of small molecular weight environmental chemicals, VHH will overcome many pitfalls encountered with conventional reagents. In the work so far, VHH antibodies often perform comparably to conventional antibodies for small molecule analysis, are amenable to numerous genetic engineering techniques, and show ease of adaption to other immunodiagnostic platforms for use in environmental monitoring. Recent reviews cover the structure and production of VHH antibodies as well as their use in clinical settings. However, no report focuses on the use of these VHH antibodies to detect small environmental chemicals (MW < 1500 Da). This review article summarizes the efforts made to produce VHHs to various environmental targets, compares the VHH-based assays with conventional antibody assays, and discusses the advantages and limitations in developing these new antibody reagents particularly to small molecule targets.

ACS Style

Candace S. Bever; Jie-Xian Dong; Natalia Vasylieva; Bogdan Barnych; Yongliang Cui; Zhen-Lin Xu; Bruce D. Hammock; Shirley J. Gee. VHH antibodies: emerging reagents for the analysis of environmental chemicals. Analytical and Bioanalytical Chemistry 2016, 408, 5985 -6002.

AMA Style

Candace S. Bever, Jie-Xian Dong, Natalia Vasylieva, Bogdan Barnych, Yongliang Cui, Zhen-Lin Xu, Bruce D. Hammock, Shirley J. Gee. VHH antibodies: emerging reagents for the analysis of environmental chemicals. Analytical and Bioanalytical Chemistry. 2016; 408 (22):5985-6002.

Chicago/Turabian Style

Candace S. Bever; Jie-Xian Dong; Natalia Vasylieva; Bogdan Barnych; Yongliang Cui; Zhen-Lin Xu; Bruce D. Hammock; Shirley J. Gee. 2016. "VHH antibodies: emerging reagents for the analysis of environmental chemicals." Analytical and Bioanalytical Chemistry 408, no. 22: 5985-6002.

Research article
Published: 18 March 2016 in Environmental Science & Technology
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A sensitive, competitive enzyme-linked immunosorbent assay (ELISA) for the detection of the antimicrobial triclosan (TCS; 2,4,4'-trichloro-2'-hydroxydiphenyl ether) was developed. Novel immunizing haptens were synthesized by derivatizing at the 4-Cl position of the TCS molecule. Compounds derived from substitutions at 4'-Cl and that replaced the 2'-OH with a Cl atom were designed as unique coating antigen haptens. Polyclonal rabbit antisera were screened against the coating antigen library to identify combinations of immunoreagents resulting in the most sensitive assays. The most sensitive assay identified was one utilizing antiserum no. 1155 and a heterologous competitive hapten, where the 2'-OH group was substituted with a Cl atom. An IC50 value and the detection range for TCS in assay buffer were 1.19 and 0.21-6.71 μg/L, respectively. The assay was selective for TCS, providing low cross-reactivity (<5%) to the major metabolites of TCS and to brominated diphenyl ether-47. A second assay utilizing a competitive hapten containing Br instead of Cl substitutions was broadly selective for both brominated and chlorinated diphenylethers. Using the most sensitive assay combination, we measured TCS concentrations in water samples following dilution. Biosolid samples were analyzed following the dilution of a simple solvent extract. The immunoassay results were similar to those determined by LC-MS/MS. This immunoassay can be used as a rapid and convenient tool to screen for human and environmental exposure.

ACS Style

Ki Chang Ahn; Anupama Ranganathan; Candace S. Bever; Sung Hee Hwang; Erika Beth Holland; Kevin Morisseau; Isaac N. Pessah; Bruce D. Hammock; Shirley J. Gee. Detection of the Antimicrobial Triclosan in Environmental Samples by Immunoassay. Environmental Science & Technology 2016, 50, 3754 -3761.

AMA Style

Ki Chang Ahn, Anupama Ranganathan, Candace S. Bever, Sung Hee Hwang, Erika Beth Holland, Kevin Morisseau, Isaac N. Pessah, Bruce D. Hammock, Shirley J. Gee. Detection of the Antimicrobial Triclosan in Environmental Samples by Immunoassay. Environmental Science & Technology. 2016; 50 (7):3754-3761.

Chicago/Turabian Style

Ki Chang Ahn; Anupama Ranganathan; Candace S. Bever; Sung Hee Hwang; Erika Beth Holland; Kevin Morisseau; Isaac N. Pessah; Bruce D. Hammock; Shirley J. Gee. 2016. "Detection of the Antimicrobial Triclosan in Environmental Samples by Immunoassay." Environmental Science & Technology 50, no. 7: 3754-3761.

Research article
Published: 18 November 2015 in Analytical Chemistry
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Single domain heavychain binders (nanobodies) obtained from camelid antibody libraries hold a great promise for immunoassay development. However, there is no simple method to select the most valuable nanobodies from the crowd of positive clones obtained after the initial screening. In this paper, we describe a novel nanobody-based platform that allows comparison of the reactivity of hundreds of clones with the labeled antigen, and identifies the best nanobody pairs for two-site immunoassay development. The output clones are biotinylated in vivo in 96-well culture blocks and then used to saturate the biotin binding capacity of avidin coated wells. This standardizes the amount of captured antibody allowing their sorting by ranking their reactivity with the labeled antigen. Using human soluble epoxide hydrolase (sEH) as a model antigen, we were able to classify 96 clones in four families and confirm this classification by sequencing. This provided a criterion to select a restricted panel of five capturing antibodies and to test each of them against the rest of the 96 clones. The method constitutes a powerful tool for epitope binning, and in our case allowed development of a sandwich ELISA for sEH with a detection limit of 63 pg/mL and four log dynamic range, which performed with excellent recovery in different tissue extracts. This strategy provides a systematic way to test nanobody pairwise combinations and would have a broad utility for the development of highly sensitive sandwich immunoassays.

ACS Style

Martín A. Rossotti; Macarena Pirez; Andres Gonzalez-Techera; Yongliang Cui; Candace S. Bever; Kin S. S. Lee; Christophe Morisseau; Carmen Leizagoyen; Shirley Gee; Bruce D. Hammock; Gualberto González-Sapienza. Method for Sorting and Pairwise Selection of Nanobodies for the Development of Highly Sensitive Sandwich Immunoassays. Analytical Chemistry 2015, 87, 11907 -11914.

AMA Style

Martín A. Rossotti, Macarena Pirez, Andres Gonzalez-Techera, Yongliang Cui, Candace S. Bever, Kin S. S. Lee, Christophe Morisseau, Carmen Leizagoyen, Shirley Gee, Bruce D. Hammock, Gualberto González-Sapienza. Method for Sorting and Pairwise Selection of Nanobodies for the Development of Highly Sensitive Sandwich Immunoassays. Analytical Chemistry. 2015; 87 (23):11907-11914.

Chicago/Turabian Style

Martín A. Rossotti; Macarena Pirez; Andres Gonzalez-Techera; Yongliang Cui; Candace S. Bever; Kin S. S. Lee; Christophe Morisseau; Carmen Leizagoyen; Shirley Gee; Bruce D. Hammock; Gualberto González-Sapienza. 2015. "Method for Sorting and Pairwise Selection of Nanobodies for the Development of Highly Sensitive Sandwich Immunoassays." Analytical Chemistry 87, no. 23: 11907-11914.

Research article
Published: 22 April 2015 in Analytical Chemistry
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Tetrabromobisphenol A (TBBPA) is a ubiquitous brominated flame retardant, showing widespread environmental and human exposures. A variable domain of the heavy chain antibody (VHH), naturally occurring in camelids, approaches the lower size limit of functional antigen-binding entities. The ease of genetic manipulation makes such VHHs a superior choice to use as an immunoreagent. In this study, a highly selective anti-TBBPA VHH T3-15 fused with alkaline phosphatase (AP) from E. coli was expressed, showing both an integrated TBBPA-binding capacity and enzymatic activity. A one-step immunoassay based on the fusion protein T3-15-AP was developed for TBBPA in 5% dimethyl sulfoxide (DMSO)/phosphate buffered saline (PBS, pH 7.4), with a half-maximum signal inhibition concentration (IC50) of 0.20 ng mL–1. Compared to the parental VHH T3-15, T3-15-AP was able to bind to a wider variety of coating antigens and the assay sensitivity was slightly improved. Cross-reactivity of T3-15-AP with a set of important brominated analogues was negligible (<0.1%). Although T3-15-AP was susceptible to extreme heat (90 °C), much higher binding stability at ambient temperature was observed in the T3-15-AP-based assay for at least 70 days. A simple pretreatment method of diluting urine samples with DMSO was developed for a one-step assay. The recoveries of TBBPA from urine samples via this one-step assay ranged from 96.7% to 109.9% and correlated well with a high-performance liquid chromatography–tandem mass spectroscopy (HPLC-MS/MS) method. It is expected that the dimerized fusion protein, VHH-AP, will show promising applications in human exposure and environmental monitoring.

ACS Style

Jia Wang; Zuzana Majkova; Candace R. S. Bever; Jun Yang; Shirley J. Gee; Ji Li; Ting Xu; Bruce D. Hammock. One-Step Immunoassay for Tetrabromobisphenol A Using a Camelid Single Domain Antibody–Alkaline Phosphatase Fusion Protein. Analytical Chemistry 2015, 87, 4741 -4748.

AMA Style

Jia Wang, Zuzana Majkova, Candace R. S. Bever, Jun Yang, Shirley J. Gee, Ji Li, Ting Xu, Bruce D. Hammock. One-Step Immunoassay for Tetrabromobisphenol A Using a Camelid Single Domain Antibody–Alkaline Phosphatase Fusion Protein. Analytical Chemistry. 2015; 87 (9):4741-4748.

Chicago/Turabian Style

Jia Wang; Zuzana Majkova; Candace R. S. Bever; Jun Yang; Shirley J. Gee; Ji Li; Ting Xu; Bruce D. Hammock. 2015. "One-Step Immunoassay for Tetrabromobisphenol A Using a Camelid Single Domain Antibody–Alkaline Phosphatase Fusion Protein." Analytical Chemistry 87, no. 9: 4741-4748.

Journal article
Published: 01 November 2014 in Biomicrofluidics
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The emerging technologies on mobile-based diagnosis and bioanalytical detection have enabled powerful laboratory assays such as enzyme-linked immunosorbent assay (ELISA) to be conducted in field-use lab-on-a-chip devices. In this paper, we present a low-cost universal serial bus (USB)-interfaced mobile platform to perform microfluidic ELISA operations in detecting the presence and concentrations of BDE-47 (2,2',4,4'-tetrabromodiphenyl ether), an environmental contaminant found in our food supply with adverse health impact. Our point-of-care diagnostic device utilizes flexible interdigitated carbon black electrodes to convert electric current into a microfluidic pump via gas bubble expansion during electrolytic reaction. The micropump receives power from a mobile phone and transports BDE-47 analytes through the microfluidic device conducting competitive ELISA. Using variable domain of heavy chain antibodies (commonly referred to as single domain antibodies or Nanobodies), the proposed device is sensitive for a BDE-47 concentration range of 10(-3)-10(4 ) μg/l, with a comparable performance to that uses a standard competitive ELISA protocol. It is anticipated that the potential impact in mobile detection of health and environmental contaminants will prove beneficial to our community and low-resource environments.

ACS Style

Arnold Chen; Royal Wang; Candace Bever; Siyuan Xing; Bruce D. Hammock; Tingrui Pan. Smartphone-interfaced lab-on-a-chip devices for field-deployable enzyme-linked immunosorbent assay. Biomicrofluidics 2014, 8, 064101 -064101.

AMA Style

Arnold Chen, Royal Wang, Candace Bever, Siyuan Xing, Bruce D. Hammock, Tingrui Pan. Smartphone-interfaced lab-on-a-chip devices for field-deployable enzyme-linked immunosorbent assay. Biomicrofluidics. 2014; 8 (6):064101-064101.

Chicago/Turabian Style

Arnold Chen; Royal Wang; Candace Bever; Siyuan Xing; Bruce D. Hammock; Tingrui Pan. 2014. "Smartphone-interfaced lab-on-a-chip devices for field-deployable enzyme-linked immunosorbent assay." Biomicrofluidics 8, no. 6: 064101-064101.

Research article
Published: 28 July 2014 in Analytical Chemistry
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Tetrabromobisphenol A (TBBPA) is a ubiquitous flame retardant. A high-throughput immunoassay would allow for monitoring of human and environmental exposures as a part of risk assessment. Naturally occurring antibodies in camelids that are devoid of light chain, show great promise as an efficient tool in monitoring environmental contaminants, but they have been rarely used for small molecules. An alpaca was immunized with a TBBPA hapten coupled to thyroglobulin and a variable domain of heavy chain antibody (VHH) T3–15 highly selective for TBBPA was isolated from a phage displayed VHH library using heterologous coating antigens. Compared to the VHHs isolated using homologous antigens, VHH T3–15 had about a 10-fold improvement in sensitivity in an immunoassay. This assay, under the optimized conditions of 10% methanol in the assay buffer (pH 7.4), had an IC50 for TBBPA of 0.40 ng mL–1 and negligible cross reactivity (<0.1%) with other tested analogues. After heating the VHH at 90 °C for 90 min about 20% of the affinity for coating antigen T3-BSA remained. The recoveries of TBBPA from spiked soil and fetal bovine serum samples ranged from 90.3% to 110.7% by ELISA and agreed well with a liquid chromatography–tandem mass spectrometry method. We conclude the many advantages of VHH make them attractive for the development of immunoassays to small molecules.

ACS Style

Jia Wang; Candace R. S. Bever; Zuzana Majkova; Julie E. Dechant; Jun Yang; Shirley J. Gee; Ting Xu; Bruce D. Hammock. Heterologous Antigen Selection of Camelid Heavy Chain Single Domain Antibodies against Tetrabromobisphenol A. Analytical Chemistry 2014, 86, 8296 -8302.

AMA Style

Jia Wang, Candace R. S. Bever, Zuzana Majkova, Julie E. Dechant, Jun Yang, Shirley J. Gee, Ting Xu, Bruce D. Hammock. Heterologous Antigen Selection of Camelid Heavy Chain Single Domain Antibodies against Tetrabromobisphenol A. Analytical Chemistry. 2014; 86 (16):8296-8302.

Chicago/Turabian Style

Jia Wang; Candace R. S. Bever; Zuzana Majkova; Julie E. Dechant; Jun Yang; Shirley J. Gee; Ting Xu; Bruce D. Hammock. 2014. "Heterologous Antigen Selection of Camelid Heavy Chain Single Domain Antibodies against Tetrabromobisphenol A." Analytical Chemistry 86, no. 16: 8296-8302.

Research article
Published: 23 June 2014 in Analytical Chemistry
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An antibody-based analytical method for the detection of a chemical flame retardant using antibody fragments isolated from an alpaca has been developed. One specific chemical flame retardant congener, 2,2′,4,4′-tetrabrominated diphenyl ether (BDE-47), is often the major poly-BDE (PBDE) congener present in human and environmental samples and that which is the most frequently detected. An alpaca was immunized with a surrogate of BDE-47 covalently attached to a carrier protein. The resulting mRNA coding for the variable domain of heavy-chain antibodies (VHH) were isolated, transcribed to cDNA, and cloned into a phagemid vector for phage display library construction. Selection of VHHs recognizing BDE-47 was achieved by panning under carefully modified conditions. The assay sensitivity for detecting BDE-47 was down to the part-per-billion (microgram per liter) level. Cross-reactivity analyses confirmed that this method was highly selective for BDE-47 and selected hydroxylated metabolites. When exposed to elevated temperatures, the camelid VHH antibodies retained more reactivity than a polyclonal antibody developed to the same target analyte. The use of this VHH antibody reagent immobilized onto a Au electrode for impedance biosensing demonstrates the increased versatility of VHH antibodies.

ACS Style

Candace R. S. Bever; Zuzana Majkova; Rajeswaran Radhakrishnan; Ian Suni; Mark McCoy; Yanru Wang; Julie Dechant; Shirley Gee; Bruce D. Hammock. Development and Utilization of Camelid VHH Antibodies from Alpaca for 2,2′,4,4′-Tetrabrominated Diphenyl Ether Detection. Analytical Chemistry 2014, 86, 7875 -7882.

AMA Style

Candace R. S. Bever, Zuzana Majkova, Rajeswaran Radhakrishnan, Ian Suni, Mark McCoy, Yanru Wang, Julie Dechant, Shirley Gee, Bruce D. Hammock. Development and Utilization of Camelid VHH Antibodies from Alpaca for 2,2′,4,4′-Tetrabrominated Diphenyl Ether Detection. Analytical Chemistry. 2014; 86 (15):7875-7882.

Chicago/Turabian Style

Candace R. S. Bever; Zuzana Majkova; Rajeswaran Radhakrishnan; Ian Suni; Mark McCoy; Yanru Wang; Julie Dechant; Shirley Gee; Bruce D. Hammock. 2014. "Development and Utilization of Camelid VHH Antibodies from Alpaca for 2,2′,4,4′-Tetrabrominated Diphenyl Ether Detection." Analytical Chemistry 86, no. 15: 7875-7882.

Research article
Published: 10 June 2014 in ACS Sustainable Chemistry & Engineering
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Due to their all-electrical nature, impedance biosensors have significant potential for use as simple and portable sensors for environmental studies and environmental monitoring. Detection of two endocrine-disrupting chemicals (EDC), norfluoxetine and BDE-47, is reported here by impedance biosensing, with a detection limit of 8.5 and 1.3 ng/mL for norfluoxetine and BDE-47, respectively. Although impedance biosensors have been widely studied in the academic literature, commercial applications have been hindered by several technical limitations, including possible limitations to small analytes, the complexity of impedance detection, susceptibility to nonspecific adsorption, and stability of biomolecule immobilization. Recent research into methods to overcome these obstacles is briefly reviewed. New results demonstrating antibody regeneration atop degenerate (highly doped) Si are also reported. Using 0.2 M KSCN and 10 mM HF for antibody regeneration, peanut protein Ara h 1 is detected daily during a 30 day trial.

ACS Style

Rajeswaran Radhakrishnan; Ian I. Suni; Candace Bever; Bruce D. Hammock. Impedance Biosensors: Applications to Sustainability and Remaining Technical Challenges. ACS Sustainable Chemistry & Engineering 2014, 2, 1649 -1655.

AMA Style

Rajeswaran Radhakrishnan, Ian I. Suni, Candace Bever, Bruce D. Hammock. Impedance Biosensors: Applications to Sustainability and Remaining Technical Challenges. ACS Sustainable Chemistry & Engineering. 2014; 2 (7):1649-1655.

Chicago/Turabian Style

Rajeswaran Radhakrishnan; Ian I. Suni; Candace Bever; Bruce D. Hammock. 2014. "Impedance Biosensors: Applications to Sustainability and Remaining Technical Challenges." ACS Sustainable Chemistry & Engineering 2, no. 7: 1649-1655.

Research article
Published: 26 July 2013 in Analytical Chemistry
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Anti-idiotypic antibodies recognize the antigenic determinants of an antibody, thus they can be used as surrogate antigens. Single-domain antibodies from camlid heavy-chain antibodies with the benefit features of small size, thermostability, and ease in expression, are leading candidates to produce anti-idiotypic antibodies. In this work, we constructed an antibody phage library from the mRNA of an alpaca immunized with an antiaflatoxin monoclonal antibody (mAb) 1C11. Three anti-idiotypic VHH antibodies were isolated and applied to immunoassay toward aflatoxin as a coating antigen. The best immunoassay developed with one of these VHH antibodies shows an IC50 of 0.16 ng/mL toward aflatoxin B1 and cross-reactivity toward aflatoxin B2, G1, and G2 of 90.4%, 54.4%, and 37.7%, respectively. The VHH-based immunoassay was successfully applied to the analysis of peanuts, corn, and rice, which are the predominant commodities regularly contaminated by aflatoxins. A good correlation (r2 = 0.89) was found between the data obtained from the conventional ELISA and the ELISA based on a VHH coating antigen for the analysis of aflatoxins in peanuts and feedstuff. The use of biotechnology in developing the surrogate, the absence of standard aflatoxin and organic solvents in the synthesis procedures, and the reproducibility of the VHH antibody makes it an ideal strategy for replacing conventional synthesized antigens.

ACS Style

Yanru Wang; Peiwu Li; Zuzana Majkova; Candace R. S. Bever; Hee Joo Kim; Qi Zhang; Julie E. Dechant; Shirley J. Gee; Bruce D. Hammock. Isolation of Alpaca Anti-Idiotypic Heavy-Chain Single-Domain Antibody for the Aflatoxin Immunoassay. Analytical Chemistry 2013, 85, 8298 -8303.

AMA Style

Yanru Wang, Peiwu Li, Zuzana Majkova, Candace R. S. Bever, Hee Joo Kim, Qi Zhang, Julie E. Dechant, Shirley J. Gee, Bruce D. Hammock. Isolation of Alpaca Anti-Idiotypic Heavy-Chain Single-Domain Antibody for the Aflatoxin Immunoassay. Analytical Chemistry. 2013; 85 (17):8298-8303.

Chicago/Turabian Style

Yanru Wang; Peiwu Li; Zuzana Majkova; Candace R. S. Bever; Hee Joo Kim; Qi Zhang; Julie E. Dechant; Shirley J. Gee; Bruce D. Hammock. 2013. "Isolation of Alpaca Anti-Idiotypic Heavy-Chain Single-Domain Antibody for the Aflatoxin Immunoassay." Analytical Chemistry 85, no. 17: 8298-8303.