Pharmacokinetics, Metabolite Measurement, and Biomarker Identification of Dermal Exposure to Permethrin Using Accelerator Mass Spectrometry.

Impregnating military uniforms and outdoor clothing with the insecticide permethrin is an approach to reduce exposure to insect borne diseases and to repel pests and disease vectors such as mosquitos and sandflies, but the practice exposes wearers to prolonged dermal exposure to the pesticide. Key metabolite(s) from a low dose dermal exposure of permethrin were identified using accelerator mass spectrometry. Metabolite standards were synthesized and a high performance liquide chromatography (HPLC) elution protocol to separate individual metabolites in urine was developed. Six human subjects were exposed dermally on the forearm to 25 mg of permethrin containing 1.0 µCi of 14C for 8 h. Blood, saliva and urine samples were taken for 7d. Absorption/elimination rates and metabolite concentrations varied by individual. Average absorption was 0.2% of the dose. Serum concentrations rose until 12-24 h postdermal application then rapidly declined reaching predose levels by 72 h. Maximum saliva excretion occurred 6 h postdosing. The maximum urinary excretion rate occurred during 12-24 h; average elimination half-life was 56 h. 3-Phenoxybenzyl alcohol glucuronide was the most abundant metabolite identified when analyzing elution fractions, but most of the radioactivity was in still more polar fractions suggesting extensive degradative metabolism and for which there were no standards. Analyses of archived urine samples with the ultra performance liquid chromatography-accelerator mass spectrometry-mass spectrometry (UPLC-AMS-MS) system isolated a distinct polar metabolite but it was much diminished from the previous analyses a decade earlier.

Highly specific nanobody against herbicide 2,4-dichlorophenoxyacetic acid for monitoring of its contamination in environmental water.

2,4-dichlorophenoxyacetic acid (2,4-D), a widely used herbicide, is a small organic chemical pollutant in the environment. To develop a nanobody-based immunoassay for monitoring trace levels of 2,4-D, a step-wise strategy for the generation of nanobodies highly specific against this small chemical was employed. Firstly, we synthesized three novel haptens mimicking 2,4-D and assessed their influence on the sensitivity and specificity of the existing antibody-based assay. Polyclonal antibodies (pAb) from rabbits showed good sensitivity and moderate specificity for 2,4-D, pAb from llama based on selected haptens showed similar performance when compared to those from rabbits. Secondly, nanobodies derived from llama were generated for 2,4-D by an effective procedure, including serum monitoring and one-step library construction. One nanobody, NB3-9, exhibited good sensitivity against 2,4-D (IC50 = 29.2 ng/mL) had better specificity than the rabbit pAb#1518, with no cross-reactivities against the 2,4-D analogs tested. Thirdly, one-step fluorescent enzyme immunoassay (FLEIA) for 2,4-D based on a nanobody-alkaline phosphatase (AP) fusion was developed with IC50 of 1.9 ng/mL and a linear range of 0.4-8.6 ng/mL. Environmental water samples were analyzed by FLEIA and LC-MS/MS for comparison, and the results were consistent between both methods. Therefore, the proposed step-wise strategy from hapten design to nanobody-AP fusion production was successfully conducted, and the resulting nanobody based FLEIA was demonstrated as a convenient tool to monitor 2,4-D residuals in the environment.

Generation of functional single-chain fragment variable from hybridoma and development of chemiluminescence enzyme immunoassay for determination of total malachite green in tilapia fish.

To determine malachite green (MG) and its major metabolite, leucomalachite green (LMG) residual levels in tilapia fish, chemiluminescent enzyme immunoassay (CLEIA) was developed based on a single-chain variable fragment (scFv)-alkaline phosphatase (AP) fusion protein. At first, VH and VL gene sequences were cloned from hybridoma cell lines secreting monoclonal antibody against LMG, and then thoroughly by database-assisted sequence analysis. Finally, the productive VH and VL were assembled to an intact scFv sequence and engineered to produce scFv-AP fusion protein. The fusion protein was further identified as a bifunctional reagent for immunoassay, then a sensitive one-step CLEIA against LMG was developed with a half-maximal inhibitory concentration (IC50) and limit of detection (LOD) of 1.3 and 0.04 ng/mL, respectively. The validation results of this novel competitive CLEIA was in line with those obtained by classical HPLC method for determination of total MG in spiked and field incurred samples.

Construction of Immunomagnetic Particles with High Stability in Stringent Conditions by Site-Directed Immobilization of Multivalent Nanobodies onto Bacterial Magnetic Particles for the Environmental Detection of Tetrabromobisphenol-A.

Bacterial magnetic particles (BMPs) are an attractive carrier material for immunoassays because of their nanoscale size, dispersal ability, and membrane-bound structure. Antitetrabromobisphenol-A (TBBPA) nanobodies (Nbs) in the form of monovalence (Nb1), bivalence (Nb2), and trivalence (Nb3) were biotinylated and immobilized onto streptavidin (SA)-derivatized BMPs to construct the complexes of BMP-SA-Biotin-Nb1, -Nb2, and -Nb3, respectively. An increasing order of binding capability of BMP-SA-Biotin-Nb1, -Nb2, and -Nb3 to TBBPA was observed. These complexes showed high resilience to temperature (90 °C), methanol (100%), high pH (12), and strong ionic strength (1.37 M NaCl). A BMP-SA-Biotin-Nb3-based enzyme linked immunosorbent assay (ELISA) for TBBPA dissolved in methanol was developed, showing a half-maximum inhibition concentration (IC50) of 0.42 ng mL-1. TBBPA residues in landfill leachate, sewage, and sludge samples determined by this assay were in a range of

Development of an immunoassay for the detection of carbaryl in cereals based on a camelid variable heavy-chain antibody domain.

BACKGROUND: The variable domain of camelid heavy-chain antibodies (VHH) is increasingly being adapted to detect small molecules in various matrices. The insecticide carbaryl is widely used in agriculture while its residues have posed a threat to food safety and human health. RESULTS: VHHs specific for carbaryl were generated from an alpaca immunized with the hapten CBR1 coupled to keyhole limpet hemocyanin. An enzyme-linked immunosorbent assay (ELISA) based on the VHH C1 and the coating antigen CBR2-BSA was developed for the detection of carbaryl in cereals. This assay, using an optimized assay buffer (pH 6.5) containing 10% methanol and 0.8% NaCl, has a half-maximum signal inhibition concentration of 5.4 ng mL-1 and a limit of detection (LOD) of 0.3 ng mL-1 for carbaryl, and shows low cross reactivity (≤0.8%) with other tested carbamates. The LOD of carbaryl using the VHH-based ELISA was 36 ng g-1 in rice and maize and 72 ng g-1 in wheat. Recoveries of carbaryl in spiked rice, maize and wheat samples were in the range of 81-106%, 96-106% and 83-113%, respectively. Relative standard deviations of repeatability and intra-laboratory reproducibility were in the range of 0.8-9.2% and 2.9-9.7%, respectively. CONCLUSION: The VHH-based ELISA was highly effective in detecting carbaryl in cereal samples after simple sample extraction and dilution. © 2019 Society of Chemical Industry.

Nanobody-based binding assay for the discovery of potent inhibitors of CFTR inhibitory factor (Cif).

Lead identification and optimization are essential steps in the development of a new drug. It requires cost-effective, selective and sensitive chemical tools. Here, we report a novel method using nanobodies that allows the efficient screening for potent ligands. The method is illustrated with the cystic fibrosis transmembrane conductance regulator inhibitory factor (Cif), a virulence factor secreted by the opportunistic pathogen Pseudomonas aeruginosa. 18 nanobodies selective to Cif were isolated by bio-panning from nanobody-phage library constructed from immunized llama. 8 out of 18 nanobodies were identified as potent inhibitors of Cif enzymatic activity with IC50s in the range of 0.3-6.4 µM. A nanobody VHH219 showed high affinity (KD = 0.08 nM) to Cif and the highest inhibitory potency, IC50 = 0.3 µM. A displacement sandwich ELISA (dsELISA) with VHH219 was then developed for classification of synthetic small molecule inhibitors according their inhibitory potency. The developed assay allowed identification of new inhibitor with highest potency reported so far (0.16 ±â€¯0.02 µM). The results from dsELISA assay correlates strongly with a conventional fluorogenic assay (R = 0.9998) in predicting the inhibitory potency of the tested compounds. However, the novel dsELISA is an order of magnitude more sensitive and allows the identification and ranking of potent inhibitors missed by the classic fluorogenic assay method. These data were supported with Octet biolayer interferometry measurements. The novel method described herein relies solely on the binding properties of the specific neutralizing nanobody, and thus is applicable to any pharmacological target for which such a nanobody can be found, independent of any requirement for catalytic activity.

One-step immunoassay for the insecticide carbaryl using a chicken single-chain variable fragment (scFv) fused to alkaline phosphatase.

Immunoassays provide a high-throughput method for monitoring pesticides in foods and the environment. Due to easy generation and capable of being manipulated, chicken single-chain variable fragment (scFv) is attractive in the development of immunoassays for pesticides. Two scFvs (X1 and X2) against the insecticide carbaryl were generated from a chicken immunized with hapten C1 conjugated to keyhole limpet hemocyanin and fused with alkaline phosphatase (AP) to develop a rapid one-step enzyme-linked immunosorbent assay for this pesticide. X2-AP showed higher binding affinity to carbaryl than X1-AP. The X2-AP-based ELISA had a half-maximum signal inhibition concentration of 15 ng mL-1 and a limit of detection of 1.6 ng mL-1. This assay showed negligible cross-reactivity with other carbamate pesticides (<0.1%) and low cross-reactivity with 1-naphthol (5%). The average recoveries of carbaryl spiked in soil, apple and pear samples by the one-step assay ranged from 90% to 114% and agreed well with those of high-performance liquid chromatography. The chicken scFv-based assay showed promise as a high-throughput screening tool for carbaryl in environmental and food matrices.

Development of a one-step immunoassay for triazophos using camel single-domain antibody-alkaline phosphatase fusion protein.

Triazophos is mainly used in Asian and African countries for the control of insects in agricultural production. Camelid variable domains of heavy-chain antibodies (VHHs) show great promise in monitoring environmental chemicals such as pesticides. To improve the rate of success in the generation of VHHs against triazophos, genes specifically encoding VHH fragments from the unique allotype IgG3a of an immunized Camelus bactrianus were amplified by using a pair of novel primers and introduced to construct a diverse VHH library. Five out of seven isolated positive clones, including the VHH T1 with the highest affinity to triazophos, were derived from the allotype IgG3a. A one-step enzyme-linked immunosorbent assay (ELISA) using VHH T1 genetically fused with alkaline phosphatase (AP) had a half-maximum inhibition concentration of 6.6 ng/mL for triazophos. This assay showed negligible cross-reactivity with a list of important organophosphate pesticides (< 0.1%). The average recoveries of triazophos from water, soil, and apple samples determined by the one-step ELISA ranged from 83 to 108%, having a good correlation with those by a gas chromatography mass spectrometry (R2 = 0.99). The VHH-AP fusion protein shows potential for the analysis of triazophos in various matrices.

Quantitative Detection of Fipronil and Fipronil-Sulfone in Sera of Black-Tailed Prairie Dogs and Rats after Oral Exposure to Fipronil by Camel Single-Domain Antibody-Based Immunoassays.

The insecticide fipronil can be metabolized to its sulfone in mammalian species. Two camel single-domain antibodies (VHHs) F1 and F6, selective to fipronil and fipronil-sulfone, respectively, were generated and used to develop enzyme linked immunosorbent assays (ELISAs) for the detection of the two compounds in the sera of black-tailed prairie dogs and rats. The limits of detection of fipronil and fipronil-sulfone in the rodent sera by the corresponding ELISAs were 10 and 30 ng mL-1, and the linear ranges were 30-1000 and 75-2200 ng mL-1. ELISAs showed a good recovery for fipronil and fipronil-sulfone cospiked in the control sera of the black-tailed prairie dogs (90-109%) and rats (93-106%). The VHH-based ELISAs detected fipronil and fipronil-sulfone in the sera of the rodents that received a repeated oral administration of fipronil. The average concentration of fipronil-sulfone was approximately 3.2-fold higher than fipronil in the prairie dog sera (1.15 vs 0.36 µg mL-1) and rat sera (1.77 vs 0.53 µg mL-1). ELISAs agreed well with a liquid chromatography-mass spectrometry method for the quantification of both fipronil and fipronil-sulfone in real serum samples. Fipronil-sulfone was identified as the predominant metabolite of fipronil in the black-tailed prairie dog and rat sera.

Strong and oriented conjugation of nanobodies onto magnetosomes for the development of a rapid immunomagnetic assay for the environmental detection of tetrabromobisphenol-A.

Variable domain of heavy chain antibody (nanobody, Nb) derived from camelids is an efficient reagent in monitoring environmental contaminants. Oriented conjugates of Nbs and bacterial magnetic particles (BMPs) provide new tools for the high-throughput immunoassay techniques. An anti-tetrabromobisphenol-A (TBBPA) Nb genetically integrated with an extra cysteine residue at the C terminus was immobilized onto BMPs enclosed within the protein membrane, using a heterobifunctional reagent N-succinimidyl-3-(2-pyridyldithiol) propionate, to form a solid BMP-Nb complex. A rapid and sensitive enzyme-linked immunosorbent assay (ELISA) based on the combination of BMP-Nb and T5-horseradish peroxidase was developed for the analysis of TBBPA, with a total assay time of 30 min and a half-maximum signal inhibition concentration (IC50) of 1.04 ng/mL in PBS (pH 10, 10% methanol and 0.137 moL/L NaCl). This assay can even be performed in 100% methanol, with an IC50 value of 44.3 ng/mL. This assay showed quantitative recoveries of TBBPA from spiked canal water (114-124%) and sediment (109-113%) samples at 1.0-10 ng/mL (or ng/g (dw)). TBBPA residues determined by this assay in real canal water samples were below the limit of detection (LOD) and in real sediments were between

Development of an Indirect Competitive Enzyme-Linked Immunosorbent Assay for Glycocholic Acid Based on Chicken Single-Chain Variable Fragment Antibodies.

Glycocholic acid (GCA) is an important metabolite of bile acids, whose urine levels are expected to be a specific diagnostic biomarker for hepatocellular carcinoma (HCC). A high-throughput immunoassay for determination of GCA would be of significant advantage and useful for primary diagnosis, surveillance, and early detection of HCC. Single-chain variable fragment (scFv) antibodies have several desirable characteristics and are an attractive alternative to traditional antibodies for the immunoassay. Because chicken antibodies possess single heavy and light variable functional domains, they are an ideal framework for simplified generation of recombinant antibodies for GCA detection. However, chicken scFvs have rarely been used to detect GCA. In this study, a scFv library was generated from chickens immunized with a GCA hapten coupled to bovine serum albumin (BSA), and anti-GCA scFvs were isolated by a phage-displayed method. Compared to the homologous coating antigen, use of a heterologous coating antigen resulted in about an 85-fold improvement in sensitivity of the immunoassay. This assay, under optimized conditions, had a linear range of 0.02-0.18 µg/mL, with an IC50 of 0.06 µg/mL. The assay showed negligible cross-reactivity with various related bile acids, except for taurocholic acid. The detection of GCA from spiked human urine samples ranged from 86.7% to 123.3%. These results, combined with the advantages of scFv antibodies, indicated that a chicken scFv-based enzyme-linked immunosorbent assay is a suitable method for high-throughput screening of GCA in human urine.

Nanobody Based Immunoassay for Human Soluble Epoxide Hydrolase Detection Using Polymeric Horseradish Peroxidase (PolyHRP) for Signal Enhancement: The Rediscovery of PolyHRP?

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.

Development of Tetramethylenedisulfotetramine (TETS) Hapten Library: Synthesis, Electrophysiological Studies, and Immune Response in Rabbits.

There is a need for fast detection methods for the banned rodenticide tetramethylenedisulfotetramine (TETS), a highly potent blocker of the γ-aminobutyric acid (GABAA ) receptors. General synthetic approach toward two groups of analogues was developed. Screening of the resulting library of compounds by FLIPR or whole-cell voltage-clamp revealed that, despite the structural differences, some of the TETS analogues retained GABAA receptor inhibition; however, their potency was an order of magnitude lower. Antibodies raised in rabbits against some of the TETS analogues conjugated to protein recognized free TETS and will be used for the development of an immunoassay for TETS.

Sensitive Immunoassay for Detection and Quantification of the Neurotoxin, Tetramethylenedisulfotetramine.

Tetramethylenedisulfotetramine (TETS, tetramine) is a formerly used and highly neurotoxic rodenticide. Its lethality, recent history of intentional use for mass poisoning, and the absence of a known antidote raise public health concerns. Therefore, rapid, high throughput, and sensitive methods for detection and quantification of TETS are critical. Instrumental analysis method such as GC/MS is sensitive but not rapid or high throughput. Therefore, an immunoassay selective to TETS was developed. The assay shows an IC50 of 4.5 ± 1.2 ng/mL, with a limit of detection of 0.2 ng/mL, comparable to GC/MS. Performance of the immunoassay was demonstrated by a recovery study using known concentrations of TETS spiked into buffer and human and mouse serum matrices giving recoveries in the range of 80-120%. The assay demonstrated good correlation in TETS recovery with established GC/MS analysis. The immunoassay was then used to quantify TETS concentration in the serum of mice exposed to 2× LD50 dose of TETS and to monitor kinetics of TETS clearance from blood over a short period of time. TETS concentration in the serum reached 150 ng/mL without significant change over 4 h post-treatment. Results obtained with the immunoassay had good correlation with GC/MS analysis. Overall, this immunoassay is an important tool to rapidly detect and quantify levels of TETS from biological samples with high sensitivity. The assay can be adapted to multiple formats including field or hospital use.

Exploring adduct formation between human serum albumin and eleven organophosphate ester flame retardants and plasticizers using MALDI-TOF/TOF and LC-Q/TOF.

Organophosphate (OP) and organophosphate ester (OPE) adducts of albumin are valuable biomarkers for retrospective verification of exposure. In the present study, our goal was to determine whether OPE flame retardants (OPE FRs) and OPE plasticizers can covalently bind to human serum albumin (HSA), which would allow the resulting adducts to be used to evaluate exposure. Eleven OPE FRs and plasticizers were examined in a HSA-adduct in vitro assay. Pure HSA was incubated with the target OPEs, as well as with an OP insecticide (profenofos) positive control. After enzymatic cleavage with pepsin or Glu-C, the digested albumin was analyzed by matrix-assisted laser desorption ionization tandem time-of-flight mass spectrometry (MALDI-TOF/TOF-MS) and liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-Q-ToF-MS). Under optimized HSA assay conditions, tyrosine adducts were formed at Y411 and Y148/Y150 with a characteristic mass shift for phosphorylation (Δm/z 166) for the profenofos positive control. However, no such phosphorylated peptides were detected for the 11 target OPEs. This negative result suggests that these OPEs have very different affinities from the OP insecticide. They are less reactive or they may specifically interact with other proteins.

Hydroxy-fipronil is a new urinary biomarker of exposure to fipronil.

Occupational medical surveillance is highly desirable in manufacturing facilities where exposure to chemicals is significant. The insecticide fipronil is generally considered safe for humans but with increasing use, exposure to fipronil is of concern. Identification of urinary metabolites of fipronil may allow development of affordable, cheap and rapid procedures for human exposure evaluation. In this study we developed a fast and easy approach for synthesis of hydroxy-fipronil, a potential urinary metabolite of fipronil. This standard was used to develop a sensitive analytical LC-MS/MS method with a limit of quantification (LOQ) of 0.4ng/mL. Fipronil sulfone, a known metabolite, and hydroxy-fipronil were quantified in urine samples from rats treated with a fipronil containing diet. Fipronil sulfone concentration centered around 20ng/mL, while the concentration of hydroxy-fipronil was dose-dependent ranging in 10-10,000ng/mL and thus being a more sensitive marker of fipronil exposure. A fipronil immunoassay with cross-reactivity to hydroxy-fipronil showed a good correlation in signal intensity with LC-MS data. It was also used to demonstrate the applicability of the method for sample screening in the evaluation of exposure levels.

Nanobody-based enzyme immunoassay for ochratoxin A in cereal with high resistance to matrix interference.

A sensitive indirect competitive nanobody-based enzyme linked immunosorbent assay (Nb-ELISA) for ochratoxin A (OTA) with high resistance to cereal matrix interference was developed. Nanobodies against OTA (Nb15, Nb28, Nb32, Nb36) were expressed in E. coli cells and their thermal stabilities were compared with that of an OTA-specific monoclonal antibody 6H8. All nanobodies could still retain their antigen-binding activity after exposure to temperature 95°C for 5min or to 90°C for 75min. Nb28 that exhibited the highest sensitivity in ELISA was selected for further research. An indirect competitive ELISA based on Nb28 was developed for OTA, with an IC50 of 0.64ng/mL and a linear range (IC20-IC80) of 0.27-1.47ng/mL. Cereal samples were analyzed following a 2.5 fold dilution of sample extracts, showing the good resistance to matrix interference of the Nb-ELSIA. The recovery of spiked cereal samples (rice, oats, barley) ranged from 80% to 105% and the Nb-ELISA results of OTA content in naturally contamined samples were in good agreement with those determined by a commercial ELISA kit. The results indicated the reliablity of nanobody as a promising immunoassay reagent for detection of mycotoxins in food matrix and its potential in biosensor development.

Developmental effects of fipronil on Japanese Medaka (Oryzias latipes) embryos.

Pesticides in urban runoff are a major source of pollutants in aquatic ecosystems. Fipronil, a phenylpyrazole insecticide, found in structural pest control products, turf grass control, and home pet flea medication, has recently increased in use and is commonly detected in urban runoff. However, little is known about the effects of fipronil on aquatic organisms at early developmental stages. Here, we evaluated toxicity of fipronil to embryos of Japanese Medaka (Oryzias latipes, Qurt strain) using a high-throughput 96-well plate toxicity test. Male and female embryos (<6 h post fertilization) were exposed to concentrations of fipronil ranging from 0.1 to 910 µg L-1 for 14 days or until hatching. Embryos were subjected to gross and microscopic examinations of developmental adverse effects as well as transcriptome analysis using RNA-seq. Results indicated a positive dose-response in reduced hatching success, increased gross deformity (tail curvature) at a lowest-observed-effect concentration (LOEC) of 200 µg L-1 and delayed hatching (∼1 day at the highest concentration, LOEC = 600 µg L-1). The transcriptome analysis indicated that fipronil exposure enhanced expression of titin and telethonin, which are responsible for muscle development. It is therefore possible that the formation of a tail curvature is due to asymmetrical overgrowth of muscle. Our results indicate that sub-lethal effects occur in embryonic stages of an aquatic vertebrate following exposure to high concentrations of fipronil, although no adverse effects at the highest published environmentally relevant concentration (6.3 µg L-1) were observed.