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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 small environmental chemicals (MW <1,500 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.

Phenylpyrazole insecticides such as fipronil have been used as replacements for organophosphates. The wide application of fipronil raises concern about environmental contamination and risk for fish, birds, other non-targeted beings and human health. A sensitive, competitive indirect heterologous enzyme-linked immunosorbent assay (ELISA) was developed. Antibodies with different specificities to fipronil and its metabolites were produced. Two ELISAs having IC50 values of 0.58 ± 0.06 and 2.6 ± 0.4 ng/mL were developed. Design of different haptens and coating antigens resulted in two assays with distinct cross-reactivity patterns for structurally related compounds: 96%, 38% and 101% vs 39%, 1.4% and 25% for fipronil-sulfide, fipronil-detrifluoromethylsulfonyl and fipronil-desulfinyl, respectively. Performance of the immunoassays was demonstrated by a recovery study from spiked water, human serum and urine matrices, giving recovery values in the range of 85–111% for different concentrations. The assays demonstrated good correlation in fipronil recovery with conventional LC-MS/MS analysis. The generic assay 2265 has the sensitivity to measure fipronil and its analogs in serum at levels relevant for exposure monitoring. The assays were used to analyze human urine samples obtained from exposure studies and serum samples from rats treated with fipronil-containing diet.

Arachidonic acid (ARA) is metabolized by cyclooxygenase (COX) and cytochrome P450 to produce proangiogenic metabolites. Specifically, epoxyeicosatrienoic acids (EETs) produced from the P450 pathway are angiogenic, inducing cancer tumor growth. A previous study showed that inhibiting soluble epoxide hydrolase (sEH) increased EET concentration and mildly promoted tumor growth. However, inhibiting both sEH and COX led to a dramatic decrease in tumor growth, suggesting that the contribution of EETs to angiogenesis and subsequent tumor growth may be attributed to downstream metabolites formed by COX. This study explores the fate of EETs with COX, the angiogenic activity of the primary metabolites formed, and their subsequent hydrolysis by sEH and microsomal EH. Three EET regioisomers were found to be substrates for COX, based on oxygen consumption and product formation. EET substrate preference for both COX-1 and COX-2 were estimated as 8,9-EET > 5,6-EET > 11,12-EET, whereas 14,15-EET was inactive. The structure of two major products formed from 8,9-EET in this COX pathway were confirmed by chemical synthesis: ct-8,9-epoxy-11-hydroxy-eicosatrienoic acid (ct-8,9-E-11-HET) and ct-8,9-epoxy-15-hydroxy-eicosatrienoic acid (ct-8,9-E-15-HET). ct-8,9-E-11-HET and ct-8,9-E-15-HET are further metabolized by sEH, with ct-8,9-E-11-HET being hydrolyzed much more slowly. Using an s.c. Matrigel assay, we showed that ct-8,9-E-11-HET is proangiogenic, whereas ct-8,9-E-15-HET is not active. This study identifies a functional link between EETs and COX and identifies ct-8,9-E-11-HET as an angiogenic lipid, suggesting a physiological role for COX metabolites of EETs.omega-6 fatty acids | epoxyeicosatrienoic acids | metabolism | cyclooxygenase | angiogenesis A rachidonic acid (ARA) is an omega-6 fatty acid that is metabolized by three major classes of enzymes, cyclooxygenases (COXs), lipoxygenases, and cytochrome P450s (CYPs), to produce an array of biologically active metabolites (1-3). The CYP pathway transforms ARA into four epoxyeicosatrienoic acids (EETs) in addition to hydroxylated metabolites (1). EETs have several biological actions, and are considered antihypertensive, antiinflammatory, neuroprotective, cardioprotective, and analgesic (4). EETs also play a role in angiogenesis (5-10), the formation of new blood vessels from preexisting vessels that is important for many physiological and pathological processes, including cancer (11).EETs can enhance tumor growth and metastasis through their angiogenic activity (12); however, this activity is transient due to their metabolic instability. EETs are further metabolized by epoxide hydrolases (EHs), primarily soluble epoxide hydrolase (sEH), to their corresponding diols (4, 13), which are generally not biologically active (14) (Fig. 1A). Inhibition of sEH prolongs EET biological activity, potentiating their angiogenic activity, leading to increased tumor growth and metastasis in some systems (12, 15) (Fig. 1B).ARA can also be metabolized by COX to form proangiogenic and proinflammator...

Nanoluciferase (Nluc), the smallest luciferase known, was used as the fusion partner with a nanobody against aflatoxin B1 to develop a bioluminescent enzyme immunoassay (BLEIA) for detection of the aflatoxin B1 in cereal. Nanobody (clone G8) against aflatoxin B1 was fused with nanoluciferase and cloned into a pET22b expression vector, and then transformed into Escherichia coli. The nanobody fusion gene contained a hexahistidine tag for purification by immobilized metal affinity chromatography, yielding a biologically active fusion protein. The fusion protein G8-Nluc retained binding properties of the original nanobody. Concentration of the coelenterazine substrate and buffer composition were also optimized to provide high intensity and long half-life of the luminescent signal. The G8-Nluc was used as a detection antibody to establish a competitive bioluminescent ELISA for the detection of aflatoxin B1 in cereals successfully. Compared to classical ELISA, this novel assay showed more than 20-fold improvement in detection sensitivity, with an IC50 value of 0.41 ng/mL and linear range from 0.10 to 1.64 ng/mL. In addition, the entire BLEIA detection procedure can be completed in one step within 2 h, from sample preparation to data analysis. These results suggest that nanobody fragments fused with nanoluciferase might serve as useful and highly sensitive dual functional reagents for the development of rapid and highly sensitive immunoanalytical methods.

A first total synthesis of (-)-ent-plakortolide I and seco-plakortolide E was accomplished from (S)-2-methylglycidol. The relevant key reactions involve a diastereoselective Mukaiyama aldol reaction, a regioselective hydroperoxysilylation, and elaboration of the 1,2-dioxane ring by intramolecular Michael addition of a hydroperoxide group to a butenolide. This synthesis allowed the revision of the absolute configuration of plakortolide I and structural revision of plakortolide E.

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 *

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 raises 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, 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 4h 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.