Smartphone-based paper microfluidic competitive immunoassay for the detection of α-amanitin from mushrooms.

α-Amanitin is often considered the most poisonous mushroom toxin produced by various mushroom species, which are hard to identify from edible, non-toxic mushrooms. Conventional detection methods require expensive and bulky equipment or fail to meet high analytical sensitivity. We developed a smartphone-based fluorescence microscope platform to detect α-amanitin from dry mushroom tissues. Antibody-nanoparticle conjugates were captured by immobilized antigen-hapten conjugates while competing with the free analytes in the sample. Captured fluorescent nanoparticles were excited at 460 nm and imaged at 500 nm. The pixel numbers of such nanoparticles in the test zone were counted, showing a decreasing trend with increasing analyte concentration. The detection method exhibited a low detection limit (1 pg/mL), high specificity, and selectivity, allowing us to utilize a simple rinsing for toxin extraction and avoiding the need for high-speed centrifugation. In addition, this assay's short response time and portable features enable field detection of α-amanitin from amanitin-producing mushrooms.

Comparison of soybean peroxidase with horseradish peroxidase and alkaline phosphatase used in immunoassays.

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.

Competitive and noncompetitive immunoassays for the detection of benzothiostrobin using magnetic nanoparticles and fluorescein isothiocyanate-labeled peptides.

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. Graphical abstract Development of competitive and noncompetitive fluorescence immunoassays for benzothiostrobin by using monoclonal antibody coupling with magnetic nanoparticles as concentration elements and peptides conjugated with fluorescein isothiocyanate as tracers.

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.

Detection of the Antimicrobial Triclosan in Environmental Samples by Immunoassay.

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.

VHH antibodies: emerging reagents for the analysis of environmental chemicals.

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. Graphical Abstract Overview of the production of VHHs to small environmental chemicals and highlights of the utility of these new emerging reagents.

Method for Sorting and Pairwise Selection of Nanobodies for the Development of Highly Sensitive Sandwich Immunoassays.

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.

Early diagnosis of amanitin exposure (amatoxicosis) in a dog with a point-of-care diagnostic test.

OBJECTIVE: To describe the rapid diagnosis, treatment, and clinical course of a dog that ingested an amanitin-containing mushroom. CASE SUMMARY: A 2-month-old female intact Australian Shepherd presented with diarrhea and vomiting, along with a possible mushroom exposure. Upon presentation, the dog's urine was collected and tested positive by a point-of-care rapid diagnostic test specific for detecting amanitins, the causative agents of amatoxicosis. NEW OR UNIQUE INFORMATION PROVIDED: This is the first reported case of amatoxicosis that was diagnosed using a point-of-care test prior to starting treatment. An early diagnosis helps to guide early treatment decisions in this frequently fatal toxicosis.

Lateral flow immunoassay (LFIA) for the detection of lethal amatoxins from mushrooms.

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.

Rapid, Sensitive, and Accurate Point-of-Care Detection of Lethal Amatoxins in Urine.

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 ß-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.

A Rapid Extraction Method Combined with a Monoclonal Antibody-Based Immunoassay for the Detection of Amatoxins.

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 ß-amanitin (ß-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.

Development and Characterization of Monoclonal Antibodies to Botulinum Neurotoxin Type E.

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.

A New Conjugation Method Used for the Development of an Immunoassay for the Detection of Amanitin, a Deadly Mushroom Toxin.

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.

Effects of triclosan in breast milk on the infant fecal microbiome.

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.

Impedance Biosensors: Applications to Sustainability and Remaining Technical Challenges.

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.