Development of a Portable Cell-Based Biosensor for the Ultra-Rapid Screening for Boscalid Residues in Lettuce.

Fungal plant pathogens have posed a significant threat to crop production. However, the large-scale application of pesticides is associated with possible risks for human health and the environment. Boscalid is a widely used fungicide, consistently implemented for the management of significant plant pathogens. Conventionally, the detection and determination of boscalid residues is based on chromatographic separations. In the present study, a Bioelectric Recognition Assay (BERA)-based experimental approach combined with MIME technology was used, where changes in the electric properties of the membrane-engineering cells with anti-boscalid antibodies were recorded in response to the presence of boscalid at different concentrations based on the maximum residue level (MRL) for lettuce. The membrane-engineering Vero cells with 0.5 µg/mL of antibody in their surface were selected as the best cell line in combination with the lowest antibody concentration. Furthermore, the biosensor was tested against another fungicide in order to prove its selectivity. Finally, the BERA cell-based biosensor was able to detect the boscalid residue, below and above the MRL, in spiked lettuce leaf extracts in an entirely distinct and reproducible manner. This study indicates that the BERA-based biosensor, after further development and optimization, could be used for the routine, high-throughput detection of boscalid residue in lettuce, and not only that.

Development and In-House Validation of an Enzyme-Linked Immunosorbent Assay and a Lateral Flow Immunoassay for the Dosage of Tenofovir in Human Saliva.

Highly active antiretroviral therapy (HAART) includes very potent drugs that are often characterized by high toxicity. Tenofovir (TFV) is a widely used drug prescribed mainly for pre-exposure prophylaxis (PreP) and the treatment of human immunodeficiency virus (HIV). The therapeutic range of TFV is narrow, and adverse effects occur with both underdose and overdose. The main factor contributing to therapeutic failure is the improper management of TFV, which may be caused by low compliance or patient variability. An important tool to prevent inappropriate administration is therapeutic drug monitoring (TDM) of compliance-relevant concentrations (ARCs) of TFV. TDM is performed routinely using time-consuming and expensive chromatographic methods coupled with mass spectrometry. Immunoassays, such as enzyme-linked immunosorbent assays (ELISAs) and lateral flow immunoassays (LFIAs), are based on antibody-antigen specific recognition and represent key tools for real-time quantitative and qualitative screening for point-of-care testing (POCT). Since saliva is a non-invasive and non-infectious biological sample, it is well-suited for TDM. However, saliva is expected to have a very low ARC for TFV, so tests with high sensitivity are required. Here, we have developed and validated a highly sensitive ELISA (IC50 1.2 ng/mL, dynamic range 0.4-10 ng/mL) that allows the quantification of TFV in saliva at ARCs and an extremely sensitive LFIA (visual LOD 0.5 ng/mL) that is able to distinguish between optimal and suboptimal ARCs of TFV in untreated saliva.

Correction: Addante-Moya et al. Assessment of the Optimum Linker Tethering Site of Alternariol Haptens for Antibody Generation and Immunoassay Development. Toxins 2021, 13, 883.

In the original publication [...].

Design of a novel hapten and development of a sensitive monoclonal immunoassay for dicamba analysis in environmental water samples.

Weed resistance to glyphosate has been a driving force behind the increased use of alternative herbicides in agriculture. Recently, dicamba-tolerant recombinant plants were introduced to the market, which may result in residues of this agrochemical contaminating environmental waters. Given that restrictions on the use of dicamba have consequently been established by regulatory agencies, it is therefore also desirable to conduct extensive controls on dicamba residues. Immunoassays are currently the most powerful bioanalytical technology for the rapid monitoring of chemical residues and contaminants. In the present study, a novel hapten was designed maintaining unaltered all the antigenic moieties of the target molecule, and this was used to generate high-affinity monoclonal antibodies against dicamba for the first time. Additionally, a collection of haptens with different linker composition or linker tethering site was synthesized and conjugated to proteins. Using these novel immunoreagents, a direct competitive enzyme-linked immunosorbent assay with a limit of detection for dicamba of 0.24 ng/mL was developed and validated. Analysis of water samples from different origins afforded recovery values between 90 % and 120 %, and coefficients of variation below 20 % were obtained. These results indicate that the developed immunochemical assay is suitable for the rapid determination of dicamba residues in environmental water samples.

Rapid Immunochemical Methods for Anatoxin-a Monitoring in Environmental Water Samples.

Algal blooms that contaminate freshwater resources with cyanotoxins constitute, nowadays, a global concern. To deal with this problem, a variety of analytical methods, including immunochemical assays, are available for the main algal toxins, for example, microcystins, nodularins, and saxitoxins, with the remarkable exception of anatoxin-a. Now, for the first time, highly sensitive, enantioselective immunoassays for anatoxin-a have been validated using homemade monoclonal antibodies. Two competitive enzyme-linked immunosorbent assays were developed in different formats, with detection limits for (+)-anatoxin-a of 0.1 ng/mL. Excellent recovery values between 82 and 117%, and coefficients of variation below 20%, were observed using environmental water samples fortified between 0.5 and 500 ng/mL. In addition, a lateral-flow immunochromatographic assay was optimized for visual and instrumental reading of results. This test showed a visual detection limit for (+)-anatoxin-a of 4 ng/mL. Performance with a reader was validated in accordance with the European guidelines for semiquantitative rapid methods for small chemical contaminants. Thus, at a screening target concentration of 2 ng/mL, the probability of a blank sample to be classified as "suspect" was as low as 0.2%. Finally, the optimized direct enzyme immunoassay was validated by comparison with high-performance liquid chromatography-tandem mass spectroscopy data and showed a good correlation (r = 0.995) with a slope of 0.94. Moreover, environmental water samples containing more than 2 ng/mL of anatoxin-a were detected by the developed dipstick assay. These results provide supplementary and complementary strategies for monitoring the presence of anatoxin-a in water.

Chemical strategies for triggering the immune response to the mycotoxin patulin.

Mycotoxins represent a major concern for human and animal health because of their harmful effects and high occurrence in food and feed. Rapid immunoanalytical methods greatly contribute to strengthening the safety of our food supply by efficiently monitoring chemical contaminants, so high-affinity and specific antibodies have been generated for almost all internationally regulated mycotoxins. The only exception is patulin, a mycotoxin mainly produced by Penicillium expansum for which such a target has not yet been achieved. Accordingly, no point-of-need tests commonly used in food immunodiagnostics are commercially available for patulin. In the present study, three functionalized derivatives conforming to generally accepted rules in hapten design were firstly tested to generate suitable antibodies for the sensitive immunodetection of patulin. However, these conventional bioconjugates were unable to elicit the desired immune response, so an alternative strategy that takes advantage of the high electrophilic reactivity of patulin was explored. Patulin was reacted with 4-bromothiophenol, and the obtained adduct was used to produce antibodies with nanomolar affinity values. These results demonstrated for the first time that targeting the adduct resulting from the reaction of patulin with a thiol-containing compound is a promising approach for developing user-friendly immunoanalytical techniques for this elusive mycotoxin.

Assessment of the Optimum Linker Tethering Site of Alternariol Haptens for Antibody Generation and Immunoassay Development.

Immunochemical methods for mycotoxin analysis require antigens with well-defined structures and antibodies with outstanding binding properties. Immunoreagents for the mycotoxins alternariol and/or alternariol monomethyl ether have typically been obtained with chemically uncharacterized haptens, and antigen conjugates have most likely been prepared with mixtures of functionalized molecules. For the first time, total synthesis was performed, in the present study, to obtain two haptens with opposite linker attachment locations. The functionalized synthetic haptens were purified and deeply characterized by different spectrometric methods, allowing the preparation of bioconjugates with unequivocal structures. Direct and indirect competitive enzyme-linked immunosorbent assays, using homologous and heterologous conjugates, were employed to extensively evaluate the generated immunoreagents. Antibodies with high affinity were raised from conjugates of both haptens, and a structure-activity relationship between the synthetic haptens and the specificity of the generated antibodies could be established. These results pave the way for the development of novel highly sensitive immunoassays selective of one or two of these Alternaria mycotoxins.

Immunochemical method for penthiopyrad detection through thermodynamic and kinetic characterization of monoclonal antibodies.

Immunoassays are nowadays being employed for rapid contaminant analysis in clinical, environmental, and agrochemical samples. A thorough characterization of the antibody‒antigen interaction can bring light to the immunoreagent selection process in order to develop sensitive and robust tests. Thus, determination of equilibrium and reaction rate constants is usually recommendable. However, this can be quite tricky for low molecular weight compounds, and competitive strategies are commonly followed to estimate apparent affinity values. In the present study, a collection of monoclonal antibodies to penthiopyrad was raised for the first time, and apparent equilibrium constants were assessed by the Langmuir model using three different competitive enzyme-linked immunosorbent assay formats. The obtained KD values from antibody-coated assays were quite close to the corresponding KD values calculated from surface plasmon resonance (SPR) evaluation. These studies were employed to select a pair of immunoreagents for immunoassay development. The KD value for penthiopyrad of the selected antibody obtained by SPR was 0.28 nM. The optimized direct assay showed an IC50 value for penthiopyrad of 0.42 nM (0.15 ng mL-1) in buffer. The limit of quantification for grape, must, and wine samples was 10 ng mL-1. An excellent correlation was found when immunochemical results were compared with those from LC-MS/MS. As an application case, it was determined that 58% of penthiopyrad was still found in wine after fermentation.

Enzyme and lateral flow monoclonal antibody-based immunoassays to simultaneously determine spirotetramat and spirotetramat-enol in foodstuffs.

Spirotetramat is employed worldwide to fight insect pests due to its high efficiency. This chemical is quickly metabolized by plants into spirotetramat-enol, so current regulations establish that both compounds must be determined in foodstuffs for monitoring purposes. Nowadays, immunochemical methods constitute rapid and cost-effective strategies for chemical contaminant analysis at trace levels. However, high-affinity binders and suitable bioconjugates are required. In this study, haptens with opposite functionalisation sites were synthesized in order to generate high-affinity monoclonal antibodies. A direct competitive enzyme-linked immunosorbent assay with an IC50 value for the sum of spirotetramat and spirotetramat-enol of 0.1 µg/L was developed using selected antibodies and a novel heterologous bioconjugate carrying a rationally-designed hapten. Studies with fortified grape, grape juice, and wine samples showed good precision and accuracy values, with limits of quantification well below the maximum residue limits. Excellent correlation of results was observed with a standard reference chromatographic method. As a step forward, a lateral flow immunoassay was developed for onsite screening analysis of spirotetramat in wine. This assay was successfully validated according to Regulation 519/2014/EU for semi-quantitative methods at concentrations in line with the legal levels of spirotetramat and spirotetramat-enol in grapes, with a satisfactory false suspect rate below 2%.

Immunoanalytical methods for ochratoxin A monitoring in wine and must based on innovative immunoreagents.

Immunochemical methods are highly deployed in analytical laboratories worldwide for monitoring the incidence of mycotoxins in the food chain. Nevertheless, most conventional immunoassays for ochratoxin A (OTA), including commercial kits, show limitations to robustly determine this mycotoxin in grape-derived products below regulated levels (2 ng/mL). Herein, two rapid tests for sensitive OTA determination in wine and must were developed capitalizing on a collection of bioconjugates from innovative synthetic haptens and monoclonal antibodies with subnanomolar affinity. The ELISA (LOD = 8 pg/mL) showed excellent performance in recovery studies, and it was applied to survey commercial wines and musts for OTA contamination. Concerning LFIA, validation according to the Commission Regulation 519/2014 showed that samples exceeding 2 ng/mL were properly scored as uncompliant. More importantly, illegal samples provided a complete inhibition of the test signal, making this test an easy-to-use, rapid, and convenient screening method for in-house control of OTA in wineries.

Monoclonal antibodies with subnanomolar affinity to tenofovir for monitoring adherence to antiretroviral therapies: from hapten synthesis to prototype development.

Approximately 32 million people have died of HIV infection since the beginning of the outbreak, and 38 million are currently infected. Among strategies adopted by the Joint United Nations Programme on HIV/AIDS to end the AIDS global epidemic, the treatment, diagnosis, and viral suppression of the infected subjects are considered crucial for HIV prevention and transmission. Although several antiretroviral (ARV) drugs are successfully used to manage HIV infection, their efficacy strictly relies on perfect adherence to the therapy, which is seldom achieved. Patient supervision, especially in HIV-endemic, low-resource settings, requires rapid, easy-to-use, and affordable analytical tools, such as the enzyme-linked immunosorbent assay (ELISA) and especially the lateral flow immunoassay (LFIA). In this work, high-affinity monoclonal antibodies were generated to develop ELISA and LFIA prototypes for monitoring tenofovir (TFV), an ARV drug present in several HIV treatments. TFV was functionalized by inserting a carboxylated C5-linker at the phosphonic group of the molecule, and the synthetic derivative was conjugated to proteins for mice immunization. Through a rigorous screening strategy of hybridoma supernatants, a panel of monoclonal antibodies strongly binding to TFV was obtained. Following antibody characterization for affinity and selectivity by competitive ELISA, a LFIA prototype was developed and tentatively applied to determine TFV in simulated urine. The point-of-care test showed ultra-high detectability (the visual limit of detection was 2.5 nM, 1.4 ng mL-1), excellent selectivity, and limited proneness to matrix interference, thus potentially making this rapid method a valuable tool for the on-site assessment of patient adherence to ARV therapy.

Click Chemistry-Assisted Bioconjugates for Hapten Immunodiagnostics.

Bioorthogonal reactions have revolutionized the way low-molecular-weight compounds are coupled to biomolecules. Organic chemistry, polymer science, and chemical biology are among the disciplines that have benefited the most from this breakthrough. Despite the reliability of the click chemistry concept for the efficient and chemoselective functionalization of biomacromolecules with haptens at preferred positions, the fact that azide-alkyne cycloaddition reactions originate new chemical moieties as part of the linker may have delayed their application in the immunodiagnostic field. Using the mycotoxin ochratoxin A as a model compound, we herein demonstrate for the first time that bioconjugates arising from the ligation between an azido-bearing hapten and an alkyne-modified carrier protein are able to elicit the generation of high-affinity monoclonal antibodies suitable for the development of rapid methods for the immunodetection of small organic molecules.

Direct competitive immunosensor for Imidacloprid pesticide detection on gold nanoparticle-modified electrodes.

A direct competitive immunosensor for the electrochemical determination of Imidacloprid (IMD) pesticide on gold nanoparticle-modified screen-printed carbon electrodes (AuNP-SPCE) is here reported for the first time. Self-obtained specific monoclonal antibodies are immobilized on the AuNP-SPCE taking advantage of the AuNPs biofunctionalization abilities. In our biosensor design, free IMD in the sample competes with IMD conjugated with horseradish peroxidase (IMD-HRP) for the recognition by the antibodies. After that, 3,3',5,5'-Tetramethylbenzidine (TMB) is enzymatically oxidized by HRP, followed by the oxidized TMB reduction back at the surface of the SPCE. This process gives an associated catalytic current (analytical signal) that is inversely proportional to the IMD amount. The main parameters affecting the analytical signal have been optimized, reaching a good precision (repeatability with a RSD of 6%), accuracy (relative error of 6%), stability (up to one month), selectivity and an excellent limit of detection (LOD of 22 pmol L-1), below the maximum levels allowed by the legislation, with a wide response range (50-10000 pmol L-1). The detection through antibodies also allows to have an excellent selectivity against other pesticides potentially present in real samples. Low matrix effects were found when analysing IMD in tap water and watermelon samples. The electrochemical immunosensor was also validated with HPLC-MS/MS, the reference method used in official laboratories for IMD analysis, through statistical tests. Our findings make the electrochemical immunosensor as an outstanding method for the rapid and sensitive determination of IMD at the point-of-use.

Synthetic Haptens and Monoclonal Antibodies to the Cyanotoxin Anatoxin-a.

Early warning systems for monitoring toxic events may benefit from the availability of monoclonal antibodies enabling the sensitive and specific detection of anatoxin-a, a cyanotoxin involved in numerous cases of animal poisoning resulting from toxic algal blooms in freshwaters. Through the synthesis of three functionalized derivatives of anatoxin-a, we have succeeded in generating the first-ever reported immunoreagents (bioconjugates and antibodies) suitable for the development of immunoanalytical approaches aimed at rapid and onsite detection of this harmful cyanotoxin.

Highly sensitive monoclonal antibody-based immunoassays for the analysis of fluopyram in food samples.

Monoclonal antibody-based techniques have become a useful analytical technology in the agro-food sector. Nowadays, residues of the recently registered fungicide fluopyram are increasingly being found in quality control programs. In the present study, novel chemical derivatives of this pesticide were prepared and specific and high-affinity monoclonal antibodies to fluopyram were raised for the first time. Moreover, immunoassays to fluopyram were developed in two alternative enzyme-linked immunosorbent assay formats, using homologous and heterologous assay conjugates, with limits of detection below 0.05 µg L-1. The optimized immunoassays were applied to the analysis of fluopyram in fortified plums and grapes of four different varieties as well as in in-house prepared musts and wines. Recoveries were between 76.3% and 109.6% and coefficients of variation were below 20%. Quantification limits were well below the maximum residue limits. Immunoassay performance was statistically validated with a reference chromatographic technique using samples from fluopyram-treated plum and grape cultivars.

A monoclonal antibody-based immunosensor for the electrochemical detection of imidacloprid pesticide.

Imidacloprid (IMD) is one of the most used pesticides worldwide as a systemic insecticide as well as for pest control and seed treatment. The toxic and potential carcinogenic character of IMD makes its monitoring of great relevance in the field of agriculture and environment, so sensitive methodologies for in field analysis are strongly required. In this context, we have developed a competitive immunoassay for the determination of IMD using specific monoclonal antibodies followed by electrochemical detection on screen-printed carbon electrodes (SPCE). The optimized immunosensor exhibited a good reproducibility (RSD of 9%) and a logarithmic response in the range 50-10 000 pM of IMD, with an estimated detection limit (LOD) of 24 pM, which was below the maximum levels allowed by the legislation. High-Performance Liquid Chromatography-Mass Spectrometry-Mass Spectrometry (HPLC-MSMS) and Enzyme-Linked Immunosorbent Assay (ELISA) analysis were also performed for comparison purposes, where the electrochemical immunosensor exhibited a wider range of response and a lower detection limit. Matrix effects below 6.5% were obtained using tap water samples. All these characteristics make our electrochemical immunosensor a valid and advantageous tool for the in field determination of IMD.

Combined heterologies for monoclonal antibody-based immunoanalysis of fluxapyroxad.

Nowadays, instrumental methodologies and rapid bioanalytical techniques complement each other for the analysis of toxic chemical compounds. Fluxapyroxad was commercialized a few years ago as a fungicide and today it is being used worldwide to control a variety of pests. In the present study, the development of monoclonal antibody-based immunochemical methods for the analysis of this chemical in food samples was evaluated for the first time. Novel haptens were synthesized and protein bioconjugates were prepared. High-affinity and specific monoclonal antibodies to fluxapyroxad were generated from two haptens with alternative linker tethering sites. Haptens with linker site heterology and a structurally heterologous hapten with a minor modification of the molecule conformation and volume but with a significant alteration of the electronic density of the pyrazole moiety were evaluated for immunoassay development. Direct and indirect competitive immunoassays were characterized and optimized, showing IC50 values for fluxapyroxad of 0.14 and 0.05 ng mL-1, respectively. The combination of two heterologies was particularly adequate in the indirect format. The two developed immunoassays showed excellent recoveries and coefficients of variation in fluxapyroxad-fortified plums and four varieties of grapes. Finally, a good correlation was found between the indirect immunoassay and UPLC-MS/MS when fruit samples with incurred residues of fluxapyroxad were analyzed. These monoclonal antibody-based immunochemical methods hold great promise for fluxapyroxad monitoring.

Hapten Design and Antibody Generation for Immunoanalysis of Spirotetramat and Spirotetramat-enol.

Spirotetramat-a tetramic acid insecticide-is rapidly metabolized or degraded to give spirotetramat-enol; so, common residue definitions include the sum of both compounds. In the present study, two spirotetramat-functionalized derivatives (haptens) have been designed to generate immunoreagents to these molecules for rapid immunochemical analysis. Haptens have been synthesized with alternative linker tethering sites and, for the first time, high-affinity antibodies have been generated with different specificities to these active principles. Two sensitive assays have been developed using the same antibody in different formats, and by using linker-site heterologous haptens, the selectivity of the final immunoassay could be improved. A generic immunoassay with sensitivity similar to spirotetramat and spirotetramat-enol and a specific assay of spirotetramat-enol have been developed. The described antibody and bioconjugates showed great potential for sensitive immunosensor development and analysis of this complex analyte.

Immunochemical rapid determination of quinoxyfen, a priority hazardous pollutant.

In 2013, quinoxyfen was included in the list of priority hazard pollutants of the European Water Framework Directive due to its toxicity to aquatic organisms. However, few analytical methods for the analysis of this fungicide have been reported and no rapid immunochemical methods have been published so far. In the present study, immunoreagents for quinoxyfen analysis were generated for the first time and an enzyme-linked immunosorbent assay was developed. Two carboxylated derivatives of quinoxyfen were designed on the basis of the minimum energy conformation of the target compound. Active esters of those novel compounds were prepared using N,N'-disuccinimidyl carbonate, and purified for covalent coupling to proteins. Matrix-assisted laser desorption mass spectrometry of the prepared bioconjugates showed optimum hapten-to-protein molar ratios. Moreover, high-affinity antibodies specific of quinoxyfen were raised. As proof of concept, an immunoassay was evaluated using a heterologous conjugate, which afforded sensitivity values in the low nanomolar range. Moreover, excellent recoveries and coefficients of variation were obtained from the analysis of environmental water samples fortified with quinoxyfen. A limit of quantification of 60 µg/L was determined. The prepared bioconjugates and antibodies could be valuable immunoreagents for the development of a variety of rapid immunosensors for quinoxyfen determination in environmental samples.

Development of immunosorbents for the analysis of forchlorfenuron in fruit juices by ion mobility spectrometry.

The advantages of using smart materials as immunosorbents in the analysis of complex matrices by ion mobility spectrometry (IMS) have been highlighted in this study. A novel analytical method has been proposed for the sensitive, selective, and fast determination of residues of the plant growth regulator forchlorfenuron in fruit juices. Three different monoclonal antibodies (s3#22, p2#21, and p6#41) were employed for the production of immunosorbents, based on Sepharose gel beads, which were characterized in terms of loading capacity, solvent resistance, and repeatability for its use in solid-phase extraction (SPE). Immunosorbents that were prepared with antibody p6#44 provided the best performance, with a loading capacity of 0.97 µg, a 10% (v/v) 2-propanol tolerance, and a reusability of at least eight uses. The SPE procedure involved the use of a column with 0.15 g Sepharose beads, containing 0.5 mg antibody, which was loaded to 20 mL of the sample, washed with 2 mL of water plus 2 mL of 10% (v/v) 2-propanol, and eluted with 2 mL of 2-propanol. The cleaned extract was directly analyzed by IMS, giving a limit of detection of 2 µg L-1 with a relative standard deviation of 7.6%. Trueness was assessed by the analysis of blank grape and kiwifruit juice samples spiked with forchlorfenuron concentrations from 10 to 400 µg L-1, with recoveries from 80 to 115%. The analytical performance of the proposed immunosorbent was compared with conventional extraction and cleanup methods, such as QuEChERS and C18-based SPE, giving the cleanest extracts for accurate determinations of forchlorfenuron by IMS. Graphical abstract ᅟ.