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Large-scale, rapid, and inexpensive serological diagnoses of severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) are of great interest in reducing virus transmission at the population level; however, their development is greatly plagued by the lack of available point-of-care methods, leading to low detection efficiency. Herein, an ultrasensitive smartphone-based electrochemical immunoassay is reported for rapid (less than 5 min), low-cost, easy-to-implement detection of the SARS-CoV-2 nucleocapsid protein (SARS-CoV-2 N protein). Specifically, the electrochemical immunoassay was fabricated on a screen-printed carbon electrode coated with electrodeposited gold nanoparticles, followed by incubation of anti-N antibody (Ab) and bovine serum albumin as the working electrode. Accompanied by the antigen-antibody reaction between the SARS-CoV-2 N protein and the Ab, the electron transfer between the electroactive species [Fe(CN)

The article contains sections titled: 1. Enzymatic Analysis Methods 1.1. Introduction 1.2. Enzymes: Basic Kinetics 1.3. Enzyme Assays: Practical Aspects 1.3.1. General Considerations 1.3.2. Spectrometric Methods 1.3.3. Other Methods 1.3.4. Effects of pH, Buffer, Composition, and Temperature 1.3.5. Sources and Activity of Enzymes 1.4. Enzyme Assays: Determination of Substrates 1.4.1. General Considerations 1.4.2. Determination of Glucose 1.4.3. Determination of Ethanol 1.5. Enzyme Assays: Determination of Inhibitors 1.5.1. Inhibitors of Cholinesterase Enzymes 1.5.2. Other Inhibition Methods 1.6. Enzyme Assays: Determination of Activators 1.6.1. Determination of Metal Ions by Metalloenzymes 1.6.2. Other Activator Analyses 1.7. Immobilized Enzymes 1.7.1. Introduction 1.7.2. Properties of Immobilized Enzymes 1.7.3. Application of Immobilized Enzymes 2. Immunoassays in Analytical Chemistry 2.1. Introduction 2.2. Polyclonal, Monoclonal, and Recombinant Antibodies 2.3. Sample Conditioning 2.4. Immunoassays 2.4.1. Radioimmunoassay 2.4.1.1. Isotopic Dilution Radioimmunoassay 2.4.1.2. Immunoradiometric Assay (IRMA) 2.4.2. Nonisotopic Homogeneous Immunoassay 2.4.2.1. Latex Particle Agglutination Immunoassay 2.4.2.2. Enzyme-Multiplied Immunoassay Technique (EMIT) 2.4.2.3. Apoenzyme Reconstitution Immunoassay System (ARIS) 2.4.2.4. Fluorophore-Labeled Homogeneous Immunoassay 2.4.2.5. Homogeneous Fluorescence Polarization Immunoassay (FPIA) 2.4.2.6. Homogeneous Liposome Immunoassay 2.4.3. Nonisotopic Heterogeneous Immunoassay 2.4.3.1. Enzyme-Linked Immunosorbent Assay (ELISA) 2.4.3.2. Sandwich Immunoassay 2.4.3.3. Immunoassays using Immobilized Affinity Ligands 2.4.3.4. Immunoassays with Biotin and Avidin 2.4.3.5. Magnetic Particle Immunoassay 2.5. Immunoaffinity Techniques 2.6. Immunoassays on Test Strips and other Planar Structures 2.7. Characterization and Interference 2.8. Future Immunological Applications and Techniques

This work presented a point-of-care (POC) photoelectrochemical (PEC) biosensing for the detection of human papillomavirus-16 (HPV-16) on a portable electrochemical detection system by using CRISPR-Cas12a trans-cleaving the G-quadruplex for the biorecognition/amplification and a hollow In

This article reports current research efforts towards designing bespoke microscale extraction approaches exploiting the versatility of 3D printing for fast prototyping of novel geometries of sorptive devices. This is demonstrated via the so-called 3D printed spinning cup-based platform for immunoextraction of emerging contaminants using diclofenac as a model analyte. A new format of rotating cylindrical scaffold (containing a semispherical upper cavity) with enhanced coverage of biorecognition elements, and providing elevated enhancement factors with no need of eluate processing as compared with other microextraction stirring units is proposed. Two distinct synthetic routes capitalized upon modification of the acrylate surface of stereolithographic 3D printed parts with hexamethylenediamine or branched polyethyleneimine chemistries were assayed for covalent binding of monoclonal diclofenac antibody.Under the optimized experimental conditions, a LOD of 108 ng L−1 diclofenac, dynamic linear range of 0.4–1,500 µg L–1, and enrichment factors > 83 (for near-exhaustive extraction) were obtained using liquid chromatography coupled with UV–Vis detection. The feasibility of the antibody-laden device for handling of complex samples was demonstrated with the analysis of raw influent wastewaters with relative recoveries ranging from 102 to 109%. By exploiting stereolithographic 3D printing, up to 36 midget devices were fabricated in a single run with an estimated cost of mere 0.68 euros per 3D print and up to 16 €/device after the incorporation of the monoclonal antibody. Graphical abstract

With the introduction of gas-based contactless electrochemical biosensors lies the prospects of separating the sensing interface from the bioassembly platform, enhancing stability, and exploring si...

This work presented an innovative and rationally engineered palindromic molecular beacon (PMB) based "Z-scheme" photoelectrochemical (PEC) biosensing protocol for the selective screening of kanamycin (Kana) through DNA hybridization-induced conformational conversion. Interestingly, the ingeniously designed PMB integrated the multifunctional elements including recognition region, primer-like palindromic fragment, and polymerization-nicking template. The cosensitized structures consisted of CdS quantum dot functionalized hairpin DNA2 (QD-HP2) and region-selectively deposited gold nanoparticles onto {001} facets of bismuth oxychloride (BiOCl-Au). Compared with BiOCl-Au alone, the attachment of CdS QDs onto BiOCl-Au (i.e., BiOCl-Au-CdS QDs) exhibited evidently enhanced photocurrent intensity thanks to the synergistic effect of Z-scheme BiOCl-Au-CdS QDs. After incubation with target Kana, Kana-aptamer binding could induce the exposure of PMB region for hairpin DNA1 (HP1). The exposed palindromic tails hybridized with each other (like a molecular machine) to consume the substrates (dNTPs) and fuels (enzyme) for the releasing of numerous nick fragments (Nick). The as-generated nick fragments could specifically hybridize with the complementary region of QD-HP2, thus resulting in decreasing photocurrent because of the increasing spatial distance for electron transfer between two-type photosensitizers. Under optimum conditions, the PMB-based sensing system exhibited satisfying photocurrent responses toward target Kana within the working range from 50 to 5000 fM at a low detection limit of 29 fM. Impressively, the concept of a palindromic fragment-mediated primer-free biosensing strategy offers a new avenue for advanced development of efficient and convenient biodetection systems.

International audience; Because the broadly consumed pain killer diclofenac (DCF) is a recognized pollutant, monitoring of its concentration is routinely performed in surface waters. As a valuable alternative to chromatographic and immunochemical assays, we developed a piezoelectric immunosensor to quantify DCF, first in buffer (PBS) and then in river water samples. A sensing layer comprising DCF was built up on the surface of silica-coated quartz sensors using a robust coupling chemistry. Binding of a highly affine monoclonal anti-DCF antibody was monitored in real time by quartz crystal microbalance with dissipation (QCM-D) measurements from which were determined a dissociation constant KD of 0.24 nM and an acoustic antibody surface coverage of 1120 ng/cm2 at saturation. On the other hand, an optical antibody surface coverage of 260 ng/cm2 was determined by combined nanoplasmonic sensing measurement, giving a hydration percentage of 75% for the antibody monolayer. DCF assay was further set up following a competitive format for which binding of antibody to the sensing layer is inhibited by DCF in solution. The piezoelectric sensor response expressed as frequency shift ΔF was inversely related to the concentration of DCF with a dynamic range of 15–46 nM and a limit of detection (LoD) of 9.5 nM (2.8 μg/L) in PBS. This piezoelectric immunosensor was eventually applied to the assay of DCF in surface water samples taken at three different locations in the Seine and Marne rivers. The calculated concentration of DCF in these samples was in good agreement with official data published by the French center of water analysis eaufrance

Surface plasmon resonance (SPR) sensors are well-established and widely used in the field of environmental and life sciences. To overcome the limitation of SPR sensors to applications in the laboratory environment initial studies on a low-cost nanostructured sensor substrate fabricated by nanoimprint lithography for the development of a SPR-based on-site biosensor system were conducted. For this purpose, diclofenac molecules were successfully immobilized both on planar and nanostructured gold sensor substrates, which was proved by XPS and SPR measurements. For the latter substrates, a specific binding between an anti-diclofenac antibody and immobilized diclofenac can be observed in form of a localized surface plasmon resonance shift in the optical transmission spectrum. The results show that our low-cost sensor substrate is wellsuited as transducer element for future SPR-based biosensors.

Benzo[a]pyrene, which is produced during the incomplete combustion of organic material, is an abundant noxious pollutant because of its carcinogenic metabolic degradation products. The high-affinity (KD ≈3 nm) monoclonal antibody 22F12 allows facile bioanalytical quantification of benzo[a]pyrene even in complex matrices. We report the functional and X-ray crystallographic analysis of 22F12 in complex with 3-hydroxybenzo[a]pyrene after cloning of the V-genes and production as a recombinant Fab fragment. The polycyclic aromatic hydrocarbon is bound in a deep pocket between the light and heavy chains, surrounded mainly by aromatic and aliphatic amino acid side chains. Interestingly, the hapten-antibody interface is less densely packed than expected and reveals polar, H-bond-like interactions with the polycyclic aromatic π-electron system, which may allow the antibody to maintain a large, predominantly hydrophobic binding site in an aqueous environment while providing sufficient complementarity to its ligand.

Plasmonic enzyme-linked immunosorbent assays (ELISA) using the localized surface plasmon resonance (LSPR) of metal nanoparticles has emerged as an appealing alternative to conventional ELISA counterparts for ultrasensitive naked-eye detection of biomolecules and small contaminants. However, batchwise plasmonic ELISA involving end-point detection lacks ruggedness inasmuch as the generation or etching of NP is greatly dependent on every experimental parameter of the analytical workflow. To tackle the above shortcomings, this paper reports on an automatic flow methodology as a reliable detection scheme of hydrogen peroxide related enzymatic bioassays for ultrasensitive detection of small molecules. Here, a competitive ELISA is combined with the in-line generation of plasmonic gold nanoparticles (AuNPs) followed by the real-time monitoring of the NP nucleation and growth rates and size distribution using a USB miniaturized photometer. Glucose oxidase was labeled to the secondary antibody and yielded hydrogen peroxide that acted as the measurand and the reducing agent of the Au(III)/citrate system in the flow network. High-throughput plasmonic assays were feasible by assembling a hybrid flow system composed of two microsyringe pumps, a perfluoroalkoxy alkane reaction coil, and a 26-port multiposition valve and operated under computer-controllable flow conditions. The ultratrace determination of diclofenac in high matrix samples, e.g., seawater, without any prior sample treatment was selected as a proof-of-concept application of the flow-based platform for determination of emerging contaminants via plasmonic ELISA. The detection limit (0.001 μg L-1) was 1 order of magnitude lower than that endorsed by the first EU Watch List for diclofenac as a potentially emerging contaminant in seawater and also than that of a conventional colorimetric ELISA, which in turn is inappropriate for determination of diclofenac in seawater at the levels endorsed by the EU regulation. The proposed automatic fluidic approach is characterized by the reproducible timing in AuNPs nucleation and growth along with the unsupervised LSPR absorbance detection of AuNPs with a dynamic range for diclofenac spanning from 0.01 to 10 μg L-1. Repeatability and intermediate precision (given as normalized signal readouts) in seawater were

With the introduction of gas-based contactless electrochemical biosensors lies the prospects of separating the sensing interface from the bioassembly platform, enhancing stability, and exploring signal transduction mechanism, all intimately linking to development of immunoassay. Herein, we report on a H

Beyond extraordinary in vivo accurate gene editing and regulation capabilities, the CRISPR-Cas-associated biotechnology has created a new era of in vitro nucleic acid sensing due to its inherent high-efficiency enzyme cleavage activity and robustness. However, most of the existing CRISPR-Cas systems are largely involve fluorescent reporters or lateral flow strips biosensor and can't fully explore their potential applications (sensitivity and field-deployable) due to the lack of effective signal transduction and wireless data transmission with smartphone readout. Herein, a CRISPR-Cas12a-mediated instrument-free based on flexible interdigitated electrodes-modified piezoresistive block [abrasive paper-molded microstructure polydimethylsiloxane (PDMS) and MXene (Ti3C2Tx)-PEDOT:PSS film] was designed for point-of-care of human papillomavirus (HPV)-related DNA with integrated-module smartphone visual readout. Biotin/thiol-modification single-stranded DNA (ssDNA), acts as a linker between Au@Pt nanoparticles (Au@PtNPs) and streptavidin-coated magnetic bead (MB), are non-specifically cleaved by Cas12a when the guide RNA binds to the target HPV-DNA. Released and separated Au@PtNPs efficiently catalyzes H2O2 to O2, and further compress the Ti3C2Tx-PEDOT:PSS/PDMS in a 3D-printed home-made pressure-tight vessel, thus causing the increasing current of the whole circuit thanks to contacting deformation of Ti3C2Tx-PEDOT:PSS/PDMS module and interdigital electrode. After integrating the Bluetooth device and wireless sensing technology in the circuit, the current signal of the target DNA can be wirelessly collected and recorded in real-time and further transmitted/displayed to the mobile terminal of the smartphone. This all-in-one detection mode not only bridges the technological gap between biological signal conduction, wireless transmission, and smartphone interface but also improves the portability and the sensitivity (more than one order of magnitude lower than that of traditional CRISPR-Cas12a biosensors). We expect that a powerful CRISPR-Cas12a system coupled with piezoresistive sensor with wireless transmission technology will become a great demonstration and widely used in real-time wireless biomedical analysis, portable point-of-care health monitoring, and molecular diagnostics.

Mycotoxins, highly toxic secondary metabolites produced by many invading species of filamentous fungi, contaminate different agricultural commodities under favorable temperature and humidity conditions. Herein, we successfully devised a novel signal-on photoelectrochemical immunosensing platform for the quantitative monitoring of mycotoxins (aflatoxin B1, AFB1, used as a model) in foodstuffs on the basis of silver nanolabels-assisted ion-exchange reaction with CdTe quantum dots (QDs) mediated hole-trapping. Initially, a competitive-type immunoreaction was carried out on a high-binding microplate by using silver nanoparticle (AgNP)-labeled AFB1-bovine serum albumin (AFB1-BSA) conjugates as the tags. Then, the carried AgNPs with AFB1-BSA were dissolved by acid to release numerous silver ions, which could induce ion-exchange reaction with the CdTe QDs immobilized on the electrode, thus resulting in formation of surface exciton trapping. Relative to pure CdTe QDs, the formed exciton trapping decreased the photocurrent of the modified electrode. In contrast, the detectable photocurrent increased with the increase of target AFB1 in a dynamic working range from 10 pg mL(-1) to 15 ng mL(-1) at a low limit of detection (LOD) of 3.0 pg mL(-1) under optimal conditions. In addition, the as-prepared photoelectrochemical immunosensing platform also displayed high specificity, good reproducibility, and acceptable method accuracy for detecting naturally contaminated/spiked blank peanut samples with consistent results obtained from the referenced enzyme-linked immunosorbent assay (ELISA) method.

Herein we describe the development of a homogeneous competitive colorimetric immunoassay using antigen-functionalized magnetic beads (MBs) and antibody-immobilized gold nanoparticles (AuNPs) combined with the established gold staining method for the determination of microcystin-leucine-arginine (MC-LR) in surface water. Solid phase extraction proved to be the most beneficial sample preparation method in order to reduce matrix effects which might lead to agglomeration of the AuNPs and failing of the assay. The developed method could detect MC-LR in a linear range from 0.51–11.68 μg L−1 with a limit of detection (LOD) of 0.38 μg L−1. The recovery rates of the toxin in spiked water samples were in the range from 88.8% to 117.5%. This proposed immunoassay constitutes a simple and cost-effective method for the detection of sanitary relevant concentrations around 1 μg L−1 of MC-LR in fresh waters without any expensive or challenging equipment.

Pressure-based portable bioassays incorporating biomolecular-recognition with gas-generation have been widely developed, but most involve low-sensitivity and are therefore unsuitable for routine use. Herein, a tactile chemomechanical transduction strategy based on sponge electrode-modified barium titanate-polypyrrole-reduced graphene oxide (BTO-PPy-rGO, piezoelectric and piezoresistive composite nanomaterial) was designed for point-of-care immunoassay/aptasensor of antibiotics, using portable digital multimeter visual readout. The disposable syringe was linked with target-induced gas-producing reactions, which transduce biomolecules signals to the volume of the syringe in real time. The weight of the same volume of liquid as the syringe was applied to the BTO-PPy-rGO/sponge electrode, and the detectable signal was derived from the resistance change. The resulting tactile flexible platform displayed successive and desirable mechanical compliance while generating extremely stable and reliable electric signal output during deformation, which lays a foundation for developing general applicability, ease of operation, yet highly sensitive portable bioassays. We expect to improve the automation of the entire testing process in the follow-up work to maximize its inherent advantages.

Eutrophication of water bodies can promote cyanobacterial (blue-green algae) blooms, which has become a source of increasing concern for both recreational and drinking water use. Many bacterial species can produce toxins that pose threats to wildlife, domestic animals and humans. Microcystin-leucine-arginine (MC-LR) is the most frequent and most toxic microcystin congener. For the first time, lab-scale investigations were performed to test the application of a recombinant plant-derived anti-MC-LR antibody immobilized on an immunoaffinity support material to selectively extract the toxin from spiked freshwater samples. As a comparison, its hybridoma-derived counterpart (murine monoclonal antibody) was evaluated. The antibody-doped material was prepared via an optimized sol-gel process; its stability and binding efficiency of MC-LR in spiked freshwater samples were thoroughly tested using the ELISA and orthogonal LC-MS methods. For removal, two column-based procedures with sequential or continuous cyclic sample addition and a suspension mode (moving adsorbent) were tested. Noteworthy the results obtained with a crude antibody fraction were fully compatible with the highly purified preparation. This study paves the way for further investigation being focused on novel applications of plant-derived anti-MC-LR antibodies in bioremediation to selectively deplete the toxin from freshwater: a green and promising technology without secondary pollution.

An affinity sensor based on capacitive transduction was developed to detect benzo(a)pyrene (BaP) in river water. Two types of recognition elements, the synthetic receptor analogues molecularly imprinted polymers (MIPs) and natural monoclonal antibody (mAb) were tested for this type of biosensor. Different polymerization strategies to obtain MIPs were compared. One approach comprised a preliminary batch synthesis of beads that were subsequently coupled covalently to an electrode surface. Another approach consisted of the in-situ synthesis of MIPs directly onto the electrode surface using electropolymerization. The latter approach provided the best results. To choose the optimal recognition element mAb and MIP-modified electrodes different sets were evaluated with regards to their sensitivity, selectivity, linear range and re-usability. The mAb-modified electrodes were considerably more sensitive toward BaP (ng L−1 range vs µg L−1 range for the MIP-modified one), and showed a broader linear working range than the MIP-modified electrodes. The latter revealed more suitable for group-selective determination of PAHs. The developed capacitive sensor was tested for the detection of BaP in naturally-contaminated water samples collected in different places of Ghent, Belgium. The results obtained with the sensor were coherent and reproducible, and were in a good agreement with the confirmation technique, HPLC-FlD.

The non-steroidal anti-inflammatory drug (NSAID) diclofenac (DCF) is found worldwide in the aqueous environment. Therefore, it has raised increased public concern on potential long-term impact on human health and wildlife. The importance of DCF has been emphasized by the European Union recently by including this pharmaceutical in the first watch list of priority hazardous substances in order to gather Union-wide monitoring data. Rapid and cheap methods of analysis are therefore required for fresh and wastewater monitoring with high sample load. Here, for the first time, well-characterized monoclonal antibodies (mAbs) against DCF were generated and a highly sensitive ELISA developed. The best antibody (mAb 12G5) is highly affine (KD = 1.5 × 10(-10) M), stable to potential matrix interferences such as pH value (pH range 5.2-9.2), calcium ion concentration (up to 75 mg/L), and humic acid content (up to 20 mg/L). The limit of detection (LOD, S/N = 3) and IC50 of the ELISA calibration curve were 7.8 and 44 ng/L, respectively. The working range was defined between 11 and 180 ng/L. On average, about 10 % cross-reactivity (CR) was found for DCF metabolites 5-OH-DCF, 4'-OH-DCF, and DCF-acyl glucuronide, but other structurally related NSAIDs showed binding

A novel (invertase) enzymatic hydrolysate-triggered displacement reaction strategy with multifunctional silica beads, doped with horseradish peroxidase-thionine (HRP-Thi) conjugate, was developed for competitive-type electrochemical immunoassay of small molecular aflatoxin B1 (AFB1). The competitive-type displacement reaction was carried out on the basis of the affinity difference between enzymatic hydrolysate (glucose) and its analogue (dextran) for concanavalin A (Con A) binding sites. Initially, thionine-HRP conjugates were doped into nanometer-sized silica beads using the reverse micelle method. Then monoclonal anti-AFB1 antibody and Con A were covalently conjugated to the silica beads. The immunosensor was prepared by means of immobilizing the multifunctional silica beads on a dextran-modified sensing interface via the dextran-Con A binding reaction. Gold nanoparticles functionalized with AFB1-bovine serum albumin conjugate (AFB1-BSA) and invertase were utilized as the trace tag. Upon target AFB1 introduction, a competitive-type immunoreaction was implemented between the analyte and the labeled AFB1-BSA on the nanogold particles for the immobilized anti-AFB1 antibody on the electrode. The invertase followed by gold nanoparticles hydrolyzed sucrose into glucose and fructose. The produced glucose displaced the multifunctional silica beads from the electrode based on the classical dextran-Con A-glucose system, thus decreasing the catalytic efficiency of the immobilized HRP on the electrode relative to that of the H2O2-thionine system. Under optimal conditions, the detectable electrochemical signal increased with the increasing target AFB1 in a dynamic working range from 3.0 pg mL(-1) to 20 ng mL(-1) with a detection limit of 2.7 pg mL(-1). The strong bioconjugation with two nanostructures also resulted in a good repeatability and interassay precision down to 9.3%. Finally, the methodology was further validated for analysis of naturally contaminated or spiked AFB1 peanut samples, giving results matched well with those from a commercialized AFB1 enzyme-linked immunosorbent assay kit. Importantly, the system provides a signal-on competitive-type immunosensing platform for ultrasensitive detection of small molecules.

Aflatoxin B

Because multianalyte methods are highly desirable in order to keep analysis time and costs low, the biosensor development increasingly focuses on parallel analysis of several mycotoxins. Here, we describe an indirect competitive immunoassay on regenerable, reusable glass microchips for the parallel determination of aflatoxins, ochratoxin A, deoxynivalenol, and fumonisin B1 in oat extracts, using a fully automated flow-through device with chemiluminescence readout.

We report on a specially designed diclofenac-ELISA for the determination of diclofenac in the presence of release-active forms of diclofenac in lactose dissolved in water solutions according to a predefined schedule in single-blind experiments. In accordance with the objective of this project, a number of experiments were conducted to determine the optimal ELISA conditions for detecting potential modulatory effects of release-active forms of diclofenac depending on their ability to affect the binding of diclofenac to anti-diclofenac antibodies. As a feature, the diclofenac antibodies were previously incubated with manufactured pharmaceutical samples containing release-active forms of diclofenac or placebo. For comparison of the sample types, measured in ELISA optical densities were chosen. For statistic analysis, Student's two-sample t-test and single-factor ANOVA were applied. The extremely low concentrations of diclofenac of 0.01, 0.05 and 0.1 ng mL− 1 seem most appropriate for routine assay performance. The source of diclofenac used for standard solution preparation is not important but it could be important as the source of diclofenac for release active form of diclofenac preparation. As an outcome, the ELISA appeared to be suitable for the detection of the modifying effects of release-active forms of diclofenac toward the pharmaceutical substance in vitro.

This paper describes the development of a molecularly imprinted polymer-based solid phase extraction (MISPE) method coupled with enzyme-linked immunosorbent assay (ELISA) for determination of the PAH benzo[a]pyrene (B[a]P) in vegetable oils. Different molecularly imprinted polymers (MIPs) were prepared using non-covalent 4-vinylpyridine/divinylbenzene co-polymerization at different ratios and dichloromethane as porogen. Imprinting was done with a template mixture of phenanthrene and pyrene yielding a broad-specific polymer for PAHs with a maximum binding capacity (Q) of ~32 μg B[a]P per 50 mg of polymer. The vegetable oil/n-hexane mixture (1:1, (v/v)) was pre-extracted with acetonitrile, the solvent evaporated, the residue reconstituted in n-hexane and subjected to MISPE. The successive washing with n-hexane and isopropanol revealed most suitable to remove lipid matrix constituents. After elution of bound PAHs from MISPE column with dichloromethane, the solvent was evaporated, the residue reconstituted with dimethyl sulfoxide and diluted 100-fold with methanol/water (10:90, (v/v)) for analysis of B[a]P equivalents with an ELISA. The B[a]P recovery rates in spiked vegetable oil samples of different fatty acid composition were determined between 63% and 114%. The presence of multiple PAHs in the oil sample, because of MIP selectivity and cross-reactivity of the ELISA, could yield overestimated B[a]P values.

SummaryThree Critically Endangered Gyps vultures endemic to South Asia continue to decline due to the use of diclofenac to treat livestock. High nephrotoxicity of diclofenac to Gyps vultures, leading to death, has been established by experiment and observation, in four out of five Gyps vulture species which occur in South Asia. Declines have also been observed in South Asia’s four other non-Gyps vulture species, but to date there has been no evidence about the importance of diclofenac as a potential cause. Neither is there any evidence on the toxicity of diclofenac to the Accipitridae other than vultures. In this study, gross and microscopic lesions and diclofenac tissue levels in Steppe Eagles Aquila nipalensis found at a cattle carcass dump in Rajasthan, India, show evidence of the toxicity of diclofenac for this species. These findings suggest the possibility that diclofenac is toxic to other accipitrid raptors and is therefore a potential threat to much wider range of scavenging species in South Asia.

Characterization of interactions between antigens and antibodies is of utmost importance both for fundamental understanding of the binding and for development of advanced clinical diagnostics. Here, fluorescence line-narrowing (FLN) spectroscopy was used to study physicochemical interactions between 3-hydroxybenzo[a]pyrene (3OH-BaP, as antigen) and a variety of solvent matrices (as model systems) or anti-polycyclic aromatic hydrocarbon antibodies (anti-PAH). We focused the studies on the specific physicochemical interactions between 3OH-BaP and different, previously obtained, monoclonal and recombinant anti-PAH antibodies. Control experiments performed with non-binding monoclonal antibodies and bovine serum albumin (BSA) indicated that nonspecific interactions did not affect the FLN spectrum of 3OH-BaP. The spectral positions and relative intensities of the bands in the FLN spectra are highly dependent on the molecular environment of the 3OH-BaP. The FLN bands correlate with different vibrational modes of 3OH-BaP which are affected by interactions with the molecular environment (pi-pi interactions, H-bonding, or van-der-Waals forces). Although the analyte (3OH-BaP) was the same for all the antibodies investigated, different binding interactions could be identified from the FLN spectra on the basis of structural flexibility and conformational multiplicity of the antibodies' paratopes.

Liposomes loaded with water-soluble and water-insoluble quantum dots (QD) were for the first time applied as labels in different heterogeneous immunoassays for the determination of food contaminants, using mycotoxin zearalenone (ZEN) as a model. A great deal of work was devoted to the optimal choice of phospholipids for the liposomes preparation and to the factors which are important for the stability and size of obtained liposomes. Thin-film hydration and reverse-phase evaporation techniques were evaluated in terms of stability of the obtained liposomes and their efficiency for QD loading. Conjugation of liposomes with proteins and the influence of cross-linkers to the nonspecific interaction of the obtained liposomes with the surface of microtiter plates and cartridges were investigated and 3-(2-pyridyldithio)propionic acid N-hydroxysuccinimide ester was found as the optimal cross-linker. The limits of detection (LOD) for ZEN of fluorescence-labeled immunosorbent assays were 0.6 μg kg(-1), 0.08 μg kg(-1), and 0.02 μg kg(-1), using QD, liposomes loaded with water-soluble QD, and water-insoluble QD, respectively. Similarly, the developed qualitative on-site tests using the different QD labels and taking into account the EU maximum residues level for ZEN in unprocessed cereals showed cutoff levels of 100, 50, and 20 μg kg(-1).

An automated flow-through multi-mycotoxin immunoassay using the stand-alone Munich Chip Reader 3 platform and reusable biochips was developed and evaluated. This technology combines a unique microarray, prepared by covalent immobilization of target analytes or derivatives on diamino-poly(ethylene glycol) functionalized glass slides, with a dedicated chemiluminescence readout by a CCD camera. In a first stage, we aimed for the parallel detection of aflatoxins, ochratoxin A, deoxynivalenol, and fumonisins in cereal samples in a competitive indirect immunoassay format. The method combines sample extraction with methanol/water (80:20, v/v), extract filtration and dilution, and immunodetection using horseradish peroxidase-labeled anti-mouse IgG antibodies. The total analysis time, including extraction, extract dilution, measurement, and surface regeneration, was 19 min. The prepared microarray chip was reusable for at least 50 times. Oat extract revealed itself as a representative sample matrix for preparation of mycotoxin standards and determination of different types of cereals such as oat, wheat, rye, and maize polenta at relevant concentrations according to the European Commission regulation. The recovery rates of fortified samples in different matrices, with 55–80 and 58–79 %, were lower for the better water-soluble fumonisin B1 and deoxynivalenol and with 127–132 and 82–120 % higher for the more unpolar aflatoxins and ochratoxin A, respectively. Finally, the results of wheat samples which were naturally contaminated with deoxynivalenol were critically compared in an interlaboratory comparison with data obtained from microtiter plate ELISA, aokinmycontrol® method, and liquid chromatography–mass spectrometry and found to be in good agreement.

We have developed a simple and sensitive competitive enzyme-linked immunosorbent assay (ELISA) to determine aflatoxin B1 (as a model small analyte) and using streptavidin-polymeric horseradish peroxidase complex (SApolyHRP) as a label for signal amplification. The performance of the assay was evaluated by comparing it with the classical indirect competitive ELISA using HRP labeled anti-mouse IgG as the tracer antibody. The results indicate that the SApolyHRP-based competitive ELISA exhibits a typically 2.4-fold steeper slope of the linear working range of the calibration curve compared to the monomeric HRP based classical ELISA, i.e., the sensitivity was increased. The SApolyHRP conjugate causes a typically 19-fold stronger signal generation in comparison to the traditional HRP labeled anti-mouse IgG at the same concentration (25 ng mL−1). Moreover, the SApolyHRP-based assay has a much wider linear range and a 3.8-fold better signal-to-noise ratio. Considering its simplicity, sensitivity and ease of operation, this competitive ELISA is considered to be a promising tool for small molecule immunodetection.

Photon-upconverting nanoparticles (UCNPs) emit light of shorter wavelength under near-infrared excitation and thus avoid optical background interference. We have exploited this unique photophysical feature to establish a sensitive competitive immunoassay for the detection of the pharmaceutical micropollutant diclofenac (DCF) in water. The so-called upconversion-linked immunosorbent assay (ULISA) was critically dependent on the design of the upconversion luminescent detection label. Silica-coated UCNPs (50 nm in diameter) exposing carboxyl groups on the surface were conjugated to a secondary anti-IgG antibody. We investigated the structure and monodispersity of the nanoconjugates in detail. Using a highly affine anti-DCF primary antibody, the optimized ULISA reached a detection limit of 0.05 ng DCF per mL. This performance came close to a conventional enzyme-linked immunosorbent assay (ELISA) without the need for an enzyme-mediated signal amplification step. The ULISA was further employed for analyzing drinking and surface water samples. The results were consistent with a conventional ELISA as well as liquid chromatography-mass spectrometry (LC-MS).

Plants have emerged as commercially relevant production systems for pharmaceutical and nonpharmaceutical products. Currently, the commercially available nonpharmaceutical products outnumber the medical products of plant molecular farming, reflecting the shorter development times and lower regulatory burden of the former. Nonpharmaceutical products benefit more from the low costs and greater scalability of plant production systems without incurring the high costs associated with downstream processing and purification of pharmaceuticals. In this review, we explore the areas where plant-based manufacturing can make the greatest impact, focusing on commercialized products such as antibodies, enzymes, and growth factors that are used as research-grade or diagnostic reagents, cosmetic ingredients, and biosensors or biocatalysts. An outlook is provided on high-volume, low-margin proteins such as industrial enzymes that can be applied as crude extracts or unprocessed plant tissues in the feed, biofuel, and papermaking industries.

International audience; We investigated the assembly of Gold nanoparticles (AuNPs) on Gold and Silicon sensors with two final objectives: (i) understanding the factors governing the interaction and (ii) building up a nanostructured piezoelectric immunosensor for diclofenac, a small-sized pharmaceutical pollutant. Different surface chemistries were devised to achieve AuNPs assembly on planar substrates. These surface chemistries included amines to immobilize AuNPs via electrostatic interaction, or a mixture of amines and thiols to covalently attach the AuNPs. We also generated PEG-amine terminated surfaces to benefit from the well-known non-biofouling properties of PEG-coated surfaces. The functional substrates and the resulting gold nanoparticle layers were characterized in detail by Surface IR, contact angle measurements and Scanning Electron Microscopy (SEM). The mechanism of adsorption is discussed herein considering the nature of the terminal groups and their charge at the pH of AuNPs adsorption. The coverage and the dispersion of AuNPs were strongly dependent on the anchoring points on the surfaces; the optimal were reached when the attachment layer offered multiple interaction points, in particular, for NH2/SH and PEG/NH2 terminated surfaces, where the percentage of isolated particles was up to 78 %. In addition, PEG-coated surfaces led to a stable AuNPs layer resistant to ultrasounds and to further functionalization of the immobilized nanoparticles. These surfaces were used to engineer quartz crystal microbalance (QCM) biosensors for diclofenac detection. The AuNPs nanostructured substrates significantly enhanced the biosensor sensitivity as compared to planar substrates (up to 6 times higher). This enhancement presages a higher sensitivity in the competitive detection of diclofenac on these systems. More importantly, despite the biorecognition and the drastic regeneration conditions, SEM images show that gold nanoparticles layers are stable and reliable, which paves the way for their use as nanostructured platforms for multiple applications.

In answer to the ever-increasing need to perform the simultaneous analysis of environmental hazards, microcarrier-based multiplex technologies show great promise. Further integration with biofunctionalized quantum dots (QDs) creates new opportunities to extend the capabilities of multicolor flow cytometry with their unique fluorescence properties. Here, we have developed a competitive microbead-based flow cytometric immunoassay using QDs fluorescent labels for simultaneous detection of two analytes, bringing the benefits of sensitive, rapid and easy-of-manipulation analytical tool for environmental contaminants. As model target compounds, the cyanobacterial toxin microcystin-LR and the polycyclic aromatic hydrocarbon compound benzo[a]pyrene were selected. The assay was carried out in two steps: the competitive immunological reaction of multiple targets using their exclusive sensing elements of QD/antibody detection probes and antigen-coated microsphere, and the subsequent flow cytometric analysis. The fluorescence of the QD-encoded microsphere was thus found to be inversely proportional to target analyte concentration. Under optimized conditions, the proposed assay performed well within 30 min for the identification and quantitative analysis of the two environmental contaminants. For microcystin-LR and benzo[a]pyrene, dose-response curves with IC(50) values of 5 μg L(-1) and 1.1 μg L(-1) and dynamic ranges of 0.52-30 μg L(-1) and 0.13-10 μg L(-1) were obtained, respectively. Recovery was 92.6-106.5% for 5 types of water samples like bottled water, tap water, surface water and seawater using only filtration as sample pretreatment.

This work reports a novel electrochemical immunoassay protocol with signal amplification for determination of proteins (human IgG here used as a model target analyte) at an ultralow concentration using DNA-based hybridization chain reaction (HCR). The immuno-HCR assay consists of magnetic immunosensing probes, nanogold-labeled signal probes conjugated with the DNA initiator strands, and two different hairpin DNA molecules. The signal is amplified by the labeled ferrocene on the hairpin probes. In the presence of target IgG, the sandwiched immunocomplex can be formed between the immobilized antibodies on the magnetic beads and the signal antibodies on the gold nanoparticles. The carried DNA initiator strands open the hairpin DNA structures in sequence and propagate a chain reaction of hybridization events between two alternating hairpins to form a nicked double-helix. Numerous ferrocene molecules are formed on the neighboring probe, each of which produces an electrochemical signal within the applied potentials. Under optimal conditions, the immuno-HCR assay presents good electrochemical responses for determination of target IgG at a concentration as low as 0.1 fg mL(-1). Importantly, the methodology can be further extended to the detection of other proteins or biomarkers.

A sensitive electrochemical approach for the detection of thrombin was designed by using densely packed hierarchical dendritic gold microstructures (HDGMs) with secondary and tertiary branches as matrices, and thionine-functionalized mesoporous silica nanospheres as signal tags. To prepare the signal tags, the positively charged thionine (as an indicator) was initially adsorbed onto the mesoporous silica nanoparticles (MSNs). Then [AuCl4]− ions were in situ reduced on the thionine-modified MSNs by ascorbic acid to construct nanogold-decorated MSNs (GMSNs). The formed GMSNs were employed as label of the aminated aptamers. The assay was carried out in PBS, pH 7.4 with a sandwich-type assay mode by using the assembled thionine in the GMSNs as indicators. Compared with the pure silica nanoparticles, mesoporous silica could provide a larger surface for the immobilization of biomolecules and improve the sensitivity of the aptasensor. Under optimal conditions, the electrochemical aptasensors exhibited a wide linear range from 0.001 to 600 ng mL−1 (i.e. 0.03 pM to 0.018 μM thrombin) with a low detection limit (LOD) of 0.5 pg mL−1 (≈15 fM) thrombin at 3σ. No obvious non-specific adsorption was observed during a series of analyses to detect target analyte. The precision, selectivity and stability of the aptasensors were acceptable. Importantly, the methodology was evaluated with thrombin spiked samples in blank fetal calf serum, and the recoveries were 94.2–112%, indicating an exciting potential for thrombin detection.

Diclofenac, a non-steroidal anti-inflammatory drug (NSAID), has caused catastrophic vulture declines across the Indian sub-continent. Here, an indirect ELISA is used to detect and quantify diclofenac in 1251 liver samples from livestock carcasses collected across India between August 2007 and June 2008, one to two years after a ban on diclofenac manufacture and distribution for veterinary use was implemented. The ELISAs applicability was authenticated with independent data obtained using LC-ESI/MS. Of 1251 samples, 1150 (91.9%) were negative for diclofenac using both methods, and 60 (4.8%) were positive at 10-4348 and 10-4441 μg kg-1 when analysed by ELISA and LC-ESI/MS, respectively. The residue level relationship in the 60 positive samples was highly significant (p < 0.001, r2 = 0.644). Data suggest that this immunological assay could be used not only for cost effective sample screening, but also for residue level semi-quantification., Financial support came from the UK Government’s Darwin Initiative and the RSPB

Due to its highly carcinogenic and mutagenic effect on humans, a maximum tolerable limit of 10 ng/L of benzo[a]pyrene (B[a]P) in drinking water was set by the European Commission (Council Directive 98/83/EC). Although several polyclonal and monoclonal antibodies (mAb) for the detection of B[a]P and other polycyclic aromatic hydrocarbons (PAH) have been developed by others, a traditional enzyme-linked immunosorbent assay (ELISA) with a limit of quantification of 10 ng/L for monitoring B[a]P has not been developed. With this in mind, several single-chain variable fragment (scFv) antibodies were created using existing mAbs against the extremely hydrophobic hapten B[a]P, and their heavy and light chains recombined to make unique variable light (V(L)) and heavy (V(H)) chain combinations. Their binding behaviour was investigated using microtiter plate ELISA and surface plasmon resonance techniques. Specifically, the coding sequences for V(L) and V(H) chains of 10 murine anti-B[a]P antibody producing hybridoma cell lines were isolated by degenerate oligonucleotide primer sets, cloned in phagemid pIT2 and transferred into Escherichia coli HB2151. To systematically investigate the interaction of the V(L) and V(H) domains, three high-affinity B[a]P-specific and one nonspecific clone were selected and recombined to build a set of 16 different V(L) and V(H) combinations. On the basis of our data, it was shown that the V(H) plays the major role for specific binding of B[a]P, whilst the V(L) can, in some cases, increase the final sensitivity of the assay by one order of magnitude. Furthermore, the sequence analysis of scFvs indicates that the complementarity determining region H3 plays a major role in affinity, whilst cross-reactivity to seven other PAHs demonstrates the importance of the V(L) in providing cross-reactivity.

We have developed a column gel-based immunoassays for the model analyte benzo[a]pyrene (B[a]P), and have evaluated three different kinds of labels, i.e., horseradish peroxidase, (HRP), colloidal gold (AuNPs), and quantum dots (QDs) with respect to rapid visual on-site testing. In case of HRP, a derivative of B[a]P-BA (benzo[a]pyrenebutyric acid) was directly coupled to HRP. In case of QDs and AuNPs, protein conjugates of B[a]P-BA were synthesized and used for surface functionalization. With the latter, previous coverage of the gold surface with 11-mercaptoundecanoic acid turned out to be advantageous in terms of antibody recognition and of preventing nonspecific binding to the gel layer. The assays based on the use of particle labels requires 4 consecutive working steps only, while those based on HRP require 5 additions of reagent. The lower limit of detection for B[a]P is 5 ng L−1 in case of using HPR or QDs as a label, but 25 ng L−1 when using AuNPs. We believe that the use of QDs constitutes the most promising label for qualitative and semi-quantitative gel-based immunoassays.

A quantitative immunochemical rapid test for sensitive determination of benzo[a]pyrene (BAP) as a model analyte was developed making use of a handheld reader for results evaluation. The covalent immobilization of antibodies to different Sepharose gels, i.e., CNBr-activated Sepharose 4B and CNBr-activated Sepharose 4 Fast Flow was compared with adsorption to a polyethylene support. The lowest limits of detection (LOD) were 4 ng L −1 and 40 ng L −1 , respectively, using optimized assay conditions. The developed test was applied to food supplements (garlic, black radish and maca), including a pretreatment procedure. LOD of 9 ng kg −1 and linear range of 13–80 ng kg −1 were obtained. Results of BAP determination in naturally contaminated samples were confirmed by high-performance liquid chromatography coupled to fluorescence detection and a good correlation was achieved. We suggest that the developed test format can be used to quantitative detection of the low molecular weight analytes, such as mycotoxins, pesticides, other pollutants in food and environmental samples.

A novel flow-through multiplexed immunoassay protocol for simultaneous electrochemical determination of carcinoembryonic (CEA) and alpha-fetoprotein (AFP) in biological fluids was designed using biofunctionalized magnetic graphene nanosheets (MGO) as immunosensing probes and multifunctional nanogold hollow microspheres (GHS) as distinguishable signal tags. The probes were fabricated by means of co-immobilization of primary anti-CEA (Ab(1)) and anti-AFP (Ab(2)) antibodies on the Fe(3)O(4) nanoparticle-coated graphene nanosheets (MGO-Ab(1,2)). The reverse-micelle method was used for the synthesis of distinguishable signal tags by encapsulation of horseradish peroxide (HRP)-thionine and HRP-ferrocene into nanogold hollow microspheres, respectively, which were utilized as labels of the corresponding GHS-Ab(1) and GHS-Ab(2). A sandwich-type immunoassay format was employed for the online detection of CEA and AFP by coupling a flow-through detection cell with an external magnet. The assay was based on the catalytic reduction of H(2)O(2) at the various peak potentials in the presence of the corresponding mediators. Experimental results revealed that the multiplexed electrochemical immunoassay enabled the simultaneous monitoring of AFP and CEA in a single run with wide working ranges of 0.01-200 ng mL(-1) for AFP and 0.01-80 ng mL(-1) for CEA. The detection limits (LODs) for both analytes at 1.0 pg mL(-1) (at 3s(B)) were very low. No obvious nonspecific adsorption and cross-talk were observed during a series of analyses to detect target analytes. Intraassay and interassay coefficients of variation were10%. Importantly, the methodology was evaluated for the analysis of clinical serum specimens, receiving a good correlation between the flow-through multiplexed electrochemical immunoassay and an electrochemiluminescence method as a reference.

Recent research has looked to develop innovative and powerful novel biofunctionalized nanometer-sized silica particles, controlling and tailoring their properties in a very predictable manner to meet the needs of specific applications. The silica shells of these particles facilitate a wide variety of surface reactions and allow conjugation with biomolecules like proteins and DNA. There exist a multitude of possible applications of fabricated nanoparticles in biotechnology and medicine. In particular, they have proved to be highly useful for biosensing, assay labelling, bioimaging, and in research on a variety of molecular tags in cellular and molecular biology. Techniques commonly rely on the use of silica-coated semiconductor quantum dots, organic dyes, magnetic particles, and Raman active particles. Inorganic-biological hybrid particles combine the properties of both materials, i.e., the spectroscopic characteristics of the entrapped nanocrystal, and the biomolecular function of the conjugated entity. Rather than being exhaustive, this review focuses on selected examples to illustrate novel concepts and promising applications. Approaches described include the encoding of silica nanoparticles with different groups, and conjugation with various biological entities. Further, promising applications in bioanalysis are considered and discussed.

A newly flow-through electrochemical immunosensor for monitoring IgG in human serum has been developed by using core-shell SiO 2 /Au nanocomposites and poly(amidoamine) G4 dendrimer as matrices. The ferrocenecarboaldehyde-labeled anti -IgG biomolecules were initially chemisorbed onto the nanoparticle surface, and then glucose oxidase (GOx), as a blocking reagent instead of bovine serum albumin (BSA), was backfilled onto the modified surface. The formation of the antibody–antigen complex by a simple one-step immunoreaction between the immobilized anti -IgG and IgG in sample solution introduced a barrier of direct electrical communication between the immobilized GOx and the base surface, and decreased the immobilized GOx toward the catalytic oxidation of glucose. The performance and factors influencing the performance of the immunosensor were evaluated. Under optimal conditions, the linear range of the developed immunosensor by using GOx as enhancer was from 5.0 × 10 −6 to 9.6 × 10 −4 mol/L with a detection limit of 8.0 × 10 −7 mol/L IgG (at 3 δ ), while the detection limit by using BSA was 1.5 × 10 −5 mol/L IgG (at 3 δ ) with the linear range from 3.5 × 10 −5 to 1.2 × 10 −3 mol/L. The selectivity, reproducibility and stability of the proposed immunosensor were acceptable. The IgG contents in 37 human serum samples obtained by the proposed method are identical with the data of clinical laboratory.

The hybridoma screening is a key step for the successful generation of high-affinity analyte-specific monoclonal antibodies (MAbs), particularly if the target of the antibody is a low-molecular weight analyte. This work presents an advanced screening method that makes use of antibody microarrays generated by contact printing of the hybridoma cell supernatant samples on glass chips initially coated with capture antibodies. The noncompetitive immunoassay is based on the specific binding of an analyte-horseradish peroxidase conjugate and is performed in an automated fashion using a chemiluminescence readout system. Compared to the standard ELISA screening, the work load is reduced due to a higher degree of automation. The quality of the generated data is comparable to data generated by a previously optimized microplate-based immunoassay method. Among a reference set of 373 hybridoma cell supernatant samples, three out of four high-affinity MAbs were identified as true positive, whereas none of the samples were detected as a false positive.

Aflatoxins are a group of very carcinogenic mycotoxins that can be found on a wide range of food commodities including nuts, cereals, and spices. In this study, the first LC-MS/MS stable isotope dilution assay (SIDA) for the determination of aflatoxins in foods was developed. The development of this method was enabled by easily accessible isotope-labeled (deuterated) aflatoxins B2 and G2, which were synthesized by catalytic deuteration of aflatoxin B1 and G1, purified, and well-characterized by NMR and MS. All four aflatoxins of interest (B1, B2, G1, and G2) were quantified in food samples by using these two labeled internal standards. The response factors (RF) of the linear calibrations were revealed to be matrix independent for labeled aflatoxin B2/aflatoxin B2 and labeled aflatoxin G2/aflatoxin G2. For labeled aflatoxin B 2/aflatoxin B 1 and labeled aflatoxin B2/aflatoxin G1 matrix-matched calibration was performed for the model matrices almonds and wheat flour, showing significant differences of the RFs. Limits of detection (LOD) were determined by applying a statistical approach in the presence of the two model matrices, yielding 0.31 microg/kg (aflatoxin B1), 0.09 microg/kg (aflatoxin B2), 0.38 microg/kg (aflatoxin G1), and 0.32 microg/kg (aflatoxin G2) for almonds (similar LODs were obtained for wheat flour). Recovery rates were between 90 and 105% for all analytes. Coefficients of variation (CV) of 12% (aflatoxin B1), 3.6% (aflatoxin B2), 14% (aflatoxin G1), and 4.8% (aflatoxin G2) were obtained from interassay studies. For further validation, a NIST standard reference food sample was analyzed for aflatoxins B1 and B2. The method was successfully applied to determine trace levels of aflatoxins in diverse food matrices such as peanuts, nuts, grains, and spices. Aflatoxin contents in these samples ranged from about 0.5 to 6 microg/kg.

This study compares diverse microplate-based hybridoma screening methods for the generation of hapten-(aflatoxin-) specific monoclonal antibodies (MAbs). Standard indirect enzyme-linked immunosorbent assay (ELISA) screenings (with immobilization of hapten-protein conjugate and use of enzyme-labeled anti-mouse IgG as tracer) were compared with direct ELISAs (with antibody immobilization and use of a hapten-enzyme conjugate as tracer). Although direct ELISA is rarely used for routine hybridoma screenings, it showed considerable advantages compared to the indirect assays. Standard indirect ELISA screening can lead to a considerable number of false positives (up to about 50% false positives of all 373 supernatants tested) if the antibody concentrations in the supernatants are too high. Direct ELISAs gave useful screening results for the different supernatant dilutions chosen. At most 3 false positives were detected out of 373 supernatants. However, the sensitivity of the direct ELISA screening is generally lower compared to indirect ELISA, and individual high-affinity MAbs might be classified as false negative. Therefore, a modified indirect ELISA screening was also developed. It includes pre-incubation of the supernatants in anti-mouse IgG-coated microplates which are then transferred into the (indirect) hapten conjugate-coated microplates. This screening method leads to excellent results with good overall selectivity and sensitivity. It can also be conveniently combined with the direct ELISA screening. Using these improved screening methods, aflatoxin-specific MAbs could be generated with IC50 values down to 3 ng/l (aflatoxin concentration).