Konjac gel foods that mainly consist of deacetylated konjac glucomannan (Da-KGM) are considered to have the same health benefits as native konjac glucomannan (KGM); however, no definitive data support this notion. The objective of this study was to compare the effects of Da-KGM and KGM on the hyperlipidemia and liver steatosis induced by high-fat diet feeding and to investigate the underlying molecular mechanisms. C57BL/6 mice were fed (1) normal chow diet, (2) high-fat diet, (3) HFD with KGM, or (4) HFD with Da-KGM for 10 weeks. KGM, but not Da-KGM, showed decreased fat accumulation, improved blood and liver lipid profiles, and prevention of liver lipid droplet deposition compared with HFD. Compared with Da-KGM, KGM increased the outputs of fecal bile acid (KGM 22.5 ± 2.34 mg/g vs Da-KGM 19.3 ± 1.87 mg/g), fat (KGM 5.56 ± 0.68 mg/g vs Da-KGM 4.42 ± 0.57 mg/g) and cholesterol (KGM2.67 ± 0.43 mg/g vs Da-KGM 1.78 ± 0.28 mg/g), fecal concentrations of total short-chain fatty acids (KGM 103 ± 14.8 μmol/g vs Da-KGM 74.5 ± 8.49 μmol/g), and improved hepatic antioxidant status and upregulated CYP7A1 and LDLR gene expression. These findings suggest that deacetylation of KGM negatively affects its fermentation characteristics and its inhibition of lipid absorption, which thereby reduces Da-KGM’s health benefits.Keywords: deacetylated konjac glucomannan; high-fat diet; konjac gel foods; konjac glucomannan; lipid metabolism;

•Irradiation in dosage of 100 kGy leads to excellent dispersibility in KGM.•100 kGy-KGM demonstrates excellent protective effect against oxidative damage.•Possible mechanism for anti-oxidative activity in 100 kGy-KGM was analyzed.•Irradiation was proven an effective method to improve properties of KGM.Konjac glucomannan (KGM) is an important functional polysaccharide in food research. However, unstable dispersibility of KGM inhibits its in-depth study and wide application. In this study, a degraded KGM (100 kGy-KGM), which showed excellent dispersibility and specific physicochemical properties, were obtained by γ-irradiation in a dosage of 100 kGy. We investigated the protective effect of 100 kGy-KGM against H2O2 induced oxidative damage in LO2 cells. Our results demonstrated that pretreatment of LO2 cells with 100 kGy-KGM not only significantly increased cellular survivals and activities of GSH-Px and CAT, but also reduced levels of LDH, MDA and intracellular accumulation of ROS. The marked protective effect against oxidative damage and excellent dispersibility in 100 kGy-KGM allowed its possible use as an antioxidant. Our study provided fundamental knowledge to understand the structure-functions relationships of degraded-KGM, which could result in a theoretical guidance for the future application of KGM.

Melatonin (MT) is an endogenous hormone that is used in medicinal foods for all kinds of purposes. Monitoring the content of MT is necessary to guarantee the quality of medicinal foods, as well as to prevent it from being abused. In this work, an easy-to-use and low-cost competitive indirect ELISA with a flexible working range for monitoring MT was developed. Four haptens were synthesized and coupled to carrier proteins as immunogens to compare their ability to generate antibodies against MT. After screening the antisera and optimizing the working conditions, a homologous ELISA with a limit of detection (LOD) of 30.85 ng mL−1 and a wide working range from 66.2 ng mL−1 to 65.9 μg mL−1, and a heterologous ELISA with an LOD of 0.21 ng mL−1 and a working range from 1.0 ng mL−1 to 104.4 ng mL−1 for MT were developed. Using the homologous format, the developed ELISA is easy-to-use for the quantitative determination of MT in a medicinal food matrix, with the recovery ranging from 84% to 113.6%. Good correlation between the results of the ELISA and standard liquid chromatography (LC) was obtained. The proposed two ELISA formats are ideal tools for screening a large number of medicinal food samples for MT prior to standard instrumental analysis.

The axial chiral herbicide propisochlor is used to control weeds. Different enantiomers of a compound usually have different biological activities. It is unclear how the toxicities of the propisochlor enantiomers differ. Propisochlor enantiomers, separated by high-performance liquid chromatography, were tested on SP2/0 myeloma cells. Cytotoxicity and apoptosis were measured, and interactions between the enantiomers were evaluated. The rac-propisochlor, pure R-(+) isomer, and pure S-(−) isomer inhibited cell proliferation and induced apoptosis. The rac-propisochlor, R-(+) isomer, and S-(−) isomer half maximal effective concentration values after 24 h of incubation were 111 ± 0.15, 68 ± 0.09, and 99 ± 0.21 μM, respectively. R-(+) isomer induced the most apoptosis. R-(+) isomer was ∼1.63 times more cytotoxic than rac-propisochlor and ∼1.46 times more cytotoxic than S-(−) isomer. Antagonistic cytotoxic interactions were found between R-(+) and S-(−) isomers. This is the first time the toxicities of these enantiomers and antagonism between the enantiomers have been reported. The antagonism indicates that the ecotoxicological effects of the enantiomers should be investigated.

A polyclonal antibody (PcAb)-based enzyme-linked immunosorbent assay (ELISA) for the determination of benzoic acid (BA) was developed. For this, 4-aminobenzoic acid was conjugated to keyhole limpet hemocyanin (KLH) by diazotization and used as an immunogen to produce a PcAb against BA. Two spectrophotometric formats, indirect and direct competitive ELISA (ci- and cd-ELISA, respectively), were developed, and the effects of heterologous coatings and assay conditions on assay performance were examined. Under optimal conditions, comparative sensitivities were obtained for a heterologous ci-ELISA and homologous cd-ELISA. The cd-ELISA showed a detection limit (LOD) of 1.25, 2.5 and 2.5 μg mL–1, linear range of 5.0–530.0, 10.0–1,060.0 and 10.0–1,060.0 μg mL–1, in carbonated drink, sausage and jelly, respectively. The assay time was 55 min (simple sum of incubations without time for the necessary intermediate manipulations). This assay exhibited good specificity for BA and its salt, sodium benzoate, and no cross-reactivity with structurally related food additives. Averaged recoveries from BA-spiked carbonated drink, sausage and jelly using this proposed dc-ELSIA ranged from 85.8 to 104.6 %. In addition, good correlation was observed between results from cd-ELISA and standard HPLC analyses (R2 = 0.9484). Overall, these results indicated that this cd-ELISA could be used effectively for simple, rapid and low-cost screening of BA abuse in food samples.

The high level of acrylamide in widely consumed processed foods poses a potentially significant risk to human health, which has led to an increasing demand for rapid, simple, and selective analytical methods. In the present work, several haptens for acrylamide were designed in an attempt to prepare antibodies with acrylamide affinity, but they failed their purpose. However, a polyclonal antibody was produced against 4-mercaptophenylacetic acid (4-MPA)-derivatized acrylamide, which showed high binding affinity to the derivative. As acrylamide easily reacted with 4-MPA at high derivation yield, a competitive indirect enzyme-linked immunosorbent assay (ciELISA) for acrylamide via a preanalysis derivatization was developed. The derivatization and ELISA conditions were fully optimized to produce a method for acrylamide assay that exhibited an IC50 of 2.86 μg/kg, limit of detection at 0.036 μg/kg, and linear range of 0.25–24.15 μg/kg. The results of preanalysis recovery tests of acrylamide-spiked food samples and screening of blind food samples by both ciELISA and HPLC-MS/MS indicated the proposed ciELISA’s good accuracy and reliability. This method was thus deemed suitable for routine acrylamide screening in food samples at low cost.

To deepen our understanding of the physiochemical principles that govern hapten–antibody recognition, ofloxacin enantiomers were chosen as a model for epitope prediction of small molecules. In this study, two monoclonal antibodies (mAbs) mAb-WR1 and mAb-MS1 were raised against R-ofloxacin and S-ofloxacin, respectively. The enantioselective mAbs have a high sensitivity and specificity, and the enantioselectivity is not affected by heterologous coating format reactions. The epitopes of the ofloxacin isomers were predicted using the hologram quantitative structure–activity relationship (HQSAR) and comparative molecular field analysis (CoMFA) approaches. The results consistently show that the epitope of the chiral hapten should be primarily composed of the oxazine ring and the piperazinyl ring and mAbs recognize the hapten from the side of this moiety. The enantioselectivity of mAbs is most likely due to the steric hindrance caused by the stereogenic center of the epitope. Modeling of chiral hapten–protein mimics reveals that ofloxacin isomers remain upright on the surface of the carrier protein. Suggestions to improve the enantioselectivity of antibodies against ofloxacin isomers were also proposed. This study provided a simple, efficient, and general method for predicting the epitopes of small molecules via molecular modeling. The epitope predictions for small molecules may create a theoretical guide for hapten design.

To develop a more sensitive immunoassay for malachite green (MG) and leucomalachite green (LMG), we identified the immunocomplex binding phage-borne peptides for use in the noncompetitive phage anti-immunocomplex assay (PHAIA). An anti-LMG monoclonal antibody (mAb) was used to select immunocomplex binding peptides from a circular random eight-amino-acid phage-displayed library. After three rounds of panning-elution, five peptides that bound the LMG–mAb immunocomplex were obtained. One of the phage-borne peptide clones that resulted in an assay with the highest sensitivity was chosen for further research. The concentration of LMG producing 50% of the saturated signal and the limit of detection of the assay were 7.02 and 0.55 ng/mL, respectively, with a linear range of 1.35 to 21.56 ng/mL. The PHAIA based on the same antibody was 16 times more sensitive compared to the competitive immunoassay. PHAIA was used to analyze LMG, MG, and two mixtures of spiked fish samples, with validation by high-performance liquid chromatography (HPLC) with fluorescence detector. Results showed a good correlation (R2LMG = 0.9841; R2MG = 0.993; R2Mixture = 0.9903) between the data of PHAIA and HPLC, thus the assay was an efficient method for monitoring food safety.

The demanding task of monitoring illegal dyes in foodstuffs requires sensitive detection methods. Here, a surface plasmon resonance (SPR) sensor, combined with a 75 ± 5 nm thick molecularly imprinted polymer (MIP) nanofilm as a recognition element, is used for the selective detection of Sudan dyes. The MIP-SPR sensor chip was fabricated by anchoring the MIP nanofilm of Sudan I on a gold chip via surface-initiated in situ polymerization. The surface morphology and thickness of the MIP films were characterized by atomic force microscopy (AFM) and scanning electron microscopy (SEM). The adsorption properties of the sensor chip were studied through application as an SPR sensor. The MIP-SPR sensor exhibited excellent adsorption capacity, and high sensitivity and selectivity for Sudan dyes. The SPR response was linearly proportional to the concentration of Sudan I over the range 50–400 ng mL−1. Based on a signal-to-noise ratio of three, a detection limit of 30 ng mL−1 was established with a 400 s response time. Non-imprinted SPR sensors were compared in terms of selectivity with MIP-SPR sensors and it was found that all the recognition coefficients (α) for Sudan I–IV were greater than 1. The MIP-SPR sensor also showed reproducible response over three equilibration–adsorption–regeneration cycles.

•Immunochromatographic assay for fluoroquinolone antibiotic ofloxacin is proposed.•Native antiserum instead of immunoglobulins may be conjugated with colloidal gold.•Non-treated milk and meat homogenate after centrifugation may be tested.An immunochromatographic assay was developed to detect fluoroquinolone antibiotic ofloxacin based on the competitive binding of ofloxacin and the membrane-immobilized ofloxacin–protein conjugate to colloidal gold-labeled antibodies in the course of the labeled antibodies, and to test sample flow through the membrane. The specific feature of labeling by colloidal gold is that native antiserum is used instead of purified immunoglobulins or specific antibodies. This makes the synthetic procedure easier, with no sacrifice in the detection limit. The proposed test makes it possible to detect down to 30 ng mL−1 of ofloxacin, which corresponds to the demands of food safety assessment. The assay time is 10 min. The assay provides reliable information on the ofloxacin content in milk without the sample preparation and in chicken and pork meat with the minimum sample preparation (the separation of the insoluble fraction of the homogenate by centrifugation). The high degree of detection of ofloxacin in foodstuffs by the proposed assay (70–112%) was shown by a comparison with the data obtained with the use of a commercial immunoenzymatic kit.

To study the preparation and separation of Konjac oligosaccharides, Konjac Glucomannan was degraded by the combination of γ-irradiation and β-mannanase, and then the degradation product was separated by ultrafiltration. To our interest, for most of Konjac oligosaccharides obtained by this method, the molecular mass was lower than 2200 Da. In addition, the 1000 Da molecular weight cut off membrane could effectively separate the Konjac oligosaccharides. In conclusion, the combination of γ-irradiation and β-mannanase was an efficient method to obtain Konjac oligosaccharides, and the oligosaccharides of molecular mass lower than 1000 Da could be effectively separated by ultrafiltration.Highlights► Konjac oligosaccharides were prepared by γ-irradiation and β-mannanase. ► It was analyzed by gel permeation chromatography and ion exchange column in HPLC. ► The oligosaccharides could be effectively separated by ultrafiltration.

Coumaphos, an organophosphorus pesticide (OP) used worldwide, has raised serious public concerns due to its positive association with major types of cancer. Herein, a novel method for attomolar coumaphos detection was developed on the basis of an electrochemical displacement immunoassay coupled with oligonucleotide sensing. An optimized displacement immunoassay was constructed to improve the binding efficiency of an antigen–antibody pair, and a guanine-rich single-strand DNA label, in combination with oligonucleotide sensing, was used to amplify the detection signal with “direct” relationship to the analyte. As a result, coumaphos was sensitively determined from the enhanced catalytic cycle of guanine-Ru(bpy)32+ by chronoamperometry. The limit of detection (LOD) was down to 0.18 ng L–1 (S/N = 3), which is equal to 49.6 amol in a sample solution of 100 μL. In comparison with conventional methods, the proposed method has the lowest LOD and better accessibility to high-throughput sensing systems. Besides, it can complete the whole analysis process in under 50 min and exhibits good performance of excellent selectivity to the OPs. With regard to the advantages of rapidity, convenience, low cost, and ease of operation, the proposed method has provided a promising platform capable of fast and in-field OP detection, which may make the system promising for potential applications in the detection of other small molecules.

A multi-analytes method for monitoring of organophosphorus pesticides (OPs) using a combination of broad-specificity direct competitive enzyme-linked immunosorbent assay (dcELISA) and high-performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) was developed. The reaction formats of dcELISA as well as the matrix effects of vegetable samples by different treatments were studied. The dcELSIA based on horseradish peroxidase-labelled monoclonal antibody and solid-phase extraction can analyse 42 samples in duplicate simultaneously for 12 OPs with a limit of detection at 20 μg L−1 within 40 min, with good accuracy and reproducibility. For screening purpose, the dcELISA can distinguish positive samples from hundreds of negative samples at a rapid, high-throughput and low cost manner. The positive samples can be following confirmed by HPLC–MS/MS for the kinds and the relative amounts of OPs. The method is suitable for monitoring of OP contamination in vegetables samples with high-efficiency and low cost.Highlights► A direct competitive ELISA for organophosphorus pesticides was developed. ► HPLC–MS/MS multi-analytes analysis for organophosphorus pesticides was developed. ► ELISA and HPLC–MS/MS are combined for monitoring of organophosphorus pesticides. ► The methods are rapid, high-throughput and with high-efficiency and low cost.

Solid-phase extraction (SPE) and direct competitive chemiluminescence enzyme immunoassay (dcCL-EIA) were combined for the detection of organophosphorus pesticides (OPs) in environmental water samples. dcCL-EIA based on horseradish peroxidase labeled with a broad-specificity monoclonal antibody against OPs was developed, and the effects of several physicochemical parameters on dcCL-EIA performance were studied. SPE was used for the pretreatment of water samples to remove interfering substances and to concentrate the OP analytes. The coupling of SPE and dcCL-EIA can detect seven OPs (parathion, coumaphos, phoxim, quinalphos, triazophos, dichlofenthion, and azinphos-ethyl) with the limit of quantitation below 0.1 ng/mL. The recoveries of OPs from spiked water samples ranged from 62.5% to 131.7% by SPE–dcCL-EIA and 69.5% to 112.3% by SPE–HPLC–MS/MS. The screening of OP residues in real-world environmental water samples by the developed SPE–dcCL-EIA and their confirmatory analysis using SPE–HPLC–MS/MS demonstrated that the assay is ideally suited as a monitoring method for OP residues prior to chromatographic analysis.

A single-chain variable fragment (scFv) linked alkaline phosphatase (AP) fusion protein for detection of O,O-diethyl organophosphorus pesticides (O,O-diethyl OPs) was produced and characterized. The scFv gene was prepared by cloning VL and VH genes from hybridoma cells secreting monoclonal antibody with broad specificity for O,O-diethyl OPs. The amplified VL and VH regions were assembled using a linker (Gly4Ser)3 by means of splicing overlap extension polymerase chain reaction to obtain the scFv gene, which was cloned into the expression vector pLIP6/GN containing an AP gene to produce the scFv–AP fusion protein in Escherichia coli strain BL21. The protein was purified by antigen-conjugated immunoaffinity chromatography and characterized by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, Western blotting, and competitive direct enzyme-linked immunosorbent assay (cdELISA). The fusion protein is bifunctional, retaining both antigen binding specificity and AP enzymatic activity. Analysis of spiked and blind river water and Chinese cabbage samples demonstrated that the fusion protein based cdELISAFP exhibited good sensitivity and reproducibility.

An immunoaffinity chromatographic (IAC) method for the selective extraction and concentration of 13 organophosphorus pesticides (OPs, including coumaphos, parathion, phoxim, quinalphos, dichlofenthion, triazophos, azinphos-ethyl, phosalone, isochlorthion, parathion-methyl, cyanophos, disulfoton, and phorate) prior to analysis by high-performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) was developed. The IAC column was prepared by covalently immobilizing a monoclonal antibody with broad specificity for OPs on CNBr-activated Sephrose 4B. The column capacity ranged from 884 to 2641 ng/mL of gel. The optimum elution solvent was 0.01 M phosphate-buffered saline containing 80% methanol. The breakthrough volume of the IAC column was found to be 400 mL. Recoveries of OPs from spiked environmental samples by IAC cleanup and HPLC–MS/MS analysis ranged from 60.2 to 107.1%, with a relative standard deviation below 11.1%. The limit of quantitation for 13 OPs ranged from 0.01 to 0.13 ng/mL (ng/g). The application of IAC cleanup coupled to HPLC–MS/MS in real environmental samples demonstrated the potential of this method for the determination of OP residues in environmental samples at trace levels.

A monoclonal antibody-based competitive indirect enzyme-linked immunosorbent assay (ELISA) with improved sensitivity and specificity for the determination of furaltadone metabolite 5-methylamorpholino-3-amino-2-oxazolidone (AMOZ) was described. AMOZ was derivatized with 2-(3-formylphenoxy)acetic acid and coupled with bovine serum albumin to form a novel immunogen. BABL/c mice were immunized and monoclonal antibody specific to the nitrophenyl derivative of AMOZ (NP-AMOZ) was produced and characterized. Four other haptens with different heterology to the immunizing hapten were synthesized and coupled to ovalbumin as coating antigens to study the effect of heterologous coating on assay sensitivity. Under the optimized heterologous coating format, the competitive indirect ELISA showed very high sensitivity to NP-AMOZ, with an IC50 of 0.14 μg/L and limit of detection of 0.01 μg/L. The assay showed high specificity toward NP-AMOZ, and negligible cross-reactivity with analogous compounds was observed. The average recoveries of AMOZ from spiked fish and shrimp samples were estimated to range from 81.0 to 104.0%, with coefficients of variation below 20%. Good correlation was obtained between the results of ELISA analysis and of standard liquid chromatography–tandem mass spectrometry analysis. These results indicated that the proposed ELISA is ideally suited as a monitoring method for AMOZ residues at trace level.

Due to the large-scale use of organophosphorus pesticides (OPs), their contamination in the environment is widespread throughout the world, especially in developing countries. With increasing public concern, there is an urgent requirement for simple, rapid, high-throughput and highly sensitive analytical methods for the screening of OP residues in the environment. In this work, a monoclonal antibody-based sensitive one-step direct competitive time-resolved fluorescence immunoassay (TRFIA) with broad specificity to a class of OPs was developed. After optimization of the assay conditions, this method can detect thirteen OPs with a limit of detection below 10 ng mL−1. The recoveries of OPs from spiked environmental water samples ranged from 74.8% to 121.3%, with the coefficient of variation (CV) ranging from 6.4% to 15.1%. The correlation coefficient between the TRFIA results and standard HPLC-MS/MS results was 0.964. The proposed TRFIA was capable of high-throughput analysis of a large number of samples prior to chromatographic analysis, with good sensitivity, simplicity and rapidity.

An immunizing hapten (4-(carboxymethoxy)phenyl)bis(4-(diethylamino)phenyl)methylium for brilliant green (BG), a triphenylmethane dye with a potential illegal use in fish feeding, was synthesized and used to produce polyclonal antibody (PcAb) against BG. Unexpectedly, the obtained PcAb showed high cross-reactivity (CR) to malachite green (MG) and crystal violet (CV) in an indirect competitive enzyme-linked immunosorbent assay (icELISA). After screening against three heterologous coating antigens, the icELISA exhibited good sensitivity and uniform response to BG (IC50 of 1.98 ng mL−1 and CR of 100%), MG (IC50 of 1.61 ng mL−1 and CR of 105%) and CV (IC50 of 1.34 ng mL−1 and CR of 142%) when using (4-(carboxymethoxy)phenyl)bis(4-(dimethylamino)phenyl)methylium as the coating hapten. Therefore, a broad-specificity icELISA for simultaneous determination of BG, MG and CV was developed. The recoveries of single analyte and mixture of three analytes from spiked grass carp tissues were estimated ranging from 74.94% to 110.39%. A statistically significant correlation of results was obtained between the developed icELISA and previously established HPLC approaches with the food-relevant three triphenylmethane dyes concentration range 1.83–200 ng mL−1 (R2 = 0.9224), indicating good accuracy of the icELISA and suitability for the broad-specific detection of the three triphenylmethane dyes in grass carp tissues.Graphical abstractHighlights► A broad-specificity ELISA for three triphenylmethane dyes was developed. ► The effect of hapten structures on assay performance was studied. ► The developed ELISA showed good sensitivity and uniform response to three analytes. ► Good accuracy and reproducibility were obtained in spiked fishes samples.

A simple, rapid and high-throughput fluorescent polarization immunoassay (FPIA) for simultaneous determination of organophosphorus pesticides (OPs) using a broad-specificity monoclonal antibody was developed. The effects of tracer structure, tracer concentration, antibody dilution, methanol content and matrix effect on FPIA performance were studied. The FPIA can detect 5 OPs simultaneously with a limit of detection below 10 ng mL−1. The time required for the equilibrium of antibody–antigen interaction was less than 10 min. The recovery from spiked vegetable and environmental samples ranged from 71.3% to 126.8%, with the coefficient of variations ranging from 3.5% to 14.5%. The developed FPIA was applied to samples, followed by confirmation with high-performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) analysis. The developed FPIA demonstrated good accuracy and reproducibility, and is suitable for rapid and high-throughput screening for OP contamination with high-efficiency and low cost.Graphical abstractHighlights► A broad-specificity FPIA for organophosphorus pesticides (OPs) was developed. ► The effect of tracer structure and several physicochemical parameters were studied. ► The FPIA can detect 5 OPs simultaneously with a limit of detection below 10 ng mL−1. ► Recovery tests from vegetable and environmental water samples were studied. ► Good accuracy and reproducibility were obtained.

The type of hapten linkage to a carrier protein can play an important role in determining the nature of the resulting antibody response. Generic haptens using three types of linkers were synthesized (a monocarboxylic acid, an unsaturated hydrocarbon and a carboxamido spacer). These haptens were conjugated to bovine serum albumin (BSA) and used as immunogens to produce broad-specificity monoclonal antibodies (MAbs) to organophosphorus pesticides (OPs). Three-dimensional (3D) structures of hapten-lysine conjugates were optimized using molecular modeling (MM) to mimic conformations of hapten-BSA conjugates. The results from MM studies revealed a change of the 3D conformation and electrostatic potential of hapten 1 when the monocarboxylic acid linker was coupled to lysine. This result was consistent with the observed high-cross-reactivity of the corresponding MAb–H1 for the OPs. The competitive indirect enzyme-linked immunosorbent assay based on MAb–H1 is ideally suited to be used as a screening method for OP contaminants.

The use of chlorpromazine (CPZ) as a sedative for livestock has been prohibited in the European Union and many other countries. In this study, a new hapten 7-amino-chlorpromazine sulfoxide (hapten 1) against CPZ was synthesized and coupled to ovalbumin (OVA) as an immunogen to produce polyclonal antibody (PAb) specific for CPZ. An heterologous hapten 7-(4-carboxyl-phenylazo)-chlorpromazine (hapten 2) was synthesized and coupled to bovine serum albumin (BSA) as coating antigen to improve the assay sensitivity. The results showed that the hapten-heterologous systems exhibited 20 times higher sensitivity than the hapten-homologous one. Based on the hapten-heterologous system, an indirect competitive enzyme-linked immunosorbent assay (ELISA) for CPZ was developed, the IC50 value was 0.58 ng mL−1 and the limit of detection was 0.03 ng mL−1. The assay showed no cross-reaction with the CPZ analogues. The average recoveries of CPZ from spiked samples were estimated to range from 77.1% to 98.6%, with a coefficient of variation (CV) of less than 10.9%. Linear regression analysis showed a good correlation between the CPZ concentrations obtained from ELISA and HPLC analysis, which suggested that the ELISA is a convenient supplementary analytical tool for monitoring CPZ.

The development of easy-to-use and rapid-monitoring immunoassay methods for organic environmental pollutants in a class-selective manner is a topic of considerable environmental interest. In this work, a heterologous competitive indirect enzyme-linked immunosorbent assay (ciELISA) based on a monoclonal antibody (MAb) with broad-specificity for organophosphorus pesticides (OPs) was applied to the detection of O,O-diethyl and O,O-dimethyl OPs in water samples. The ciELISA conditions were carefully optimized to obtain a three to five-fold improvement of sensitivity for most OPs, and thirteen OPs were determined at concentrations ranging from 0.017 to 30 ng mL−1. The determination of spiked environmental water samples showed average recoveries from 81.5% to 115.1%, with the coefficient of variation (CV) ranging from 6.1% to 20.9%, which showed satisfactory reproducibility of the developed ciELISA. To overcome the negative aspect of broad-specificity immunoassays not providing qualitative and quantitative analysis of individual OPs in blind samples, we used “percent inhibition rate” to make the developed ciELISA a semi-quantitative method, which allows the monitoring of positive samples from hundreds of negative samples. The determination of OPs in blind water samples by the developed ELISA with confirmation by HPLC-MS/MS analysis demonstrated that the assay is ideally suited as a screening method for OP residues prior to chromatographic analysis.

A monoclonal antibody (mAb) against 4-(diethoxyphosphorothioyloxy)benzoic acid (hapten 1) was raised and used to develop a broad-specificity competitive indirect enzyme-linked immunosorbent assay (ciELISA) for 14 O,O-diethyl organophosphorus pesticides (OPs). Computer-assisted molecular modeling was used to model two-dimensional (2D) and three-dimensional (3D) quantitative structure−activity relationships (QSARs) to study antibody recognition. On the basis of insights obtained from the QSAR models, two heterologous coating haptens, 4-(diethoxyphosphorothioylamino)butanoic acid (hapten 2) and 4-(diethoxyphosphorothioyloxy)-2-methylbenzoic acid (hapten 3) were designed, synthesized, and used to develop heterologous ciELISAs with significantly improved sensitivity. The heterologous ciELISA using hapten 2 as the coating hapten showed good sensitivity in a broad-specific manner for eight O,O-diethyl OPs and may be used as a screening method for the determination of these OPs. Our studies demonstrated that molecular modeling can provide insights into the spatial and electronic effects of molecular structures that are important for antibody activity, which can then be used to improve immunoassay sensitivity.

The incorporation of melamine into food products is banned but its misuse has been widely reported in both animal feeds and food. The development of a rapid screening immunoassay for monitoring of the substance is an urgent requirement. Two haptens of melamine were synthesized by introducing spacer arms of different lengths and structures on the triazine ring of the analyte molecular structure. 6-Aminocaproic acid and 3-mercaptopropionic acid were reacted with 2-chloro-4,6-diamino-1,3,5-triazine (CAAT) to produce hapten 1 [3-(4,6-diamino-1,6-dihydro-1,3,5-triazin-2-ylamino) hexanoic acid] and hapten 2 [3-(4,6-diamino-1,6-dihydro-1,3,5-triazin-2-ylthio) propanoic acid], respectively. The molecular structures of the two haptens were identified by 1H nuclear magnetic resonance spectrometry, mass spectrometry and infrared spectrometry. An immunogen was prepared by coupling hapten 1 to bovine serum albumin (BSA). Two plate coating antigens were prepared by coupling both haptens to egg ovalbumin (OVA). A competitive indirect enzyme-linked immunosorbent assay (ciELISA) was developed to evaluate homogeneous and heterogeneous assay formats. The results showed that polyclonal antibodies with high titers were obtained, and the heterogeneous immunoassay format demonstrated a better performance with an IC50 of 70.6 ng mL−1, a LOD of 2.6 ng mL−1 and a LOQ of 7.6 ng mL−1. Except for cyromazine, no obvious cross-reactivity to common compounds was found. The data showed that the hapten synthesis was successful and the resultant antisera could be used in an immunoassay for the rapid and sensitive detection of this banned chemical.

Immunoassay for low molecular weight food contaminants, such as pesticides, veterinary drugs, and mycotoxins is now a well-established technique which meets the demand for a rapid, reliable, and cost-effective analytical method. However, due to limited understanding of the molecular structure of antibody binding sites and antigenic epitopes, as well as the intermolecular binding forces that come into play, the traditional ‘trial and error’ method used to develop antibodies still remains the method of choice. Therefore, development of enhanced immunochemical techniques for specific- and generic-assays, requires new approaches for antibody design that will improve affinity and specificity of the antibody in a more rapid and economic manner. Computer-assisted molecular modeling (CAMM) has been demonstrated to be a useful tool to help the immunochemist develop immunoassays. CAMM methods can be used to help direct improvements to important antibody features, and can provide insights into the effects of molecular structure on biological activity that are difficult or impossible to obtain in any other way. In this review, we briefly summarize applications of CAMM in immunoassay development, including assisting in hapten design, explaining cross-reactivity, modeling antibody–antigen interactions, and providing insights into the effects of the mouse body temperature on the three-dimensional conformation of a hapten during antibody production. The fundamentals and theory, programs and software, limitations, and prospects of CAMM in immunoassay development were also discussed.

•Graphene oxide (GO) is proposed to be able to quench the fluorescence signal.•β-lactamase aptamer was used to replace the traditional antibody.•Based on aptamer assembled onto GO, a new, sensitive and direct determination of β-lactamase was established.•The established method was verified by ELISA.Fluorescent sensor assay (FSA) was developed by using a fluorescein-labeled aptamer assembled onto Graphene oxide (GO) in order to determine β-lactamase in milk. Under optimal conditions, FSA indicated a detection range from 1 to 46 U/mL with a limit of detection (LOD) of 0.5 U/mL (R2 = 0.999, n = 3). In addition, commercial milk samples tainted with β-lactamase were detected by the established FSA with a recovery rate between 96.04 and 119.67%. Additionally, the reliability and sensitivity of FSA was validated by enzyme-linked immunoassay (ELISA) with a high correlation of 0.993. Thus, these data, combined with the ease and speed of the assay, suggest that the developed FSA may represent a promising method for monitoring β-lactamase contamination in milk.

Polyclonal antibody (PAb) with broad-specificity for O,O-diethyl organophosphorus pesticides (OPs) against a generic hapten, 4-(diethoxyphosphorothioyloxy)benzoic acid, was produced. The obtained PAb showed high sensitivity to seven commonly used O,O-diethyl OPs in a competitive indirect enzyme-linked immunosorbent assay (ciELISA) using a heterologous coating antigen, 4-(3-(diethoxyphosphorothioyloxy)phenylamino)-4-oxobutanoic acid. The 50% inhibition value (IC50) was 348 ng mL−1 for parathion, 13 ng mL−1 for coumaphos, 22 ng mL−1 for quinalphos, 35 ng mL−1 for triazophos, 751 ng mL−1 for phorate, 850 ng mL−1 for dichlofenthion, and 1301 ng mL−1 for phoxim. The limit of detection (LOD) met the ideal detection criteria of all the seven OP residues. A quantitative structure–activity relationship (QSAR) model was constructed to study the mechanism of antibody recognition using multiple linear regression analysis. The results indicated that the frontier-orbital energies (energy of the highest occupied molecular orbital, EHOMO, and energy of the lowest unoccupied molecular orbital, ELUMO) and hydrophobicity (log of the octanol/water partition coefficient, log P) were mainly responsible for the antibody recognition. The linear equation was log(IC50) = −63.274EHOMO + 15.985ELUMO + 0.556 log P − 25.015, with a determination coefficient (r2) of 0.908.

The development of easy-to-use and rapid-monitoring immunoassay methods for organic environmental pollutants in a class-selective manner is a topic of considerable environmental interest. In this work, a heterologous competitive indirect enzyme-linked immunosorbent assay (ciELISA) based on a monoclonal antibody (MAb) with broad-specificity for organophosphorus pesticides (OPs) was applied to the detection of O,O-diethyl and O,O-dimethyl OPs in water samples. The ciELISA conditions were carefully optimized to obtain a three to five-fold improvement of sensitivity for most OPs, and thirteen OPs were determined at concentrations ranging from 0.017 to 30 ng mL−1. The determination of spiked environmental water samples showed average recoveries from 81.5% to 115.1%, with the coefficient of variation (CV) ranging from 6.1% to 20.9%, which showed satisfactory reproducibility of the developed ciELISA. To overcome the negative aspect of broad-specificity immunoassays not providing qualitative and quantitative analysis of individual OPs in blind samples, we used “percent inhibition rate” to make the developed ciELISA a semi-quantitative method, which allows the monitoring of positive samples from hundreds of negative samples. The determination of OPs in blind water samples by the developed ELISA with confirmation by HPLC-MS/MS analysis demonstrated that the assay is ideally suited as a screening method for OP residues prior to chromatographic analysis.

The use of chlorpromazine (CPZ) as a sedative for livestock has been prohibited in the European Union and many other countries. In this study, a new hapten 7-amino-chlorpromazine sulfoxide (hapten 1) against CPZ was synthesized and coupled to ovalbumin (OVA) as an immunogen to produce polyclonal antibody (PAb) specific for CPZ. An heterologous hapten 7-(4-carboxyl-phenylazo)-chlorpromazine (hapten 2) was synthesized and coupled to bovine serum albumin (BSA) as coating antigen to improve the assay sensitivity. The results showed that the hapten-heterologous systems exhibited 20 times higher sensitivity than the hapten-homologous one. Based on the hapten-heterologous system, an indirect competitive enzyme-linked immunosorbent assay (ELISA) for CPZ was developed, the IC50 value was 0.58 ng mL−1 and the limit of detection was 0.03 ng mL−1. The assay showed no cross-reaction with the CPZ analogues. The average recoveries of CPZ from spiked samples were estimated to range from 77.1% to 98.6%, with a coefficient of variation (CV) of less than 10.9%. Linear regression analysis showed a good correlation between the CPZ concentrations obtained from ELISA and HPLC analysis, which suggested that the ELISA is a convenient supplementary analytical tool for monitoring CPZ.

Due to the large-scale use of organophosphorus pesticides (OPs), their contamination in the environment is widespread throughout the world, especially in developing countries. With increasing public concern, there is an urgent requirement for simple, rapid, high-throughput and highly sensitive analytical methods for the screening of OP residues in the environment. In this work, a monoclonal antibody-based sensitive one-step direct competitive time-resolved fluorescence immunoassay (TRFIA) with broad specificity to a class of OPs was developed. After optimization of the assay conditions, this method can detect thirteen OPs with a limit of detection below 10 ng mL−1. The recoveries of OPs from spiked environmental water samples ranged from 74.8% to 121.3%, with the coefficient of variation (CV) ranging from 6.4% to 15.1%. The correlation coefficient between the TRFIA results and standard HPLC-MS/MS results was 0.964. The proposed TRFIA was capable of high-throughput analysis of a large number of samples prior to chromatographic analysis, with good sensitivity, simplicity and rapidity.

The demanding task of monitoring illegal dyes in foodstuffs requires sensitive detection methods. Here, a surface plasmon resonance (SPR) sensor, combined with a 75 ± 5 nm thick molecularly imprinted polymer (MIP) nanofilm as a recognition element, is used for the selective detection of Sudan dyes. The MIP-SPR sensor chip was fabricated by anchoring the MIP nanofilm of Sudan I on a gold chip via surface-initiated in situ polymerization. The surface morphology and thickness of the MIP films were characterized by atomic force microscopy (AFM) and scanning electron microscopy (SEM). The adsorption properties of the sensor chip were studied through application as an SPR sensor. The MIP-SPR sensor exhibited excellent adsorption capacity, and high sensitivity and selectivity for Sudan dyes. The SPR response was linearly proportional to the concentration of Sudan I over the range 50–400 ng mL−1. Based on a signal-to-noise ratio of three, a detection limit of 30 ng mL−1 was established with a 400 s response time. Non-imprinted SPR sensors were compared in terms of selectivity with MIP-SPR sensors and it was found that all the recognition coefficients (α) for Sudan I–IV were greater than 1. The MIP-SPR sensor also showed reproducible response over three equilibration–adsorption–regeneration cycles.