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Fluoroquinolones (FQs) are among the antibiotics whose widespread use in farm-raised animals results in potentially harmful residues in the end products. Additionally, most Thai farmers use antibiotics. Amoxicillin and enrofloxacin were commonly used by pig farms, and hens were given enrofloxacin to prevent immunization side effects. Moreover, antibiotic overuse has harmed food safety in the long term, and the use of low-dose antibiotics causes bacterial resistance. Herein, an indirect competitive enzyme-linked immunosorbent assay (icELISA) was used to make a fast, easy, sensitive, and cost-effective method for monitoring FQs residues. After immunizing hens with mixed multi-hapten ciprofloxacin-bovine serum albumin (CPFX-BSA) with norfloxacin-bovine serum albumin (NFX-BSA), the IgY antibody purified from egg yolk was used for the detection of FQs residues in chicken and pork samples. The efficiency of the IgY antibody showed excellent sensitivity, with 50% inhibitory concentration (IC50) of enrofloxacin at 0.05 µg/mL, far below the MRLs defined by the EU for muscle samples, which was not to exceed 100 µg/kg. The recovery range for chicken muscle samples spiked with ENFX concentrations of 1.00–0.01 µg/mL was 86.65–112.71%, similar to pork samples, which were 84.24–117.22.2%. This method has a lot of potential for analyzing fluoroquinolones in complex samples quickly, easily, and at a low cost on-site. The IgY-based ic ELISA was developed to detect ciprofloxacin (CPFX), norfloxacin (NFX), and enrofloxacin (ENFX) residues; it confirms that IgY could be a promising choice for the detection of antibiotic residues in food samples. [ABSTRACT FROM AUTHOR]

Acrylamide is a probable human carcinogen with a wide occurrence in commonly consumed food. Because of the lack of specific antibodies against acrylamide, immunoassays usually measure acrylamide derivates, which require long derivatization times at elevated temperatures. One exception is xanthylacrylamide (XAA), which can be derived from acrylamide at room temperature within 30 min. Here, we present the development of a monoclonal antibody against XAA and a competitive enzyme-linked immunosorbent assay (ELISA) based on the multicolor detection of gold nanorods (AuNRs). The biotinylated monoclonal antibodies bound streptavidin alkaline phosphatase, which induced XAA concentration-dependent silver deposition on the AuNRs. The resulting blue shift of the AuNR surface plasmon resonance peak translated into a strong gray-to-orange color change that could be conveniently distinguished by the naked eye or smartphone color detection. The assay showed a broad linear range (0.3–17.2 ng/mL), a low limit of detection (0.06 ng/mL), and a 3.6-fold sensitivity improvement compared to those of conventional tetramethylbenzidine-based ELISA. The analysis of several different food products showed excellent acrylamide concentration recoveries (84–102%) and confirmed its applicability as an effective tool for routine monitoring of acrylamide in food. Rapid and simple color analysis demonstrated a strong potential for implementation into direct consumer product testing. [ABSTRACT FROM AUTHOR]

Due to the highly similar genetic background, it is difficult to distinguish Bacillus cereus (B. cereus) with other members of B. cereus group. Herein, an antibody-based colorimetric immunoassay using Cu-doped CeO2 nanospheres as peroxidase mimics was developed for the detection of B. cereus in food. First, monoclonal antibodies (mAbs) and polyclonal antibody (pAb) with good specificity to B. cereus were prepared and characterized. Second, the regular-shaped hollow Cu/CeO2 nanospheres with highly catalytic activity and biocompatibility were synthesized as mimic nanozymes to capture secondary antibody. Finally, a sandwich colorimetric immunoassay for the specific and sensitive detection of B. cereus was developed, showing linear detection range from 3.2 × 102 to 1 × 105 CFU/mL and a limit detection of 1.7 × 102 CFU/mL. The developed immunoassay holds great potential as an effective tool for detecting B. cereus in food poisoning. [ABSTRACT FROM AUTHOR]

The tadalafil‐like compounds have appeared recently as adulterants in drinks and healthcare dietary supplements sourced from medicinal and edible food, which may cause illness and even death. In this work, the rationality of haptens was explored by computational chemistry and molecular simulation theories such as frontier molecular orbital (FMO)‐based softness (S), three‐dimensional (3D) structure, surface electrostatic potential (ESP), and lipophilic potential (LP). An antiserum from hapten H5 with the highest softness and maintaining the appropriate three‐dimensional (3D) structure showed the optimal immunoassay performance, indicating an increasing softness was a critical factor for effective hapten. Based on the antibody induced by hapten H5, an indirect competitive enzyme‐linked immunosorbent assay (icELISA) method for detecting multiple tadalafil‐like adulterants was established. The icELISA showed a limit of detection (LOD), 50% inhibition concentration (IC50), and a working range of 0.004–0.396, 0.89–4.27, and 0.094–16.71 ng/ml for tadalafil, amino tadalafil, acetamino tadalafil, nortadalafil, and N‐desmethyl ent‐tadalafil, respectively. The spiked recoveries of tadalafil‐like adulterants in samples ranged from 84.9% to 116.2%. The results of the icELISA and HPLC‐MS/MS methods had a good correlation for real samples with the R2 of 0.9955. Specially, this work not only provided a convenient immunoassay method for measuring tadalafil‐like adulterants in spirit drinks and dietary supplements in group‐screening manner, but also suggested that softness was likely to be a general theory for rational hapten design. Practical Application: Rapid monitoring of tadalafil‐like adulterants in food samples is very necessary and important for consumers, regulatory agencies, and the food industry. [ABSTRACT FROM AUTHOR]

The conazole fungicide propiconazole is frequently found in vegetables although usage is not allowed. To overcome the high-cost and time-consuming labour requirements of instrumental methods, we developed a simple and visual lateral flow immunoassay for the sensitive determination of propiconazole. A hapten was carefully designed to raise a monoclonal antibody against propiconazole. Bal b/c mice were immunised with the hapten-carrier protein conjugate and a specific monoclonal antibody (mAb) was produced. Based on this mAb, a sensitive immunochromatographic strip assay (ICA) was established for rapid screening of propiconazole in vegetable samples. After optimisation of analytical parameters, the ICA strip showed a detection limit of 0.13 ng g−1 and a linear range from 0.5 to 80 ng g−1 using a strip reader. The assay also can be read by the naked eye with a visual limit of detection of 80 ng g−1. The recoveries for spiked vegetable samples by ICA ranged from 85.2% to 114.9%, with a coefficient of variation less than 11.7%. The assay time is within 45 min for a single sample including the sample pre-treatment. For spiked and blind samples, the detection capability of ICA was equivalent to liquid chromatography-mass spectrometry. [ABSTRACT FROM AUTHOR]

Citrulline is one of the major precursors of ethyl carbamate in soy sauce, and the accumulation of citrulline is attributed to the metabolism of arginine by bacteria with the arginine deiminase (ADI) pathway. However, key strains and factors affecting citrulline accumulation are not yet clear. In this study, two key strains of Pediococcus acidilactici and Weissella confusa were isolated from soy sauce moromi , and the regularity of citrulline formation was studied. Results showed that the conversion rates from arginine to citrulline (A / C rate) and the citrulline accumulation ability of W. confusa and P. acidilactici significantly increased in the presence of different concentrations of NaCl, indicating that salt stress was the main factor for citrulline accumulation. The inconsistent expression of arc genes by salt stress was the reason for citrulline accumulation for P. acidilactici , but for W. confusa , it may be due to the influence of arginine/citrulline on the transportation system: the intracellular citrulline could neither transport to extracellular space nor convert into ornithine. Environmental factors greatly influenced citrulline accumulation of the two key bacteria; A / C rate and citrulline formation in both strains decreased at low temperature (15°C) under high salt stress, but opposite effects were observed for the two key strains under anaerobic light condition. Moreover, quercetin and gallic acid significantly decreased the A / C rate and citrulline accumulation ability of the two key strains. The optimal quercetin and gallic acid as suggested by simulation experiment were 100 and 10 mg/l, respectively, and the lowest A / C rate of 28.4% and citrulline level of 1326.7 mg/l were achieved in the simulation system. This study explored the main factors for citrulline formation by the two key strains and proposed a targeted way to control citrulline in soy sauce. [ABSTRACT FROM AUTHOR]

Oxidase‐mimicking nanozymes are more desirable than peroxidase‐mimicking ones since H2O2 can be omitted. However, only a few nanomaterials are known for oxidase‐like activities. In this work, we compared the activity of Mn2O3, Mn3O4 and MnO2 and found that Mn2O3 had the highest oxidase activity. Interestingly, the activity of Mn2O3 was even inhibited by H2O2. The oxidase‐like activity of Mn2O3 was not much affected by the presence of proteins such as bovine serum albumin (BSA), but the physisorption of antibodies to Mn2O3 was not strong enough to withstand the displacement by BSA. We then treated Mn2O3 with 3‐aminopropyltriethoxysilane to graft an amine group, which was used to conjugate antibodies using glutaraldehyde as a crosslinker. A one‐step indirect competitive ELISA (icELISA) was developed for the detection of isocarbophos, and an IC50 of 261.7 ng/mL was obtained, comparable with the results of the standard two‐step assay using horseradish peroxidase (HRP)‐labeled antibodies. This assay has the advantage of significant timesaving for rapid detection of large amounts of samples. This work has discovered a highly efficient oxidase‐mimicking nanozyme useful for various nano‐ and analytical applications. [ABSTRACT FROM AUTHOR]

Having the benefits of low cost, excellent stability and tolerance to extreme conditions, nanozymes are a potential alternative of horseradish peroxidase (HRP) or other enzymes for bioanalytical chemistry, especially immunoassays. CeO2 nanoparticles have oxidase-mimicking activity and can avoid the use of unstable H2O2. For robust assays, the effect of proteins on the activity of CeO2 needs to be carefully studied. Herein, we studied the adsorption and desorption of bovine serum albumin (BSA) from CeO2. The CeO2 nanoparticles exhibited a higher protein adsorption capacity compared to the other tested metal oxide nanoparticles. Although the oxidase-like activity of CeO2 was inhibited by BSA, low concentrations of phosphate and fluoride ions boosted the activity of protein-capped CeO2. CeO2 was still active under strong acidic conditions and at high temperature, while HRP lost its activity. For immunoassay development, we covered CeO2 with an amine-modified silane for covalent conjugation to antibodies. A one-step indirect competitive ELISA for fenitrothion was developed, and an IC50 value of 35.6 ng mL−1 and a limit of detection of 2.1 ng mL−1 were obtained. [ABSTRACT FROM AUTHOR]

Amyloid beta peptides (Aβ1-42) have been found to be associated with the cause of Alzheimer's disease (AD) and dementia. Currently, methods for detecting Aβ1-42 are complicated and expensive. The present study is aimed at developing an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) to detect Aβ1-42 by using a polyclonal antibody from alpaca, an application used in urine samples. The serum was collected from the alpaca after immunizing it with Aβ1-42 at 500 μg/injection 5 times. The ic-ELISA was developed and showed a half-maximal inhibitory concentration (IC50) of 103.20 ng/ml. The limit of detection (LOD) was 0.39 ng/100 μl. The cross-reactivity was tested with Aβ1-40 and 8 synthesized peptides that had sequence similarities to parts of Aβ1-42. The cross-reactivity of Aβ1-40 and peptide 1 (DAEFRHDSGYE) was 55% and 69.4%, respectively. The ic-ELISA was applied to analyze Aβ1-42 in the urine and precipitated protein urine samples. This method can be used for detecting a normal level of total soluble Aβ (approximately 1 ng in 5 mg of precipitated urine protein) and can be used for detecting the early stages of AD. It is considered to be an easy and inexpensive method for monitoring and diagnosing AD. [ABSTRACT FROM AUTHOR]

Immunochromatographic assay (ICA) has been used widely for the onsite monitoring of illegal additives due to its simplicity, speed, and low cost. However, a scanner is commonly required for ICA to achieve quantitative results. In this work, we developed a visual semi-quantitative ICA for sibutramine, a banned additive in diet foods, without the need for a scanner for measurement. Monoclonal antibodies specific for sibutramine were raised and conjugated with upconversion nanoparticles (UCNPs) as the luminescent tracer. ICA was developed by employing multiple test lines to achieve the semi-quantitative detection of sibutramine. Based on the optimal conditions, the cutoff levels (limit of quantitation, LOQ) of T1 line, T2 line, T3 line, and T4 line were 0.02 μg/mL, 0.15 μg/mL, 1.0 μg/mL, and 7.5 μg/mL, respectively, in buffer system. The ICA demonstrated a LOQ at 0.2 mg/kg for sibutramine in diet food samples. The assay (including pretreatment) can be finished within 30 min without the aid of other instruments, except a laser pen. No false positive or false negative results were observed. The results indicated that the proposed method was reliable, simple, and rapid for the screening of sibutramine abuse in diet food samples. [ABSTRACT FROM AUTHOR]

In this study, a heterologous competitive indirect enzyme-linked immunosorbent assay (ciELISA) for the determination of estriol (E3) in milk was developed. Rabbit polyclonal antibody specific for E3 was raised by introducing a carboxyl spacer arm from bromoacetic acid of E3 as immunizing hapten. A heterologous ciELISA using a coating hapten containing a carboxyl spacer arm from 6-ketoestriol of E3. The assay exhibited the required sensitivity for detection of E3 with limit of detection of 0.07 ng mL−1 and a working range from 0.41 ng mL−1 to 15.78 ng mL−1. Good recoveries were obtained ranging from 83.2% to 125.8% for E3 spiked milk samples. A satisfactory correlation (R2 = 0.994) between the results of ciELISA analysis and that of LC–MS/MS analysis indicated good accuracy and reproducibility of the proposed method, which was ideally suited as a rapid screening tool for routine E3 monitoring. [ABSTRACT FROM AUTHOR]

Hydrocortisone (HYD) is one of the corticosteroids that illegally used in cosmetic products, which could lead to a potential health problem. In this work, HYD was conjugated to bovine serum albumin via a succinic anhydride spacer arm to yield an immunogen. New Zealand rabbits were immunized to obtain a polyclonal antibody against HYD. Based on the pAb, a heterologous competitive indirect enzyme-linked immunosorbent assay (ciELISA) by using progesterone carboxylic acid as a competitive hapten was developed. The assay showed an IC50 of 0.50 ng mL−1, detection limit of 0.04 ng mL−1 and a linear range of 0.10–2.00 ng mL−1. The assay was applied to determine HYD in cosmetic samples. Recoveries from 82.5% to 118.9% and CV from 1.3% to 9.5% were obtained using a simple pretreatment. Good correlation between the results of ciELISA and standard LC–MS/MS was obtained, suggesting its potential usage in HYD monitoring in cosmetic samples. [ABSTRACT FROM AUTHOR]

In this study, we develop a sensitive indirect competitive chemiluminescent enzyme immunoassay (ic-CLEIA) to detect cadmium (Cd) in cereals. A luminol-hydrogen peroxide chemiluminescence system catalysed by horseradish peroxidase (HRP) was used as the signal detecting system. The method showed a half-maximal inhibition concentration (IC50) of 0.5 ng/mL and a limit of determination (LOD) of 0.06 ng/mL. The assay indicated an effective linear range from 0.16 to 1.56 ng/mL. There were no obvious cross-reactivities with other metal ions, including Hg2+, Pb2+, As2+, Ag+, Fe3+, Cu2+, Cr2+, Au3+, Ca2+, Mg2+, Mn2+ and Zn2+. Recovery rates ranged from 79.2% to 120.0% for rice, maize, wheat, brown rice and glutinous rice samples. Twenty-five practical cereal samples were analysed, and the comparative analysis of the results showed a strong correlation (r = 0.984) between the CLEIA and the GFAAS methods. The results indicated that the ic-CLEIA developed was acceptable to screen for cadmium in cereal samples. [ABSTRACT FROM AUTHOR]

The herbicide propisochlor can be introduced into the water cycle via agricultural activities, thus possibly leading to harmful ecological effects. It is significant for the development of a rapid method for propisochlor analysis in water. In this work, a rapid homogeneous immunoassay based on fluorescence polarization immunoassay (FPIA) was firstly developed for the determination of propisochlor in pond water. The developed FPIA assay can be completed within only 14 min; the 50% inhibition concentration (IC50) was 125.1 ng/mL and the limit of detection was 8.4 ng/mL. The recoveries of propisochlor in pond water ranged from 80.0% to 86.6%. A high performance liquid chromatography confirmation showed a good correlation with the developed FPIA (R2 = 0.9748). The developed FPIA exhibited satisfactory rapidity, high sensitivity and excellent specificity, meeting the standards of the screening assay of propisochlor in pond water on site. The molecular modeling showed that the alkoxyalkyl of chloroacetamide herbicides exerted important effects on the herbicide-antibody binding. [ABSTRACT FROM AUTHOR]

A simple and rapid method of fluorescence polarization immunoassay (FPIA) for detection of Alternaria mycotoxin tenuazonic acid (TeA) was described. In this study, the tracers TeAH-FITC and TeAH-DTAF were synthesized from hapten 5-(sec-butyl)-3-(1-hydrazonoethyl)-4-hydroxy-1H-pyrrol-2(5H)-one (TeAH) and different fluoresceins isothiocyanate isomer I (FITC) or 5-([4,6-dichlorotriazin-2-yl]amino)fluorescein hydrochloride (DTAF), respectively. The established FPIA showed an IC50 value of 3.0 μg/mL with a detection limit of 0.13 μg/mL for TeAH based on the tracer of TeAH-FITC, and with the same properties of 7.3 and 0.93 μg/mL based on TeAH-DTAF. The former gave better assay sensitivity than the latter, that the 2,4-dichloro-s-triazine group of TeAH-DTAF may affect the specific recognition of tracer and antibody. Models of the lowest energy conformation and the molecular electrostatic potential isosurface studies revealed that the -C=N-N- and -CHCH3(CH2CH3) groups were the characteristic chemical groups in anti-TeAH antibody-analyte recognition via molecular modeling, which could create a theoretical guide for hapten design of small molecules and provide the traditional theory with empirical proof. [ABSTRACT FROM AUTHOR]

In this paper, we report the development of a sensitive and specific monoclonal antibody-based immunodiagnostic method for the detection of iso-tenuazonic acid (ITeA), an Alternaria mycotoxin, in food samples. The ITeA was derivatized with hydrazine hydrate to produce the antigen (E)-3-(1-hydrazonoethyl)-4-hydroxy-5-isobutyl-1H-pyrrol-2(5H)-one (ITeAH) which was further reacted with glyoxalic acid to generate the hapten (E)-2-((Z)-(1-(4-hydroxy-5-isobutyl-2-oxo-2,5-dihydro-1H-pyrrol-3-yl)ethylidene) (ITeAHGA) which was used as an immunogen after conjugation to bovine serum albumin (BSA). A highly specific monoclonal antibody selectively binding to ITeAH was generated via the hybridoma technique and subsequently used to construct a heterologous indirect competitive enzyme-linked immunosorbent assay (icELISA) using ITeAH as the competitive antigen for the detection of ITeA with a limit of detection (LOD) of 0.5 ng/mL. Under the optimum conditions, the developed icELISA is highly sensitive (IC = 7.8 ng/mL) with recovery rates ranged from 82.3 to 109.8% for spiked food samples. The comparative analysis of results revealed a good correlation between the icELISA and the standard HPLC-MS/MS method, confirming the suitability of the developed icELISA for screening and detection of mycotoxin ITeA in food samples. [ABSTRACT FROM AUTHOR]

A high-performance liquid chromatography with fluorescence detection (HPLC-FLD) method for ethyl carbamate (EC) determination in Chinese soy sauce was developed and used to predict EC precursors, and extraction, derivatization, and chromatographic conditions were optimized systematically. Under optimal conditions, the limit of detection, linear range, and recoveries were 3.91 μg/L, 12.87-274.87 μg/L, and 81.5-95.4%, respectively. The method precision was less than 8.9% (RSD) and no significant difference was found between EC determinations measured by HPLC-FLD and GC/MS. Using the proposed HPLC-FLD method, the EC contents in Chinese soy sauces ranged from not detected to 64.88 μg/L ( n = 19). Except for one soy sauce brand, the EC contents in low-salt solid-state fermented (LSF) soy sauces were lower than those in high-salt liquid fermented (HLF) soy sauces and were mainly affected by alcohol content. Moreover, the free ornithine and total arginine contents were significantly correlated with EC content in the soy sauce products. The results of this work provide a foundation for further study of EC formation and inhibition in Chinese soy sauces. [ABSTRACT FROM AUTHOR]

Folic acid (FA) is an important B vitamin naturally found in foods and also used in dietary supplements and food fortification. To monitor FA content is important to guarantee food quality and also to prevent its abuse in dietary supplements. In this work, we developed a simple and robust lateral flow immunochromatographic assay (ICA) for the routine screening of FA in food samples. FA was conjugated to carrier protein as immunogen and used to produce specific polyclonal antibody (pAb). The pAb was conjugated with gold nanoparticle and used to develop a lateral flow ICA. After the careful optimization of ICA conditions, the assay showed an IC of 51.8 ng mL and linear range from 23.4 to 114.5 ng mL for FA in assay buffer, with the assist of a DY6510 strip reader within 8 min. Using milk powder as a matrix model, after a 15-fold dilution to eliminate matrix effect, the ICA showed intra-assay recoveries from 94.2 to 107.0% with coefficient of variance (CV) below 15% and inter-assay recoveries from 91.4% to 111.3% with CV below 18%. Good agreement was obtained between the results of ICA and ciELISA and standard HPLC. It is an ideal screening tool for the fast monitoring of FA in a simple, fast, and low-cost manner. [ABSTRACT FROM AUTHOR]

A VHH antibody (or nanobody) is the antigen binding fragment of heavy chain only antibodies. Discovered nearly 25 years ago, they have been investigated for their use in clinical therapeutics and immunodiagnostics, and more recently for environmental monitoring applications. A new and valuable immunoreagent for the analysis of small molecular weight environmental chemicals, VHH will overcome many pitfalls encountered with conventional reagents. In the work so far, VHH antibodies often perform comparably to conventional antibodies for small molecule analysis, are amenable to numerous genetic engineering techniques, and show ease of adaption to other immunodiagnostic platforms for use in environmental monitoring. Recent reviews cover the structure and production of VHH antibodies as well as their use in clinical settings. However, no report focuses on the use of these VHH antibodies to detect small environmental chemicals (MW < 1500 Da). This review article summarizes the efforts made to produce VHHs to various environmental targets, compares the VHH-based assays with conventional antibody assays, and discusses the advantages and limitations in developing these new antibody reagents particularly to small molecule targets. [ABSTRACT FROM AUTHOR]

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, linear range of 5.0-530.0, 10.0-1,060.0 and 10.0-1,060.0 μg mL, 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 ( R = 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. [ABSTRACT FROM AUTHOR]

A simple and rapid method for determination of carbofuran was developed using the fluorescence polarisation immunoassay (FPIA). Three tracers with different lengths of bridge (0-, 2- and 6-carbon bridge) between the hapten molecule 4-[[(2,3-dihydro-2,2-dimethyl-7-benzofuranyloxy)carbonyl]-amino]-butanoic acid (BFNB) and 5-aminofluorescein (AF), fluoresceinthiocarbamyl ethylenediamine (EDF), fluoresceinthiocarbamyl hexylenediamine (HDF), were synthesised and their binding response with anti-carbofuran-specific antibody were evaluated. The physicochemical parameters were optimised for the FPIA. The AF-labelled BFNB conjugate (BFNB-AF) was found to be the optimal tracer for FPIA of carbofuran. The detection limit of carbofuran, IC50value and the working range were 2.3, 48.8 and 7.4−202.2 µg/L, respectively; and the reaction time was only 10 min. The average recovery from spiked water and vegetable samples was 86.9−95.4% and the mean coefficient of variation was 6.2% for inter-assay and 8.7% for intra-assay, which showed good reproducibility for FPIA. Thus, the developed FPIA method exhibited the potential for the rapid and accurate determination of carbofuran in agricultural and environmental samples. [ABSTRACT FROM AUTHOR]

An indirect competitive chemiluminescent enzyme immunoassay method was developed for screening malachite green (MG). Assay conditions, including the concentration of coating, antibody dilution, incubation time, dilution buffer, ionic strength and pH, were optimised. Under the optimised conditions, coating antigen concentration was 125 ng/mL; and dilution fold of antibody was 40,000. The 50% inhibition values of 0.22 ng/mL for MG were achieved with a limit of detection of 0.01 ng/mL, the linear range was from 0.03 ng/mL to 3.27 ng/mL. The assay showed a little cross-reactivity of 3.4%, 2.7% and 1.0% with methylene blue, brilliant green and crystal violet, respectively, and negligible cross-reactivity with other analogues of MG. The developed method has been used to quantify MG in seafood samples. The recovery of MG ranged from 82.43% to 108.0% at four concentrations (0.1, 1, 3 and 5 ng/mL), and the coefficients of variation were less than 13%. A comparison results between the high-performance liquid chromatography and the developed assay showed good relativity. The results indicated that the assay was a sensitive and stable method for screening MG. [ABSTRACT FROM PUBLISHER]

In this study, an indirect competitive chemiluminescent enzyme immunoassay (ic-CLEIA) for determining aflatoxin M1(AFM1) in milk products has been developed. A luminol–hydrogen peroxide chemiluminescence system catalysed by horseradish peroxidase (HRP) was used as the signal detecting system. The effects of several factors, including concentration and pH of phosphate buffer, dilution ratio of antibody and antigen and other relevant variables on the immunoassay, were studied and optimised by single-factor experiments. The developed method presented an IC50of 0.05 ng/mL, a detection limit of 0.01 ng/mL and a linear range from 0.018 to 0.13 ng/mL. This method has been successfully applied to the evaluation of AFM1in milk products, the recoveries ranging from 71.9% to 109.0%. A good correlation with the commercial available ELISA kit for AFM1(r= 0.9978) was obtained, indicating that the ic-CLEIA method developed can be used to determine AFM1in real samples. [ABSTRACT FROM PUBLISHER]

The expression efficiency was improved for the recombinant single-chain variable fragment (scFv) against clenbuterol (CBL) obtained from mouse and expressed in the methylotrophic yeast Pichia pastoris GS115, by redesigning and synthesizing the DNA sequence encoding for CBL-scFv based on the codon bias of P. pastoris. The codons enco4ding 124 amino acids were optimized, in which a total of 156 nucleotides were changed, and the G+C ratio was simultaneously decreased from 53 to 47.2 %. Under the optimized expression conditions, the yield of the recombinant CBL-scFv (41 kDa) antibodies was 0.223 g L in shake culture. Compared to the non-optimized control, the expression level of the optimized recombinant CBL-scFv based on preferred codons in P. pastoris demonstrated a 2.35-fold higher yield. Furthermore, the recombinant CBL-scFv was purified by Ni-NTA column chromatography, and the purity was 95 %. The purified CBL-scFv showed good CBL recognition by a competitive indirect enzyme-linked immunoassay. The average concentration required for 50 % inhibition of binding and the limit of detection for the assay were 5.82 and 0.77 ng mL, respectively. [ABSTRACT FROM AUTHOR]

A Pichia pastoris (P. pastoris) cell surface display system of Bombyx mori acetylcholinesterase (BmAChE) was constructed and its bioactivity was studied. The modified Bombyx mori acetylcholinesterase gene (bmace) was fused with the anchor protein (AGα1) from Saccharomyces cerevisiae and transformed into P. pastoris strain GS115. The recombinant strain harboring the fusion gene bmace-AGα1 was induced to display BmAChE on the P. pastoris cell surface. Fluorescence microscopy and flow cytometry assays revealed that the BmAChE was successfully displayed on the cell surface of P. pastoris GS115. The enzyme activity of the displayed BmAChE was detected by the Ellman method at 787.7 U/g (wet cell weight). In addition, bioactivity of the displayed BmAChE was verified by inhibition tests conducted with eserine, and with carbamate and organophosphorus pesticides. The displayed BmAChE had an IC50 of 4.17×10−8 M and was highly sensitive to eserine and five carbamate pesticides, as well as seven organophosphorus pesticides. Results suggest that the displayed BmAChE had good bioactivity. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Aristolochic acid (AA) toxicity has been shown in humans regarding carcinogenesis, nephrotoxicity, and mutagenicity. Monitoring the AA content in drug homologous and healthy foods is necessary for the health of humans. In this study, a monoclonal antibody (mAb) with high sensitivity for aristolochic acid I (AA-I) was prepared. Based on the obtained mAb, a chemiluminescent immunoassay (CLEIA) against AA-I was developed, which showed the 50% decrease in the RLUmax (IC50) value of 1.8 ng/mL and the limit of detection (LOD) of 0.4 ng/mL. Carbon dots with red emission at 620 nm, namely rCDs, were synthesized and employed in conventional indirect competitive enzyme-linked immunosorbent assay (icELISA) to improve the assay sensitivity of a fluoroimmunoassay (FIA). Oxidized 3,3′′,5,5′′-tetramethylbenzidine dihydrochloride (oxTMB) can quench the emission of the rCDs through the inner-filter effect; therefore, the fluorescence intensity of rCDs can be regulated by the concentration of mAb. As a result, the assay sensitivity of FIA was improved by five-fold compared to CLEIA. A good relationship between the results of the proposed assays and the standard ultra-high performance liquid chromatography-triple quadrupole mass spectrometer (UPLC-QQQ-MS/MS) of real samples indicated good accuracy and practicability of CLEIA and FIA. [ABSTRACT FROM AUTHOR]

Tylosin and tilmicosin (T&T) residues in livestock products have received extensive attention from consumers. Time-resolved fluorescence immunochromatographic assay (TRFICA), as a fast, efficient and sensitive immunoassay method, has played an increasingly important role in the food safety field. Therefore, herein a quantitative and visual TRFICA was established for simultaneously detecting T&T in milk in a group-screening manner. Under the optimal conditions, the standard curve range of developed TRFICA based on the T&T was 1.87~7.47 ng/mL, and the half-maximal inhibition concentrations (IC50) were 4.06 ng/mL and 3.74 ng/mL, respectively. The limits of detection (LOD) of the TRFICA method were from 1.72 ng/mL to 1.39 ng/mL, and the visual cut-off values were 31.25 ng/mL and 62.50 ng/mL for T&T in milk, respectively. Moreover, the stability experiments showed that the strips could be stored at 4 °C for more than 6 months, the total detection time was less than 13 min, and the cross-reactivities (CRs) with related compounds were less than 0.1%, which concluded that the developed TRFICA method could be used in real milk sample detection. [ABSTRACT FROM AUTHOR]

Nanobody (Nb), a new type of biorecognition element generally from Camelidae, has the characteristics of small molecular weight, high stability, great solubility and high expression level in E. coli. In this study, with 19-nortestosterone (19-NT), an anabolic androgenic steroid as target drug, three specific Nbs against 19-NT were selected from camel immune library by phage display technology. The obtained Nbs showed excellent thermostability and organic solvent tolerance. The nanobody Nb2F7 with the best performance was used to develop a sensitive indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) for 19-NT detection. Under optimized conditions, the standard curve of ic-ELISA was fitted with a half-maximal inhibitory concentration (IC50) of 1.03 ng/mL and a detection limit (LOD) of 0.10 ng/mL for 19-NT. Meanwhile, the developed assay had low cross- reactivity with analogs and the recoveries of 19-NT ranged from 82.61% to 99.24% in spiked samples. The correlation coefficient between ic-ELISA and the ultra-performance liquid chromatography/mass spectrometry (UPLC-MS/MS) method was 0.9975, which indicated that the nanobody-based ic-ELISA could be a useful tool for a rapid analysis of 19-NT in animal urine samples. [ABSTRACT FROM AUTHOR]

A series of haptens were rationally designed for producing monoclonal antibodies specific for EC and a simple fluorescence immunoassay platform was developed for the sensitive determination of EC based on alkaline phosphatase (ALP)-triggered Cu+ quenching of CdSe quantum dots (QDs). It was noted that Cd as a fluorescence substrate in CdSe QDs can be selectively substituted by Cu+ that resulted in a more significant fluorescence quenching in comparison with Cu2+. Meanwhile, because ALP catalyzed ascorbic acid phosphate and then assisted the transformation of Cu2+ to Cu+, the change in fluorescence intensity was found to be proportional to ALP concentration. After simple magnetic separation, the sensitivity and linear range of the established assay were improved approximately 53-fold and an order of magnitude, respectively, when compared with the conventional ELISA. The proposed platform was able to both amplify the signal and eliminate matrix interferences, making it a promising to determine EC as well as other contaminants in complex food matrix in a highly sensitive and simple manner. [ABSTRACT FROM AUTHOR]

In this work, a monoclonal antibody-based indirect competitive enzyme-linked immunosorbent assay (icELISA) was established to detect tylosin and tilmicosin in milk and water samples. A sensitive and specific monoclonal antibody was prepared by rational designed hapten, which was achieved by directly oxidizing the aldehyde group on the side chain of tylosin to the carboxyl group. Under the optimized conditions, the linear range of icELISA for tylosin and tilmicosin were 1.3 to 17.7 ng/mL and 2.0 to 47.4 ng/mL, with half-maximal inhibition concentration (IC50) values of 4.7 and 9.6 ng/mL, respectively. The cross-reactivity with other analogues of icELISA was less than 0.1%. The average recoveries of icELISA for tylosin and tilmicosin ranged from 76.4% to 109.5% in milk and water samples. Besides, the detection results of icELISA showed good correlations with HPLC-MS/MS. The proposed icELISA was satisfied for rapid and specific screening of tylosin and tilmicosin residues in milk and water samples. [ABSTRACT FROM AUTHOR]

Histamine (HA) is an important food contaminant generated during food fermentation or spoilage. However, an immunoassay for direct (derivatization free) determination of HA has rarely been reported due to its small size to induce the desired antibodies by its current hapten-protein conjugates. In this work, despite violating the classical hapten design criteria which recommend introducing a linear aliphatic (phenyl free) linker into the immunizing hapten, a novel haptens, HA-245 designed and synthesized with a phenyl-contained linker, exhibited significantly enhanced immunological properties. Thus, a quality-improved monoclonal antibody (Mab) against HA was elicited by its hapten-carrier conjugates. Then, as the linear aliphatic linker contained haptens, Hapten B was used as linker-heterologous coating haptens to eliminate the recognition of linker antibodies. Indirect competitive ELISA (ic-ELISA) was developed with a 50% inhibition concentration (IC50) of 0.21 mg/L and a limit of detection (LOD) of 0.06 mg/L in buffer solution. The average recoveries of HA from spiked food samples for this ic-ELISA ranged from 84.1% and 108.5%, and the analysis results agreed well with those of referenced LC-MS/MS. This investigation not only realized derivatization-free immunoassay for HA, but also provided a valuable guidance for hapten design and development of immunoassay for small molecules. [ABSTRACT FROM AUTHOR]

Nanobodies are one-tenth the size of conventional antibodies and are naturally obtained from the atypical heavy-chain-only antibodies present in camelids. Their small size, high solubility, high stability, and strong resilience to organic solvents facilitate their use as novel analytical reagents in immunochemistry. In this study, specific nanobodies against pesticide carbofuran were isolated and characterized from an immunized library via phage display platform. We further established an indirect competitive enzyme-linked immunosorbent assay (ELISA) using nanobody Nb316 to detect carbofuran in vegetable and fruit samples. The results showed a half-maximal inhibitory concentration (IC50) of 7.27 ng/mL and a detection limit of 0.65 ng/mL. A simplified sample pretreatment procedure omitting the evaporation of organic solvent was used. The averaged recovery rate of spiked samples ranged between 82.3% and 103.9%, which correlated with that of standard UPLC–MS/MS method. In conclusion, a nanobody with high specificity for carbofuran was characterized, and a nanobody-based sensitive immunoassay for simple and rapid detection of carbofuran in real samples was validated. [ABSTRACT FROM AUTHOR]

Immunoassay for pesticides is an emerging analytical method since it is rapid, efficient, sensitive, and inexpensive. In this study, a recombinant antigen-binding fragment (Fab) against a broad set of O,O-diethyl organophosphorus pesticides (DOPs) was produced and characterized. The κ chain and Fd fragment were amplified via PCR and inserted into the vector pComb3XSS and the soluble Fab on phagemid pComb3XSS was induced by isopropyl β-d-thiogalactoside in E. coli TOP 10F’. SDS-PAGE, Western blotting, and indirect competitive ELISA results indicated that Fab maintained the good characteristics of the parental mAb. To better understand antibody recognition, the three-dimensional (3D) model of Fab was built via homologous modeling and the interaction between Fab and DOPs was studied via molecular docking and dynamics simulations. The model clearly explained the interaction manner of Fab and DOPs, and showed that the Arg-L96 and Arg-H52 were mainly responsible for antibody binding. This work provided a foundation for further mutagenesis of Fab to improve its characteristics. [ABSTRACT FROM AUTHOR]