Arctigenin Attenuates Tumor Metastasis Through Inhibiting Epithelial-Mesenchymal Transition in Hepatocellular Carcinoma via Suppressing GSK3ß-Dependent Wnt/ß-Catenin Signaling Pathway In Vivo and In Vitro.

Arctigenin (ARG) has been reported to be a bioactive lignan from Arctium lappa exerting various activities including anti-cancer and immune-regulation. The present study aimed to investigate the anti-metastasis activity and mechanism of ARG against hepatocellular carcinoma in vitro and in vivo. The results showed that ARG exhibited a significant cytotoxicity on Hep G2 and SMMC 7721 cells (but not on normal liver cells LO2). In addition, the migration and invasion of Hep G2 and SMMC 7721 cells were also remarkably repressed. Furthermore, ARG attenuated Wnt/ß-catenin signaling activation, resulting in the down-regulation of ß-catenin target genes including c-Myc, cyclin D1, MMP-9, and ZO-1. Noticeably, ARG attenuated the activation of Wnt/ß-catenin through a GSK3ß-dependent pathway. Besides, we also found that ARG potentially inhibited epithelial-mesenchymal transition by up-regulating the epithelial and down-regulating the mesenchymal marker proteins. In vivo, intraperitoneal injection of ARG not only significantly inhibited the growth of subcutaneous transplanted tumor but also dramatically alleviated the tumor metastasis in liver. Our data demonstrated that ARG exerted anti-epithelial-mesenchymal transition and anti-metastasis activities against hepatocellular carcinoma, which might make it a candidate as a preventive agent for cancer metastasis.

A universal multi-wavelength fluorescence polarization immunoassay for multiplexed detection of mycotoxins in maize.

Multi-analyte immunoassays have attracted increasing attention due to their short assay times, low sample consumption, and reduced detection costs per assay. In this work, we describe a homologous and high-throughput multi-wavelength fluorescence polarization immunoassay (MWFPIA) for the multiplexed detection of mycotoxins. Three typical Fusarium mycotoxins, deoxynivalenol (DON), T-2 toxin and fumonisin B1 (FB1), were labeled with different dyes. Tracers and specific monoclonal antibodies (mAbs) were employed in the MWFPIA to simultaneously detect the three mycotoxins. Under optimal conditions, the limits of detection using MWFPIA were 242.0 µg kg(-1) for DON, 17.8 µg kg(-1) for T-2 toxin and 331.5 µg kg(-1) for FB1, providing sufficient sensitivity to meet the action levels of these three contaminants in maize as set by the European Union. The use of a methanol/water (2:3, v/v) mixture for sample pretreatment allowed recoveries ranging from 76.5-106.3%, with coefficients of variation less than 21.7%. The total time of analysis, including sample preparation, was less than 30 min. Twenty naturally contaminated maize samples were tested using MWFPIA and HPLC-MS/MS, with correlation coefficients (R(2)) of 0.97 for DON and 0.99 for FB1. By changing the targets of interest, homologous MWFPIA, a method with high sensitivity, a simple procedure and a short analysis time, can easily be extended to other chemical contaminants. Thus, MWFPIA represents a versatile strategy for food safety analysis.

Fluorescence polarization immunoassay using IgY antibodies for detection of valnemulin in swine tissue.

Immunoglobulin Y (IgY) is derived from egg yolk and has been identified as a cheap and high-yield immunoreagent. The application of IgY in immunoassays for the detection of chemical contaminants in food samples has rarely been reported. In this work, we describe a rapid and sensitive fluorescence polarization immunoassay (FPIA) for valnemulin (VAL) using IgY which was produced using a previously prepared immunogen. Three fluorophore-labeled VAL tracers were synthesized and the sensitivity of the best tracer (VAL-DTAF) in the optimized FPIA with antibody IgY100 demonstrated an IC50 value of 12 ng mL(-1) in buffer. After evaluation of several extraction procedures, acidified acetonitrile was selected to extract VAL from swine tissue. The recoveries of VAL in spiked swine tissue at three levels (50, 100, and 200 µg kg(-1)) were higher than 79% with coefficients of variation (CVs) lower than 12%. The limit of detection (LOD) of the FPIA in swine tissue was 26 µg kg(-1) and was lower than the maximum residue limit (MRL) of VAL set by the European Union. The study showed that IgY could be a good substitute for IgG when developing a high-throughput assay for chemical residues.

Development of a screening fluorescence polarization immunoassay for the simultaneous detection of fumonisins B1 and B2 in maize.

This paper reports the development of a screening fluorescence polarization immunoassay (FPIA) for the simultaneous detection of fumonisins B1 (FB1) and B2 (FB2) in maize. Three FB1 tracers including FB1-fluorescein isothiocyanate isomer I (FB1-FITC), FB1-5-([4,6-dichlorotriazine-2-yl]amino)-fluorescein (FB1-5-DTAF), and FB1-Texas Red-X succinimidyl ester (FB1-TRX) were synthesized and studied to select appropriate tracer-antibody pairs using seven previously produced monoclonal antibodies (mAbs). An FPIA employing the pair of FB1-FITC and mAb 4B9 showing 98.9% cross-reactivity (CR) toward FB2 was used to simultaneously detect FB1 and FB2. Maize flour samples were extracted with methanol/water (2:3, v/v). After optimization, the FPIA revealed a limit of detection (LOD) of 157.4 µg/kg for FB1 and an LOD of 290.6 µg/kg for FB2, respectively. Recoveries were measured for spiked samples of FB1 or FB2 separately, ranging from 84.7 to 93.6%, with a coefficient of variation (CV) of <9.9%. Total time needed for FPIA including sample pretreatment was <30 min. The FPIA was used to screen naturally contaminated maize samples. Results detected by FPIA showed good agreement with that of HPLC-MS/MS with a fit of R(2) = 0.99 for the simultaneous detection of FB1 and FB2. The established method offered a rapid, simple, sensitive, and high-throughput screening tool for the detection of fumonisins in maize.

Hapten synthesis, monoclonal antibody production and development of a competitive indirect enzyme-linked immunosorbent assay for erythromycin in milk.

Erythromycin is an antibiotic used extensively in veterinary practice worldwide for treatment, prevention and growth promotion. In this work, monoclonal antibodies (Mabs) against erythromycin were produced and used to develop a competitive indirect enzyme-linked immunosorbent assay (ciELISA) for the determination of erythromycin in milk. A novel carboxyphenyl derivative of erythromycin (ERO-CMO) was synthesized and conjugated with bovine serum (BSA) for use as the immunogen or ovalbumin (OVA) as the coating antigen. Four hybridoma cell lines were isolated, which produced Mabs that competed with erythromycin. The 6C1 and 5B2 Mabs had IC50 values for erythromycin of 14.40 and 0.94 µg L(-)(1), respectively. These Mabs demonstrated high cross-reactivity to the macrolides containing 14-membered rings, but not to oleandomycin. No cross-reactivity was observed for 12 macrolides that contained 15 or 16-membered lactone rings or for 2 pleuromutilins. The ciELISA developed using the 5B2 Mab afforded recovery values that ranged from 76.9% to 85.7% with only a 10-fold sample dilution prior to analysis.

Simultaneous determination of multiple (fluoro)quinolone antibiotics in food samples by a one-step fluorescence polarization immunoassay.

This paper describes a rapid one-step fluorescence polarization immunoassay (FPIA) for the simultaneous determination of multiple (fluoro)quinolone antibiotics (FQs) in food samples. Several fluorescent tracers were synthesized and evaluated in the FPIA method based on a broad-specificity of monoclonal antibodies toward FQs. The heterogeneous tracer, SAR-5-FAM, was considered as the optimal choice to prepare the immunocomplex single reagent, which allows a rapid and sensitive displacement reaction by addition of analytes. Optimized single-reagent FPIA exhibited broad cross-reactivities in the range of 7.8-172.2% with 16 FQs tested and was capable of determining most FQs at the level of maximum residue limits. Recoveries for spiked milk and chicken muscle samples were from 77.8 to 116%, with relative standard deviation lower than 17.4%. Therefore, this method could be applicable in routine screening analysis of multiple FQ residues in food samples.

A proof-of-concept receptor-based assay for sulfonamides.

The gene encoding the dihydropteroate synthase (DHPS) of Streptococcus pneumonia has been cloned, sequenced, and expressed in Escherichia coli. The protein has been purified and used to develop a novel microplate assay for the detection of sulfonamides. The assay was based on the competition between sulfonamides and horseradish peroxidase (HRP)-labeled sulfonamide derivative, 4-(4-aminobenzenesulfonylamino) benzoic acid (CS) for the immobilized protein. Under optimized conditions, nine sulfonamides and p-aminobenzoic acid (PABA) could be detected below 100 ng/ml and 28 sulfonamides used in the study could be detected with IC50 values ranging from 426 to 50,000 ng/ml. It is concluded that this method offers a robust and rapid alternative to other methods for the screening of sulfonamides.

A monoclonal antibody-based ELISA for multiresidue determination of avermectins in milk.

Due to the widespread use and potential toxicity of avermectins (AVMs), multi-residue monitoring of AVMs in edible tissues, especially in milk, has become increasingly important. With the aim of developing a broad-selective immunoassay for AVMs, a broad-specific monoclonal antibody (Mab) was raised. Based on this Mab, a homologous indirect enzyme-linked immunosorbent assay (ELISA) for the rapid detection of AVMs in milk was developed. Under the optimized conditions, the IC50 values in assay buffer were estimated to be 3.05 ng/mL for abamectin, 13.10 ng/mL for ivermectin, 38.96 ng/mL for eprinomectin, 61.00 ng/mL for doramectin, 14.38 ng/mL for emamectin benzoate. Detection capability (CCß) of the ELISA was less than 5 ng/mL and 2 ng/mL in milk samples prepared by simple dilution and solvent extraction, respectively. The optimized ELISA was used to quantify AVMs in milk samples spiked at different amounts. The mean recovery and coefficient of variation (CV) were 95.90% and 15.42%, respectively. The Mab-based ELISA achieved a great improvement in AVMs detection. Results proved this broad-selective ELISA would be useful for the multi-residue determination of AVMs in milk without purification process.

Simultaneous determination of trace levels of 10 quinolones in swine, chicken, and shrimp muscle tissues using HPLC with programmable fluorescence detection.

A HPLC method using a modified sample preparation procedure was optimized and validated for the quantification of 10 quinolones (QNs), including marbofloxacin, ciprofloxacin, norfloxacin, lomefloxacin, danofloxacin, enrofloxacin, sarafloxacin, difloxacin, oxolinic acid, and flumequine, in swine, chicken, and shrimp tissues. In this method, only a small mass (