Development of a sensitive and rapid fluorescence polarization immunoassay for high throughput screening eight glucocorticoids in beef.

In this study, a sensitive, rapid, homogeneous, and high-throughput fluorescence polarization immunoassay (FPIA) for the rapid screening of eight glucocorticoids (GCs) in beef samples was successfully established. Two tracers including 5-aminofluorescein-labeled dexamethasone (5-AF-DMS) and fluorescein isothiocyanate-labeled dexamethasone (FITC-DMS) were studied to select appropriate antibody-tracer pairs using four previously produced broad-specific monoclonal antibodies. An optimal combination of the antibody 12D9 and the tracer FITC-DMS was selected. Under optimal detection conditions, the half inhibitory concentrations of dexamethasone (DMS), betamethasone (BMS), prednisolone (PNS), hydrocortisone (HCS), beclomethasone (BCMS), cortisone (CS), 6-α-methylprednisone (6-α-MPNS), fludrocortisone acetate (HFCS) were 1.00, 2.17, 3.49, 12.45, 1.20, 5.66, 6.85 and 3.45 ng/mL, respectively. The average recoveries of the proposed method in beef samples ranged from 77.3-91.7% with the coefficient of variation less than 12%. The developed FPIA was time-saving that could be completed within 10 min. The FPIA was applied to beef samples and showed a good correlation with liquid chromatography-tandem mass spectrometry (LC-MS/MS) (R2 = 0.9894). Thus, the proposed method provides a rapid, reliable, sensitive, and high-throughput screening tool for the simultaneous screening of eight GCs in beef, which shows great potential in the food safety analysis.

A simple and rapid immunochromatography test based on readily available filter paper modified with chitosan to screen for 13 sulfonamides in milk.

In this study, we developed a novel, simple, rapid, and low-cost colloidal gold-based immunochromatography method, with filter paper replacing nitrocellulose membrane as the substrate. To obtain adequately immobilized protein, chitosan was used to functionalize the filter paper. After conditions and parameters were optimized, the novel immunochromatography method was applied for detection of sulfonamide residues in milk. Quantitative detection was accomplished using a smartphone and Photoshop software (Adobe Inc., San Jose, CA), allowing us to screen 13 sulfonamides with a limit of detection ranging from 0.42 to 8.64 µg/L and recovery ranging from 88.2 to 116.9% in milk. The proposed novel method performed similarly to the conventional method that uses a nitrocellulose membrane as the transport medium, and it had lower cost and better usability because of the inexpensive and easily available filter paper.

Highly sensitive chromatographic time-resolved fluoroimmunoassay for rapid onsite detection of streptomycin in milk.

Antibiotic residues are major contaminants in milk because of their use in agriculture and animal husbandry. In particular, streptomycin, an aminoglycoside antibiotic, is a potential risk to consumers because of its ototoxicity, anaphylaxis, and growth inhibition. Herein, monoclonal antibodies for streptomycin were conjugated with europium microspheres to serve as detection probes for the development of a chromatographic time-resolved fluoroimmunoassay to detect streptomycin residues in milk. The method had a low detection limit of 0.58 µg/kg, a linear range of 0.8 to 6.25 µg/kg, and substantial recovery, from 85.6 to 108.3%. It showed slight cross-reactivity with another aminoglycoside analog. Strong correlations between the results of established chromatographic time-resolved fluoroimmunoassay and ultra-performance liquid chromatography-tandem mass spectrometry indicated that the established fluoroimmunoassay is a reliable method for rapid onsite detection of streptomycin in milk and it has great potential in food safety monitoring.

Site-directed mutations of anti-amantadine scFv antibody by molecular dynamics simulation: prediction and validation.

A recombinant single-chain variable fragment (scFv) antibody was produced from a hybridoma cell strain secreting the monoclonal antibody for amantadine (AMD), and then its recognition mechanisms for AMD were studied using the molecular docking and molecular dynamics. Complex dockings revealed that three regions are involved in antibody recognition; framework 2 of the VL chain (LFR2) GLU40 and TYR42, complementarity-determining region of the VL chain (LCDR3) TYR116, and framework 2 of the VH chain (HFR2) HIS40 and TRP52 were the key amino acid residues. The results of molecular dynamics show that the most important amino acid residues in the interaction between AMD and scFv are HIS40 and TYR116. On the basis of the results of virtual mutation, the scFv antibody was evolved by directional mutagenesis of amino acid residue GLY107 to PHE. Indirect competitive ELISA (icELISA) results indicated that the scFv mutant had highly increased affinity for AMD with up to 3.9-fold improved sensitivity. Thus, the scFv antibody can be applied for mechanistic studies of intermolecular interactions, and our work offered affinity maturated antibodies by site mutations, which were beneficial for valuable anti-AMD antibody design and preparation in future.

Immunochromatographic fluorometric determination of clenbuterol with enhanced sensitivity.

A method is described to enhance the sensitivity of an immunochromatographic assay for clenbuterol (CLE) by making use of dually-labeled gold nanoparticles (GNPs), background fluorescence blocking, and immunomagnetic separation. The GNPs were labeled with biotinylated antibody and streptavidin, respectively, and dually labeled GNPs were obtained via the biotin-streptavidin interaction to amplify the detection signal. The fluorescent signal was blocked by dually labeled GNPs and decreased as the dually labeled GNPs aggregation increases on nitrocellulose membrane, which derived from fluorescent polyvinylchloride card. However, fluorescence (measured at excitation/emission wavelengths of 518/580 nm) recovers when CLE reacts with dually labeled GNPs. Immunomagnetic separation was first applied for sample pretreatment. This can offset the matrix effect and improves the sensitivity and accuracy of the assay. Under the optimal conditions, the limits of detection of CLE visually were 0.25 µg·L-1. In addition, clenbuterol can be quantified in swine urine with a 0.03 µg·L-1 detection limit. This is 60-fold lower than current immunochromatography. Response is linear in the 0.06-0.59 µg·L-1 concentration range, and the recoveries from spiked swine urine range from 81 to 115%." Graphical abstract Schematic presentation of the strategies for improving sensitivity of immunochromatographic assay. It includes immunomagnetic separations, dually-labeled gold nanoparticles and background fluorescence blocking. The assay was applied to detect clenbuterol (CLE) in swine urine with an excellent performance.

Highly luminescent green-emitting Au nanocluster-based multiplex lateral flow immunoassay for ultrasensitive detection of clenbuterol and ractopamine.

A multiplex lateral flow immunoassay sensor based on highly luminescent green-emitting Au nanoclusters (AuNCs-MLFIA sensor) was successfully established for the simultaneous and quantitative determination of clenbuterol (Clen) and ractopamine (RAC) in swine urine. The antigens of Clen and RAC were dispersed on a nitrocellulose membrane as two test lines, and the Au nanoclusters were synthesized from 6-aza-2-thiothymine and l-arginine to obtain highly green luminescence and ultra-small nanoparticles (Arg/ATT/AuNCs). Free carboxyl groups on Arg/ATT/AuNCs enabled conjugation with biomolecules to afford an indicator for the biosensor. The AuNCs-MLFIA sensor is based on the indirect competition assay and could successfully detect samples within 18 min without sample pretreatment, qualitative results can be obtained by visual inspection under a UV lamp. The limits of detection of Clen and RAC by the naked eye were both 0.25 µg L-1. In addition, the AuNCs-MLFIA sensor allowed quantitative detection combined with a portable fluorescence reader. The half-maximal inhibitory concentrations of Clen and RAC were 0.06 and 0.32 µg L-1, respectively, with detection limits of 0.003 and 0.023 µg L-1. Thirty blind-spiked swine urine samples were analyzed by the AuNCs-MLFIA sensor and liquid chromatography-tandem mass spectrometry, and the results of the two methods showed a significant correlation. The newly developed AuNCs-MLFIA sensor overcomes several limitations of conventional LFIA sensors, including their low sensitivity, limitation to quantify analytes, and single-analyte detection.

Determination of aflatoxin and zearalenone analogs in edible and medicinal herbs using a group-specific immunoaffinity column coupled to ultra-high-performance liquid chromatography with tandem mass spectrometry.

Six aflatoxins (AFs; AF B1, B2, G1, G2, M1 and M2) and six zearalenone (ZEN) analogs (ZEN, zearalanone, α-zeralanol, ß-zeralanol, α-zearalenol, and ß-zearalenol) were simultaneously extracted from edible and medicinal herbs using a group-specific immunoaffinity column (IAC) and then identified by ultra-high-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). The IAC was prepared by coupling N-hydroxysuccinimide-activated Sepharose 4B Fast Flow gel with two group-specific monoclonal antibodies. The column capacities to six AFs and six ZEN analogs ranged from 100.2 ng to 167.1 ng and from 59.5 ng to 244.4 ng, respectively. The IAC-UPLC-MS/MS method was developed and validated with three different matrices (Chinese yam [Dioscorea polystachya], Platycodon grandiflorum and coix seed [Semen Coicis]). Recoveries of twelve analytes from edible and medicinal herbs were in the range of 64.7%-112.1%, with relative standard deviations below 13.7%. The limits of quantification were in the range from 0.08 µg kg-1 to 0.2 µg kg-1. The method was proven to be sensitive and accurate, and suitable for the determination of real samples.