Evaluation of the molecular recognition of monoclonal and polyclonal antibodies for sensitive detection of 2,4,6-trinitrotoluene (TNT) by indirect competitive surface plasmon resonance immunoassay

Detection of TNT is an important environmental and security concern all over the world. We herein report the performance and comparison of four immunoassays for rapid and label-free detection of 2,4,6-trinitrotoluene (TNT) based on surface plasmon resonance (SPR). The immunosensor surface was constructed by immobilization of a home-made 2,4,6-trinitrophenyl–keyhole limpet hemocyanin (TNPh–KLH) conjugate onto an SPR gold surface by simple physical adsorption within 10 min. The immunoreaction of the TNPh–KLH conjugate with four different antibodies, namely, monoclonal anti-TNT antibody (M-TNT Ab), monoclonal anti-trinitrophenol antibody (M-TNP Ab), polyclonal anti-trinitrophenyl antibody (P-TNPh Ab), and polyclonal anti-TNP antibody (P-TNP Ab), was studied by SPR. The principle of indirect competitive immunoreaction was employed for quantification of TNT. Among the four antibodies, the P-TNPh Ab prepared by our group showed highest sensitivity with a detection limit of 0.002 ng/mL (2 ppt) TNT. The lowest detection limits observed with other commercial antibodies were 0.008 ng/mL (8 ppt), 0.25 ng/mL (250 ppt), and 40 ng/mL (ppb) for M-TNT Ab, P-TNP Ab, and M-TNP Ab, respectively, in the similar assay format. The concentration of the conjugate and the antibodies were optimized for use in the immunoassay. The response time for an immunoreaction was 36 s and a single immunocycle could be done within 2 min, including the sensor surface regeneration using pepsin solution. In addition to the quantification of TNT, all immunoassays were evaluated for robustness and cross-reactivity towards several TNT analogs.      

Determination of trace elements in suspensions and filtrates of drinking and surface water by wavelength-dispersive X-ray fluorescence spectrometry

An X-ray fluorescence method (XRF) is presented that allowed low detection limits (at the 0.1–23 ng mL⁻¹ level) to be obtained for Cr, Mn, Fe, Ni, Zn, Sr, Pb, Bi and Br in water. The samples were prepared using a thin layer method. Trace elements were determined via the calibration curve and standard addition. Absorption effects and inhomogenities in prepared samples were checked for using the emission–transmission method and internal standards, respectively. The results from the XRF method were compared with the results from the inductively coupled plasma atomic emission spectrometry method.      

Novel gel-based rapid test for non-instrumental detection of ochratoxin A in beer

A rapid easy-to-use immunoassay was optimised for the non-instrumental detection of ochratoxin A (OTA) in beer. The analytical method involves preconcentration on the immunoaffinity layer inside a column followed by direct competitive ELISA detection in the same layer. The visual cut-off value, i.e. the lowest OTA concentration resulting in no colour development, was 0.2 μg L^-1. Assay validation was performed using samples spiked with OTA. Thirty-seven naturally contaminated samples were screened with the gel-based method developed and no false-negative results were obtained. The method described offers a simple, rapid and cost-effective screening tool, thus contributing to better health protection of consumers. Figure Gel-based immunoassay of spiked beer samples.     

Characterization and application of quantum dot nanocrystal–monoclonal antibody conjugates for the determination of sulfamethazine in milk by fluoroimmunoassay

Quantum dot (Qdot) nanocrystals have been increasingly used as fluorescence labels in fluoroimmunoassays recently because of their excellent optical characteristics. In this paper, a new monoclonal antibody (MAb) against sulfamethazine (SMZ) was successfully produced and linked to Qdot nanocrystals by covalent coupling. The Qdot–MAb conjugates were characterized by SDS-PAGE and high-performance capillary electrophoresis (HPCE). An enzyme-linked immunosorbent assay (ELISA) method was utilized to evaluate the antigen–antibody binding affinity and then a novel direct competitive fluorescence-linked immunosorbent assay (cFLISA) for the detection of SMZ in milk by using Qdots as fluorescent labels was evaluated. The results showed that the 50% inhibition values (IC₅₀) of the cFLISA were 4.3 ng/mL in milk and 5.2 ng/mL in PBS, and the limits of detection (LODs) were 0.6 ng/mL in milk and 0.4 ng/mL in PBS, respectively. The recoveries of SMZ from spiked milk samples at levels of 10–100 ng/mL ranged from 94 to 106%, with coefficients of variation (CVs) of 2.1–9.2%. Figure Shematic diagram of the direct cFLISA procedure Jianzhong Shen and Fei Xu contributed equally to this work.     

Determination of vancomycin in human plasma using high-performance liquid chromatography with fluorescence detection

A high-performance liquid chromatographic (HPLC) method with fluorescence detection for the quantification of vancomycin in human plasma was developed and validated. The method includes an extraction of vancomycin by deproteinization with acetonitrile. The analyses were carried out at 258 nm as the emission wavelength while exciting at 225 nm on a reversed-phase column (30 cm × 4 mm i.d. × 10 μm Waters Associates μBondapak C18) using a mobile phase composed of methanol and phosphate buffer at pH 6.3. Vancomycin was quantitatively recovered from human plasma samples (>96%) with high values of precision. The separation was completed within 27 min. The calibration curve was linear over the range from 5 to 1,000 ng/mL with the detection and quantification limits of 2 ng/mL and 5 ng/mL, respectively. This method is suitable for the routine assay of plasma samples. Figure The effect of the deproteinization solvent on the signal of the interference peak at retention time of 15.0 min. The peak which interferes with the peaks of Erythromycin and Vancomycin has been disappeared by using 2 mL acetonitrile as the deproteinization solvent.     

Simultaneous quantification of cilostazol and its primary metabolite 3,4-dehydrocilostazol in human plasma by rapid liquid chromatography/tandem mass spectrometry

A simple, rapid, sensitive and selective liquid chromatography/electrospray tandem mass spectrometry method was developed and validated for the simultaneous quantification of cilostazol and its primary metabolite 3,4-dehydrocilostazol in human plasma using mosapride as an internal standard. The method involves a simple one-step liquid-liquid extraction with a diethyl ether and dichloromethane mixture (7:3). The analytes were chromatographed using an isocratic mobile phase on a reversed-phase C₁₈ column and analyzed by mass spectrometry in the multiple reaction monitoring mode using the respective [M+H]⁺ ions, m/z 370/288 for cilostazol, m/z 368/286 for 3,4-dehydrocilostazol and m/z 422/198 for the internal standard. The assay exhibited a linear dynamic range of 5–2,000 ng/mL for cilostazol and 5–400 ng/mL for 3,4-dehydrocilostazol in human plasma. The lower limit of quantitation was 5 ng/mL for both cilostazol and its metabolite. Acceptable precision and accuracy were obtained for concentrations over the standard curve ranges. A run time of 2.5 min for each sample made it possible to analyze more than 400 human plasma samples per day. The validated method has been successfully used to analyze human plasma samples for application in pharmacokinetics, bioavailability or bioequivalence studies.      

Fluorescence determination of metoprolol in human plasma by trilinear decomposition-based calibration techniques

In this study a new spectrofluorimetric method for the direct determination of metoprolol in human plasma is presented and discussed. It is based on the use of fluorescence excitation–emission matrices (EEMs) and second-order calibration performed with parallel factor analysis (PARAFAC) or alternating trilinear decomposition (ATLD). This methodology enables accurate and reliable discrimination of the analyte signal, even in the presence of unknown and uncalibrated fluorescent component(s), which is often referred to as the second-order advantage. No separation or sample pretreatment steps were required. Satisfactory results were obtained. Metoprolol recoveries in plasma were determined as 87±2% and 90±4% with PARAFAC and ATLD, respectively. All RSD values of intra- and interday assays were below 5%. Figure A three-dimensional plot of EEMs for a plasma sample and metoprolol solution     

Quantification of atrazine and its metabolites in urine by on-line solid-phase extraction–high-performance liquid chromatography–tandem mass spectrometry

We have developed a method using on-line solid-phase extraction–high-performance liquid chromatography–tandem mass spectrometry (SPE-HPLC-MS/MS) and isotope dilution quantification to measure atrazine and seven atrazine metabolites in urine. The metabolites measured were hydroxyatrazine, diaminochloroatrazine, desisopropylatrazine, desethylatrazine, desethylatrazine mercapturate, atrazine mercaturate and atrazine itself. Our method has good precision (relative standard deviations ranging from 4 to 20% at 5, 10 and 50 ng/mL), extraction efficiencies of 67 to 102% at 5 and 25 ng/mL, relative recoveries of 87 to 112% at 5, 25, 50 and 100 ng/mL limits of detection (LOD) ranging from 0.03 to 2.80 ng/mL. The linear range of our method spans from the analyte LOD to 100 ng/mL (40 ng/mL for atrazine and atrazine mercapturate) with R ² values of greater than 0.999 and errors about the slope of less than 3%. Our method is rapid, cost-effective and suitable for large-scale sample analyses and is easily adaptable to other biological matrices. More importantly, this method will allow us to better assess human exposure to atrazine-related chemicals. Figure A schematic representation showing the elution of the analytes from the solid-phase extraction cartridge onto the analytical column for chromatographic separation prior to MS/MS analysis     

Application of a new anti-zearalenone monoclonal antibody in different immunoassay formats

Monoclonal antibodies against zearalenone (ZEA) were raised in mice according to the hybridoma technology and applied in different immunochemical techniques. More specifically, three formats based on the competitive direct enzyme immunoassay principle were developed: an enzyme-linked immunosorbent assay (ELISA), a flow-through gel-based immunoassay column and a flow-through membrane-based immunoassay. In ELISA, the 50% inhibitory concentration (IC50) was 0.8 ng/mL, and the limit of detection for ZEA standard solutions was 0.1 ng/mL. The antibodies showed a high ZEA (100%) and α-zearalenol (α-ZOL) (69%) recognition, while cross-reactivities with α-zearalanol, zearalanone, β-zearalenol and β- zearalanol were 42%, 22%, <1% and <1%, respectively. For standard solutions, a cut-off level at 10 ng/mL could be established for the gel- and membrane-based enzyme immunoassays. Assay time of both non-instrumental tests was 25 min for 10 samples. By including a simple sample extraction procedure, the methods were applied to wheat with IC50s in ELISA of 80 and 120 μg/kg (dilution up to 5% and 15% (v/v) of wheat matrix, respectively). The cut-off level of the gel- and membrane-based immunoassays was established at 100 μg/kg. Potentials and limitations of the developed methods were compared. The possible application for multi-mycotoxin analysis of the ELISA method based on a single monoclonal antibody was investigated. Therefore, principal component analysis and partial least squares regression data modelling were used to separate the immunoassay responses of two cross-reactants (ZEA and α-ZOL).      

Application of anodized aluminum in fluorescence detection of biological species

Thin nanoporous alumina obtained by anodization of aluminum films offers promising advantages for application in fluorescence-based biological sensors including convenient preparation, increased density of binding sites, and improved collection efficiency of fluorescence. These advantages are illustrated in the detection of streptavidin using biotin covalently bound to the surface of alumina nanopores. Fluorescence intensity enhancement as high as 7 times is observed in nanopores in comparison to flat glass surface.