Tight Molecular Recognition of Benzo[a]pyrene by a High-Affinity Antibody.

Benzo[a]pyrene, which is produced during the incomplete combustion of organic material, is an abundant noxious pollutant because of its carcinogenic metabolic degradation products. The high-affinity (KD ≈3 nm) monoclonal antibody 22F12 allows facile bioanalytical quantification of benzo[a]pyrene even in complex matrices. We report the functional and X-ray crystallographic analysis of 22F12 in complex with 3-hydroxybenzo[a]pyrene after cloning of the V-genes and production as a recombinant Fab fragment. The polycyclic aromatic hydrocarbon is bound in a deep pocket between the light and heavy chains, surrounded mainly by aromatic and aliphatic amino acid side chains. Interestingly, the hapten-antibody interface is less densely packed than expected and reveals polar, H-bond-like interactions with the polycyclic aromatic π-electron system, which may allow the antibody to maintain a large, predominantly hydrophobic binding site in an aqueous environment while providing sufficient complementarity to its ligand.

Detection of the carcinogenic water pollutant benzo[a]pyrene with an electro-switchable biosurface.

The toxic nature of polycyclic aromatic hydrocarbons (PAHs), in particular benzo[a]pyrene (B[a]P), neccessitates the monitoring of PAH contamination levels in food and the environment. Here we introduce an indirect immunoassay format using electro-switchable biosurfaces (ESB) for the detection of B[a]P in water. The association of anti-B[a]P antibodies to microelectrodes is analyzed in real-time by measuring changes in the oscillation dynamics of DNA nanolever probes, which are driven to switch their orientations by high-frequency electrical actuation. From the association kinetics, the active concentration of anti-B[a]P, and hence the B[a]P contamination of the sample, can be determined with picomolar sensitivity. The detection limit of the assay improves with measurement time because increasingly accurate analyses of the binding kinetics become possible. It is demonstrated that an exceedance of the permissible 10 ng/L (40 pM) limit for B[a]P is detectable in an unprecedented short assay time (<1 h), using a simple three-step workflow involving minimal sample preparation. The reproducibility was satisfying with standard deviations below 5%. Further, the utility of the assay for practical applications is exemplified by analyzing a river water sample.

Analysis of benzo[a]pyrene in vegetable oils using molecularly imprinted solid phase extraction (MISPE) coupled with enzyme-linked immunosorbent assay (ELISA).

This paper describes the development of a molecularly imprinted polymer-based solid phase extraction (MISPE) method coupled with enzyme-linked immunosorbent assay (ELISA) for determination of the PAH benzo[a]pyrene (B[a]P) in vegetable oils. Different molecularly imprinted polymers (MIPs) were prepared using non-covalent 4-vinylpyridine/divinylbenzene co-polymerization at different ratios and dichloromethane as porogen. Imprinting was done with a template mixture of phenanthrene and pyrene yielding a broad-specific polymer for PAHs with a maximum binding capacity (Q) of ~32 µg B[a]P per 50 mg of polymer. The vegetable oil/n-hexane mixture (1:1, (v/v)) was pre-extracted with acetonitrile, the solvent evaporated, the residue reconstituted in n-hexane and subjected to MISPE. The successive washing with n-hexane and isopropanol revealed most suitable to remove lipid matrix constituents. After elution of bound PAHs from MISPE column with dichloromethane, the solvent was evaporated, the residue reconstituted with dimethyl sulfoxide and diluted 100-fold with methanol/water (10:90, (v/v)) for analysis of B[a]P equivalents with an ELISA. The B[a]P recovery rates in spiked vegetable oil samples of different fatty acid composition were determined between 63% and 114%. The presence of multiple PAHs in the oil sample, because of MIP selectivity and cross-reactivity of the ELISA, could yield overestimated B[a]P values.

Application of a heterogeneous immunoassay for the quality control testing of release-active forms of diclofenac.

We report on a specially designed diclofenac-ELISA for the determination of diclofenac in the presence of release-active forms of diclofenac in lactose dissolved in water solutions according to a predefined schedule in single-blind experiments. In accordance with the objective of this project, a number of experiments were conducted to determine the optimal ELISA conditions for detecting potential modulatory effects of release-active forms of diclofenac depending on their ability to affect the binding of diclofenac to anti-diclofenac antibodies. As a feature, the diclofenac antibodies were previously incubated with manufactured pharmaceutical samples containing release-active forms of diclofenac or placebo. For comparison of the sample types, measured in ELISA optical densities were chosen. For statistic analysis, Student's two-sample t-test and single-factor ANOVA were applied. The extremely low concentrations of diclofenac of 0.01, 0.05 and 0.1 ng mL(-1) seem most appropriate for routine assay performance. The source of diclofenac used for standard solution preparation is not important but it could be important as the source of diclofenac for release active form of diclofenac preparation. As an outcome, the ELISA appeared to be suitable for the detection of the modifying effects of release-active forms of diclofenac toward the pharmaceutical substance in vitro.

Probing the physicochemical interactions of 3-hydroxy-benzo[a]pyrene with different monoclonal and recombinant antibodies by use of fluorescence line-narrowing spectroscopy.

Characterization of interactions between antigens and antibodies is of utmost importance both for fundamental understanding of the binding and for development of advanced clinical diagnostics. Here, fluorescence line-narrowing (FLN) spectroscopy was used to study physicochemical interactions between 3-hydroxybenzo[a]pyrene (3OH-BaP, as antigen) and a variety of solvent matrices (as model systems) or anti-polycyclic aromatic hydrocarbon antibodies (anti-PAH). We focused the studies on the specific physicochemical interactions between 3OH-BaP and different, previously obtained, monoclonal and recombinant anti-PAH antibodies. Control experiments performed with non-binding monoclonal antibodies and bovine serum albumin (BSA) indicated that nonspecific interactions did not affect the FLN spectrum of 3OH-BaP. The spectral positions and relative intensities of the bands in the FLN spectra are highly dependent on the molecular environment of the 3OH-BaP. The FLN bands correlate with different vibrational modes of 3OH-BaP which are affected by interactions with the molecular environment (π-π interactions, H-bonding, or van-der-Waals forces). Although the analyte (3OH-BaP) was the same for all the antibodies investigated, different binding interactions could be identified from the FLN spectra on the basis of structural flexibility and conformational multiplicity of the antibodies' paratopes.

Highly sensitive and specific determination of mercury(II) ion in water, food and cosmetic samples with an ELISA based on a novel monoclonal antibody.

Mercury is one of the most toxic heavy metals present in the environment. In this study, a highly sensitive and specific monoclonal antibody (mAb)-based indirect competitive enzyme-linked immunosorbent assay (ELISA) for the determination of Hg(2+) was developed. A new bifunctional ligand, 6-mercaptonicotinic acid (MNA), which contains a pyridine ring bearing a carboxylic group and a mercapto group, was selected for the preparation of immunogen. After immunization of mice and performing the hybridoma technique, the obtained mAb was characterized for its binding affinity and selectivity for Hg(2+). Based on this novel mAb, an ELISA was established. At optimal experimental conditions, the standard curve of the ELISA for Hg(2+) was constructed in concentration range of 0.1-100 ng mL(-1). The values of IC(50) and LOD of the assay were found to be 1.12 and 0.08 ng mL(-1). The cross-reactivity was lower than 2% with MNA, CH(3)Hg, and CH(3)Hg-MNA and was 11.5% and 4.4% for Hg(+) and Au(3+), respectively. No cross-reactivity was found with other metal ions such as Cu(2+), Sn(2+), Ni(2+), Mn(2+), Pb(2+), Zn(2+), Cd(2+), Fe(2+), Co(2+), Mg(2+), Ca(2+), and anions such as Cl(-), NO(3)(-), NO(2)(-), HCO(3)(-), F(-), and SO(4)(2-), indicating that the assay displays not only high sensitivity but also high selectivity. Different kinds of samples including water, milk, green vegetable, kelp, facial cleanser, and night cream were spiked with Hg(2+) and the extracts were analyzed by ELISA. Acceptable recovery rates of 80.0-113.0% and coefficients of variation of 1.9-18.6% were obtained. A linear relationship between ELISA and cold-vapor atomic fluorescence spectroscopy (CV-AFS) as indicated by a correlation coefficient of 0.97 for liquid samples (water samples) and 0.98 for other samples was obtained. The proposed mAb-based ELISA provides a feasible analytical method for highly sensitive and specific, fast, simple, and accurate determination of uncomplexed trace Hg(2+) in environmental and food samples.

Screening and characterization of new monoclonal anti-benzo[a]pyrene antibodies using automated flow-through microarray technology.

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental pollutants, which can cause cancer in humans. The maximum tolerable limit of benzo[a]pyrene (B[a]P) in drinking water was set to 10 ng/L by the European Commission (Council Directive 98/83/EC), because of its highly carcinogenic and mutagenic effect on humans. In the present investigation, mice were immunized with B[a]P-bovine serum albumin conjugates and 110 generated hybridoma cell lines screened by different techniques to identify clones that produce anti-B[a]P antibodies. Subsequently, a new automated flow-through biochip noncompetitive direct chemiluminescence immunoassay (CLEIA) was compared with conventional indirect and direct enzyme-linked immunosorbent assays (ELISAs). It was demonstrated that the microchip-based screening method compared to ELISA was fast and very sensitive with use of only nanoliter volumes of supernatant. Forty clones could be evaluated in less than 5 min. Six high affinity monoclonal antibodies with different cross-reactivities (CR) for individual PAHs were identified by the chip-based assay and indirect microtiter plate ELISA. In comparison, the direct ELISA in the microtiter plate failed to identify three of these clones. The four antibodies with the highest affinity had half maximum inhibitory concentrations (IC(50) values) between 0.31 and 0.92 µg/L for B[a]P. Affinity constants of these four antibodies were determined by surface plasmon resonance using a water soluble B[a]P-peptide. The observed CR pattern of the four monoclonal antibodies for 16 tested PAHs was quite different. Only one specific antibody for B[a]P was observed, while others were more suitable for class-specific PAH determination.