Immunochemical methods for biomonitoring of chlorophenols as potential biomarkers of exposure

The thesis presents the development of immunochemical methods for detection of trichlorophenols (TCP) in environmental and biological samples. An indirect enzyme linked immunosorbent assay (ELISA) for 2,4,5-TCP has been developed after a rational design of the immunizing hapten chemical structure, and the screening of 12 competitor haptens. The effect of the conjugation degree of the competitors and their homology with the target analyte, the physicochemical parameters (pH, ionic strength), the concentration of detergent, the time of incubation and the specificity were studied. Two immunoassays (2,4,5- and 2,4,6-TCP ELISAs) were evaluated for the analysis of water, milk, serum and urine. Drinking water was analyzed directly after buffering the sample. The strong matrix effects in milk samples requires the sample clean up. Human serum can be analyzed after protein precipitation with absolute ethanol. The strong matrix effect of urine and its variability for samples from different individuals suggested the introduction of a purification step prior to ELISA. The C18- solid phase extraction (SPE) is an effective clean up method to remove an important part of the nonspecific interferences present in urine. The C18-SPE-ELISA method allows accurate quantification of TCPs in urine of occupationally exposed persons. SPE based on immunosorbents (immunoaffinity extraction, IAE) have been developed in single and 96-column formats. IAE is an effective clean up method to remove all nonspecific urinary interferences. The IAE step was optimized regarding sample volume, loading level, type of urine hydrolysis washing and elution conditions. The selectivity of the immunosorbents can be modulated by the washing conditions. The immunosorbents have sufficient capacity to effectively extract 2,4,6-TCP from urine samples of occupationally exposed persons and the general population. The HTS-IAE-ELISA method allows the processing of 100 samples/day with very good precision and accuracy. The method was validated with GC-MS and applied to the biomonitoring of three groups of population from Catalonia.A quenching fluorescence immunoassay based on the laser-induced fluorescence detection in microdroplets (LIF-microdroplet-QFIA) for 2,4,6-TCP has been developed as a novel biodetection system. This approach offers significant improvement in method detectability compared to the microplate immunoassays and is the first application urine samples that can be directly analyzed after sample dilution

Biomonitoring Human Exposure to Organohalogenated Substances by Measuring Urinary Chlorophenols Using a High-Throughput Screening (HTS) Immunochemical Method

9 pages, 5 tables, 3 figures.The widespread contamination of the environment by persistent organochlorinated substances is well-known. High-throughput immunochemical methods may improve routine assessment of the exposure of the population to these chemicals by analyzing urinary biomarkers. Trichlorophenols (TCP) have often been considered as biomarkers of many organochlorinated compounds. With the aim to assess exposure of the population to these substances a high-throughput immunosorbent solid-phase extraction (HTS-IS-SPE) procedure coupled to ELISA for simultaneous analyses of 2,4,6-TCP immunoreactivity equivalents (2,4,6-TCP-IR equiv) in multiple hydrolyzed urine samples has been developed. Around 100 urine samples can be processed simultaneously with an inter- and intra-assay precision lower than 23% CV and a limit of detection of 0.3 μg L-1.The analyses by HTS-IS-SPE−ELISA and HTS-IS-SPE−GC/MS of urine samples (N = 117) collected from three different population groups point to a broad exposure of the Catalonian population to organohalogenated substances including the recently emerging organobrominated pollutants. Environment and edible products seem to be the most likely sources of exposure, since excretion of 2,4,6-TCP-IR equiv has been found to be independent from the occupational sector. An excellent correlation was observed between the 2,4,6-TCP-IR equiv determined by HTS-IS-SPE−ELISA and the concentrations measured by HTS-IS-SPE−GC/MS (R2 = 0.912). The results show that immunochemical screening methods, based on the quantification of urinary biomarkers, can be excellent tools for exposure assessment. The HTS-IS-SPE−ELISA presented here has proved to be efficient, precise, accurate, rapid, and specific, which opens up the possibility for a broad variety of applications where routine testing of large number of samples is required.This work has been supported by MCyT (AGL 2002-04653-C04-03 and AGL2004-20341-E) and by EC (NM2-CT-2003-505485). M.N. thanks the Spanish Ministry of Education for her fellowship of the FPU program. We gratefully acknowledge Xavier Guardino, Jordi Obiols, and Enric Gadea (Instituto de Seguridad y Higiene en el Trabajo, Ministerio de Trabajo y Asuntos Sociales) as well as Angels Valbuena and Ana Oliete (Centre de Seguretat i Condicions de Salut en el Treball, Departament de Treball, Generalitat de Catalunya) for the helpful discussions and for kindly providing us with urine samples used on the evaluation/validation studies. The AMR group is a Grup de Recerca de la Generalitat de Catalunya and has support from the Departament d'Universitats, Recerca i Societat de la Informació de la Generalitat de Catalunya (expedient 2005SGR 00207).Peer reviewe

Competitive quenching fluorescence immunoassay for chlorophenols based on laser-induced fluorescence detection in microdroplets

8 pages, 7 figures, 1 table.-- PMID: 12530822 [PubMed].-- Available online Nov 2, 2002.An improved biomonitoring system for the analysis of 2,4,6-trichlorophenol (TCP) in urine samples has been developed. The principle of the biosensor device is the detection of laser-induced fluorescence (LIF) in single microdroplets by a homogeneous quenching fluorescence immunoassay (QFIA). The competitive immunoassay occurs in microdroplets (d = 58,4 μm) produced by a piezoelectric generator system with 10-μm-diameter orifice. A continuous Ar ion laser (488 nm) excites the fluorescent tracer; its fluorescence is detected by a spectrometer attached to a 512 × 512 cooled, charge-coupled device camera. Fluorescence is quenched by specific binding of TCP polyclonal antibodies to the fluorescent tracer (hapten A−fluorescein); the quenching effect is diminished by the presence of the analyte. Thus, an increase in the signal is produced in a positive dose-dependent manner when TCP is present in the sample. In 10 mM PBS buffer, the IC50 of the LIF-microdroplet QFIA is 0.45 μg L-1 reaching a LOD of 0.04 μg L-1. The QFIA with the same reagents performed in microtiter plate format achieved a LOD of 0.36 μg L-1 in buffer solution. Performance in human urine was similar to that observed in the buffer. A LOD of 1.6 μg L-1, with a dynamic range between 4 and 149.5 μg L-1 in urine, was obtained without any sample treatment other than dilution with the assay buffer. The detectability achieved is sufficient for occupational exposure risk assessment.This research was supported by the Superfund Basic Research Program from the National Institute of Environmental Health Sciences (Grant 5P42ES04699, NIH with funding provided by EPA), by the EC Program (Contract QLRT-2000-01670), and by the Spanish Government through CICYT (BIO2000-0351-P4-05). UCD is a NIEHS Environmental Health Center P30 ESO5707.Peer reviewe

Synthesis and characterization of multifunctional silica core-shell nanocomposites with magnetic and fluorescent functionalities.

Multifunctional core-shell nanocomposites with a magnetic core and a silica shell doped with lanthanide chelate have been prepared by a simple method. First, citric acid-modified magnetite nanoparticles were synthesized by a chemical coprecipitation method. Then the magnetite nanoparticles were coated with silica shells doped with terbium (Tb(3+)) complex by a modified Stöber method based on hydrolyzing and condensation of tetraethyl orthosilicate (TEOS) and a silane precursor. These multifunctional nanocomposites are potentially useful in a variety of biological areas such as bio-imaging, bio-labeling and bioassays because they can be simultaneously manipulated with an external magnetic field and exhibit unique phosphorescence properties