Nathalie Fischer-Durand

Characteristics of immunosorbents used as a new approach to selective solid-phase extraction in environmental analysis

SummaryThe trace-level determination of organic pollutants in complex matrices is difficult and often not reliable because theccurrent extraction procedures are non-selective. New extraction sorbents involving antigen-antibody interactions, called immunosorbents (ISs), have been synthesised in order to trap a group of structurally related pollutants. The IS capacity is always high for the analyte-antigen used to make the antibodies, but can be low for some related compounds. In this work, we show the relationship that exists between capacity, break-through volume and recovery of analytes because of the competition between the structurally related compounds for antibody sites. Breakthrough due to the overloading of the column should be avoided because calibration curves are no longer linear. The capacity of two ISs, one made for trapping the triazine pesticide group and the second for the phenylurea, group, have been optimised by selecting silica with 50 nm pore size. Calibration curves are linear for all the compounds in a mixture of ten phenylureas up to a concentration of 5 to 10 μg L−1 for each compound when handling 50 mL water samples through a precolumn packed with 0.22 g of IS. Under these conditions, reliable quantitative results are obtained because calibration curves are similar when compounds are alone or in a mixture. Application to the clean-up of soil extracts illustrates the high selectivity and the high potential of these new sorbents in environmental analysis.

Synthesis of metal-carbonyl-dendrimer-antibody immunoconjugates: Towards a new format for carbonyl metallo immunoassay

We report the preparation of metal‐carbonyl–dendrimer–antibody conjugates. These metal‐carbonyl‐multilabeled antibodies are designed to be used in a new solid‐phase‐format carbonyl metallo immunoassay (CMIA). A fourth‐generation polyamidoamine dendrimer was labeled with 10–25 (η5‐cyclopentadienyl)iron dicarbonyl (η1‐N‐succinimidyl) entities. An antibody was chemically modified at its carbohydrate chains by a site‐directed process used to preserve the antigen–antibody binding site. The antibody was then coupled with the dendrimer labeled with 10 metal carbonyl groups. An average of 1.4 labeled dendrimers were grafted per antibody molecule. These metal‐carbonyl–dendrimer–antibody conjugates were used as new universal detection reagents that recognize their specific antigens. The antigens were spotted onto nitrocellulose membranes and detected by using the conjugates in combination with Fourier transform infrared spectroscopy. A detection level in the range 5–200 pmol per membrane was achieved. This approach opens the way to a new CMIA format.

Site-specific conjugation of metal carbonyl dendrimer to antibody and its use as detection reagent in immunoassay.

We describe here the conjugation of polyclonal goat anti-rabbit antibody to generation 4 polyamidoamine (G4-PAMAM) dendrimers carrying (i) (η(5)-cyclopentadienyl) iron dicarbonyl succinimidato complexes as infrared (IR) probes, (ii) nitroaniline entities as nuclear magnetic resonance (NMR) probes, (iii) acetamide groups for surface neutralization, and (iv) hydrazide-terminated spacer arms for the reaction with aldehyde. To preserve a high binding affinity, the conjugation was performed on the carbohydrate moieties located on the Fc fragment. The resulting conjugates were characterized by Fourier transform-IR, ultraviolet (UV), and high-mass matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. On the basis of relative concentration ratios of IR probes and antibody, an average labeling of 30 IR probes per antibody was reached (i.e., more than twice the value obtained with our previous strategy that generated no spacer arm). Immunoassays revealed that the antibody-dendrimer conjugates retained 55.1% of immunoreactivity on average with respect to underivatized antibody. Finally, the conjugates were used to quantify their antigen by solid-phase carbonyl metallo immunoassay (CMIA). Results showed a significant enhancement of the IR signal, demonstrating the efficiency of the new conjugation strategy and the potential of the new antibody-dendrimer conjugates as universal immunoanalytical reagents.

Development and Validation of an Indirect Enzyme Immunoassay for the Detection of the Herbicide Isoproturon in Water Matrices

Abstract An indirect enzyme immunoassay (EIA) for the detection of the phenylurea herbicide isoproturon is described. The specific antibodies did not cross-react with other structurally related compounds. The concentration of isoproturon that inhibits 50% of antibody-antigen binding (IC50) was 0.64 ng/mL. The sensitivities were 0.07 ng/mL (IC80) and 0.02 ng/mL (IC90) respectively, when the crude serum was used in the assay. Matrix effects were observed when river water samples were analyzed showing recoveries as high as 150%. The IC50 was increased to 0.81 ng/mL. To overcome these difficulties, a novel method of anatibody purification was developed to reduce the heterogeneity of the medium when the test was performed with complex surface water matrices. This technique involved the extraction of the specific anti-isoproturon antibodies from the crude anti-serum. The refined fraction gave an IC50 not higher than 0.29 ng/mL and an IC90 of 0.01 ng/mL, when assayed with river water samples. The method was vali...

Atrazine-based self-assembled monolayers and their interaction with anti-atrazine antibody: building of an immunosensor.

As a part of our objective to build an immunosensor for the detection of the pesticide atrazine (ATZ) in environmental samples, we studied the self-assembling process of the disulfide derivative of the pesticide atrazine on a gold substrate. Atrazine-based self-assembled monolayers were characterized by ellipsometry, scanning tunneling microscopy, polarization-modulation infrared reflection-absorption spectroscopy (PM IRRAS), X-ray photoelectron spectroscopy and quartz crystal microbalance (QCM) measurements. Two different time constants for the adsorption process were observed, depending on the experimental method used. The QCM data reflect adsorption kinetics of the original disulfide compound, whereas ellipsometry and ex situ PM IRRAS refer to the formation of thiolate (ATZS) monolayers. In situ QCM data demonstrated the suitability of such monolayers for the detection of atrazine in aqueous samples. Exposure of the ATZS sensing surface to an anti-atrazine antibody (anti-ATZ IgG) resulted in complete coverage of the surface by antibody, whereas approximately half of the antibody molecules were displaced from the QCM sensor surface by further addition of atrazine into the solution.

Purified polyclonal anti‐phenylurea antibodies for an improved immunoaffinity chromatography

A one‐step technique for the purification of isoproturon antibody has been developed to overcome the common elution problems encountered when high‐affinity polyclonal antibodies are used in immunoaffinity columns. Because of the non‐uniform affinity, desorption of the analyte can be problematic. Two herbicide derivatives were coupled to Sepharose CL solid support using a flexible linker, β‐lactoblobulin. Using a structurally‐related chlortoluron ligand showed a lower affinity to the target antibody rather than the isoproturon derivative ligand and resulted in an improved capacity of the affinity chromatography gel. The recovery of the specifically adsorbed antibodies was achieved using a carboxylic derivative of the analyte isoproturon in a water‐ethylene glycol buffer (70/30). The purification efficiences of this procedure were characterized by dot blot, fast protein liquid chromatography and ELISA. The chlortoluron‐β‐lactoglobulin purified IgG showed a six times higher titer than the pure isoproturon‐sp...

Development of a Novel Dry Process to Functionalize Membranes for the Covalent Attachment of Antibodies Used in Immunochemical – Based Environmental Strip Tests

Abstract The controlled attachment of antibodies is a prime requirement for developing membrane-based immunoassays used in field tests. A novel dry process was developed to functionalize a nitrocellulose membrane by introducing amino groups on the surface, allowing an oriented covalent linkage of antibodies through their oxidized carbohydrate moieties. A non-equilibrium low pressure plasma of NH3 and NH3/H2 mixtures was used to incorporate an average of 2.4 amine functions per nm2 of porous surface. A mechanism of the functionalization process was proposed using on-line emission spectroscopy and mass spectrometry analysis to monitor the reaction process. The modification of the physical properties and the chemical composition of the treated material was also investigated by X-ray Spectrophotometric (XPS) analysis and scanning electron microscopy. The immobilization of radiolabelled antibodies resulted in an average binding capacity of 60 μg/cm2. Their activity was retained as shown by an enzyme linked imm...