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P Brossier

Publications and source records attributed to P Brossier.

At least 19 recordsLinked to original sources
PubMed2001

Gold nanoparticle-based quantitative electrochemical detection of amplified human cytomegalovirus DNA using disposable microband electrodes.

An electrochemical DNA detection method has been developed for the sensitive quantification of an amplified 406-base pair human cytomegalovirus DNA sequence (HCMV DNA). The assay relies on (i) the hybridization of the single-stranded target HCMV DNA with an oligonucleotide-modified Au nanoparticle probe, (ii) followed by the release of the gold metal atoms anchored on the hybrids by oxidative metal dissolution, and (iii) the indirect determination of the solubilized AuIII ions by anodic stripping voltammetry at a sandwich-type screen-printed microband electrode (SPMBE). Due to the enhancement of the AuIII mass transfer by nonlinear diffusion during the electrodeposition time, the SPMBE allows the sensitive determination of AuIII in a small volume of quiescent solution. The combination of the sensitive AuIII determination at a SPMBE with the large number of AuIII released from each gold nanoparticle probe allows detection of as low as 5 pM amplified HCMV DNA fragment.

Cytomegalovirus↗
PubMed2000

Hybridization assay at a disposable electrochemical biosensor for the attomole detection of amplified human cytomegalovirus DNA.

A disposable electrochemical biosensor for the detection of DNA sequences related to the human cytomegalovirus (HCMV) is described. The sensor relies on the adsorption of an amplified human cytomegalovirus DNA strand onto the sensing surface of a screen-printed carbon electrode, and to its hybridization to a complementary single-stranded biotinylated DNA probe. The extent of hybrids formed was determined with streptavidin conjugated to horseradish peroxidase. The peroxidase label was indirectly quantified by measuring the amount of the chromophore and electroactive product 2,2'-diaminoazobenzene generated from the o-phenylenediamine substrate. The intensity of differential pulse voltammetric peak currents resulting from the reduction of the enzyme-generated product was related to the number of target HCMV-amplified DNA molecules present in the sample, and the results were compared to those obtained with standard methods, i.e., agarose gel electrophoresis quantification and colorimetric hybridization assay in a microtiter plate. A detection limit of 0.6 amol/ml of HCMV-amplified DNA fragment was obtained with the electrochemical DNA biosensor. The electrochemical method was 23,000-fold more sensitive than the gel electrophoresis technique and 83-fold more sensitive than the colorimetric hybridization assay in a microtiter plate.

Biosensing Techniques↗
PubMed1999

Carbonyl metallo immuno assay: a new application for Fourier transform infrared spectroscopy.

We describe here the development of a new, non-isotopic immunological assay termed CMIA (carbonyl metallo immunoassay) that uses metal carbonyl complexes as tracers and Fourier transform infrared spectroscopy (FT-IR) as the detection method. This assay is based on the particular spectral features of these complexes, which show very strong absorption bands in the 1,800-2,200 cm(-1) spectral range where proteins and organic molecules do not absorb. In Section 1, the optimisation of the quantitative detection of these tracers is detailed. In Section 2, the implementation of mono-CMIA is described, including the CMIA assays of three antiepileptic drugs (carbamazepine, phenobarbital, phenytoin). Finally, extension to the simultaneous double- and triple-CMIA of these drugs is reported.

Anticonvulsants↗
PubMed1996

Quantitative analysis of mixtures of metal-carbonyl complexes by Fourier-transform infrared spectroscopy: application to the simultaneous double immunoassay of antiepileptic drugs by the nonisotopic carbonyl metalloimmunoassay method.

The feasibility of a double immunoassay of haptens by the nonisotopic carbonyl metalloimmunoassay (CMIA) method is demonstrated. Three different pairings of antiepileptic medications from the groups carbamazepine, diphenylhydantoin, and phenobarbital (for each of which a mono-CMIA is already available) were assayed by double CMIA. The assay method employs as tracers metal-carbonyl complexes that give very strong signals in the range of 1850-2200 cm-1 in the infrared spectrum, permitting quantitative analysis by Fourier-transform infrared spectroscopy. The fact that the signals are individually assignable and of comparable intensity permits quantitative analysis of mixtures of two tracers. The analysis may proceed in one of two ways: in the simpler case, there is no peak overlap with the two tracers and the quantitative analysis can be performed by simply measuring the absorbance of characteristic peaks of the two tracers. In the second case, in which there is partial or total overlap of peaks, a stepwise calculation provides rapid quantification of the two tracers. These findings allowed us to perform the double CMIA of two antiepileptics in which experimental conditions and time of analysis were strictly identical to those for mono-CMIA. We show here that there is a very good correlation between the results obtained in mono- and double-immunoassay by the CMIA method (correlation coefficient > 0.990).

Animals↗
PubMed1996

[Study of an anti-serum against phenytoin with two organometallic tracers].

In order to develop a new immunoassay procedure based on the use of organometallic labels, an antiserum is raised against antiepileptic drug: diphenyl-hydantoin (phenytoin). Immunization was performed in rabbits with phenytoin coupled to bovine-serum albumin. The evaluation of the specificity and the affinity constants of the antiserum is studied by radioimmunoassay against different antiepileptic drugs and metallotracers (phenytoin labelled with organometallic fragments derived respectively from cobaltocene and ferrocene). The results of this study indicate that metallotracers cross-react slightly with phenytoin (cross reaction percent about 11 for the two compounds). The affinity constants of the antiserum for phenytoin and for metallohaptens derived from cobaltocene and from ferrocene are respectively 0.03 nM-1, 0.05 nM-1 and 0.06 nM-1.

Affinity Labels↗
PubMed1995

Production of specific antibodies and development of a non-isotopic immunoassay for carbamazepine by the carbonyl metallo-immunoassay (CMIA) method.

As part of our ongoing work to extend the range of applications of the non-isotopic carbonyl metalloimmunoassay (CMIA), previously developed in our laboratory, we describe here the first CMIA study of carbamazepine. The CMIA method uses a metal carbonyl complex as a non-isotopic tracer, and in this case we chose to employ the dicobalt hexacarbonyl moiety (Co2(CO)6) attached to an alkyne. Two organometallic tracers, 3 and 7, were synthesized, differentiated by the nature and length of the spacer arm of the Co2(CO)6 moiety. Two different coupling methods were subsequently used to synthesize the immunogens 1 and 2, the first one used a carbodiimide, while the second, employed dimethyl adipimidate as coupling agent. Titer values of the antisera obtained by injection of these immunogens into rabbits, were determined by CMIA, using one of the organometallic complexes, 3 or 7, as tracer. Both antisera had higher titer values with the long-chain tracer, 7, than with the short-chain tracer, 3. However these titer values were very different: low for antiserum 1 and high for antiserum 2. The cross-reactivity of antiserum 2 with other antiepileptic drugs was negligible. For competition curves, there was good sensitivity with the antibody 2/3 pairing, while a broad assay range was obtained with antibody 2/7 pairing. These results demonstrate the viability of CMIA as an immunoassay method for carbamazepine, and open the way to development of a simultaneous multiassay by CMIA of the principal antiepileptic drugs.

Animals↗
PubMed1994

Application of the non-radioisotopic carbonyl metalloimmunoassay (CMIA) to diphenylhydantoin.

As part of our ongoing work to develop the new non-isotopic assay method carbonyl metalloimmunoassay (CMIA), whose efficacy has already been proven in the laboratory for phenobarbital and cortisol, we here present the steps involved in establishing CMIA of 5,5 diphenylhydantoin (DPH), one of the most commonly used antiepileptic medications. First, anti-DPH antibodies were obtained by injection of the immunogen DPH-3-valerate-BSA into rabbits. The titer value and specificity of these antibodies were examined by RIA using [14C]-DPH as tracer, and an antibody batch selected for its high titer value and good specificity for metabolites of DPH and other antiepileptic drugs. Next the organometallic complex Cr(CO)3-DPH, chosen as the CMIA tracer, was synthesized and shown to conserve a high recognition value for anti-DPH antibodies (CR = 200%). Isopropyl ether was selected as the best organic solvent for use in separating the free and bound fractions of the tracer. Employing the Cr(CO)3-DPH complex as tracer and FT-IR spectroscopy as the detection method, we were able to obtain a titration curve by CMIA using an amount of tracer identical to that used in RIA. The titer value obtained in CMIA is approximately twice that obtained by RIA. These results demonstrate the feasibility of DPH assay by the CMIA method.

Animals↗
PubMed1993

Use of Fourier transform infrared spectroscopy for the simultaneous quantitative detection of metal carbonyl tracers suitable for multilabel immunoassays.

We describe here a new approach for multilabel immunoassays. The starting point of this approach is a new nonradioisotopic immunoassay (carbonyl metallo immunoassay) based on the use of metal carbonyl complexes as tracers and Fourier transform infrared (FT-ir) spectroscopy as the detection method. We show here that the judicious choice of the organometallic tracers associated with multicomponent analysis of FT-ir spectroscopic data provides the simultaneous rapid and reliable quantitation of pmol of the organometallic tracers. The feasibility of this approach is demonstrated in the case of the two major antiepileptic drugs phenobarbital and carbamazepine.

Carbamazepine↗
PubMed1993

Atomic absorption spectrometric detection of biotin-cymantrene as a metallo-tracer for the avidin-biotin system.

Biotin-cyclopentadienylmanganese tricarbonyl (biotin-cymantrene; biotin-Cy) is proposed as a universal metallo-tracer for immunoassays. Associated with the possibility of detecting this type of label (organometallic moieties) along with different analytical procedures, this reagent appears to be a universal system with the advantages of being inexpensive, stable and directly detected (no substrate is required). The optimum conditions for detecting the organometallic label biotin-Cy with a Zeeman atomic absorption spectrometer and the affinity of biotin-Cy for the streptavidin ligand immobilized on the walls of microtitre plates, wells or tubes are described. The results obtained demonstrate the possibility of assaying a wide range of clinical parameters because the detection limit of biotin-Cy is about 1 pg per assay. Based on the competition between immobilized bovine serum albumin adsorbed on the wall and free bovine serum albumin for specific polyclonal antibodies, the streptavidin-metallo-biotin system was tested on a competitive immunoassay model and a satisfactory calibration graph was obtained.

Animals↗
PubMed1992

Carbonylmetalloimmunoassay (CMIA) a new type of non-radioisotopic immunoassay. Principles and application to phenobarbital assay.

A new non-radioisotopic immunoassay procedure, which we have termed carbonylmetalloimmunoassay (CMIA), is described. The tracers used in this approach are organometallic carbonyl complexes that can be detected at femtomole levels (300-700 fmol) by Fourier transform infrared (FT-IR) spectroscopy. The validity of the technique has been tested in a phenobarbital assay using as the marker a cyclopentadienylmanganese (I) tricarbonyl (cymantrene) moiety, ethyl acetate extraction to separate the free and bound organometallic fractions, and FT-IR spectroscopy to detect the CO stretching modes of the organometallic label. Typical dilution and standard curves obtained with this CMIA procedure are presented. The method was of comparable sensitivity to a [14C] radioimmunoassay (RIA) for the detection of phenobarbital. A comparison of the results for phenobarbital assays by both CMIA and RIA showed that higher titres were obtained using the CMIA method. The standard curves suggest that CMIA is a reliable and reproducible immunoassay procedure for phenobarbital.

Cross Reactions↗
PubMed1991

Potentiality of an organometallic labelled streptavidin--biotin system in metalloimmunoassay.

Biotin labelled with a cymantrene moiety (cyclopentadienyl manganese tricarbonyl complex) is described for the first time. Because this metallo-biotin retains full recognition for the specific glycoprotein avidin (or streptavidin), the labelled streptavidin-biotin system is proposed for use in a solid-phase competition-type metalloimmunoassay in which bovine serum albumin (BSA) is used as a model of applications. Atomic absorption spectrometry is utilized for the detection of the cymantrene-labelled biotin.

Bacterial Proteins↗
PubMed1991

[An antiserum directed against phenobarbital with an organometallic tracer: production and characterization].

In order to develop a new immunoassay procedure based on the use of organometallic labels, antisera are raising against sedative-hypnotic drug: phenobarbital. Immunization was performed in rabbits with parasuccinylamidophenobarbital coupled to bovine-serum-albumin. The evaluation of the specificity and the affinity constants of antisera are studied against different hypnotic or antiepileptic drugs and the metallotracer (phenobarbital labelled with an organometallic fragment derived from cymantrene). The most important result of this study indicates that the organometallic moiety of the metallotracer did not disturb neither the inhibition (% inhibition = 110) nor the affinity constant (Ka = 2.10(8) L/M) of the phenobarbital.

Animals↗
PubMed1990

A new powerful association for immunoassay: organometallic label conjugated to solvent separation method.

The convenient use of solvents as a method to separate free (F) and bound (B) fractions in drug immunoassays which utilize organometallic moieties as labels, has been developed. Two types of drugs labelled with an organometallic complex and denominated metallohaptens (or metallotracers) are studied as model assays: desipramine (tricyclic antidepressant) and phenobarbital (antiepileptic agent). Different organic solvents have potentialities to extract these metallohaptens. To illustrate this solvent procedure we describe antiserum dilution curves and we compare the results with those obtained for the same samples using radiotracers in conjunction with dextran coated charcoal as separation agent. This immunoassay method which associates a metallotracer and B/F solvent separation appears to be fast, cheap, without the step of centrifugation.

Desipramine↗
PubMed1989

Infrared immunoassay.

An immunoassay method based on the labelling of an antigen with a transition-metal carbonyl organometallic marker and detection of the label by Fourier transform infrared (FT-IR) spectroscopy is described. The viability of this novel non-isotopic approach, termed infrared immunoassay (IRIA), has been evaluated in the quantitative determination of the tricyclic antidepressant nortriptyline.

Animals↗
PubMed1986

Metalloimmunoassay of tricyclic antidepressant drugs: production and characterization of antiserum.

Metalloimmunoassay is a new immunoassay using an organometallic complex as the label. For the development of this assay, antibodies were produced in rabbits immunized with N-succinyldesipramine-bovine serum albumin conjugate. Antisera thus obtained were shown to be specific for the tricyclic antidepressant group of drugs and are tested as regard as desipramine or nortriptyline labelled by an organometallic moiety (designated metallohaptens). The evaluation of the affinity constants shows that antisera cross-react almost equally well with both imipramine labelled with tritium (radiotracer) and metallohaptens using either ferrocene or cymantrene as labels (metallotracers). Detection of metallohaptens is performed by a ZEEMAN atomic absorption spectrophotometer (ZAAS).

Animals↗