Pierre Brossier

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.

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.

A new application of bioorganometallics: the first simultaneous triple assay by the carbonylmetalloimmunoassay (CMIA) method

The first example of simultaneous triple carbonylmetalloimmunoassay (CMIA), a non-isotopic immunological method, is presented herein. This method, originally developed for mono-assays, uses various transition metal carbonyl complexes as labels and sensitive quantitation of these tracers by Fourier transform infrared (FT-IR) spectroscopy, thanks to the characteristic νCO bands appearing in the 1800–2200 cm−1 region. We show here that this method offers an advance in the challenging field of simultaneous multi-immunoassay, with the example of the assay of three antiepileptic drugs (carbamazepine, phenobarbital and diphenylhydantoin).

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.

Redox cationic or procationic labeled drugs detected at a perfluorosulfonated ionomer film-coated electrode

With the aim of developing a new type of immunoassay with electrochemical detection, a Nafion® film-modified electrode was used as an amplifying sensor to detect redox cationic labeled drugs by square-wave voltammetry. A series of amino drugs was labeled with three model redox labels, i.e. cationic cobaltocenium and procationic ferrocene and nitroxide groups. Several parameters were involved in the optimization of the analytical properties of the sensor, i.e. film processing and film thickness, accumulation time, ionic strength and ethanol content. The preconcentration of the labeled drugs was impeded by the competitive accumulation of hydrophobic amines. The accumulation process of the procationic labeled drugs was influenced significantly by the potential applied during accumulation, and their strong non-specific binding with proteins was observed in the presence of serum. Therefore the choice of cobaltocenium as a label was preferred. Optimized conditions were defined, which took into account these various aspects. The avidin-biotin system was proposed as an applicable extension of this approach.

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.

Comparison of two means of attachment of an organometallic acid on gold surfaces by combining X-ray photoelectron spectroscopy and IR reflection spectroscopy

Two methods of immobilization of the butyric 4-oxocyclopentadienylmanganese tricarbonyl acid I on gold surface are presented: either through the assembly of a disulfide derived from the acid, or by coupling on a cystamine monolayer; the efficiency of these two ways are evaluated by IR reflection spectroscopy and X-ray photoelectron spectroscopy.

New immunoassay techniques using Nafion-modified electrodes and cationic redox labels or enzyme labels

Abstract Two new competitive immunoassay techniques with sensitive electrochemical detection at Nafion film modified electrodes are described and compared. Method A implies the use of an antigen labeled with a cationic redox group in homogeneous immunoassay. Method B involves an enzyme-labeled antigen, in connection with an anionic substrate + cationic electroactive product of the enzymatic reaction in heterogeneous assay. A 5 min accumulation step precedes the electrochemical detection in both methods. Method B can be applied to the determination of any kind of antigen, whereas method A is restricted to haptens. Phenytoin, an anti-epileptic drug, was chosen as a model antigen and labeled by cobaltocenium and alkaline phosphatase to illustrate methods A and B, respectively. The enzymatic reaction was performed with N-ferrocenoyl)-6-amino-2,4-dimethylphosphate as substrate in method B. The dianionic substrate was repulsed from the Nafion film, whereas the corresponding enzymatically generated alcohol was selectively entrapped within the film as a ferricinium salt by applying a potential of 0.6 V vs. solAg AgCl during the accumulation step performed at pH 7.4. Both methods were tested for the determination of phenytoin in clinical serum samples.

Covalent immobilization of oligonucleotides on p-aminophenyl-modified carbon screen-printed electrodes for viral DNA sensing

DNA-sensing platforms were prepared by covalently attaching oligonucleotide capture probes onto p-aminophenyl-functionalized carbon surfaces and applied to the determination of an amplified herpes virus DNA sequence in an electrochemical hybridization assay.

Quantitative IR Analysis in the Picomole Range of Organometallic-Labeled Biomolecules in KBr Disks

Quantitative IR studies of materials dispersed in KBr disks are often unreliable because of variable sample homogeneity due to incomplete mechanical mixing. The inherent difficulties in this type of measurement are exacerbated when one is examining the extremely small amounts of material encountered with biological samples. We report here a simple, quantitative KBr-disk procedure for the IR detection of biologically important molecules at physiological levels (picomoles). This analytical technique is based on ones labeling the biomolecules with transition metal carbonyl fragments, such as Cr(CO)3, Mn(CO)3, and Co2(CO)6, and then taking advantage of the rapid multi-scanning capability of an FTIR spectrometer to detect the CO stretching vibrations of the organometallic labels in BKr disks of the solids from the subsequent biochemical precipitations. Metal carbonyl complexes containing terminal CO groups were selected as the IR markers because the v(CO) modes exhibit intense absorptions in the 2150-1900 cm−1 range, a region which is normally devoid of peaks associated with the vibrational modes of biomolecules, such as v(OH), v(NH), v(CH), v(C=O), v(C=C), and v(C-C). This non-radioisotopic procedure has already been successfully applied in estradiol receptor and immunoassay.