[Contribution of simultaneous multiple immunoassays to biology]. / Apport des immunoanalyses multiple simultanées á la biologie.

During the last years, research in the area of immunoanalysis has been oriented towards the marketing of more reliable, faster and low cost kits. In this field, simultaneous multiple immunoanalysis that offer long-time ignored original and specific advantages, has undergone tremendous improvements triggered by striking technological innovations and the advent of lanthanide chelate based markers. Simultaneous multiple immunoassays have turned from semi-quantitative methods restricted to pharmacology to quantitative methods for medical biology. We chose first to describe several commercial systems among the most representative then we stressed on the main emerging systems based on optical or electrochemical detection as well as some immunosensor devices. The array of simultaneous multiple immunoassays currently available appears attractive enough so as to take a significant area within the analytical arsenal at the biologist disposal.

Reaction of hen egg white lysozyme with Fischer-type metallocarbene complexes. Characterization of the conjugates and determination of the metal complex binding sites.

The introduction of heavy atoms into protein crystals is sometimes rendered difficult and tedious because of the poor specificity of the available reagents for particular target residues. On the other hand, transition organometallic chemistry offers an almost untouched field for this purpose. In particular, Fischer-type metallocarbene complexes of the general formula (CO)5W=C(OR1)R2 may be attractive reagents because they contain the heavy element tungsten and specifically target amino groups to form stable, covalent aminocarbene adducts. With a small protein such as hen egg white lysozyme (HEWL) with a limited number of potential binding sites, it was possible to form protein-aminocarbene conjugates that have an average of one aminocarbene moiety per protein molecule. RP-HPLC combined with matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) MS analysis of the conjugates revealed that they were mixtures of the native protein, monoaminocarbenes and diaminocarbenes. Tryptic proteolysis experiments performed on the protein conjugates combined with MALDI-TOF-MS analysis of the aminocarbenic peptides allowed us to determine that lysines 13, 33, 97 and 116 were involved in the reaction of HEWL with (CO)5W=C(OMe)Me.

New and efficient routes to biomolecules substituted with cyclopentadienyltricarbonylrhenium and -technetium derivatives.

The small, compact, robust, and nonpolar units of [CpM(CO)3] (M = Re, Tc) coupled with biomolecules may be considered as bioorganometallic entities of potential interest in the field of medicinal chemistry. However, the short half-life of useful radionuclides (186Re t1/2 = 3.7 d, 188Re t1/2 = 16.8 h, 99mTc t1/2 = 6 h), the risks inherent in their use, and their cost have led chemists to search for novel synthetic strategies that allow the rapid introduction of the [CpM(CO)3] moiety as a late step in the course of synthesizing the target molecule. The present paper describes different strategies recently reported in the literature to tackle this problem.

Labelling and binding of poly-(L-lysine) to functionalised gold surfaces. Combined FT-IRRAS and XPS characterisation.

We compare herein the interfacial reactivity of self-assembled monolayers (SAMs) of 11-mercaptoundecanoic acid (MUA), 1-undecanethiol (UDT) and 11-mercaptoundecanol (MUD) on gold surfaces towards aqueous solutions of poly-(L-lysine) (PL). Liquid-phase labelling of PL with the alkyne dicobalt hexacarbonyl cluster 1 combined with analysis of the substrates by Fourier transform infrared reflection-absorption spectroscopy (FT-IRRAS) and X-ray photoelectron spectroscopy (XPS) revealed that irreversible binding of PL occurred in all cases. However, the mechanism of binding involved differed markedly from one monolayer to the other. The main mode of interaction of PL to MUA SAM was of electrostatic nature between the terminal carboxylate of MUA and the ammonium groups of PL. For a similar number of bound thiolate molecules, the UDT adsorbed layer was found less continuous than the MUA one, allowing a higher fraction of PL to directly bind to the gold surface. As for MUD, very little thiolate molecules were adsorbed, leaving bare gold surface areas for non specific adsorption of PL.

Use of heavy-metal clusters in the design of N-succinimidyl ester acylation reagents for side-chain-specific labeling of proteins.

New heavy transition metal carbonyl markers for protein labeling, containing an "Mn(CO)11" (M = Ru, Os, n = 3; M = Ir, n = 4) moiety, were prepared by reaction of "lightly stabilized" clusters with an N-succinimidyl ester functionalized phosphine, namely N-succinimidyl 3-diphenylphosphine-propionate (DPPS). The reaction of Os3(CO)11(DPPS) with the model amino acid beta-alanine was performed and led to the expected amide. From the reaction of Mn(CO)11(DPPS) with bovine serum albumin (BSA) in mixed organic/aqueous medium, conjugates bearing a fairly high number of metal carbonyl fragments were obtained, thus demonstrating the usefulness of this class of reagents for the selective and covalent graft of heavy metal clusters to side chain of proteins.

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.

Side-chain selective and covalent labelling of proteins by organometallic complexes of heavy transition metals--possible application in radio-crystallography of proteins.

Organo-rhenium and organo-tungsten N-succinimidyl and N-sulfosuccinimidyl esters react at amino groups of proteins to form stable amide bonds between the protein and the heavy transition metal complexes. We show here that factors such as pH of the reaction medium and nature of the reagent (coordinating metal, surrounding ligands, type of ester) had a marked influence on the final number of coupled organometallic groups per protein molecule, defined as the coupling ratio. By operating with stoichiometric quantities of reagent with respect to the number of aqino groups, up to 30 organometallic groups were introduced into the model protein, bovine serum albumin (BSA). BSA conjugates were characterized by gel electrophoresis in non-denaturating conditions. Hen egg-white lysozyme organo-tungsten conjugates were prepared in the same manner and the peptides resulting from their tryptic hydrolysis were analyzed by reverse-phase HPLC. A tentative assignment of the preferential amino sites of conjugation is suggested from the latter results.

Cyclopentadienyl iron dicarbonyl (eta 1-N-phthalimidato) complexes containing an isothiocyanate function: synthesis and application to protein side-chain selective labeling.

The two first transition metal carbonyl isothiocyanates were prepared in high yield within two steps from photolysis of CpFe(CO)2I and 3- or 4-aminophthalimide in the presence of diisopropylamine followed by reaction with thiophosgene/triethylamine. Their reaction with a model amino acid, i.e. beta-alanine, was performed and led to the expected thioureas. When reacted with bovine serum albumin in aqueous medium, conjugates bearing 6-10 iron-carbonyl fragments were obtained and characterized by Fourier transform infrared spectroscopy, thus demonstrating the usefulness of these reagents for the selective and covalent labeling of proteins.

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).

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.

[eta 5-Cyclopentadienyl]metal tricarbonyl pyrylium salts: novel reagents for the specific conjugation of proteins with transition organometallic labels.

New specific reagents for the conjugation of organo transition metal species to proteins are described. These reagents are pyrylium salts bearing a (eta 5-C5H4)M(CO)3 (M = Mn and Re) at position 4. They couple with simple amines (n-butylamine and tert-butylamine) and to lysine side chains of proteins (bovine serum albumin and lysozyme) with varying yields. In almost all cases, the final conjugated species is a pyridinium salt, with the exception of lysozyme, for which the reaction ends at the divinylogous amide form. Differences in reactivity for bovine serum albumin and lysozyme can be explained in terms of differences of isoelectric point and steric local environment around the reactive lysine residue.

Labeling of proteins by organometallic complexes of rhenium. (I). Synthesis and biological activity of the conjugates.

We describe herein a totally new pathway for the introduction of rhenium in the form of low oxidation state organometallic complexes covalently attached to various proteins. The synthesis of several rhenium conjugates takes advantage of the specificity of N-succinimidyl esters for amino residues. Conjugation experiments were carried out under various conditions, and analysis of the conjugates was performed by FT-IR spectroscopy. Yields were optimized and reached 50%. Furthermore, the conjugate resulting from the coupling of N-succinimidyl 4-[eta 5-cyclopentadienylrhenium tricarbonyl] 4-oxobutanoate to an anti-hTSH monoclonal antibody retained a satisfactory immunoreactivity. Finally, IR detection of conjugates adsorbed onto nitrocellulose membranes was achieved and response was found to be related to the coupling extent of the conjugate.

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.

Transition metal carbonyl labeling of proteins. A novel approach to a solid-phase two-site immunoassay using Fourier transform infrared spectroscopy.

Labeling of bovine serum albumin (BSA) and anti-human thyroid stimulating hormone (hTSH) monoclonal antibodies (mAbs) was performed using (N-succinimidyl 4-pentynoate)hexacarbonyldicobalt (NSCo2(CO)6). Conditions of coupling were different depending on the protein to be labeled, denaturation of the mAbs occuring with high percentages of organic solvent in the reaction mixture. The influence of reaction time and initial concentration of NSCo2(CO)6 was examined. They were both shown to affect the final coupling rate of the metal carbonyl probe. Preservation of the immunoreactivity toward 125I-hTSH was observed for five conjugates having different NSCo2(CO)6: mAb molar ratios when compared to unmodified and peroxidase-labeled mAbs. Finally, a preliminary study of the quantitative detection of the metal carbonyl mAbs on microtiter wells was achieved using Fourier transform infrared spectroscopy.

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.

Fourier transform infrared spectroscopic method for the quantitative trace analysis of transition-metal carbonyl-labeled bioligands.

A quantitative FT-IR spectroscopic method has been developed for the trace analysis in chlorinated organic solvents of transition-metal carbonyl-labeled bioligands. In order to illustrate the widespread analytical potential of the method, three derivatives of the female hormonal steroid 17 beta-estradiol, containing Cr(CO)3, Cp2Mo2(CO)4 (Cp = eta 5-C5H5), and Co2(CO)6 as labels, and the anticonvulsant drug phenobarbital, labeled with (eta 5-C5H4)Mn(CO)3, were examined. The cobalt carbonyl marker proved to be the best sulted for quantitative analysis purposes, and the minimum tracer quantity detectable for this particular marker (64 scans, 4-cm-1 resolution, 3.5 min) was optimized in CCl4 solution at about 300 fmol (or 0.3 pmol, 180 pg) by using an ultralow volume (23.0 microL), gold light-pipe IR solution cell and a liquid nitrogen cooled, InSb (indium antimonide) IR detector. The repeatability of this radically different analytical procedure over the concentration range 1.0 x 10(-6) to 5.0 x 10(-8) M was good (coefficient of variance less than or equal to 6%) and the method provides the basis for a new immunological test--carbonylmetalloimmunoassay (CMIA).

(N-succinimidyl 4-pentynoate)hexacarbonyldicobalt: a transition-metal carbonyl complex having similar uses to the Bolton-Hunter reagent.

The synthesis of the transition-metal carbonyl complex (N-succinimidyl 4-pentynoate)hexacarbonyldicobalt [[(C4H4O2N)O(CO)CH2CH2C identical to CH]Co2(CO)6] is described. This cobalt carbonyl complex is structurally similar to the Bolton-Hunter conjugation reagent and has been successfully employed as a nonradioactive tracer for labeling the drug carbamazepine. The metal carbonyl tracer can be detected at extremely low concentrations (ca. 1 pmol) by FT-IR spectroscopy in the v(CO) region (2150-1800 cm-1). The cobalt carbonyl labeled carbamazepine retains good recognition for anti-carbamazepine antibodies. This novel labeling procedure, which can be broadly termed carbonylmetalloimmunoassay (CMIA), has considerable potential for assaying a wide range of biological materials.

Organometallic derivatives of estradiol as bioligands: targetted binding of the estradiol receptor.

The complexation of estrogens by transitional metal units e.g. (alkyne)Co2(CO)6 and (alkyne)Mo2Cp2(CO)4, at the 17 alpha-position brings about a dramatic change in the chemical behavior of these compounds with respect to that of the free ligands. The 17 beta-OH function becomes particularly labile, even in weakly acidic medium, giving rise to carbenium ion-like species, from which, depending on the metal and the nucleophile, substitution, elimination and rearrangement take place. This situation provides the basis for a new type of active site directed-reagent for estradiol receptor. The hypothesis of vicinal space positioning of an acidic and a nucleophilic group in the estradiol receptor cavity is examined in the light of the amino-acid composition of the steroid binding domain. The requirement of the sulfhydryl group of a cysteine residue is suspected in the first step of the receptor inactivation process.