| PATENT ASSIGNEE'S COUNTRY | Belgium. |
| UPDATE | 09.00 |
| PATENT NUMBER | This data is not available for free |
| PATENT GRANT DATE | 26.09.00 |
| PATENT TITLE |
System for qualitatively and/or quantitatively analyzing preferably biological substances using enhanced chemiluminescence, and method and analysis kit using same |
| PATENT ABSTRACT |
The field of the present invention is that of identification and analysis of chemical and/or biological species of the enzyme/substrate, enzyme/inhibitor or antigen/antibody etc. type. The problem on which the invention is based is to provide a system for qualitative and/or quantitative analysis of biological substances by amplified chemiluminescence which allows an actual significant improvement in the emission of light resulting from passage of a chemiluminescent reagent to the excited state. This problem has been solved by means of a system according to the invention, which involves a ligand a) which can be coupled with the substances to be analysed, a chemiluminescent reagent b) of the luminol type, an enzyme c), a substrate d) which oxidizes the enzyme c), and at least one amplifier e), this system being characterized in that the amplifier e) is chosen from the family of halogenophenol (iodophenol) esters. The invention also relates to an analytical method using this system and to an analytical kit comprising the said system. |
| PATENT INVENTORS | This data is not available for free |
| PATENT ASSIGNEE | This data is not available for free |
| PATENT FILE DATE | 09.05.98 |
| PATENT CT FILE DATE | 18.10.96 |
| PATENT CT NUMBER | This data is not available for free |
| PATENT CT PUB NUMBER | This data is not available for free |
| PATENT CT PUB DATE | 01.05.97 |
| PATENT FOREIGN APPLICATION PRIORITY DATA | This data is not available for free |
| PATENT REFERENCES CITED |
Blum et al, NATO ASI Ser., Ser. 2 38 (Biosensors for Direct Monitoring of Environmental Pollutants in Field), 271-280 (1997). Analytical Letter, vol. 27, No. 6, Jan. 1, 1994 pp. 1189-1122, XP 000568630, Hori et al, "A New . . . Luminometer". |
| PATENT CLAIMS |
What is claimed is: 1. A composition for qualitative and/or quantitative analysis of biological substances by amplified chemiluminescence comprising a) at least one ligand capable of coupling to the biological substance, b) at least one cyclic diacylated dihydrazide fused to an aromatic chemiluminescence reagent, c) an oxido reduction luminescence enzyme, d) at least one oxidizing agent specific to enzyme c) and e) at least one amplifier of luminescence of the formula ##STR13## wherein R.sup.0 is selected from the group consisting of ##STR14## and polyorganosiloxane, R.sup.1 is selected from the group consisting of alkyl of 1 to 10 carbon atoms, alkylcarboxyl of 2 to 10 carbon atoms, alkylamino and aminoalkyl of 1 to 10 carbon atoms, aryl, alkylaryl and aralkyl, R is selected from the group consisting of haloqen, alkyl of 1 to 30 carbon atoms, aryl of 6 to 30 carbon atoms, aralkyl of 7 to 30 carbon atoms, alkylaryl of 7 to 30 carbon atoms, ##STR15## --CH.dbd.--Z, ##STR16## and alkyl of 1 to 6 carbon atoms, wherein W is hydrogen or carboxyl and Z is carboxyl or 2,4-dinitrophenyl, Y is selected from the group consisting of --CH.sub.2, --O-- and --N.dbd.N-- and V is hydrogen or Y is selected from the group consisting of --O--, --S-- and --S--S and V is --OH, one of A and B is hydrogen and the other is halogen or A is hydrogen or halogen and R and B together form ##STR17## and X is hydrogen or halogen. 2. A composition of claim 1 wherein R is ##STR18## Y is selected from the group consisting of --CH.sub.2, --O-- and --N.dbd.N-- and V is hydrogen or Y is selected from the group consisting of --O--, --S-- and --S--S-- and V is --OH. 3. A composition of claim 1 wherein R is selected from the group consisting of ##STR19## --CH.dbd.CH--Z, --CH.sub.2 --CH.sub.2 --C--OCH.sub.2 --CH.sub.3 and alkyl of 1 to 6 carbon atoms, W is hydrogen or carboxyl and Z is carboxyl or 2,4-dinitrophenyl. 4. A composition of claim 1 wherein the e) amplifier is at least 40% by weight of the composition. 5. A composition of claim 1 wherein the e) amplifier is at least 90% by weight of the composition. 6. A composition of claim 1 wherein e) is a mixture of at least two esters where R.sup.0 is ##STR20## 7. A composition of claim 6 wherein one R.sup.1 is selected from the group consisting of alkyl of 1 to 10 carbon atoms, alkylcarboxy of 2 to 10 carbon atoms and aminoalkyl and alkylamino of 1 to 10 carbon atoms and the second R.sup.1 is selected from the group consisting of phenyl, aryl, alkylaryl and aralkyl. 8. A composition of claim 1 wherein component b) has the formula wherein R.sup.4 is NH.sub.2 and R.sup.5, R.sup.6 and R.sup.7 are individually selected from the group consisting of hydrogen, alkyl of 1 to 6 carbon atoms, alkenyl of 2 to 6 carbon atoms, --OH, alkoxy of 1 to 6 carbon atoms, carboxyl and --NH.sub.2 or R.sup.5 is --NH.sub.2 and R.sup.4, R.sup.6 and R.sup.7 are individually selected from the group consisting of hydrogen, alkyl of 1 to 6 carbon atoms, alkenyl of 2 to 6 carbon atoms, --OH, alkoxy of 1 to 6 carbon, atoms, --COOH and --NH.sub.2 or R.sup.4 or R.sup.5 are amino unsubstituted or substituted by phenyl and R.sup.6 and R.sup.7 are individually selected from the group consisting of hydrogen, alkyl of 1 to 6 carbon atoms, alkenyl of 2 to 6 carbon atoms, --OH, alkoxy of 1 to 6 carbon atoms, --COOH and --NH.sub.2. 9. A composition of claim 1 wherein the chemiluminescence enzyme c) is selected from the group consisting of oxidoreductase and xanthine oxidase. 10. A composition of claim 9 wherein enzyme c) is selected from the group consisting of horseradish peroxidase, micro peroxidse, lacto peroxidase and peroxidase extracted from Arthromyces ramosus. 11. A composition of claim 1 wherein the oxidizing agent d) is selected from the group consisting of hydrogen peroxide, an alkali metal perborate and an alkaline earth metal perborate. 12. A composition of claim 1 in an aqueous solution containing an alkali metal halide or alkaline earth metal halide. 13. A composition of claim 12 wherein the halide is present in an amount of 0.1 M to saturation. 14. A composition of claim 1 wherein ligand a) comprises at least one of the elements of a pair of substances selected from the group consisting of antigen/antibody, enzyme/substrate, enzyme/inhibitor, receptor/ligand, lectin/sugar and nucleic acid/complementary nucleic acid which can be coupled. 15. An analysis kit comprising a composition of claim 1 and the substances to be analyzed are contained: on or in solid supports selected from the group consisting of whole or divisible microtitration plates, test tubes of synthetic polymer or glass biological polymers, immunohistology sections, membranes and or in liquid media of the solution, emulsion or dispersion type, forming part of the analysis medium. |
| PATENT DESCRIPTION |
TECHNICAL FIELD The field of the present invention is that of analytical chemistry, a field of interest, more particularly, for identification and analysis of chemical species, preferably biological species, and in particular high molecular weight species, such as nucleic acids, or also biopolymers of a protein nature, e.g.: enzymes/substrates and antigens/antibodies. More precisely, the present invention relates to: firstly, a system for qualitative and/or quantitative analysis of substances, preferably biological substances, by amplified chemiluminescence, secondly, an analytical method using this system, and thirdly, an analytical kit comprising the said system. Substances or biological species, more particularly but not limitatively, envisaged by the invention are the nucleic acids RNA and DNA and any genetic structure containing them, as well as compounds which are capable of being involved in immunological antigen (Ag)/antibody (Ab) reactions, and furthermore products which couple to one another in the context of a mechanism of recognition and enzymatic reaction: enzyme/substrate, enzyme/inhibitor, receptor/ligand and lectin/sugar. PRIOR ART To detect, identify or analyse this type of molecule or structure, it is conventional to use their property of bioaffinity, that is to say their peculiar capacity for coupling specifically with their complements by mechanisms of genetic hybridization or immunological or enzymatic recognition. Chemiluminescence, and more specifically also that which is so-called amplified, is one of the analytical techniques which proceeds by this principle and which is currently used in the laboratory. Detection by amplified chemiluminescence can be found in the context of well-known analytical procedures of the "western blot" type or of the "ELISA" type as regards proteins, or also "southern & northern blot" type as regards RNA and DNA. This technique of amplified chemiluminescence is an advantageous and promising alternative compared with conventional immunological analyses in which radioactive isotopes are used as the marker and indicator of the coupling under consideration. In fact, radioanalysis is starting to become out-of-date, since it has a large number of disadvantages, including, in particular: hazardous manipulation, low useful storage life of the radioactively labelled substance, difficulty of radioactive labelling, and treatment of radioactive waste. Unfortunately, the development of these analytical techniques by amplified chemiluminescence has been slowed down because of the limited sensitivity of the systems which currently exist. This sensitivity depends in part on the light emitted in the course of and at the end of the luminescence reaction which indicates the couplings under consideration. At this stage of the description, it appears useful to state the concepts by recording the principles of detection by amplified chemiluminescence. Firstly, luminescence can be defined as being the emission of light resulting from the restoration of part of the energy emanating from a substance in an excited state. In chemiluminescence, the excitation results from a chemical reaction. The latter is triggered following coupling of a ligand, directly or indirectly, with one or more substances, preferably biological substances, to be analysed. This ligand is one of the elements of an analytical system comprising, in addition, a chemiluminescent reagent, an enzyme or catalytic subunits of chemiluminescence, and a substrate which is specific to this enzyme or the catalytic subunit or subunits and is capable of being converted under the effect thereof into at least one initiator of the excitation of the luminescent reagent, this excitation being accompanied by the production of light. One of the systems used the most in amplified chemiluminescence is that at the heart of which is found chemical reactions of reagents of the cyclic diacylhydrazide type, such as luminol or isoluminol. In such a system, the enzymes used are, for example, peroxidases, which are capable of converting a substance called an oxidant, such as H.sub.2 O.sub.2, into an initiator of the oxidation of luminol. Since this oxidation produces too few photons, the excitation of luminol cannot be detected with a significant sensitivity, and it is therefore necessary to amplify this oxidation reaction. Among the most effective amplifiers known, there may be mentioned those which belong to the family of halogenophenols and their derivatives. However, as already indicated above, the performances achieved with the aid of such amplifiers remain notably insufficient, in particular with regard to the amount of light emitted after the reaction. There are several previous technical proposals which deal in vain with this problem. Thus, the patent EP 0 116 454 discloses a system for chemiluminescence analysis in which the luminescent reaction takes place between a peroxidase, an oxidant and a chemiluminescent reagent in the form of luminol: 2,3-dihydro-1,4-phthalazinedione. The invention protected in this patent proposes the use of an amplifier consisting of, in particular, 4-iodophenol, 4-bromophenol, 4-chlorophenol and other phenol derivatives, such as 4-hydroxycinnamic acid, 2-naphthol, 6-bromonaphth-2-ol and 4-hydroxyphenyl disulphide, among others. These amplifiers allow an improvement in the emission of the light and, more particularly, the signal/background noise ratio. However, the better signal/noise ratio with the aid of these amplifiers remains insufficient with regard to the sensitivity of the method. This causes an increase in the volume of amplifiers used to obtain a sufficient sensitivity. This results in possible disturbances in the reaction and an undeniable increase in the cost of the analysis. To attempt to perfect this known system, new chemiluminescence amplifiers were subsequently proposed through the international patent application PCT WO 91/05 872. The latter are intended to be used in a system comprising luminol as the reagent, a peroxidase as the enzyme and H.sub.2 O.sub.2 as the oxidizing substrate. The said means of amplification consist of inactive precursors chosen from the family of esters of p-halogenophenols, e.g. iodophenols. The activation of these precursors of amplifiers is realized with the aid of an esterase when all the components of the chemiluminescence system are brought together, in order to obtain the production of light, which will serve as a detection indicator. According to the inventors mentioned in this PCT application, the coupling realized in this way between a chemiluminescence enzyme and an enzyme which activate the amplifier is the cause of an amplification phenomenon of a nature which improves the sensitivity of the analysis. However, even here, the improvements obtained remain insufficient. The patent application EP 0 516 948 also discloses chemiluminescence amplifiers for the system luminol/H.sub.2 O.sub.2 /peroxidase which consist of esters of the formula ArOX, in which X is a masking group which can be activated under the action of a hydrolytic enzyme and Ar is an aromatic group. These are also inactive precursors of amplifiers, which are capable of being converted into active products under the effect of an enzyme, which can be, for example, an esterase in the case where these pre-amplifiers are esters. Disodium phosphate esters of iodophenol, acetic esters of diphenol and, finally, exolydic derivatives of iodophenol and diphenol are mentioned in particular in this application. The amplification means described in this European patent application do not produce better results than those according to the application PCT WO 91/05 872 mentioned above. The previous reference "Analytical Letters, 27(6), 1109-1122, (1994)" by H. HORI et al. also describes new phenolic amplifiers of chemiluminescence in the context of their luminol/H.sub.2 O.sub.2 /peroxidase reaction. These new amplifiers are 4-hydroxybenzylidene-cyclopentenediones on the one hand and 4-hydroxybenzylidene-malononitriles on the other hand. The improvements in the emission of light which are obtained with these amplifiers remain inferior to those found for p-iodophenol described before. Furthermore, the related study in this article reveals a change in trend in the context of development of new amplifiers. In fact, the works on inactive esters derived from halogenophenols are no longer in vogue. Finally, the international patent application PCT WO 94/23 060 proposes a method for amplification of the chemiluminescence reaction involving luminol, a peroxidase, an oxidizing substrate (.dbd.H.sub.2 O.sub.2) and an amplifier intended for increasing the signal/noise ratio of luminous emissions. The amplifier consists of a combination of an organoboron compound and a non-boron organic compound. The organoboron compound corresponds to the formula ArB(OR).sub.2, in which Ar=a substituted or unsubstituted aromatic group. The non-boron organic compound of the amplification is a halogenophenol derivative of the type of those mentioned above in the other references of the prior art. Once again, the improvement in the signal/noise ratio obtained with the invention described in this application WO 94/23 060 is not of significant interest. BRIEF DESCRIPTION OF THE INVENTION In this state of knowledge, one of the essential objects of the present invention is to provide a system for qualitative and/or quantitative analysis of substances, preferably biological substances, by amplified chemiluminescence which allows an actual and significant improvement in the emission of light resulting from passage of a chemiluminescent reagent to the excited state. Another essential object of the invention is to provide a system for amplified chemiluminescence of the type of that above, which is simple to use and economical. Another essential object of the invention is to provide a system for analysis by chemiluminescence of the type of those comprising luminol or analogues as the luminescent reagent, a peroxidase as the luminescence enzyme, a peracid or peroxide, such as H.sub.2 O.sub.2, as the oxidizing substrate and, in addition, an amplifier belonging to the family of phenol derivatives, which system should be perfectly effective at a low dose, with respect to the level of luminescence obtained. Another essential object of the invention is to provide a method for the qualitative and/or quantitative analysis of substances, preferably biological substances, by chemiluminescence which obtains the abovementioned light emission and sensitivity performances. Another essential object of the invention is to provide a kit for analysis by chemiluminescence using the said system and the said method. In the search to achieve these objects, the applicant has succeeded in demonstrating, after several research works and experiments and quite surprisingly and unexpectedly, that it is appropriate to choose the amplifier from the sub-family of esters derived from phenol, the said esters being distinguished by their characteristic of being not readily hydrolysable. It follows from this that the present invention firstly relates to a system for qualitative and/or quantitative analysis of substances, preferably biological substances, by amplified chemiluminescence, a system of the type of those which involve, essentially: a) at least one ligand which is capable of coupling with the substance or substances to be analysed, b) at least one chemiluminescent reagent which belongs to the family of cyclic diacylated dihydrazides fused to an aromatic radical, c) at least one enzyme, d) at least one oxidizing substrate which is specific to the enzyme (c) and is capable of being converted, under the effect thereof, into at least one initiator of the oxidation of reagent (b) with production of light, e) and at least one amplifier of the luminescence reaction, characterized: in that the amplifier (e) corresponds to the following general formula: ##STR1## in which: R.sup.0 is a functionalized or non-functionalized linear or branched C.sub.1 -C.sub.10 radical, R.sup.0 being preferably selected from the following radicals: ##STR2## where R.sup.1 corresponds: 1i.box-solid. to a linear or branched C.sub.1 -C.sub.10 alkyl radical, advantageously a methyl, a propyl, a butyl, a pentyl or a hexyl; 2i.box-solid. to a C.sub.1 -C.sub.10 alkylcarboxyl substituent, advantageously alkylmonocarboxyl, the substituents malonyl, succinyl, glutaryl, adipyl, heptanoyl, maleyl or fumaryl being particularly preferred alkylmonocarboxyls; 3i.box-solid. to alkylamine or aminoalkyl; 4i.box-solid. to aryl, aralkyl or alkylaryl, preferably phenyl; * or a radical of a (poly)organosiloxane and/or (poly)organosiloxane nature: R is a radical chosen from the group consisting of: halogens, iodine being more particularly selected, linear or branched alkyls containing 1 to 30 carbon atoms, C.sub.1 -C.sub.30 aryls or C.sub.1 -C.sub.30 aralkyls or alkylaryls; the halogens, the phenyls, the phthalates and the following radicals being adopted more specifically: ##STR3## Y representing --CH.sub.2 --, --O-- or --N.dbd.N-- and V representing hydrogen, or Y representing --O--, --S-- or --S--S-- and V representing hydroxyl; ##STR4## W representing hydrogen or carboxyl; ##STR5## --CH.dbd.CH--Z, Z representing carboxyl or 2,4-dinitrophenyl; CH.sub.2 CH.sub.2 COOC.sub.2 H.sub.2 ; or a C.sub.1 -C.sub.6 alkyl; A represents hydrogen, B represents a halogen or a C.sub.1 -C.sub.6 alkyl and R represents a halogen; A represents halogen; B represents hydrogen and R represents a halogen or a phenyl, or A represents hydrogen or a halogen and R and B together represent a chain which completes a naphthalene nucleus which, read in the direction from R to B, has the formula: ##STR6## X representing hydrogen or a halogen, such that the compound of the formula (I) is a beta-naphthol of the formula: ##STR7## R.sup.0, R, A and B being substituted or unsubstituted; and in that it contains no hydrolytic enzyme which is capable of lysis of the bond between the oxygen and R.sup.0 in --OR.sup.0. In its most general aspect, the present invention thus provides a perfected system of chemiluminescence or chemiluminometric analysis. It is based on the astonishing discovery of particular amplifiers which are derived from esters of halogenophenols (e.g. iodophenols) and which significantly improve the sensitivity of the luminescent reaction under consideration. In the context of the present description, the term "improved" indicates that the total luminous emission of the luminescent reaction according to the invention and/or the signal/background noise ratio of the said reaction are superior to those achieved by the previously known systems. Furthermore, in contrast to that advocated by some doctrines of the prior art, the chemiluminescent system according to the invention does not comprise hydrolytic enzymes intended to allow cleavage of the bond between the oxygen and the radical R.sup.0 in the formula (I) to render the amplifier active. Moreover, the essence of the invention is radically opposed to that concept, since the aim is to obtain a minimum level of hydrolysis, which is synonymous with high performance of the amplification of the luminescence. DETAILED DESCRIPTION OF THE INVENTION According to a preferred characteristic of the invention: ##STR8## in formula (1) of the amplifier (e). In this case, the content of the non-hydrolysed ester: ##STR9## of the amplifier (e) is preferably greater than or equal to 40% by weight, preferably 60% by weight, and more preferably 90% by weight. The present invention thus proceeds from advantageous and judicious selection of certain amplifier derived from phenols, and more especially consisting of esters of phenol (halogenophenols e.g.) with carboxylic acids, in particular mono-, di- or polycarboxylic acids, which are optionally functionalized, preferably by amine functional groups or also by aromatic radicals which carry one or more carboxylic acid functions. The amplifier (e) used can be in the form of compounds of the formula (1) of the same nature or, advantageously, can comprise a mixture of at least one compound of the formula (1), preferably the esters in which: ##STR10## According to an interesting variant of the invention, in the case concerning a mixture of esters of the formula (1) as the amplifier (e), at least one of these esters preferably contains a substituent R.sup.1 chosen from the radicals (i), (2i) and (3i), as defined above, and at least one other of these esters is distinguished by a substituent R.sup.1 chosen from the radicals (4i) as defined above. Regarding the ligand contained in the system according to the invention, this advantageously consists of at least one of the elements of the following pairs of substances which can be coupled: antigen/antibody, enzyme/substrate, enzyme/inhibitor, receptor/ligand lectin/sugar nucleic acid/complementary nucleic acid. These terms enzymes, substrates, antigens and antibodies can designate various products, such as proteins, hormones, haptens, steroids and metabolites, among others. The luminescent chemical reagent (b) is a product which can lead to the excited state in the course of a chemiluminescence reaction, for example initiated by the product (OH') of an enzymatic reaction, and which then returns to the non-excited state after having emitted light or by emitting light. In the context of the invention, the chemiluminescent reagent (b) is preferably a product belonging to the family of 2,3-dihydro-1,4-phthalazinedione (DPD). More particularly, this DPD corresponds to the following general formula (2): ##STR11## in which: R.sup.4 represents a substituted or unsubstituted amine radical, and each of the radicals R.sup.5, R.sup.6 and R.sup.7 represents H, a substituted or unsubstituted C.sub.1 -C.sub.6 alkyl, a substituted or unsubstituted C.sub.1 -C.sub.6 alkenyl, a hydroxyl, a C.sub.1 -C.sub.6 alkoxy, a substituted or unsubstituted carboxyl or a substituted or unsubstituted amine radical, or R.sup.5 represents a substituted or unsubstituted amine radical, and each of the radicals R.sup.4, R.sup.6 and R.sup.7 represents H, a substituted or unsubstituted C.sub.1 -C.sub.6 alkyl, a substituted or unsubstituted C.sub.1 -C.sub.6 alkenyl, a hydroxyl, an alkoxy, a substituted or unsubstituted carboxyl or a substituted or unsubstituted amine radical, or R.sup.4 and R.sup.5 are taken together and represent an amino derivative which is unsubstituted or substituted by a benzo radical, and each of the radicals R.sup.6 and R.sup.7 represents H, substituted or unsubstituted C.sub.1 -C.sub.6 alkyl, substituted or unsubstituted C.sub.1 -C.sub.6 alkenyl, hydroxyl, alkoxy, substituted or unsubstituted carboxyl or a substituted or unsubstituted amine radical, luminol and isoluminol being particularly preferred. In the context of the present invention, the term "substituted amino" used in the legend of the formula (2) above also designates amido radicals. In the case where a preferred reagent (b), that is to say luminol where R.sup.4, R.sup.5, R.sup.6 =H and R.sup.7 =NH.sub.2, is present, this amino radical can serve as the coupling bridge of (b) with the ligand, directly or by the intermediary of a spacer compound of the type of those which are known and suitable, e.g. hemisuccinates, hemiglutarates, hemimaleates, carboxymethyl and glucuronyl derivatives and mercaptoacetates. After direct or indirect coupling of the ligand coupled to (b) with the species to be analysed, the analytical procedure will thus consist of reacting the chemiluminescent reagent (b), which has been fixed, with the other components of the system in a manner such that the coupling is indicated through the chemiluminescence reaction. The fixing of (b) on to the ligand represents only one of the method variants which can be envisaged. In the context of the invention, the term enzyme designates both complete proteins and catalytic subunits. The enzyme (c) preferably corresponds to an oxidoreduction enzyme as defined by the International Union of Biochemistry. This relates in particular to the enzymes of class 1, or oxidoreductases, of the International Union of Biochemistry classification. An advantageous example of an oxidoreductase according to the invention is xanthine oxidase. The enzyme (c) is more preferably selected from the peroxidases, such as horseradish peroxidase, microperoxidase, the peroxidase extracted from the micro-organism Arthromyces ramosus and lactoperoxidase; horseradish peroxidase and microperoxidase being particularly preferred. Like the chemiluminescent reagent (b), the enzyme (c) can optionally be coupled to the ligand, directly or indirectly, and thus constitutes a marker. Depending on the variants, this enzyme (c) may be in solution or also immobilized on a matrix. In the preferred mode of carrying out the invention, the substrate (d) is an oxidant in the form of, preferably, a peracid or a peroxide, and more preferably hydrogen peroxide and/or an alkali metal perborate or alkaline earth metal perborate. In fact, in the case where the chemiluminescent reagent is 2,3-dihydro-1,4-phthalazinedione (specifically luminol or isoluminol), this oxidant is converted, under the effect of the enzyme (c), into in an initiator (OH.) of oxidation of the reagent (b). As above, it can be envisaged that this oxidant is fixed directly or indirectly to the ligand, thus constituting a marker. According to one alternative, it can be part of the means of indication of the coupling by chemiluminescence which are used for reaction with the marker(s) carried by the ligand. Regarding precisely the location of the components of the system according to the invention, the above shows that it is advantageous for at least one, and at most three, of these components (b) to (e) to be bonded (or carried by) the ligand (a), the other component or components being contained in the analysis medium. Whatever the marker or markers of the ligand, it is preferable, according to the invention, that during the progress of the chemiluminescent analysis the system under consideration is combined with a solution comprising at least one pH regulator and/or at least one ionic strength regulator chosen from the salts, preferably the halides (advantageously chlorides), of alkali metals and/or alkaline earth metals, KCl, KBr, NaCl, CaCl.sub.2 and CsCl being particularly preferred. It has proved useful, in fact, for the pH of the analysis medium to be alkaline. Alkalinity is favourable to the luminescence reaction and also plays a role regarding the solubility of the amplifiers of the formula (1) and of the chemiluminescent reagent of the formula (2). The pH regulation is therefore advantageously ensured by a regulator which consists of any suitable buffer, such as, for example, Tris buffer, carbonate buffer or phosphate buffer. The ionic strength is also an important parameter of the analysis medium comprising the system according to the invention. In fact, it is determines the stability of the amplifiers (e) of the formula (1) with regard to hydrolysis of the radical OR.sup.0, and more particularly the ester bond. It has been seen above that the lower the level of hydrolysis of the amplifiers (e) in the form of esters, the greater the luminescence performances. The halides mentioned above thus advantageously ensure this function of regulation of the ionic strength. To this end, their amount is from 0.1 M to saturation, preferably 0.1 to 3 M. There are of course secondary factors other than the pH and the ionic strength which are important for the chemiluminescent reaction to proceed well. There may be mentioned, among others, temperature, concentration of the components (b) to (e) of the system, the rate of mixing and the technique of measurement of the light. These are the characteristics of the method and the analysis which fit in perfectly in the context of the present description, since it is noted that the present invention also relates to a method for qualitative and/or quantitative analysis of substances, preferably biological substances, by amplified chemiluminescence, characterized in that it comprises the system as defined above. Generally, the conditions used in this method, and therefore the system described above, correspond to those used previously, in particular in the methods described in the patent EP 0 116 454 and in the application PCT WO 94/23 060. By reference, the corresponding parts of these documents are therefore integrated into the present description. To recap, it may be noted that the temperature of the analysis is advantageously between 10 and 50.degree. C., and that the concentrations of components (b) to (e) of the system are the following: reagent (b) (e.g DPD): 5 .mu.mol-200 .mu.mol/l, peroxidase (c): 0.1 ng to 5 g/l, oxidizing substrate (d): 10 .mu.mol-30 mmol/l, amplifier (e): 1 .mu.mol-100 mmol/l These concentrations are given with respect to the entire analysis medium, which advantageously comprises the aqueous buffered solution regulated a fortiori as defined above. It is important to note that the exceptional efficiency of the amplifiers (e) according to the invention enables reductions in the concentration of enzyme to be envisaged. It is in fact possible to manage with concentrations of between 0.1 ng and 1 .mu.g/l. This represents not insignificance savings in consumables. The reaction according to the preferred embodiment of the invention can be outlined as follows: ##STR12## Industrial Application Under another of its advantageous aspects, the present invention also provides a kit for qualitative and/or quantitative analysis of substances, preferably biological substances, by amplified chemiluminescence, characterized in that it comprises the system as defined above. The system, the method and the kit according to the invention have immediate and obvious applications in the field of biochemical and biological analysis. In fact, they can readily be incorporated in the context of techniques of immunological, immunoenzymatic, immunohistological, enzymatic and genetic analyses. It is important to note that the indication systems can include recognition systems of the ligand/receptor, sugar/lectin, enzyme/substrate and enzyme/inhibitor type, for example: membrane receptors, hormones, neurotransmitters and, generally, signal transducers. These can be, for example, "ELISA" techniques of the competitive or non-competitive type, of the sandwich type, of the "western, northern & southern blot" type or of the immunohistological type (antibody/marked antigen or nucleic probe). The substances to be analysed can thus be contained; on or in solid supports chosen, preferably, from the following list: whole or divisible microtitration plates, test tube of a synthetic polymer or glass, biological polymers, immunohistology sections, membranes, beads etc. or in liquid supports of the solution, emulsion or dispersion type forming part of the analysis medium. The solid supports are used in so-called heterogeneous, heterogeneous competitive or also heterogeneous two-site analytical procedures. The liquid media are more suitable for so-called homogeneous phase analytical procedures. The system and the method according to the invention can also be used to identify and analyse in a biological medium, substances such as peroxidases or endogenous peroxides (H.sub.2 O.sub.2). These are substances which correspond to one or more of the constituents of the analysis system. The examples which follow will allow better understanding of the invention and reveals all its use advantages and variants. |
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