IE59302B1 - Homogeneous process for the detection and/or determination by luminescence of an analyte in a medium in which it may be present - Google Patents

Abstract

Homogeneous process for the detection and/or determination by luminescence of an analyte in a medium susceptible of containing it by evidencing the product obtained by the reaction between the analyte and a corresponding receiver. Said process comprises 1) the addition to said medium of a first reactant comprised of an analyte receiver; 2) the addition of a second reactant comprised of at least one of the elements of the product obtained from the reaction of the analyte with at least one of its receivers, one of the two reactants being coupled with a luminescent compound and the other reactant comprising a heavy atom or patterns containing a heavy atom; 3) the incubation of the resulting medium either after the addition of each reactant or after the addition of both reactants; 4) excitation of the resulting medium; and 5) measuring, in balance or kinetic conditions, the signal emitted by the luminescent compound, said signal being modulated by the heavy atom effect.

Description

The present invention relates to a homogeneous process for the detection and/or determination of an analyte in a medium in which it may be present.

The determination of the presence or concentra5 tion of circulating organic or biological substances in biological liquids is an important step in the diagnosis of a large number of diseases.

One of the methods commonly used for this determination is based on the formation of a complex between the analyte, i.e. the substance to be detected or determined, and an analyte receptor, which is a substance capable of fixing specifically to the analyte. The complex thus formed is disclosed by a labeled reagent.

This method embraces the so-called processes of immunological determination by competition or by excess, described for example by R. EKINS in Monoclonal Antibodies and Development in Immunoassay, Elsevier 1981, pp. 3-21. The reagent employed is labeled in particular with the aid of a radioactive element, an enzyme or a luminescent compound, for example a fluorescent, chemoluminescent or phosphorescent compound. We are thus referring to radioimmunological processes, immunoenzymatic processes or immunological processes using luminescence (fluorescence, phosphorescence or chemo25 luminescence).

In the so-called processes of immunological determination by competition, the medium in which the target analyte may be present is incubated with a deficit of an analyte receptor in the presence of a given 3q quantity of the labeled analyte.

Competition for the receptor then takes place between the target analyte and the labeled analyte. The fraction containing the bound labeled analyte is then separated from the fraction containing the free labeled analyte, and the quantity of labeled analyte in one or other of the fractions is measured.

In the so-called processes of determination by excess, two receptors are used which have a different specificity for the target analyte, one of the receptors being labeled. These processes also require a step for separating the fraction containing the bound labeled receptor from the fraction containing the free labeled receptor .

To perform the determinations rapidly with a very high sensitivity, various means for dispensing with the separation step have been sought and so-called ’'homogeneous processes have been developed.

In the field of immunological determinations using fluorescence, there are relatively few homogeneous processes .

Every homogeneous fluorescence method is based on the fact that the binding of the labeled receptor with the analyte causes a modification of the emission characteristics of the fluorescent molecule.

In fluorescence polarization, for example, the polarization of the emitted light is measured, which varies with the size of the molecular structure carrying the fluorescent molecule.

Another homogeneous process, based on the phenomenon of energy transfer between two chromophores, is described in French Patent 2 282 115. In this process, the transfer of energy from the donor chromophore to the acceptor chromophore takes place if the emission spectrum of the former and the excitation spectrum of the latter overlap (energy compatibility) and if the distance between the two chromophores is in general less than 100 A. Similarly, the process described in French Patent 2 422 165 uses a chemoluminescent tracer and a quenching agent which is capable of modifying the emission of the light by chemoluminescence when this molecule is at a short but not collision-causing distance, which in o 3 general is less than 100 A.

However, these processes have disadvantages.

In the case of polarization, there are limitations associated with the size of the target analyte. In the process which uses energy transfer, both the analyte and the receptor have to be labeled and the emission of the acceptor’s luminescence interferes with the measurement of the donor’s luminescence. Furthermore, not all pairs of chromophores can be chosen in this case (energy compatibility ) .

There may also be mentioned the homogeneous processes of determination using fluorescence which are described in U.S. Patent 4 318 707 and European Patent Application 17908 in the name of SYVA.

The process described in U.S. Patent 4 318 707 uses particles which are capable of reducing the excitation intensity and/or of reducing the emission intensity of electronically excitable molecules.

The process according to European Patent Application 17908 is based on the capability of distributing a chromogenic substance between a fraction in which the chromogen retains its chromogenic activity and a fraction in which the chromogenic activity is inhibited, the degree of distribution depending on the concentration of the analyte in the medium of determination.

Furthermore, it is known that the presence of a heavy atom, such as an iodine atom, near or within the structure of a fluorescent compound causes its fluorescence to decrease.

* This very general effect is described in the literature, for example by E.L. WEHRY in Fluorescence”, - 4 edited by G.G. Guilbault, M. Dekker, N.Y. 1967.

According to the prior art, this effect is observed when the fluorescent molecule naturally contains a heavy atom, when heavy atoms are introduced chemically into the fluorescent molecule or when heavy atoms are present in solution in the measurement medium. The mechanism of this heavy atom effect is not very well understood in the case where the heavy atom is outside the molecule. However, in the case where the fluorescent molecule contains heavy atoms (naturally present or incorporated chemically), this phenomenon is explained by an increase in the spin-orbit couplings of the said fluorescent molecule compared with its homolog not containing a heavy atom, and this can result in an increase in the non-radiant inter-system transitions * * * S /w—*T and radiant inter-system transitions T.— »S., 11 ¢10 but also in non-radiant internal conversions S1a/v—*Sq.

A decrease in the fluorescence due to the presence of a heavy atom is observed in all cases and a modification of the phosphorescence is observed when the molecules and the conditions of measurement allow. A concrete example of this effect is the decrease in the quantum yield of fluorescein observed when the latter is chemically bonded to polyiodinated molecules such as triiodothyronine (T^) or tetraiodothyronine (T^); this is an internal heavy atom effect.

In the case where the heavy atom is not fixed by chemical bonding to the fluorescent molecule but is present in solution in the measurement medium, it is thought that the increase in the spin-orbit coupling in the luminescent molecule might be due either to collisions of the fluorescent molecule with the heavy atom or to the formation of weak complexes by charge transfer. It actually manifests itself by an increase in the intersystem transitions of the luminescent molecule, ·ι· τ '·* —►T^ and T -► Sq, S° t^iat albhough the heavy atom effect results sometimes in an increase and sometimes in a decrease in the intensity of phosphorescent molecules, it always results in a decrease in the intensity of fluorescent molecules.

This internal heavy atom effect has already been utilized in a homogeneous process of determination by fluorimetry. Here, reference may be made to European Patent Application 15695, which describes a process of this type using a conjugate formed between a ligand analog and a fluorescent compound, the ligand analog naturally carrying a heavy atom capable of quenching the said fluorescent molecule, and the said conjugate being covalently bonded to a macromolecular polysaccharide. In this case, the binding of the antibody specific with the molecule containing the heavy atom reduces the inhibition and results in an increase in the fluorescence.

It has now been found that the heavy atom effect can be used to advantage in a homogeneous process for the detection and/or determination of an analyte using luminescence, without the heavy atom being in solution in the measurement medium or fixed to the luminescent molecule.

Surprisingly, it has in fact been found that, in an immunological determination by competition or by excess, an inter-system transition takes place in the luminescent molecule when the luminescent molecule is bound to one of the reagents used, while the other reagent carries units containing at least one heavy atom.

From the general point of view, the present invention therefore relates to a process for the detection and/or determination of an analyte in a medium in which it may be present, by disclosing the reaction product of the analyte and at least one corresponding - 6 receptor, which consists in: 1) adding to said medium a first reagent consisting of a receptor for the said analyte; 2) adding to the second reagent consisting of at least one of the components of the reaction product of the analyte and at least one of its receptors, one of the two reagents being coupled with a luminescent compound and the other reagent being coupled with a heavy atom or units containing a heavy atom. 3) incubating the resulting medium after the addition of each reagent or after the addition of said reagents, 4) exciting the resulting medium and ) measuring at equilibrium or during the kinetics, the signal emitted by the luminescent compound, said signal being modulated by the heavy atom effect.

The following definitions apply in the present description : - analyte : any substance or group of analogous substances to be detected and/or determined; receptor : any substance capable of fixing specifically to a site on the said analyte ; - luminescent compound : any substance which, when excited at a given wavelength or by a given chemical compound, is capable of emitting light; - heavy atom : an atom of high atomic number, whose presence near a luminescent molecule is capable of causing an increase in the spin-orbit coupling of the latter. Examples which may be determined of heavy atoms suitable for the purposes of the invention are, in particular, halogen atoms, mercury, thallium, lead and silver ; - unit containing at least one heavy atom : any chemical substance which naturally contains at least one heavy atom or to which at least one heavy atom can be f ixed.

Furthermore, the expression component of the reaction product of the analyte and at least one of its receptors denotes the analyte or at least one of its receptors, depending on the type of method used.

The analyte can be of a biological or non-biological nature. It embraces, in particular, biological substances such as : antibodies, antigens, toxins, enzymes, proteins, for example protein A, hormones, steroids, avidin, biotin, micro-organisms and haptens and non-biological substances capable of binding specifically with a ligand, such as drugs. Examples of analytes which can be detected by the process of the invention are cited in European Patent Application 17 908, which is incorporated in the present thyroglobulin, trypsin, HBe hepatitis, transferrin , description by way of reference.

Specific examples of such analytes are: adrenocorticotropic hormone (ACTH), antidiuretic hormone (ADH) , aldosterone, albumin, cyclic AMP, androstenedione, angiotensin, anti-thyroglobulin antibodies, carbohydrate antigens CA-125, CA 19-9 and CA 15-3, cortisol, digoxin, digitoxin, estriol, ferritin, gastrin , human growth hormone (HGH), human placental lactogen (HPL), insulin, methotrexate, myoglobin, parathormone, pepsinogen, 17 alph-hydroprogesterone, thyroxine-binding globulin, glucagon, and Anti-HBe hepatitis, HBs and anti-HBs particles delta and anti-delta , IgG, IgM, IgA, C3, haptoglobulin, ceruloplasmin, alpha^-antitrypsin, rheumatoid factor, and more particularly : carcinoembryonic antigen (CEA) , alpha-fetoprotein (AFP), estradiol progesterone, testosterone, thyroid-stimulating hormone (TSH) , triiodothyronine (T3) , free triiodothyronine .,(FT3) , thyroxine (T4), free thyroxine (FT4), prolactin, luteinizing hormone (LH), follicle-stimulating hormone (FSH), total IgE.

The luminescent compound used in the process ot the invention can be chosen from the group consisting of fluorescent, chemoluminescent or phosphorescent compounds.

Fluorescent compounds which can be used are any compounds which absorb light at wavelengths above 300 nm, preferably above 400 or 450 nm, and which have an extinc4 tion coefficient greater than 10 above 400 nm.

Another class of fluorescent compounds suitable for the purposes of the invention consists in fluorescent rare earth chelates and fluorescent organic molecules with a long life time, such as pyrene derivatives.

Examples of fluorescent compounds suitable for the purposes of the invention are cited in particular in European Patent 15 695 and in U.S. Patent 3 998 943, which are incorporated in the present description by way of reference .

The particularly preferred fluorescent compounds are fluorescein, fluorescein isothiocyanate and the rare earth chelates and rare earth cryptates described by the Applicant Company in French Patent Application 84 14 799.

In the process according to the invention, it is also possible to use a chemoluminescent compound, such as luminol and acridinium esters (Methods in Enzymology 1978, 57, 424), or fluorescent compounds, such as fluorescein, excited by the reaction product of an oxaldte and hydrogen peroxide (Acc. Chem. Res. 1969, 2t 80).

Phosphorescent compounds, such as, for example, erythrosin and eosin (Biochem. J. 1979, 183, 50) , are also suitable as luminescent compounds for the purposes of the invention.

It should be noted that erythrosine and eosine, which are iodinated compounds, may be also used as units containing at least one heavy atom.

The coupling of one of the reagents with the luminescent compound is carried out by conventional coupling processes so as to produce a covalent bond between the said reagent and the luminescent compound. It is also possible to fix the luminescent compound to one of the reagents by adsorption.

The heavy atom present in one of the reagents can be introduced by direct substitution, for example in the case of halogens, by substitution in units present in the biological molecule, such as the aromatic nuclei, or by fixing units containing a heavy atom to the reagent. These units can be fixed by any of the coupling means commonly used for proteins, for example by means of chelating agents or by coupling with a disulfide bridge in the case of mercury, as described in British Patent 2 109 407. Preferably, the heavy atom is an iodine atom introduced into the second reagent by iodination, for example by the process of A.E. BOLTON and W.N. HUNTER (Biochem. J. 132» 529, 1973).

The heavy atom or the units containing one heavy atom may be also fixed on one of the reagents by means of an appropriate molecule containing functions suitable for coupling with said reagent and functions suitable for coupling with the heavy atom or the units containing one heavy atom. For instance, a polypeptide may be used as intermediate molecule, such as polvlysine, the coupling reactions with the reagent and the heavy atom or the units containing one heavy atom being carried out by the conventional coupling methods.

The use of such an intermediate molecule allows to increase the number of heavy atoms by reagent molecule without considerably affecting the immunoreactivity thereof.

Among examples of units containing at least one heavy atom are iodinated derivatives of succinimide, such as the following derivatives : -N-£3-(3,5— iodo-4-hydroxyphenyl)pro pionyloxxJ succinimide ester, -N-£3-(3-iodo-4-hydroxypheny1)prop ionyloxy7 succinimide ester, -N-£2-(4-iodophenylsulfonamido) ace toxy_/ succinimide ester, -N-£6-(4-iodophenylsulfonamido)hexyloxy_/succinimide ester, as well as the coupling products of these compounds, with a polypeptide, such as polylysine.

These iodinated organic derivatives are directly combined with the reagents used in the invention process - 10 by placing them in contact with a solution of said reagent with an appropriate buffer.

The addition of the first and second reagent within the invention process may be simultaneously or stepwise. In the case of a stepwise addition the medium is advantageously incubated between each reagent addition.

The excitation of the resulting medium is carried out during the later incubation or after this one following the measurement is effected during the kinetics or at the equilibrium,.

This excitation is carried out with the means of light energy when the luminescent compound is a fluorescent or phosphorescent compound or with the means of appropriate chemical reagents when the luminescent compound is a chemoluminescent compound.

The excitation by light energy is effected at a wavelength within the absorption spectrum of the used fluorescent or phosphorescent compound.

It should be noted that the light exciting step may be carried out under a conventional manner or by a pulsed manner, for example according to the process disclosed by WIEDER in US patent 4.058.732. In this latter case, it is necessary to use a luminescent compound having a long luminescent (fluorescent or phosphorescent) decay lifetime compared with the decay lifetime of the ambient substances, such as in particular the substances contained in the medium to be assayed and the assay material. Preferably, this decay lifetime should be higher than one microsecond. The excitation duration should be of course lower than the luminescent decay lifetime of the chosen luminescent compound. Advantageously, the rare earth chelates or rare earth cryptates, such as the ones disclosed by the applicant company in French patent application 8 414 799 may be used as fluorescent compounds having a long fluorescent decay lifetime (or a high half-lifetime).

On the other hand, it should be noted that the invention process may be carried out in liquid phase and that the measurement of the fluorescence or phosphorescence may be effected after the deposit of the reaction medium on a solid phase, - 11 such as a strip, a gel or any other suitable The process according to the invention is suitable for both the so-called excess methods and the so-called competition methods.

Thus, in the case of an excess method the process of the invention consists in: 1) adding to the said medium containing the target analyte a first reagent consisting of a receptor for the said analyte, coupled with a luminescent compound; 2) adding a second reagent consisting of one or more additional receptors for the said analyte, the said second reagent being coupled with a heavy atom or units containing a heavy atom; 3) incubating the resulting medium in the above conditions; 4) exciting the resulting medium and ) measuring the signal emitted at equilibrium or during the kinetics.

In the case of a competition method, the process of the invention consists in: 1) adding to the said medium containing the target analyte a first reagent consisting of a receptor for the said analyte, coupled with a heavy atom or units containing a heavy atom; 2) adding a second reagent consisting of the analyte coupled with a luminescent compound; 3) incubating the medium in the above conditions; 4) exciting the resulting medium and ) measuring the signal emitted at equilibrium or during the kinetics.

According to another alternative embodiment of the process of the invention in the case of a competition method, a first reagent consisting of a receptor for the said analyte is initially added to the medium containing the target analyte, the said receptor being coupled with a luminescent compound, and the analyte coupled with a heavy atom or units containing a heavy atom is added as a second reagent, the following steps being identical to those defined above.

The process according to the invention is particularly applicable to immunological determinations of antigens or haptens by excess or by competition.

For example, the determination of an antigen or hapten by competition uses, as the first reagent, a corresponding antibody labelled with fluorescein or iodinated, and a given quantity of the iodinated or fluorescein-labelled antigen.

The determination of an antigen or hapten by excess uses two antibodies with different specificities for the target antigen or hapten, one being labelled with fluorescein and the other being iodinated. Of course, in this type of determination, it is also possible to use other fluorescent compounds and other units containing at least one heavy atom, such as those mentioned above.

The present invention also relates to a kit comprising essentially: - a first reagent consisting of at least one receptor for the analyte to be determined; - a second reagent consisting of at least one of the components of the reaction product of the analyte and at least one of its receptors, one of the reagents being coupled with a luminescent compound and the other reagent being coupled with a heavy atom or units containing at least one heavy atom; - standard samples containing known quantities of the analyte to be determined, for establishing the standard curves or standard range ; and - the diluents or buffers required for the determination.

If the luminescent compound is a chemoluminescent coumpound, the kit according to the invention also contains the appropriate chemical reagents required for excitation.

The invention will now be described in greater details by means of the non-limiting examples below, in which the substance to be detected or determined is an antibody or antigen.

The following compounds were used in these examples: - rabbit gamma globulins (R—d—G) from Miles ; - sheep anti-rabbit gammaglobulins (SAR-^f-G) obtained by passing a sheep antiserum immmunized with (R-K-G) through a column of DEAEcellulose. - human serum albumin (HSA) at a concentration of 10 mg/ml in a 0.1 M phosphate buffer of pH 7.4 ; - fluorescein isothiocyanate, isomer I ; - hydroxysuccinic ester of 3-(4-hydroxyphenyl) propionic acid (NHSPP: Bolton and Hunter's reagent) ; - sodium metabisulfite ; - l,3,4,6-tetrachloro-3a,6a-diphenyl—glycoluril as an iodine generator ; - Wathman DE 52 DEAE-cellulose as an ion exchanger ; - a column of PD 10 Pharmacia filtration gel ; - a phosphate buffer (PB) ; the fluorescence measurements were made on a PERKIN ELMER LS 5, 2.5/10 nm slots, with an excitation at 495 nm, an emission at 520 nm and an expansion factor of 15 ; - the anti-prolactin monoclonal antibodies E^ and 303 contained in the kits for the immunoradiometric assays of the prolactin made by the Company ORIS INDUSTRIE SA and available in the market under the name ELSA PROL and - the anti-CEA monoclonal antibodies G 12, G 13 and G 15 contained in the kits for the immunoradiometric assays of carcinoembryonic antigen made by the Company ORIS INDUSTRIE SA and available in the market under the name ELSA-CEA, Example I: Demonstration of the heavy atom effect a) Labelling of R-Y-G with fluorecein. 1.44 mg of the fluorescent compound (FITC) were dissolved in 1 ml of water and the pH was brought to 9.5 with sodium hydroxide. mg of R-3^ G were dissolved in 2 ml of 0.05 M phosphate buffer of pH 7.4 and 200jjl of the solution containing the fluorescent compound were added. The reaction was carried out for 2 Hours at room temperature, the pH being kept at 9.5 with dilute sodium hydroxide.

The reaction mixture was then dialyzed overnight against a 0.05 M phosphate buffer of pH 7.4.

The solution was then passed through a column of Whatman DE 52 DEAE-cellulose equilibrated with 0.05 M phosphate buffer of pH 7.4.

Various fractions were eluted by increasing salinity gradient elution (NaCl). The molar ratio fluorescein/protein (R-(f-G) is determined by the formula: F/P = 495 '495 ε2θθ x 150,000 A - 0.35 A 280 495 in which: Αχ is the absorption at wavelength X; £, represents the molar absorption coefficient; 495 72,000; and £_on = 1.4 for 0.12 by weight/volume. 2oU The various fractions obtained according to the salinity of the mobile phase are indicated below: [NaCl] F/P Approximate concentration of R-ZT-G 0.05 M » 1.3 120 jig/ml 0.1 M »2.2 140 jjg/ml 0.2 M »3.5 240 pg/ml 0.4 M »5.5 180 jig/ml b) Labelling of SAR-Y-G with iodine This labelling was carried out by the chloramine T method of R . HUNTER (Proc . Soc. Exp. Biol. Med. 133 (3) 989-1970. The following ' were brought into contact for minutes: - 200 ji 1 of SAR-if-G 100 ul of KI at a concentration of 10“^ M in water; _2 200 pi of chloramine T at a concentration of 10 M in water, _2 and 200 pi of a 10 M aqueous solution of MBS were then added.

The solution obtained was charged onto a PD 10 5 column equilibrated with 0.05 M phosphate buffer of pH 7.5 (pump throughput: 16 ml/h). Detection at the column outlet was effected by measurement of the optical density at 280 nm. The fraction corresponding to the top of the first peak was collected; its concentration θ ofSAR-^G was evaluated as 0.24 mg/ml. c) Demonstration of the effect of the iodine atom prepared : pi of 0.1 M PB of pH 7.4 200 pi of HSA pi of 0.1 M PB of pH 7.4 50 pi of a fraction of R-^- G labelled with fluorescein , diluted to 1/400 in HSA 150 pi of HSA pi of SAR-lf-G labelled with iodine SAR-^-G pi of R-^G diluted to 1/400 in HSA 150 pi of HSA Incubation was carried out for 1 hour 30 minutes, 250 pi of 0.1 M phosphate buffer of pH 7.4 were added and the fluorescence was measured for an excitation at 495 nm.

The efficiency E of the inter-system transition was determined by* the formula: I free solution - I bound solution E = —- 1 ....... — I free solution - I reference solution in which I is the intensity of fluorescence.

Three solutions were reference solution: - free solution: - bound solution: /16 The results obtained with the fraction F/P = 3.5 are given in Table I below. They show that 16% of the emission energy of the fluorescein molecule has been transferred in a non-radiant manner. Since the quantity of SAR-(f-G used is an excess quantity, it is considered that all the R-(f-G are bound.

Example 2: Use of polyiodinated units Some NHSPP was dissolved in a 1:1 mixture of be’nzene/acetaldehyde to give a solution containing 1.3* 10-2 mol/1.

The following were brought into contact: NHSPP evaporated at the bottom of a tube 100 pi - SAR-2f-G 200 pi 3 mg/ml The reaction was left to proceed for 15 minutes in ice, after which the following were added: - KI (5-10-1 M) pi 20 pi _9 - chloramine T (5*10 M) and 20 ul of 5*10-2 jji or o*ru ~ M MBS were added after 1 minute.

The solution was charged onto a PD 10 column; the top of the first peak was collected; it had an optical density of 0.527 at 280 nm. The fluorescence of the three solutions, prepared under the same conditions as in Example 1, was measured for an excitation at 495 nm.

The results, which are given in Table I below, show that the efficiency of the process is enhanced by the use of polyiodinated units.

Example 3 a) Preparation of the iodinated reagent The following compounds were brought into contact in a tube: ,-3 100 yjl 100 pi 100 pi - NHSPP (10 M) - KI (10-1 M) chloramine (10“2 M) After a contact time of 3 minutes, 100 jil of MBS (10 2 M) were addedExtraction was then carried out with 2 x 2 ml of benzene containing 20 jjI of dimethylformamide. The organic phase was evaporated in another tube and 100 j»l of SAR-^-G were added. The reaction was carried out for 15 minutes in ice. The mixture was deposited on a PD 10 column. The top of the peak collected had an optical density of 0.1 at 280 nm. b) Demonstration of the heavy atom effect This fraction of SAR-i-G was used to prepare a bound solution in the same proportions as in Example 1, using the fraction R-^-G 1/400, F/P = 3.5.

The reference and free solutions were prepared under the same conditions as in Example 1. The results of fluorescence measurement are given in Table I. Example 4: The following were brought into contact: NHSPP, 10 evaporated 100 ul 2 iodine generator in CCl^, 5·10 M, evaporated 20 jil - ΚΙ 5-1Ο2 M 20 jil 0.05 M phosphate buffer of pH 7.4 40 ul After a contact time of 1 minute, extraction was carried out with 2 χ 1 ml of benzene in dimethylformamide (12). The organic phase was evaporated in another tube and 10 jil of SAR-i-G, were added, after which the reaction was left to proceed for 15 minutes in ice. The SAR- 8¾ formed were separated off by dialysis against 1 liter of 0.05 M phosphate buffer of pH 7.4.

A 1/10 dilution of SAR-i-G was used to fo^rm a free solution and a bound solution with theR-i-G, 1/400, F/P = 3.5, prepared according to Example 1.

The results obtained are reported in Table I.

TABLE I Solutions tested Example \ No. Reference solution Free solution Bound solution Efficiency 1 43.5 77.9 72.4 0.16 2 36.7 76.6 65.2 0.286 3 34.4 59.4 53.9 0.21 4 33 64.5 56.5 0.25 Example 5 The effect of labeling the R-d^-G with fluorescein was evaluated. The SAR-(f-G labelled with iodine according to Example 4 and diluted to 1/20 in HSA, and the various R-i-G fractions prepared in Example 1, were used.

The fluorescence measurements were made according to the procedure of Example 1. The following results were obtained: R-^-fe fraction, F/P Bound solution Free solution E 1.3 5.4 6.5 0.17 2.2 11.7 14.7 0.20 3.5 26.3 35.2 0.25 5.5 26.8 47.7 0.44 The fluorescence of the reference solution was 26.8.

It is noted that the effect increases with the number of fluoresceins per R-if-G. This increase is definitely associated with the delocalization of the energy of the excited state of the fluorescein by inter19 molecular transfer between fluorescein molecules. Example 6 The dilution curve was established for the antibody SAR-jf- G, labelled according to the procedure of Example 4 with the Η-γίε, F/P = 5.5, diluted to 1/400.

The following results were obtained: Dilution of SAR-V-G I EZ 1/10 26.6 44 1/20 26.7 44 1/100 43.2 9.4 1/500 46.4 2.7 1/1000 47.7 .0 It is noted that the efficiency decreases as the dilution factor increases.

Example 7: Use of the process of the invention in a method of determination by excess a) Labeling of the anti-prolactin monoclonal antibody E^ with fluorescein 0.5 ml of a solution of E^ containing 9.7 mg/ml was mixed with 0.2 mg of FITC (molecular probe) in 0.5 ml of water. The pH was adjusted to 9.3 with sodium hydroxide. The reaction was left to proceed for 3 hours at room temperature, the pH being kept constant. The solution was then neutralized to pH 7 and dialyzed for 20 hours against 2x2 liters of 0.05 M phosphate buffer of pH 7.4.

A column of about 15 ml of DEAE-cellulose gel, equilibrated with 0.05 M phosphate buffer of pH 7.4, was made up. The column was charged with the dialyzed reaction medium and elution was carried out with buffers which were respectively 0.05 M, 0.1 M, 0.2 M, 0.4 M, 0.7 M and 1 M in respect of NaCl, the pH being 7.4.

The peaks eluted with the 0.4 M NaCl and 0.7 M NaCl buffers were collected and dialyzed.

The optical density observed for the peak obtained with the 0.4 M NaCl buffer was 0.184 at 280 nm and 0.167 at 495 nm, which corresponds to an approximate antibody concentration of 90 jig/ml and to a ratio F/P of about 4. b) Labeling of the monoclonal antibody 3D3 with iodine - NHSPP (102 M) iodine generator (5*10 KI (5-10 0.05 M phosphate buffer of pH 7.4 ,-2 tical to that of used: 200 M) 40 M) 40 7.4 40 F1 F1 F1 F1 Extraction was carried out with 2 χ 1 ml of benzene at a concentration of 12 in dimethylformamide. After evaporation, 200 jil of 3D3 containing 3.3 mg/ml were added and the reaction was left to proceed for 15 minutes in ice. Separation was performed on a column of PD 10 and the fraction having an optical density of 0.485 at 280 nm was recovered; its concentration was 350 jjg/ml. c) Determination by excess fluorescent E^ (E^) at a concentration of 3 jjg/ml in a solution of HSA containing .5 mg/ml; iodine-labeled 3D3 (3D3^) at a concentration of 110 jig/ml in the same solution of HSA; 3D3 at a concentration of 110 jjg/ml in the same solution of HSA; prolactin, PRL, at a concentration of 0.6 jig/ml in the same solution of HSA.

The following three solutions were prepared: reference solution: 150 jil of HSA containing 5 mg/ml bound solution' - bound solution 50 r1 ofEi 50 r1 of 3D3 50 r1 of PRL 50 r1 of 4 50 r1 of 3D3 50 ui of PRL Each solution was incubated for 2 hours at room temperature and 100 pi of HSA and 250 pi of phosphate buffer were added.

The fluorescence measurements made according to the procedure of Example 1 gave the following results: Fluorescence reference solution bound solution^ bound solution Δ E 29.7 197.3 231.1 33.8 0.17 The same phenomenon is therefore observed in an excess method (Example 7), by labelling two antibodies having different specificities, as in a competition method (Examples 1 to 6).

Example 8: Kinetic study The determination of Example 7 was repeated at room temperature using a solution of prolactin, PRL, containing 0.6 pg/ml in 100 pi of HSA containing 5 pg/ml, and the same reagents E^ and 3D3^, but varying their concentration, and the efficiency E of the inter-system transition was measured as a function of the incubation time .

The following concentrations were used: E^ at a concentration of 3 pg/ml in a solution of HSA containing 5 pg/ml Ac^ = 3D3^ at a concentration of 360 pg/ml solution of HSA containing 5 pg/ml fEF 1/4 ' F at a concentration of 1.5 pg/ml in the same solution of HSA Ac1 β 3D31 at a concentration of 360 pg/ml in the same solution of HSA at a concentration of 0.75 pg/ml in the same solution of HSA |Ac1/3 s 3D31 at a concentration of 120 pg/ml in t the same solution of HSA ' F Ej/2 at a concentration of 1.5 pg/ml in the same solution of HSA at a concentration of 120 pg/nl in the same Ac 1/3 I solution of HSA The results obtained are shown on the graph of the attached Figure 1, on which the incubation time in minutes is plotted on the abscissa and the efficiency E in Z on the ordinate.

These results show that the process of the invention can also be used in kinetics.

Example 9: Standard curve Six solutions of PRL, containing 600, 150, 75, , 7.5 and 0 ng/ml in a solution of HSA containing 5 mg/ml, were prepared. ul of each solution were incubated in a tube * F T with 50 pi of E^ containing 3 pg/ml and 50 pi of 3D3 containing 120 pg/ml, for 30 minutes at room temperature, and 350 pi of phosphate buffer were added. The inhibition efficiency E was then measured as a function of the value of the standard medium consisting of 150 pi of HSA containing 5 mg/ml, it being known that 1 pU = 30 ng.

The results obtained are shown on the graph of the attached Figure 2, on which the concentration of prolactin, PRL, expressed in ng/tube is plotted on the abscissa and the efficiency E in % on the ordinate.

It is thus possible to establish standard curves for each protein to be determined.

Example 10 This example was carried out using the antiCEA monoclonal antibodies G 12, G 13 and G 15. a) Labelling of the anti-CEA antibody G 12 with fluorescein The protocol used was the same as in Example 7a. The dialysis and the elution of the column were carried out with 0.01 M TRIS buffer of pH 8. The peaks eluted with 0.2 and 0.4 M NaCl were recovered. The value of F/P was about 2.75 for the peak eluted at 0.2 M. The solution collected was concentrated to 800 jig/ml. b) Labelling of G 15 with iodine The protocol was identical to that of Example 4. The following products were used: NHSPP (10“2 M) _2 iodine generator (5·10 M) ΚΙ (5·10“2 M) 200 jil 40 jil 40 r1 jil 200 pi 0.05 M, phosphate buffer of pH 7.4 G 15 (5 mg/ml) The fraction having an optical density of 0,84 at 280 nm was collected; its concentration was 600 jig/ml. c) Labelling of G 13 with iodine The above procedure was followed and the fraction having an optical density of 0,35 at 280 nm was collected; its concentration was 250 jjg/ml.

A standard containing 300 ng of CEA per ml was used .

G* was diluted to 1/2000 in HSA containing 5 mg/ml.

Gjs was diluted to 1/6 in HSA.

The following three solutions were prepared: reference solution - bound solution: free solution r100 ul of HSA + 100 ul r of buffer r50 pi of G 1/2000 50 pi of G ' 1/6 100 pi /50 pi I diluted to diluted to of the standard p of diluted to 1/2000 50 1/6 100 pi of G^ diluted to of buffer These solutions were incubated for 2x1 hour at 45°C and 300 pi of phosphate buffer were added.

The fluorescence of each solution was measured for an excitation at 495 nm, giving the following results: Solution Intensity of fluorescence Efficiency reference solution: 44.5 E = 0.16 bound solution: 90.5 = 8.8 free solution: 99.3 Therefore, there is also a 16% inhibition when two antibodies of different specificities are bound to the antigen.

The same experiment was carried out with the addition, in a third incubation, of the antibody G^, the specificity of which is different from that of G 12 and G 15.

The following solutions were tested: bound solution 50 rl ofG12 diluted to 1/2000 50 r1 of diluted to 1/6 50 r1 ofG13 diluted to 1/3 100 yl of the standard free solutionr 50 jil of diluted to 1/2000 50 pi of CJ5 diluted to 1/6 50 ul of G*3 diluted to 1/3 100 ul of buffer These solutions were incubated for 3x1 hour at 45°C and 250 ul of phosphate buffer were added. The following results were obtained : Solution Intensity of fluorescence Efficiency reference solution 81 0.41 bound solution 106.7 AF = 18.2 free solution 124.9 These results show that the presence of a second iodinated antibody at another site on the antigen increases the inhibition of fluorescence of the fluorescein-labeled antibody G 12.

Example 11: Standard curve Four solutions of CEA, containing 300, 200, 100 and 0 ng/ml, were prepared. Each solution was incubated F II 2q with Gj2 diluted to 1/3000 in HSA and with Gj^ and Gj^ diluted respectively to 1/3 and 1/6 in a solution of HSA, and the fluorescence was measured.

The following results were obtained: Solution Intensity of fluorescence AF EX reference solution 54 - - solution containing 300 ng/ml 65.9 11.9 0.5 solution containing 200 ng/ml 68 9.8 0.41 solution containing 100 ng/ml 71.8 6 0.25 solution containing 0 ng/ml 77.8 0 0 Example 12 : Use of iodinated derivatives of succinimide as units containing at least one heavy atom. .

A. Preparation of iodinated derivatives of succinimide a) Compound 1 ; N-£3-(3,5—iodo-4-hydroxyphenyl)propionyloxyjsuccinimide ester of formula : 1x10* moles (26.3 mg) of . N-£3-(4-hydroxyphenyl) propiony loxy_/ succinimide ester(origin : FLUKA) were dissolved in 2.5 ml of a mixture of benzene and ethyl -4 acetate (50:50 v/v), after what 2.10 moles (86.4 og) of Iodogen® (origin: SIGMA) were added in one step, followed by 83 mg of potassium iodide in solution in 100 jil 0.05 M phosphate buffer of pH 7.4 ; a bright violet color developed instantaneously. The reaction was allowed to continue at 20°C under stirring for 15 mins (under argon). Then it was stopped by adding a saturated solution of sodium metabisulfite in water until discoloration of the reaction medium. The organic phases were separated by decanting, then dried over anhydrous MgSO^ and evaporated in vacuo. The residue was taken up in CH2CI2 or in anhydrous benzene.

The product was then purified by silica gel chromatography. The eluent was a discontinuous gradient of benzene/ethyl acetate. The expected product was eluted for a mixture of benzene and ethyl acetate (90:10 v/v).

The purity of Compound 1 was controlled by T.L.C. (eluent : toluene/ethy1 acetate 1/1 v/v) and compared with T.L.C (eluent: toluene/ethyl acetate 1/1) and the mass spectrometry were found to be in conformity with the structure of the product. Yield : 582. b) Compound 2 : N-£3~(3-iodo-4-hydroxyphenyl)propionyloxy/ succinimide ester of formula : It was proceeded as described above for compound 1, 5 using the following ingredients in the proportions indicated hereunder : - N-/_3-(4-hydroxypheny1)propionyloxy7succinimide ester : 2.103 moles (0.526 g) - Iodogen “ (Sigma) : 2.10 moles (0.864 g) -, q - KI : 4.10 moles (0.584 g) in 500 pi 0.05 M phosphate buffer of (pH: 7.4) The resulting product was put through the same purifying and control stages as compound 1. c) Compound 3 : N-£2-(4-iodophenylsulf onamido) acetoxyj succinimide ester of formula : The synthesis of compound 3 was made in two separate stages after purification and isolation from intermediate product A, according to the following reaction diagram : I^<C^>~~S02C1 * NH2-CH2'COOH —* 3<2>S02-NH-CH2-C00H A Compound 3 1) Synthesis of A: 8.1O_ moles (2.4g) of p-iodophenylsulfochloride in solution in 5 ml of dioxane were added dropwise to 7.10 moles (0.525 g) of glycocoll in aqueous solution adjusted beforehand by sodium hydroxide IM (10 ml) at pH 9 and cooled in an ice bath. When the addition is completed, the ice bath is removed and the reaction is allowed to continue for one hour at 20°C under stirring. (The pH was controlled with a pH meter and readjusted to pH 9 throughout the reaction).

At the end of the reaction, the mixture was diluted with twice its own volume of distilled water. The solution was freed of any reaction insoluble materials by filtration. The filtrate was acidified dropwise under stirring at pH 2 with hydrochloric acid 6N. The expected condensation product precipitated profusely in white flakes.

The crude product was filtered, then washed several times in distilled water, then dewatered and dried in vacuo in a drier in the presence of PjO,. for a w^°^e night.

Yield : 73Z (1.75 g).

The purity of the product was controlled by T.L.C. and by the conventional spectrophotometric methods. 2) Synthesis of compound 3: To a solution of 2 mmoles of A (0.682 g) and 2 mmoles of N-hydroxy succinimide (0.230 g) in 10 ml of THF, cooled beforehand to 0eC, were added in one step, mmoles of O.C.C. (0.412 g).

The reaction mixture was stirred and kept for one hour at that temperature. After that period of time, the cooling bath was removed and the reaction mixture was filtered on fritted glass No. 3, and the filtrate evaporated in vacuo. The residue, constituted of a white foam, was taken up with CH2C12, filtered on Celite®then rethen reevaporated in vacuo. The resulting crude product was 5 recrystallized in CHjClj· Weight obtained : 0.371 g ; Yield: 42%. (D.C.C. = dicyclohexyldiimide).

The product was identified by the conventional spectrophotometric methods and its composition confirmed by centesimal analysis, d) Compound 4: N-/_6(4-iodophenylsulfonamido)hexyloxy7succinimide ester of formula : I so2-nh-(ch2)5-coo-n.

The synthesis of Compound 4 was conducted in two stages after isolation of the intermediate product B and according to the following reaction diagram : so2ci + h2n-(ch2^-cooh—O >-so2-nh-(ch2)5cooh ON - 30 1) Synthesis of B : mmoles (1.52 g) of p-iodopheny1sulfochloride in solution in 5 ml of dioxane were added dropwise to an aqueous solution of 7 mmoles (0.918 g) of 5-amino caproic acid (Fluka), adjusted beforehand to pH9 with 10 ml of NaOH 1 M and cooled with an ice bath.

After the addition of the iodinated reagent, the ice bath was removed and the reaction was allowed to continue for 3 hours at 20 °C (the pH was also controlled throughout the reaction as in the preceding example).

The reaction mixture was then filtered on fritted glass No. 3 and the filtrate was acidified to pH 4 with a few drops of HCl 12N. The expected product B then precipitated profusely. Said product was filtered, washed on the filter with distilled water, de-watered and finally dried in a drier in vacuo over ?2θ5 ^or a whole night. Weight obtained : 1.30 g ; Yield : 68%.

The purity of the product was controlled by T.L.C. (silica) eluent : CHCl^/Methanol (3:1 v/v). M.P. was : 154 + leC, The structure of the product was confirmed by centesimal analysis and by I.R. 2) Synthesis of compound 4 It was proceeded as indicated for compound 3, using the following ingredients : - product B : 2 mmoles (0.762 g) - N-hydroxy succinimide : 3 mmoles (0.345 g) - T.L.C. : 3 mmoles (0.614 g) - THF solvent : 10 ml.

The reaction time was one hour at 4°C and one night at 20eC.

The purification of the product was identical to that of Compound 3. Weight obtained : 0.87q g; Yield : 91 Z.

Purity was controlled by T.L.C. (eluent ethyl acetate/CH2 Clg (1:1 v/v) and by centesimal analysis. The structure of the product was determined by spectrophotometry I.R. and by mass spectrometry. This was found to be conformed to the structure expected for the described compound 4.

B - Coupling of succinimide derivatives with antibodies.

Antibody 30 3 (antiprolactin antibody) was used in this example. ,-6 1.10 mole of 3D 3(10 mg/ml) in solution in 200 jjl of phosphate buffer 0.05 M (pH : 7.4) and 200 μΐ of borate buffer 0.01 M (pH : 9) were added to 2 mg of diiodinated reagent 1 (compound 1) dissolved beforehand in CHgClg and evaporated under argon so as to create a coating on the walls of the reaction tube. The coupling reaction was allowed to continue under stirring for one hour at 20°C. After that period of time, the iodine-labelled 3D3 was then purified by chromatoqraphy on a colum of PD lu and the peak corresponding to the labelled antibody (void volume of the column) was isolated. Its concentration was then measured by its absorbing power at 280 nm.

The isolated labelled antibody was used at the concentration of 150jjg/ml in the fluorescence inhibition test.

It was proceeded as indicated for compounds 3 and 4.

Following a similar process compound 2 was coupled with anti-CEA antibody G 15. For this purpose, antibody G 15 (200^1 at 6.5 mg/ml) were put into contact with 200 jjI of compound 2 (1.24 mg in 3.75 ml of CHgClg, then 200jjI evaporated at the bottom of the tube) and 200 jjl of borate buffer (pH 9). The mixture was incubated for 1 h 30 min at the room , temperature.

C - Determination by excess of the prolactin The reagents used, are as follows: - 3D31 antibody: 150 ^jg/ml by dilution in HSA at 5 g/1 - E1P antibody diluted at 1/100° (1jug/ml) in solution in rabbit gamma globulins at the concentration of 5 mg/ml - prolactin antigen (supplied by Immunotech) ; 0.5 jjg/ml in solution in HSA at 5 g/1 - Diluents: - Rabbit gamma globulin(Concentration 5 g/1) in solution in 50 mM phosphate buffer of pH : 7.4; - HSA (concentration 5 g/l) in 50 mM phosphate buffer of pH : 7.4 - Control : HSA (5 g/l) + eluting solution of purification (50 :50 v/v) The three following solutioi ns were prepared : - control solution : 50 Ul of R- -G 50 of HSA 50 f1 of control bound solution : 50 ί1 of 50 )11 of prolactir 50 F1 of 30/ - free solution : 50 r1 ofE1 50 50 F1 F1 of of HSA 3D/ Each solution was incubated for one hour, at 20° C. Then, before making any readings, 350yjl of phosphate buffer 0.05 M (pH : 7.4.) were added to each one.

The fluorescence measurements were made at 496 nm (excitation) and 520 nm (emission) with a fluorometer and the efficiency E was determined.

The results obtained are given in the following table .

D - Determination by excess of CEA antigen The above method was repeated using CEA antigen instead of prolactine and the following reagents : iodinated reagent : The solution obtained under point B with compound 2. Said solution was adjusted to 0.10 mg/ml after the purification on PD 10. fluorescent reagent : solution of antibody G 12 labelled with fluorescein and diluted to 1/2000.

The incubation was carried out for two hours at 45°C. The obtained results are also in the following table.

Deter- Iadinatad • mina- Iodinated derivative Coupling • • tion derivative reaction pH E% : of 3D^ molc/r.iole • « Coumpound 1 10/ 1 9.0 1 1 1 1 ! 1 1 50/1 9.018 i 300/1 9.0 16. 5 1 z (-1 t- Coumpound 3 30/1 9 .0 CJ 3 < 100/1 9.09 1 -3 q O£ CU Conpound 4 30/1 7.4 2. 3 100/1 7.4 5 : 100/1 9.0 7 CEA Compound 2 20/1 9.0 11 : Example 13.

Use of the coupling product between a polypeptide and an iodinated organic molecule as units containing at least one heavy atom In this example the coupling product between the polylysine and diiodinated compound 1 was prepared; said coupling product is hereinunder named reagent A. This reagent A may be represented by the following statistic formula : NH_ (cn2)2 NH2 H2)2 NH--CH-CO-(NH-CH-CO) -NH-CH-COOH Z ι n NH- (CO-(CH2)2· OH I I - 34 in which n = 327 k = 50 A - Synthesis of reagent A. mg of polylysine chlorhydrate(M.W = 48000) supplied by Sigma Chemicals were dissolved in 500 μ 1 of 0.01 M borate buffer of pH8.9; the pH of the resulting solution was thereafter adjusted to —β with sodium hydroxide (0.5 M) and added to 2.7 mg (5.2 x 10 moles) of above compound 1 previously deposited on the bottom of a test tube by evaporation of a solution into GHgC^· The reaction was continued for one hour at 20°C under stirring.

The product reaction was eluted on PD 10 (Sephadex G 25) with a 50 mM phosphate buffer. The recovered fractions were concentrated up to 90 μ 1 volume by centrifugation on Amicon cones.

B - Coupling of reagent A with antibody 3D3 120 μ 1 of 25 mM phosphate buffer of pH 5 were added to 80 μ 1 of reagent A.

Thereafter, 100 μ 1 of a solution of carbodiimide was added to 2 mg/ml of water.

After 2 or 3 minutes, 400 μ 1 of a solution of antibody 3D3 (200 μ 1 of 3D3 at 9.6 mg/ml of 50 mM phosphate buffer of pH 7.4 + 200 μ 1 of 200 mM phosphate buffer of pH 8).

The incubation was effected for 1 hour at 20°C, then one night at 4°C and 1 hour at 20°C.

The separation was carried out on a column of PD 10 using 50 mM of phosphate buffer of pH 7.4 as eluting agent.

From the void volume of the column it was collected a solution (1 ml) having an optical density of 0.742 at 280 nm. The antibody concentration of this solution was estimated to be 530 μg/ml. Said fraction was submitted to the fluorescence inhibition test described in example 12.

Claims (20)

CLAIMS 1. ) adding to the said medium containing the target analyte a first reagent consisting of a receptor for the said analyte, the said receptor being coupled with a luminescent compound ; 1) adding to the said medium containing the target analyte a first reagent consisting of a receptor for the said analyte, on which is coupled a heavy atom or units containing a heavy atom ; 1) adding to the said medium containing the target analyte a first reagent consisting of a receptor for the said analyte, coupled with a luminescent compound ; 1) adding to said medium a first reagent consisting of a receptor for the said analyte ;

1. Homogeneous process for the detection and/or determination by luminescence of an analyte in a medium in which it may be present, by revealing the reaction product of the analyte and a corresponding receptor, characterized in that it consists in: 2. ) adding, as a second reagent, the analyte on which is coupled a heavy atom or units containing a heavy atom ; 2) adding a second reagent consisting of the analyte coupled with a luminescent compound ; 2) adding a second reagent consisting of one or more additional receptors for the said analyte, the said second reagent being coupled with a heavy atom or units containing a heavy atom ;

2. Process according to claim 1, which consists of an excess method, characterized in that it consists in 2) adding a second reagent consisting of at least one of the components of the reaction product of the analyte and at least one of its receptor ; one of the two reagents being coupled with a luminescent compound and the other reagent being coupled with a heavy atom or units containing a a heavy atom ; 3. ) incubating the said medium after addition of each reagent or after the addition of both reagents ; 3) incubating the said medium after addition of each reagent or after the addition of both reagents ;

3. Process according to claim 1, which consists of a competition method, characterized in that it consists in : 3) incubating the said medium after addition of each reagent or after the addition of both reagents ; 3) incubating the resulting mechum after addition of each reagent or after the addition of both reagents ; 4. ) exciting the resulting medium and

4. Process according to claim 1, which consists of a competition method, characterized in that it consists in : 4) exciting the resulting medium and 4) exciting the resulting medium and 4) exciting the resulting medium and 5. Examples.

5. Process according to any one of claims 1 to 4, characterized in that the analyte is a biological substance, or a non-biological substance. 5) measuring the signal emitted during the kinetics or at equilibrium. 5) measuring the signal emitted at equilibrium or during the kinetics. 5) measuring the signal emitted at equilibrium or during the kinetics. 5) measuring at equilibrium or during the kinetics, the signal emitted by the luminescent compound, said signal being modulated by the heavy atom effect.

6. Process according to any one of claims 1 to 4, characterized in that the analyte is selected among biological substances such as antibodies, antigens, toxins, enzymes, proteins, hormones, steroids, avidin, biotin, micro-organisms and haptens and non-biological substances capable of binding specifically with a ligand,such as drugs.

7. Process according to one of claims 1 to 5, characterized in that the analyte is prolactin or carcinoembryonic antigen.

8. Process according to any one of claims 1 to 6, characterized in that the luminescent compound is a fluorescent, chemoluminescent or phosphoroscent compound.

9. Process according to claim 7, characterized in that the luminescent compound is a fluorescent compound chosen from the group consisting of fluorescein and rare earth cryptates and chelates.

10. Process according to any one of claims 1 to 8, characterized in that one of the reagents is labelled with fluorescein and the other is iodinated.

11. Process according to any one of claims 1 to 9, characterized in that the luminescent compound has a long luminescent decay lifetime and in that excitation of the resulting medium is a pulsed excitation.

12. Process according to claim 10, characterized in that the luminescent compound is a rare earth cryptate.

13. Process according to any one of claims 1 and 3 to 12, for the determination of antigens or haptens by the competition method, characterized in that it consists in incubating the medium containing the target antigen with the corresponding fluorescein-labelled antibody in the presence of a given quantity of iodinated antigen.

14. Process according to any one of claims 1 and 3 to 13, for the determination of antigens or haptens by the competition method, characterized in that it consists in incubating the medium containing the target antigen with the corresponding iodinated antibody in the presence of a given quantity of fluorescein-labelled antigen.

15. Process according to any one of claims 1 ( 2 and 5 to 14, for the determination of antigens or haptens by the excess method, characterized in that it consists in incubating the medium containing the target antigen with a first fluorescein-labelled antibody in the presence of a given quantity of a second iodinated antibody, or vice versa, the said antibodies having different specificities for the target antigen.

16. A kit for the homogeneous detection and/or determination in liquid phase of an analyte in a medium in which it may be present, characterized in that it contains : - a first reagent consisting of at least one receptor for the analyte to be determined ; - a second reagent consisting of at least one of the components of the reaction product of the analyte and at least one of its receptors, one of the reagents being coupled with a luminescent compound and the other reagent being coupled with a heavy atom or units containing at least one heavy atom ; - standard samples containing known quantities of the analyte to be determined, for establishing standard curves ; and - the diluents or buffers required for the determination.

17. Kit according to claim 16, characterized in that the luminescent compound is a chemoluminescent compound and in that the said kit also comprises the appropriate chemical reagents required for excitation.

18. Kit according to one of claims 16 or 17, for the determination of the prolactin or the carcinoembryonic antigen.

19. A homogeneous process according to claim 1 for the detection and/or determination by luminesence of an analyte in a medium in which it nay be present, substantially as hereinbefore described with particular reference to the accompanying

20. A kit according to claim 16 for the homogenous detection and/or determination in liquid phase of an analyte in a medium in which it may be present, substantially as hereinbefore described.