CA1279260C - 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

ABSTRACT OF THE DISCLOSURE

The invention relates to a homogeneous process for the detection and for determination by luminescence of an analyte in a medium in which it may be present in which a luminescent molecule is bound to one of the reagents used, while the other reagent carries units containing at least one heavy atom.

Description

1~7~60 ~O~iOGE~EOUS PROCESS FOR T~E D~TECTION A~D/OD~ DETE~5I.~A-TION BY LU~INESC~NCE ~F ~N A~ALYTE IN`A ~IEDIU~I IN l~HICH IT
MAY BE PRESENT.
_ 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 concentra-tion of circulating organic or biological substances in hiological liquids is an important step in the diagnosis of a large number of diseases.
One of the methods commonly used for this deter-mination is based on the formation of a complex between ~he analyte, i.e. the substance to be detected or determined, and an snalyte 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 par-ticular with the aid of a radioactive element, an enzyme or a luminescent compound, for example a fluorescent, chemoluminescent or phosphorescent compound. ~;e are thus referring to radioimmunological processes, immuno-enzymatic processes or immunological processes using luminescence (fluorescence, phosphorescence or chemo-luminescence).
In the so-called processes of immunological determination by cQmpetition, 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 quantity of the labeled analyte.
Competition for the receptor then takes place between the target analyte and the labeled analyte. The rraction 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 molecu~e.
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 e~ergy 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 bet-ween the two chromophores is in general less than 100 A.
Similarly, the process described in French Patent 2 ~22 ~Z792~iO

165 uses a chemo]uminescent tracer and a quenchino ~gent which is capah1e ol modifying the emission of the ]ight by chemoluminescence when this molecule is at a short but not co]lision-causing distance, which in 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 (en,ergy com-patibility).
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 excita-tion intensity and/or of reducing the emission intensity of electronically excitable molecules.
The process according to European Patent Applica-tion 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 de8ree 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 fluores-cence to decrease.
This very general effect is described in the literature, for example by E.L. WEHRY in "Fluorescence", ~2~ 0 edited by G.G. Guilbault, M. Dekker, ~.Y. 1967.
According to the state of the art, this effect is ol)served when the fluorescent molecule naturally con-taiils 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 fl.uorescent molecule contains heavy atoms (naturally present or incorporated chemically), this phenomenon is explained by an increase in the spin-orl)it 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~ ~Tl and radiant inter-system transitions Tl S
but also in non-radiant internal conversions S~ ~S0.
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 chemi-cally bonded to polyiodinated molecules such as triiodo-thyronine (T3) or tetraiodothyronine (T4); this is aninternal 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 actual.ly manifests itself by an increase in the inter-system transitions of the luminescent molecule, ~2~7~3Z~,O

* ~ ~
S~ ~Tl and Tl~ SO, so that although the heavy atomeffect results sometimes in an increase and sometimes in a decrease in the intensity o phosphorescent molecules, it always result~s in a decrease in the intensity of f1uorescent 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 poly-saccharide. In this case, the binding of the antibodyspecific 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 re-agent carries units containing at least one heavy atom.
From the general point of view, the present invention therefore relates to a process for the detec-tion 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 12792~0 receptor, which consists in :
1) adding to said medium a first reagent consisting of a receptor for the said analyte ;

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 receptors;one of-the two reagents being coupled with a luminescent compound and the other reagent possessing a heavy atom or units containing a heavy atom.

3) incubating said medium after the addition of each reagent or after the addition of both reagents, 4) exciting the resulting medium and 5) 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 fixed.

'7~2fiO

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-bio-logical 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-bio-logical 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.

Specific examples of such analytes are :
adeno-cortico~lopic hormone (ACTH~, anti-diuretic ,~rmone (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, ferritine, gastrine, growth hormone (HGH), placental lactogen hormone (PLH), insulin, metho-trexate, myoglobin, parathyryn, pepsinogen, 17 alpha-hydroprogesterone, thyroglobulin, thyroxine binding glo-bulin, glucagon, trypsin, HBe and Anti-HBe Hepatitis, HBs and anti-HBs hepatitis,delta - and anti-delta-par-ticles, transferrin, IgG, IgM, IgA,C3, haptoglobulin, ceruloplasmin, alpha 1 antitrypsine, rheumatoid factor, and more particularly : carcino-embryonic antigen (CEA), alpha-foetoprotein (AFP), estradiol, progesterone, testo-sterone, thyreostimulant hormone (TSH), tri-iodothyrosine (T3), free tri-iodothyrosine (FT3), thyroxine (T4), free thyroxine (FT4), prolactine, luteinizing hormone (LH), stimulating folliculo hormone (SFH), total IgE.

127~fio The luminescent compound used in the process of the invention can be chosen from the group consistin~
of fluorescent, chemolumincscent or phosphorescent com-pounds.
05 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 extinc-tion coefficient greater than lO above 400 nm.
Another class of fluorescent compounds suitable for the purposes of the invention consists of 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 l 5 695 and in U.S. Patent 3 998 943 .
The particularly preferred fluorescent compounds are fluoresce m, fluoresceLn isothiocyanate and the rare earth chelates and rare earth cryptates described by the Applicant Company, Commissariat a L'Energie Atomique in French Patent Application 84 14 799, Sept. 26, 1984.
In the process according to the invention, it is al90 possible to use a chemoluminescent compound, such as luminol and acridinium esters (Methods in Enzymology 1978, 57, 424), or fluorescent compounds, such as fluores-cein, excited by the reaction product of an oxalate and hydrogen perioxide (Acc. Chem. Res. 1969, 2, 80).
Phosphorescent compounds, such as, for example, erythrosin and eosin (Biochem. J. 1979, 183, S0), 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 lumines-cent 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.

~2~60 g 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. 133, 529, 1973). The heavy atom or the units containing at least 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 at least one heavy atom. For instance, a polypeptide may be used as intermediate molecule, such as polylysine, the coupling reactions with the reagent and the heavy atom or the units containing at least 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 without considerably affecting the immunoreactivity thereof.

127~fi'~
- 9a -Among examples of "units containing at least one heavy atom" are iodinated derivatives of succinimide, such as the following derivatives :
-N-L3-(3,5-diodo-4-hydroxyphenyl)propionyloxy7succinimide ester, -N-L3-(3-iodo-4-hydroxyphenyl)propionyloxylsuccinimide ester, -N-a-~4-iodophenylsulfonamido)acetoxy/succinimide ester, -N-L6-(4-iodophenylsulfonamido)hexyloxy7succinimide ester as well as the couplin~ products of these compounds with a polypeptide, such as polylysine.
These iodinated organic derivatives are directly combined with the reagents used in the process according to the invention by placing them in contact with a solu-tion 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.

~L27~2~0 The exciting step is carried out during the later incubation or after this one following the measurement is effected during the kinetics or at the equilibrium.
û5 This exciting step is carried out with the means of light energy when the luminescent compound is a fluorescent or phosphorescent compound and with the means of appropriate chemical reagents when the luminescent compound is a chemoluminescent.
The exciting step 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 later 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 ambiant substances, such as the substances contained in the medium to be assayed and the assay material. Preferably, this decay lifetime should be higher than one microseconde.
The excitation duration should be of course lower than the luminescent decay lifetime of the chDosen luminescent compound.
Advantageously, the rare earth chelates or rare earth cryptates, such as the ones disclosed in FR patent application 84.I4.799 (Sept. 26, 1984;
Commissariat à L'Energie Atomique) 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, such as a strip, a gel or any other suitable support.
The process according to the invention is suitable both for the io-called excess methods and for the so-called competition methods.
C

1~7~0 Thus, in the case of an excess method, the process of the invention consists in :
1) adding to the medium containing the target analyte a first reagent consisting of a receptor for the said analyte, OS coupled with a luminescent compound;
2) adding a second reagent consisting of one or more additional receptor for the said analyte, the said second reagent possessing a heaving atom or units containing a heavy atom;
3) incubating the medium in the above conditions;
4) exciting the resulting medium and 5) 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 medium containing the target analyte a first reagent consisting of a receptor for the said analyte, possessing a heavy atom or units containing a heavy atom ;
2) adding a second reagent consisting of the analyte coupled with a luminescent compound;
3) incubatin,q the medium in the above conditions ;
4) exciting the resulting medium and S) 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, the medium containing the target analyte is initially incubated with a first reagent consisting of a receptor for the said analyte, the said receptor being coupled with a luminescent coumpound, and the analyte possessing 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 determination6 of antigens or haptens by excess or by competition.

12 ~2792fiO

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.
05 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 coumpound and the other reagent possessing 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 gammaglobulins (~RG) from Miles ;
- anti-rabbit sheep gammaglobulins (~ARSG) obtained by passing a sheep antiserum immunized with ~RG through a column of DEAE-cellulose ;

13 ~ X7~

- human serum albumin (HSA) at a concentration of lO mg/ml in a O.l M phosphate buffer of pH 7.4 ;
- fluorescein isothiocyanate, isomer I ;
- hydroxysuccinic ester of 3-(4-hydroxyphenyl) propionic acid 05 (NHSPP: Bolton and Hunter's reagent) ;
- sodium metabisulfite ;
- 1,3,4,6-tetrachloro-3a,6a-diphenylglycoluril 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/IO nm slits, with an excitation at 495 nm, an emission at 520 nm and an expansion factor of 15 ;
- the anti-prolactin monoclonal antibodies El and 3D3 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 1: Demonstration of the heavy atom effect a) Labelling of Y~G with fluorescein.
1.44 mg of the fluorescent compound (FITC) were dissolved in 1 ml of water and the pH wasbrought to 9.5 with sodium hydroxide.
24 mg of ~RG were dissolved in 2 ml of 0.05 M phosphate buffer of pH 7.4 and 200 ~1 of the solution containing the fluorescent compound were added. The reaction was carried out for 2 Hours at romm temperature.

*Trademark ~L27~iO

the pH being kept at 9.5 with dilute sodium hydroxide.
The reaction mixture was then dialyzed over-night 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 (~RG) is determined by the formula:

F/p = 495 280 x 150,000 495 280 ~ 0-35 A49 in which:
~ AX is the absorption at wavelength X;
- represents the molar absorption coefficient;
- 495 = 72,000; and ~ ~280 = i 4 for 0.1% hy weight/volume.
The various fractions obtained according to the salinity of the mobile phase are indicated below:

[NaCl] F/P Approximate concentration of ~RG
-0.05 M ~1.3 120 ~g/ml 0.1 M Y2.2 140 lug/ml 0.2 M ~ 3.5 240 lug/ml 0.4 M ~5.5 180 jug/ml b) Labeling of ~ARSG with iodine This labeling was carried out by the chloramine T method of R. HU~TER (Proc. Soc. Exp. Biol. Med. 133(3), 989, 1970). The following were brought into contact for 3 minutes:
- 200 ~ul of ~ARSG at a concentration of 3.5 mg/ml;
- 100 ~1 of KI at a concentration of 10 M in water;
and 79~fiO

- 200 ~l of chloramine T at a concentration of 10 2 M
in water, and 200 lul of a 10 2 M aqueous solution of MBS were then added.
The solution obtained was charged onto a PD 10 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 of YARSG was evaluated as 0.24 mg/ml.
c) Demonstration of the effect of the iodine atom Three solutions were prepared:
- reference solution: (- 50 1 of 0.1 M PB of pH 7.4 1l - 200 pl of HSA
- free solution: - 50 ~1 of 0.1 M PB of pH 7.4 - 50 ~1 of a fraction of ~RG labeled with fluorescein (~RG)' diluted to 1/400 I in HSA
~- 150 ~1 of HSA
- bound solution: ~ 50 ~l fI~ARsG
iodine (~ARSG) - 50 ~1 of ~G diluted to ¦ 1/400 in HSA
~- 150 ~1 of HSA
Incubation was carried out for 1 hour 30 minutes, 250 ~1 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
I free solution - I reference solution in which I is the intensity of fluorescence.

12792~S0 The ~esults 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 rARsG used is an excess quantity, it is considered that all the ~RG are bound.
Example 2: Use of polviodinated units Some NHSPP was dissolved in a 1:1 mixture of benæene/acetaldehyde to give a solution containing 1.3 10 2 mol/l.
The following were brought into contact:
- NHSPP evaporated at the bottom of a tube 100 lul YARSG 200 ~l 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) -2 20 ~1 - chloramine T (5-10 M) 20 ~l and 20 JUl of 5-10 2 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 condi-tions 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 yrocess is enhanced by the use of polyiodinated units.
Example 3 a) Preparation of the iodinated reagent The following compounds were brought into con-tact in a tube:
- NHSPP (10 3 M) 100 ~1 - KI (10~1 M) 100 lul - chloramine (10-2 M) 100 ~1 After a contact time of 3 minutes, 100 ~1 of 12~2t~0 ~BS (10 2 M) were added.
Extraction was then carried out with 2 x 2 ml of benzene containing 20 pl of dimethylformamide. The organic phase was evaporated in another tube and 100 ~ul of ~ARsG 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 rARSG as used to prepare a bound solution in the same proportions as in Example 1, using the fraction ~G~ 1/400, F/P = 3.5.
The reference and free solutions were prepared separately 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 2 M, evaporated 100 ~1 - iodine generator in CC14, 5-10 M, evaporated 20 ~1 - KI 5-10 M 20 lul - phosphate buffer, 0.05 M, pH 7.440 ~1 After a contact time of 1 minute, extraction was carried out with 2 x 1 ml of benzene in dimethyl-formamide (1%). The organic phase was evaporated in another tube and 10 ~1 of ~ARsG were added, after which the reaction was left to proceed for 15 minutes in ice.
The ~ARSG formed were separated off by dialysis against 1 liter of 0.05 M phosphate buffer of pH 7.4.
A 1/10 dilution of ~ARSG was used to form a free solution and a bound solution with the ~RG~ 1/400, F/P = 3.5, prepared according to Example 1.
The results obtained are reported in Table I.

~2 7 ~ ~ 6~

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TABLE I

So]utions Reference Free Bound Efficiency \ tested solution solution solution No.
~ .._ 1 43.5 77.972.4 0.16 2 36.7 76.665.2 0.286 3 34.4 59.453.9 0.21 4 33 64.556.5 0.25 Example 5 The effect of labeling the ~RG with fluorescein was evaluated- The ~ARSG labeled with iodine according to Example 4 and diluted to 1/20 in HSA, and the various ~RFG fractions prepared in Example 1, were used.
The fluorescence measurements were made accor-ding to the procedure of Example 1. The following results were obtained:
.. _ _ ........................ ..
YRG 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 ~RG. This increase is definitely associated with the delocalization of the energy of the excited state of the fluorescein by inter-~279'~0 molecular transfer between fluorescein molecules.
Example 6 The diluti.on curve was established for the antibody ~ARSG labeled according to the procedure of Example 4 with the ~RG~ F/P = 5.5, diluted ~o 1/400.
The foll.owing results were obtained:

Dilution of ~ARSG in HSA I E%
1/10 26.6 44 l/20 26.7 44 l/100 43.2 9.4 l/500 46.4 2.7 l/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 El with fluorescein 0.5 ml of a solution of El 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 2 x 2 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 re-action 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 l M in respect of NaCl, the pH being 7.4.

127~260 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 lug/ml and to a ratio F/P
of about 4.
b) Labeling of the monoclonal antibodv 3D3 with iodine The protocol is identical to that of Example 4.
10 The following products were used:
- NHSPP (10-2 200 lul - iodine generator (5-10 2 M)40 /ul - KI (5-10 2 M) 40 Jul - phosphate buffer, 0.05 M, pH 7.4 40 ~1 Extraction was carried out with 2 x 1 ml of benzene at a concentration of 1% in dimethylformamide.
After evaporation, 200 lul 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 ~g/ml.
c) Determination by excess - fluorescent El (EFl) at a concentration of 3 ~g/ml in a solution of HSA containing 5 mg/ml;
- iodine-labeled 3D3 (3D3I) at a concentration of 110 ~g/ml in the same solution of HSA;
- 3D3 at a concentration of 110 ~g/ml in the same solution of HSA;
- prolactin, PRL, at a concentration of 0.6 ~g/ml in the same solution of HSA.
The following three solutions were prepared:
- reference solution: 150 ~1 of HSA containing 5 mg/ml /

1;~7~

- bound solutionI: ~50 ~1 of EFl ~50 /ul of 3D3I
50 ~l of PRL
- bound solution: ~50 lul of El 15 ~l of 3D3 l50 ~l of PRL
Each solution was incubated for 2 hours at room temperature and 100 lul of HSA and 250 ~1 of phosphate buffer were added.
The fluorescence measurements made according to the procedure of Example 1 gave the following results:
Fluorescence reference solution29.7 bound solutionI 197.3 bound solution 231.1 33.8 E 0.17 The same phenomenon is therefore observed in an excess method (Example 7), by labeling two antibodies having different specificities, as in a competition method (Examples 1 to 6).
Example 8: Kinetic studv The determination of Example 7 \;as repeated at room temperature using a solution of prolactin, PRL, containing 0.6 ~g/ml in 100 ~l of HSA containing 5 ~ug/ml, and the same reagents El and 3D3 , but varying their concentration, and the efficiency E of the inter-system transition was me~sured as a function of the incubation time.
The following concentrations were used:
fEl at a concentration of 3 ~g/ml in a solution of HSA containin~ 5 ~ug/ml AcI = 3D3 at a concentration of 360 pglml in a solution of HSA containing 5 ~g/ml ~ Z7~3~iO

- 2~ -~El/2 at a concentration of 1.5 ~Ig/ml in the same solution of HSA
AcI = 3D3I at a concentration of 360 ~g/ml in the same solution of HSA
~E1/4 at a concentration of 0.75 ~g/ml in the same solution of HSA
AcI/3 = 3D3I at a concentration of 120 ~g/ml in the same solution of HSA
~ElF~2 at a concentration of 1.5 ~g/ml in the same solution of HSA
AcI/3 at a concentration of 120 ~g/ml in the same 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
~n % 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, 30, 7.5 and 0 ng/ml in a solution of HSA containing 5 mg/ml, were prepared.
50 ~1 of each solution were incubated in a tube with 50 ~l of ElF containing 3 ~g/ml and 50 ~l of 3D3I
containing 120 ~g/ml, for 30 minutes at room temperature, and 350 ~1 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 ~l of HSA
containing 5 mg/ml, it being known that 1 /uU = 30 ng.
The results obtained are shown on the graph of the attached Figure 2, on which the concentration of pro-lactin, 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.

~27~3X~

Example 10 This example was carried out using the anti-CEA antibodies G 12, G 13 and G 15 a) Labeling 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 lug/ml.
b) Labeling of G 15 with iodine The protocol was identical to that of Example 4.
The following products were used:
NHSPP (10 2 M) 200 ~1 iodine generator (5-10 2 M) 40 ~1 KI (5 10 M) 40 lul phosphate buffer, 0.05 M, pH 7.4 40 ~1 G 15 (5 mg/ml) 200 ~1 The fraction having an optical density of0,84 at 280 nm was collected; its concentration was 600 ~g/ml.
c) Labeling of G 13 with iodine The above procedure was followed and the fraction having an optical densi~y of 0,35 at 280 nm was collected; its concentration was 250 ~g/ml.
A standard containing 300 ng of CEA per ml was used.
GF2 was diluted to 1/2000 in HSA containing 5 mg/ml.
GI5 was diluted to 1/6 in HSA.
The following three solutions were prepared:

79~60 - reference solution: 100 ~1 of HSA + 100 jul of buffer - bound solution: ~50 lul of GF2 diluted to ~50 ~1 of GI5 diluted to 100 ~l of the standard - free solution: 50 ~1 of GF2 diluted to 50 ~1 of G15 diluted to 100 jul of buffer These solutions were incubated for 2 x 1 hour at 45C and 300 lul of phosphate buffer were added.
The fluorescence of each solution was measured for an excitation at 495 nm, giving the following results:
SolutionIntensity of Efficiency fluorescence reference solution: 44.5 E = 0.16 bound solution: 90.5 ~F = 8.8 free solution: 99.3 Therefore, there is also a 16% ir.hibition 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 GI3, the specificity of which is different from that of G 12 and G 15.
The following solutions were tested:
bound solution ~ 50 ~l of GlF2 diluted to 1/2000 50 ~l of GI5 diluted to 1/6 50 Jul of GI3 diluted to 1/3 100 ~l of the standard lX79Z~o ~ 25 free solution ~ 50 lul of GF2 diluted to 112000 ¦ 50 ~l of GI5 diluted to 1/6 50 ~l of GI3 diluted to l/3 ~ 100 ~l of buffer These solutions were incubated for 3 x 1 hour at 45C and 250 ~l of phosphate buffer were added. The following results were obtained:
Solution Intensity of Efficiency fluorescence reference solution 81 0.41 bound solution 106.7 ~F = 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 with GF12 diluted to 1/3000 in HSA and with GI3 and GI5 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~F E%
fluorescence 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 1279X~O

Example 12 : Use of iodinated derivatives of succinimide _ A ___ aslJ;~ s containinS a-~ le2s~ on~ ne3vy a~o,~. -A. Preparation of iodinated derivatives ofsuccinimide a) Compound 1 : N-L3-(3,5-diodo-4-hydroxyphenyl)pro?ion-rl-oxylsuccinimide ester of formula :
I ~\
H0 ~ CH -CH -C00-N

1 x 10 moles (26.3 mg) of N-L3-(4-hydroxyphenyl) propionyloxy~ succinimide ester(origin : FLUKA) were dissolved in 2.5 ml of a mixture of benzene and ethyl acetate (50:50 v~v), after what 2.10 moles (86.4 mg) of Iodogen ~ (origin:SIGMA) were added in one step, followed by 83 mg of potassium iodide in solution in 100 )ul of phosphate buffer 0.05 M (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 MgS04 and evapo-rated in vacuo. The residue was taken up in CH2C12 or in anhydrous benzene.
The product was then purified by silica gel chroma-tography. 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 C.C.M.
(eluent : toluene/ethyl acetate 1/1 v/v) and compared with a control; RF - 0.7. The elementary analysis and mass spectrometry were found to be in conformity with the structure of the product. Yield : 58%.

1279~fiO

b) Compound 2 : N-L -(3-iodo-4-hydroxyphenyl)propionyloxy/
succinimide ester of formula :
I
S H0 ~ CH2-CH2-C- ~

It was proceeded as described above for compound 1, using the following ingredients in the proportions indicated hereunder :
- N-L3-(4-hydroxyphenyl)propionyloxy,7succinimide ester :
2.10 3 moles (o.526 g) - Iodogen (Sigma) : 2.10 3 moles (0.864 g) - KI : 4.10 moles (0.584 g) in 500 yl of phosphate buffer 0.05 M (pH: 7.4) The resulting product was put through the same purifying and control stages as compound 1.
c) Compound 3 : N-~2-(4-iodophenylsulfonamido)acetoxy/
succinimide ester of formula :

I ~ S02-NH-CH2-C00-o 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 ~ 52C1 t N H 2 - C H 2 - C O O H ~ I {~502-~ CH2 -COOH

12792~0 I ~ S02-NH-CH2-C00- ~ ~ H0-Compound 3 - Synthesis of A :

8.10 moles (2.4g) of p-iodophenylsulfochloride in solution in 5 ml of dioxan were added dropwise to 7.10 moles (0.525 g) of glycocoll in aqueous solution adjusted beforehand by sodium hydroxide lM (10 ml) at pH 9 and cooled in an ice bath. I~hen 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 filtra-tion. The filtrate was acidified dropwise under stirring at pH 2 with hydrochloric acid 6N. The expected conden-sation 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 P205 for a whole night.
25 Yield : 73% (1.75 g).
The purity of the product was controlled by C.C.M.
and by the conventional spectrophotometric methods.
- Synthesis of Compound 3 :
To a solution of 2 mmoles of A (0.682 g) and 2 30 mmoles of N-hydroxysuccinimide (0.230 g) in 10 ml of THF, cooled beforehand to 0C, were added in one step, 2 mmoles of dicyclohexylcarbodiimide (DCC) (0.412 g).
The reaction mixture was stirred and kept for one hour at that temperature. After that period of time, the coolin~
bath was removed and the reaction mixture was filtered ~27~2fiO

on fritted glass ~o. 3, and the filtrate evaporated in vacuo. The residue, const;tuted of a white foam, was taken up with CH2C12, filtered on Celite ~ then re-evaporated in vacuo. The resulting crude product was recrystallized in CH2C12. Weight obtained : 0.371 g ;
Yield : 42%. The product was identified by the conven-tional spectrophotometric methods and its composition confirmed by centesimal analysis.
Compound 4 :
-d) N-L6-(4-iodophenylsulfonamido)hexyloxy7succinimide ester of formula :

I ~ S02-NH-(CH2)5-C00-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 :

I ~ S02Cl + H2N-(cH2)s-cooH--~I ~ 02-NH-(CH2)5COOH

~ B
ON-N~

-~ I~S02 -NH- ( CH2 ) 5 -COO- ~N~

lZ79Z60 _l) Synthesis of B :
5 mmoles (1.52 g) of p-iodophenylsulfochloride in solution in 5 ml of dioxan 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 20C tthe 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 filtered with a lot of distilled water, de-watered and finally dried in a drier in vacuo over P205 for a whole night. Weight obtained : 1.30 g ; Yield : 68%.
The purity of the product was controlled by C.C.M. (silica) eluent : CHC13/Methanol (3:1 v/v). M.P. was : 154 + 1C.
The structure of the product was confirmed by centesimal analysis.
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-hydroxysuccinimide : 3 mmoles (0.345 g) - D.C.C. : 3 mmoles (0.614 g) - THF solvent : 10 ml.
The reaction time was one hour at 4C and one night at 20C.
The purification of the product was identical to that of Compound 3. Weight obtained : 0.840 g; Yield : 91 %.
Purity was controlled by C.C.M. (eluent ethyl acetate/CH2 C12 (l:l) and by centesimal analysis. The structure of the product was determined by spectrophotometry I.R. and ~;~7~60 ~1 by mass spectrometry. This was found to be conformed to the structure expected for the described compound 4.
B - Couplinq of succinimide derivatives with antibodies.
Antibody 3D3 (anti-prolactine antibody) was used in this example.
1.10-6 mole of 3D2 (10 mg/ml) in solution in 200 ~1 of phosphate buffer 0.05 M (pH : 7.4) and 200 ~1 of borate buffer 0.01 M(pH : 9) were added to 2 mg of iodinated reagent 1 (compound 1) dissolved beforehand in CH2C12 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 20C. After that period of time, the iodine-marked 3D3 was then purified by chromatography on a column of PD 10 and the peak corresponding to the marked antibody (void volume, of the column) was isolated. Its concentration was then measured by its absorbing power at 280 nm.
The isolated marked antibody was used at the concentration of 150 ~g/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 /ll at 6.5 mg/ml) were put into contact with 200 ~1 of compound 2 (1.24 mg in 3.75 ml of CH2C12, then 200 ~1 evaporated at the bottom of the tube) and 200 ~1 of borate buffèr (pH 9). The mixture was incubated for 1 h 30 min at the ambient temperature.
C - Dosaae by excess of the prolactine The reagents used, are as follows:
3D3I antibody : 150 ~g/ml by dilution in HSA at 5 g/l;
ElF antibody diluted at 1/100 [1 ~g/ml] in solution in rabbit gammaglobulins at the concentration of 5 mg/ml.
Prolactine antigene (supplied by Immunotech) : 0,5 ~l/ml in solution in HSA at 5g/1.

1~'Z792~
31a - Diluents :
- Rabbit gammaglobulin (conce -ration 5 g/l) in solution in phosphate buffer 50mM (pH 7.4) 1~79260 - HSA (concentration 5 g/l) in phosphate buffer 50 nM
- Control : HSA (5 g/l) + eluting solution of purification (50:50 v/v) The three following solutions were prepared:
- control solution : 50 ~1 of ~GL
50 ~1 of HSA
50 ~1 of control - bound solution : 50 ~1 of ElF
50 ~1 of prolactine 50 ~1 of 3D3I
- free solution : 50 ~1 of El 50 ~1 of HSA
50 ~1 of 3D3I
Each solution was incubated for one hour, at 20 C.
Then, before making any readings, 350 ~1 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 efficacity E was determinated.
The results obtained are given in the following table D - Determination by excess of CEA antiaen The above method was repeated using CEA antigen instead of prolactine and the following reagents:
Iodinated reaaent : 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 reaqent : solution of G 12 labelled with fluoresceine 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.

~' ,,^~,, lX79Z60 Dosage Iodinated Iodinated Coupling Measured of derivative derivative reaction E%
pH
3D3 mole/

Compound 1 10/1 9.0 11 50/1 9.0 18 300/1 9.0 16.5 Compound 3 30/1 9.0 8.2 100/1 9.0 9 Compound 4 30/1 7.4 2.3 100/1 7.4 5 100/1 9.0 7 Compound 2 20/1 9.0 11 Example 13.
Use of the couPling product between a polYPeptide and an iodinated oraanic 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:
NIH2 lH2 (ICH2)2 (1CH2)2 NH2-CH-C0-(NH-CH-CO)n-NH-CH-COOH

NH- ~-tCH2)2 ~

X~

1~79i~,0 in which n = 327 k = 50 A- Synthesis of reagent A.
5 mg (1.04 10-7 moles) of polylysine chlorhydrate (M.W = 48000) supplied by Sigma Chemicals were dissolved in 500 ~Ll of borate buffer 0.01 M pH 8.9; the pH of the resulting solution was thereafter adjusted to 12 with sodium hydroxide (0.5 M) and added to 2.7 ~g (5.2 10-6 moles) of above compound 1 previously deposited on the bottom of a test tube by evaporation of a solution of said compound 1 into CH2C12. The reaction was continued for one hour at 20 C under agitation.
The product reaction was eluted on PD 10 (Sephadex G 25) with a phosphate buffer 50 mM. The recovered fractions were concentrated up to 90 ~1 volume by centrifugation.
B - Couplinq of reagent A with antibodv 3D3 120 ~1 of phosphate buffer 25 nM pH 5 were added to 80 ~1 of reagent A and thereafter 100 ~1 of a solution of carbodiimide (2mg/ml of water).
After 2 or 3 minutes, 400 ~1 of a solution of antibody 3D3 (200 ~1 of 3D3 at 9.6 mg/ml in phosphate buffer 50 mM, pH 7.4 + 200 ~1 of phosphate buffer 200 mM
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 phosphate buffer 50 mM 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 ~g/ml. Said evolution was submitted to the fluorescence inhibition test described in example 12.
The fluorescence intensities of the different solutions were as follows:
Reference solution : 16 bound solution : 134 '1 Z79260 34a free solution : 150 The efficacity E was : 11.9%

Claims (18)

1. 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 a corresponding receptor, which consists in :
1) adding to said medium a first reagent consisting of a receptor for the said analyte ;

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 receptors ; one of the two reagents being coupled with a luminescent compound and the other reagent possessing a heavy atom or units containing a heavy atom ;

3) incubating the medium after addition of each reagent or after the addition of both reagents ;

4) exciting the resulting medium and S) measuring at equilibrium or during the kinetics, the signal emitted by the luminescent compound, said signal being modulated by the heavy atom effect.
2. The process as claimed in claim 1, which consists of an excess method and consists in :
1) adding to the 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 possessing a heavy atom or units containing a heavy atom ;
3) incubating the medium after addition of each reagent or after the addition of both reagents ;
4) exciting the resulting medium and

5) measuring the signal emitted at equilibrium or during the kinetics.

3. The process as claimed in claim 1, which consists of a competition method and consists in :
1) adding to the medium containing the target analyte a first reagent consisting of a receptor for the said analyte, possessing 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 after addition of each reagent or after the addition of both reagents ;
4) exciting the resulting medium and 5) measuring the signal emitted at equilibrium or during the kinetics.
4. The process as claimed in claim 1, which consists of a competition method and consists in :
1) adding to the 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 ;
2) adding, as a second reagent, the analyte possessing a heavy atom or units containing a heavy atom ;
3) incubating the medium after addition of each reagent or after the addition of both reagents ;
4) exciting e he resulting medium and 5) measuring the signal emitted during the kinetics or at equilibrium.
5. The process as claimed in claim 1, wherein the analyte is a biological or non-biological substance.

6. The process as claimed in claim 5, wherein the analyte is selected among the group consisting of antibodies , antigens, toxins, enzymes, proteins, hormones , steroids, avidin, biotin, microorganisms and haptens and non-biological substances capable of binding specifically with a ligand, such as drugs.

7. The process as claimed in claim 1, wherein the analyte is prolactin or carcino-embryonic antigen.

8. The process as claimed in claim 1, wherein the luminescent compound is a fluorescent, chemoluminescent or phosphorescent compound.

9. The process as claimed in claim 8, wherein the luminescent compound is a fluorescent compound chosen from the group consisting of fluorescein and rare earth cryptates and chelates.

10. The process as claimed in claim 1, wherein one of the reagents is labelled with fluorescein and the other is iodinated.

11. The process as claimed in claim 1 or 3, for the determination of antigens or haptens by the competition method, which 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.

12. The process as claimed in claim 1 or 3, for the determination of antigens or haptens by the competition method, which 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.

13. The process as claimed in any one of claims 1, 2 or 5, for the determination of antigens or haptens by the excess method, which 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.

14. Process according to claim 1, wherein the luminescent compound has a long luminescent decay lifetime and in that excitation of the resulting medium is a pulsed excitation.

15.Process according to claim 14, wherein the luminescent compound is a rare earth cryptate.

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, which contains:
- a first reagent consisting of at least one recceptor 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 coumpound and the other reagent possessing 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 - diluents or buffers required for the determination.

17. The kit as claimed in claim 16, wherein the luminescent compound is a chemoluminescent coumpound and wherein the said kit also comprises the appropriate chemical reagents required for excitation.

18. The kit as claimed in claim 16 or 17, for the determination of the prolactin or the carcinoembryonic antigen.