AU595821B2 - Homogeneous process for the luminescent determination and/or detection of an analyte in a medium susceptible of containingit - Google Patents

Description

DEMANDE IN cRNAIN (51) Classification internatioA G01N 33/542 (21) Numiro de la demande int4 (22) Date de de6ft internationa (31) Numiro de la demande pri (32) Date de priorite: (33) Pays de priorite: (71) Deposant (pour tous les E 29-33, rue de la F~d~rati dex 15 (FR).

(72) Inventeurs; et (715) Inventeurs/Diposants (W~ rard [FR/FR]; 17, impa F-30200 Bagnols-s/Ceze FRI; 37, residence les

(FR).

147~ ORGANISATION MONDIALE DE LA PROPRIETE INTELLECTUELLE Bureau international ftP L EEIN VERTU D RATjDE OOPER.TIONEN MATIERE DE BREVETS (PCT) 1des~brfevetF (11) Numero de pubication internationale: WO 87/100927 1 T (43) Date de publicaticin nternationale: 12 f~vrier 1987 (12.02-87) ernationae: PCT/FR86/00269 1: 31 juillet 1986 (3 1.07.86) oritaire: 85/1 1905 2 tofit 1985 (02.08.85)

FR

tals d~sign~s sauf US): C-M- CTIE-A-F8MIQU'E-FR/FR]; ion, BP 510, F-75752 Paris C6- Sseulement) MATHIS, G&- Lsse de la Capelle des Ladres, DAVIN, Thierry [FR/ Lavandins, F-84840 Lapalud (74) Mandataires: GILLARD, Marie-Louise etc., Cabinet Beau de Lomenie, 55, rue d'Amsterdam, F-75008 Paris (FR).

(81) Etats designes: AT (brevet europ~en), A.U, BE (brevet europ~en), CH (brevet europeen), DE (brevet europ~en), GB (brevet europ~en), IT (brevet europ~en), JP, KR, LU (br vet europ~en), NL (brevet europ~en), SE (brevet europ~en), US.

-Publie Avec rapport de recherchie internationale.

Avant 1'expiration du d~Iai pr~vY, pour la modification des revendica,-ions, sera republige si de telles modiications sont reques.

IX

(54) Title: HOMOGENEOUS PROCESS FOR THE LUMINESCENT ETei M1N-AT(-D5N ND/OR DETECTION OF AN ANALYTE IN A MEDIUM SUSCEPTIIBLE OF CONT MING IT (54)Titre: PROCEDE HOMOGENE DE DETECTION ET/OU DE DETERMIN ATIQN PAR LUMINESCENCE D'UN ANALYTE DATAS UN MILIEU SUSCEPTIBLE DE LE CONTENIR (57) 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 rzaction 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 ele ments of the. product~pbtained frmthe reaction of th- analyte with at least one of its receivers, one of the two reactants being coupled *With a luaminescent 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.

(57) Abii6 Un pro6d& homog~ne de detection et/ou de &etermination par luminescence d'un analyte dans tlf milieu susceptible de le contenir,'par mise en evidence du produit de I4 reaction entre l'analyte et un r~cepteur correspondant. Ce proc6d6 consiste, 1) A ajouter audit milieu un premier r~actif constitu6 d'un r~cepteur dudit analyte; 2) a~outer un second r~actif constiii'i d' au momns un des Al6ments du produit de la reaction de 1'analyte avec au momns I'un~de ses r~.cepteurs, l'un des deux r~actifs 6tant couple avec un cornpos6 luminescent et l'autre r6actif comportant un atoqie lourd ou des motifs contenant un atome lourd, 3) A faire incuber le rtiieu resultant soit apr~s I'addition de chaque rea~jif soit apres !'addition des deux r~actifs; 4) A exciter le milieu r~sultan(,"et 5) A mesurer, A ll~quilibre ou en cinkique, le sign~al 6mis par le compose luminescent, ledit signal 6tant rnodul, par l'effet Wlatome VkUrd. cin te onspdments made under SectiOw 49.

mdIs correct for Prt~tng.

4N

-I

y ;i 7 HOMOGENEOUS PROCESS FOR THE DETECTION AND/OR DETERMINA- TION BY LUMINESCENCE OF AN 'ANALYTE IN A MEDIUM IN WHICH 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 concentration of circulating organic or biological substances in biological liquids is an important step in the diagnosis of a large number -f 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 chemoluminescenc 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 ,.he presence of a given quantity of the labeled analyte, SCompetition for the receptor then takes place 35 between the target analyte and the labeled analyte. The i..qi 1 0, di' 1 :TF 1 C 1 1 U 1 2 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.

i 5 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 Sprocesses.

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 25 varies with the size of the molecular structure carrying S the Eluorescent 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 t'he excitation spectrum of the latter overlap (energy compatibility) and if the distance betwein the two chromophores is in general less than 100 A.

35 Similarly, the process described in French Patent 2 422 A AT I 3 165 uses a chemoluminescent tracer and a quenching agent which is capable of modi ying Lhe emission of the light by chemoluminescence when this molecule is at a short but not co Lision-causing dist.ince, 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 (energy compatibility).

There may also be mentioned the homogeneous processes of determination using fluorescence which are Sdescribed 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 4c lkea dct-d cd k eCIivy L This very general effect is described in the literature, for example by E.L. WEHRY in "Fluorescence", According to the state of the 3rt,.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 molecurs. 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 S r- T and radiant inter-system transitions T1I-- SO but also in non-radiant internal conversions S IAn-+S i 0 A decrease in the fluorescence due to the presence of a heavy atom is ouserved 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 quantu yield of fluorescein observed when the l1tter is chemically bonded to polyiodinated molecules such as triiodothyronine (T 3 or tetraiodothyronine (T 4 this is an internal heavy atom effect.

In the case where the heavy atom is not fixed by chemical bondi, g to the flflrescent 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 collisons of the fluorescent molecule with the heavy atom or to the formation of ,eak complexes by charge transfer. Ic actually manifests itself by an increase in the intersystem transitions;of tie luminescent molecule, buTl2i o a i n ,i te n l c n e s o s S A 5 SSl v-T and T SO, so that although the heavy atom 1 1 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 at6m 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.

25 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 thereforerelates 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 j- -6receptor, 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 5 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 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, ,nen 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.

i- i •t -7- Furthermore, the expression "component of hie 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 rethod 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, horitones, steroids, avidin, biotin, micro-organisms and hapteus and non-biological substances capable of binding specifically with a lgand, 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 description by way of reference.

Specific examples of such analytos are adeno-cortiocotropic hormone (ACTH), aiti-diuretic hormone (ADH), aldosterone, albumin, cyqlic 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, methotrexate, myoglobin, parathyrin, pepsinogen, 17 alpha-hydroprogesterone, thyroglobulin, thyroxine binding globlulin, glucagon, trypsin, HBe and Anti-HBe iapatitis, Hss and anti-HBs hepatitis, delta and anti-delta-particles, transferrin, IgG, IgM, IgA,C3, haptoglobulin, ceruloplasmin, alpha 1 antitrypsino, theumatoid factor, and more particularly carbino-enibryonic antigen (CEA), alpha-foetoprotein (AFP), estradiol, progesterone, testosterone, thyreostimulant hormone (TSH), tri-iodothyrosine free triiodothy /osine (FT3), thyroxine free thyroxine (FT4), prolactine, luteinizing hormone stimulating folliculo hotmone (SFH), total IgE.

LA L35 The luminescent compound used in the process of the invention can be chosen from the group. ionsisting of (~r 8 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 extinc- 4 tion coefficient greater than 10 above 400 nm.

Another class of fluorescent compounds suitable for the purposes of the invention conoistsin 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 1 5.695 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 oxalate and hydrogen perioxide (Ace. Chem. Res. 1969, 2, 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 cosine, 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 is to produce a covalent bond between the said reagent and the luminescent compound. It is also possible LtA to fix the luminescent compound to one of the reagents by adsorption.

1 9 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.

Among examples of "units containing at least one heavy atom" are iodinated derivatives of succinimide, such as the following derivatives -N-Z3-(3,5-diodo-4-hydroxyphenyl)propionylox.-7succinimide ester, -N-,3-(3-iodo-4-hydroxyphenyl)propionyloxy]succinimide ester, -N-2-(4-iodophenylsulfonamido)acetoxy/succinimide ester, 6-(4-iodophenylsulfonamido)hexyloxy7succinimide ester as well as the coupling products of these compounds with a polypeptide, such-is polylysine.

These iodinated organic derivatives are directly combined with the reagents used in the process accerding to

IAIV

t4~ I i

I

10 the invention 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 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.

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 WI.t':V9 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 d&cay lifetime should be higher than one microsecond. The excitation duration should be of course lower than the luminescent decay lifetime of the choosen luminescent compound. Advantageously, the rare earth chelates or rare cryptates, rouch as the ones disclosed in FR patent application 84.14.799 may be used as fluorescent compounds having long tfitoreig d o cay lifetime (or a high half-lifet.ime), On the other hand, it should b' the invention process may be carried out and that the measurement of the fluorest -escence may be effected after the deposit o z meiindium on a solid p e, such as a strip, a gol 'r ,Aj other suitable 'N F r 'Li~ir i I 11 support.

Thus, in the case of an excess method the process of the invention consists in 1) adding to the medium containing the target analyte first reagent consisiting of a receptor for the said analyte, 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 posses' ig a heavy atom or units containing a heavy atom 3) incubating the 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 medium containing the target analyte Sa first reagent consisting of a receptor for the said ana- Slyte, possessing a heavy atom or units containing a heavy atom 2) adding a second reagent consisting of the analyte coupled with a lumitescent compound 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.

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 th& said analyte, the said receptor being coupled with a luminescent compound, 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.

SThe process according to the invention is particularly applicable to immunological determinations of anti- A gens or haptens by excess or by composition.

L 1* 12 SFor 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 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 arktibody or antigen.

The following compounds were used in these examples: rabbit gammaglobulins (ORG) from Miles anti-rabbi, sheep gammaglobulins (ARSG) obtained by passing a sheeo antiserum immunized with 'rRG through a column of OEAE-cellulose 4 e a 0 s~t 13 human serum albumin (HSA) at a concentration of 10 mg/ml in a 0.1 M phosphate buffer of pH 7.4 fluorescein isothiocyanate, isomr I hydroxysuccinic ester of 3-(4-hydroxyphenyl) propionic acid (NHSPP: Bolton and Hunter's reagent) sodium met-bisulfite 1,3,4,6-tetrachloro-3a,6a-diphenylglycoluril as an iodine generator Wathman DE 52 DEAE-cellulose as an ion exchanger a column of ?D 10 Pharmacia filtration gel a phosphate buffer (PB) the fluorescence measurements were made on a PERKIN-ELMER LS 2.5/10 nm slits, with an excitation at 495 nm, an emission at 520 nm and an expansion factor of 15 the anti-prolactin monoclonal an' ,'.dies E 1 and 303 contained in ithe 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 R with fluorescein.

1.44 mg of the-Tluorescent 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 20 0 yl of the solution containing the fluorescent compound were added. The reaction was carried out for 2 Hours at romm temperature.

Yj 1. i fS;) '7 i v l ,i 2 C T T T r u I II 1 I[ I I 1 1 -14 the pH being kept at 9.5 with dilute sodium hydroxide.

The reaci:ion 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 (NaCI). The molar ratio fluorescein/protein RG) is determined by the formula: A F280 x 150,000 F/P 495 x A 0.35 A 495 280 495 in which:

A

X is the absorption at wavelength X; represents the molar absorption coefficient; E495 72,000; and 280 1.4 for 0.1% by weight/volume.

The various fractions obtained according to the salinity of the mobile phase are indicated below: [NaC1] F/P Approximate concehtration of yRG 0.05 M 1.3 120 pg/ml 0.1 M -2.2 140 pg/ml 0.2 M v3.5 240 pg/ml 0.4 M c5.5 180 pg/ml b) Labeling of SGwith iodine 2C 6 This labeling was carried out by the chloramine T ne ihod of R. HUNTER (Proc. Soc. Exp. Biol. Med. 133(3), "989, 1970). The following were brought into contact for 3 minutes: 200 al of ARS at a concentration of 3.5 mg/ml; 100 p 1 of KI at a concentration of 10 M in water; and 4Tc 1' i C^J- t^ *o 15 I -2 200 ul of chloramine T at a concentration of 10 2

M

in water, -2 and 200 Pl of a 10 M aqueous solution of MBS were then added.

The solution obtained was charged onto a PD column equilibrated with 0.05 M phosphate buffer of pH (pump throug.:;ut: 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 jl of 0.1 M PB of pH 7.4 200 )1 of HSA free solution: 50 i 1 of 0.1 M PB of pH 7.4 50 il of a fraction of Y'G labeled with fluorescein

RG

(fRC), diluted to 1/400 in HSA 150 P 1 of IISA bound solution: 50 of Y ASG labeled with iodine

(ARSG)

50 P 1 of fG diluted to 1/400 in HSA 150 1I of HSA Incubation was carried out for 1 hour 30 minutes, 250 Pl 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 whih- I is the intensity of fluorescence.

i 16 The results obtained with the fraction F/P 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 .ARSG used is an excess quantity, it is considered that all the RG are bound.

RG

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

The following were brought into contact: NHSPP evapora.ted at the bottom of a tube 100 pl ARSG 200 P 1 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) 20 P 1 chloramine T (5-10- M) 20 l -2 and 20 Pl of 5-10 M MBS were added af-er 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: NHSPP (10 3 M) 100 1 KI (101 M) 100 1l chloramine (10 2 M) 100 i After a contact time of 3 minutes, 100 Pl of j

A.

07 177 17 MBS (10 2 M) were added.

Extraction was then carried out with 2 x 2 ml of benzene containing 20 pl of dimethylformamide. The orga'nic phase was evaporated in another tube and 100 l of,, RSG were added. The reaction was carried out for minutes in ice. The mixture was deposited on a PD 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 f was used to prepare a boind solution in the same proportions as in Eyample 1, using the fraction F' 1/400, F/P 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 P1 iodine generator in CCl 4 5"10 2 m, evapora ted KI 510 2 M 20 Yi phosphate buffer, 0.05 M, pH 7.4 40 P 1 After a contact time of 1 minute, extraction was carried out with 2 x 1 ml of benzene in dimethylformamide The organic phase was evaporated in another tube and 10 p 1 of__/ARSG were added, after which the reaction was left to proceed for 15 minutes in ice.

The YARSG formed were separated off by dialysis against 1 liter of 0.05 M phosphate buffer of pH 7.4.

A 1/10 dilutionof RS 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.

18 TABLE I Free solution Solutions tested Example No.

Reference 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 0.2.5 Example The effect of labeling the YRG with fluorescein was evaluated. The YARSG labeled with iodine according to Example 4 and diluted to 1/20 in HSA, and the various YF fractions prepared in Example 1, were used.

RG

The fluorescence measurements were made according to the procedure of Example 1. The following results were obtained: RG fraction, F/P Bound solution Free solution E

RG

1.3 5.4 6.5 0.17 2.2 11.7 14.7 0.20 26.3 35.2 0.25 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 This increase is definitely associated with the delocalization -of the energy of the ex8 ited state of the fluorescein by inter- J0 i 1 19 molecular transfer between fluorescein molecules.

Example 6 The dilution curve was established for the antibody ARSG labeled according to the procedure of Example 4 with the F F/P 5.5, diluted to 1/400.

The following results were obtained: Dilution of YRSG in HSA I E% 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 -l ml of a solution of E 1 containing 9.7 mg/ml was mixed with 0.2 mg of FITC (molecular probe) in 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 columnewas 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 NaC1, the pH being 7.4.

-2 Q t i 20 The peaks eluted with the 0.4 M NaCI and 0.7 M NaCI buffers were collected and dialyzed.

The optical density observed for the peak obtained with the 0.4 M NaC1 buffer was 0.184 at 280 nm and 0.167 at 495 nm, which corresponds to an approximate antibody concentration of 90 g/ml and to a ratio F/P of about 4.

b) Labeling of the monoclonal antibody 3D3 with iodine The protocol is i, 'ntical to that of Example 4.

The following products were used: NHSPP (10- 2 M) 200 1 l Siodine generator (5-10- M) 40 pl KI (5-10-2 M) 40 pl phosphate buffer, 0.05 M, pH 7.4 40 ul Extraction was carried out with 2 x 1 ml of benzene at a concentration of 1% in dimethylformamide.

After evaporation, 200 p of 3D3 containing 3.3 mg/ml were added and the reaction was left to proceed for 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 pg/ml.

c) Determination by excess fluorescent E 1 (EF) at a concentration of 3 pg/ml in a solution of HSA containing .5 mg/ml; iodine-labeled 3D3 (3D3

I

at a concentration of 110 pg/ml in the same solution of HSA; 3D3 at a concentration of 110 pg/ml in the same solution of HSA; prolactin, PRL, at a concentration of 0.6 Yg/ml in the same solution of HSA.

The following threq solutions were prepared: reference solution: 150 l of HSA containing 5 mg/ml

CW~--

'v

JL.--

2 1 of PRL I F bound solution: 50 Pl of E 1 5 0 l1 of 3D3 pl of PRL Each solution was incubated for 2 hours at room temperature and 100 1 l of HSA and 250 1 l of phosphate buffer were added.

The fluorescence measurements made according to the procedure of Example 1 gave the following results: Fluorescence reference solution 29.7 bound solution I 197.3 bound solution 231.1 A 33,8 E 0,,17 The same phenomenon is therefore observed in an excess method (Example by labeling t.o antibodies having d&tferent 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 pl of HSA containing 5 pg/ml, and the same reagents E and 3D3 but varying their 1 concentration, and the efficiency E of the inter-system transition was measured as a function of the incubation time.

The followipg concentrations were used:

F

El at a concentration of 3 rg/ml in a solution of HSA containing 5 pg/ml Lh Ac =3D3 at a concentration of 360 pg/ml in a slution of HSA containing 5 pg/ml 0 M L. i L l4; 22

F

E /2 at a concentration of 1.5 pg/ml in the same solution of HSA Ac 3D3 at a concentration of 360 g/ml in the same solution of HSA

F

E1/ 4 at a concentration of 0.75 pg/ml in the same solution of HSA I I Ac 3 3D3 at a concentration of 120 g/ml in th-e same solution of HSA (E at a concentration of 1.5 ug/ml in the same solution of HSA Ac/3 at a concentration of 120 Pg/ml in the same 1/3same 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 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, 7.5 and '0 ng/ml in a solution of HSA containing mg/ml, were prepared.

P

1 of each solution were incubated in a tube with 50 of Econaining 3 g/ml and 50 of 3D31 containing 120 pg/ml, for 30 minutes at room temperature, and 350 p 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 P 1 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 prolactin, PRL, express.ed 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.

'9 1 J h_ 7 i It 32 23 Example g0 This example was carried out using the anti- CEA antibodies G 12, 0 13 and G 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 Pg/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 I) 200 P 1 iodine generator (5'10 2 M) 40 P 1 KI (5'10 2 40 p 1 phosphate buffer. 0.05 M, pH .4 40 P 1 S1, (5 mg/ml) 200 p 1 The 'action having an optical density of 0,84 at 280 nm was collected; its concentration was 600 pg/ml.

c) Labeling 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 Pg/ml.

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

GF was diluted to 1/2000 in IISA containing 12 mg/ml.

I was diluted to 1/6 in HSA.

15 The following three solutions were prepared:

A*

)r

I

K

I

I

T.

-24 reference solution: 100 Pi of HSA 100 Jl of buffer bound solution: "50 of G 2 diluted to 1/2000 50 l of I diluted to 50 of 15 1/6 100 P1 of the standard free solution: 50 p1 of CF diluted to 12 1/2000 50 r l of C 1 I diluted to 15 1/6 100 P1 of buffer These solutions were incubated for 2 x 1 hour at 45°C and 300 v! of phosphate buffer were added.

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

The same experiment was carried out with the addition, in a third incubation, of the antibody GI 13' the specificity of which is differenv from that of C 12 and G The following solutions were te ,ed: bound solutiol 50 p 1 of GI2 ied to 1/2000 P1 of G diluted to 1/6 rI of G diluted to 1/3 100 ril of the standard IV S- 25 free solution 50 il of GF diluted to 1/2000 1 of G 5 diluted to 1/6

I

l of G 1 5 diluted to 1/6 \50 fl of GI diluted to 1/3 S100 p 1 of buffer i These solutions were incubated for 3 x 1 hour at 45°C and 250 pl of phosphate buffer were added. The following results were obtained: Solution Intensity of Efficiency fluorescence reference solution 81 4 0.41 bound solution 106.7 free solution 124.9 A 18.2 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 tf CEA, containing 300, 200, 100 and 0 ng/ml, were prepared. Each solution was incubated with G dilutad to 1/3000 in HSA and with G and G 12 13 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 AF E% fluorescence reference solution, 54 solution containing 300 ng/ml 65.9 11.9 I solution containing 200 ng/mil 68 9.8 0.41 solution containing, 100 ng/ml/ 71.8 6 0.25 solution containi l f 0 ng/ml 77.8 0 0 0 CS2 i A T; I i *i~ii*i 26 Example 12 Use of iodinated derivatives of succinimide i as units containing at least one hoavy atom. A. Preparation of iodinated derivatives of succinimide a) Compound 1 N-L3-(3,5-diodo-4-hydroxyphenyl)propionyloxyZsuccinimide ester of formula 0 HO 0 C-2-CH 2

-COO-N

0 -4 1 x 10 4 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 -4 acetate (50:50 after what 2.10 moles (86.4 mg) of lodogen0 (origin:SIGMA) were added in one step, followed by 83 mg of potassium iodide in solution in 100 Yl of phosphate buffer 0.05 M (pH 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 4 and evaporated in vacuo. The residue was taken up in CHC1 2 or in anhydrous benzene.

The product was then purified by silic- 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 C.C.M.

(eluent toluene/ethyl acetate 1/1 v/v) and compared with a control; RF a 0.7. The elementary analysis and i mass spectrometry Wre found to be in conformity with ;the structure of the product. Yield 58%.

4 kJ S s S t V i i

"I

7 '11 39 27 Compound 2 :N-L3-( 3 -iodo- 4 -hydroxy'phenyl)propionyloxy7/ ;duccinimide ester of formula HO 0 CH C. O- 0 It was proceeded as described above for compound 1, using the following ingredients in the proportions indica'ted hereunder N-L3- (4-hydroxyphenyl)propionyloxy~succinimide ester 2.10 3moles (0.'526.g) Iodogen R(Sigma) :.2.10 -3moles (0.864 g) KI :4.10- moles (0.584 g) in 500 pl of phosphate buffer 0.05 M (pH: 7.4) The resulting product was put through the same purifying~ and co-trol stages as compound 1.

c) Compound S N-L"-(4-iodophenylsulfonamido)acetoxY-! succinimide ester of formula I so S 2 NH-CH 2 co 0 The synthesis of compound 3 was ma dg in two separate stages after purificatlion and isolation from intermediate product A, according to the following reaction diagram 1 0K i-0Cl NH CH 2 -COOH 1 0 ~~..SNH.CH.C

A

14Af ;4cw Ttk i 28 I -SO 2 -NH-CH -COO-N HO-N Compound 3 Synthesis of A -3 8.10 moles 2 4 g) of p-iodophenylsulfochloride in solution in 5 ml of dioxan were added dropwise to -3 7.10 moles (0.525 g) of glycocoll in aqueous solution ic 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 cf 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 P 2 0 5 for a whole night.

Yield 73% (1.75 g).

The purity of hhe product was controlled by C.C.M.

and by the conventional spectrophotometric methods.

j.j Synthesis of Compound 3 To a solution of 2 mmoles of A (0.682 g) and 2 mmoles of N-hydroxysuccinimide (0.230 g) in 10 ml of THE, cooled beforehand to O°C, 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 cooling bath was removed and the reaction mixture was filtered 1

L

J A -29 on fritted glass No. 3, and the filtrate evaporated in vacuo. The residue, constituted of a white foam, was taken up with CH 2 C 1 2 filtered on Celite then reevaporated in vacuo. The resulting crizde product was recrystallized in CH 2 C1 2' Weight obtained :0.371 g Yield The product was identified by the conventional spectrophotometric methods and its composition confirmed by centesimal. analysis.

Compound 4 d) N-Lf6- (4-iodophenylsulfonamido) hexyloxy ?succinimide ester of formula I -so 2 NH-(CH Cs-0Nh 0 The synthesis of Compound 4 was conducted i~n two stages after isolation of the intermediate product 110 and according to the following react'on diagram SO Cl H N-(CH 2 0 2 NH(CH 2 5

COOH

B

ON-

IAIi 4IW 30 1) Synthesis of B mnioles (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 "he addition of the iodinated reagent, the ice bath was removed and the reaction was allowed to continue for 3 hours at 20 0 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 HC1 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 vacuc over P 2 0 5 for a whole night. Weight obtained 1.30 g Yield 68%.

The purity of the product was controlled by C.C.M. (silica) eluent CHCl 3 /Methanol (3:1 M.P. was 154 1*C.

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 4 0 C and one night at 200C.

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/CH 2 Cl2 and by centesimal analysis. The structure of the product was determined by spectrophotometry I.R. and J-o 31 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 3D 3 (anti-prolactine antibody) was used in this example.

1.10 6 mole of 303 (10 mg/ml) in solution in 200 yl of phosphate buffer 0.05 M (pH 7.4) and 2 0 0 y l of borate buffer 0.01 M(pH 9) were added to 2 mg of iodinated regaent 1 (compound 1) dissolved beforehand in CH 2 C1 2 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 0 C. After that period of time, the iodine-marked 30 3 was then purified by chromatography on a colum 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 Jg/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 pl at mg/ml) were put into contact with 2 0 0 jl of compound 2 (1.24 mg in 3.75 ml of CH 2 C1 2 then 200 l evaporated at the bottom of the tube) and 200 l of borate buffer (pH The mixture was incubated for I h 30 min at the ambient temperature.

C Dosage by excess of the prolactine The reagents used, are as follows 3D3 antibody 150 g/ml by dilution in HSA at 5 g/l; EF antibody diluted at 1/1000 f lyg/ml J in solution in rabbit gammaglobulins at the concentration of 5 mg/ml.

prolactine antigen (supplied by Immunotech) 0,5 g/ml in solution in HSA at Diluents Rabbit gammaglobulig (concentration5 g/l) in solution in phosphate buffer 50mM (pH 7.4) Tot. 32 HSA (concentration 5g/1) in phosphate buffer 50 mM Control HSA (53/1) eluting solution of purification (50:50 v/v) The three following solutions were prepared control solution 50 1 of GL 50 Y1 ofd HSA j1 of control

F

bound solution 50 1 of E1 l of prolactine 1 of 303 free solution 50 1 of E 1 l of HSA yl of 303 Each solution was incubated for one hour, at 200 C. Then, before making any readings, 3 50ypl of phosphate buffer 0.05 M (pH 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 antigen The above method was repeated using CEA antigen instead of prolactine and the following reagents iodinated reagent The sclution obtained under point B with coumpound 2. Said solution, was ajusted to 0.10 mg/ml after the purification on PD Fluorescent reagent solutiotr of G 12 labelled with fluoresceine and diluted to 1/2000.

The incubation was carried out for two hours at 450 C.

The obtained results are also in the following table.

-IN SA(ocetaio gl)i hopae ufr 0m otrl HS ltigsluino puiicto (5050 33 lodinated Dosage lodinated derivative Coupling measured derivative roaction pH E% 3D3 mole mole 3 Coumpound 1 10/1 9.0 11 S50/1 9.0 18 300/1 9.0 16. 3 Sz Coumpound 3 30/ 1 9 .0 8.2 10 0/1 9.0 9 0' 1 Copound 4 30/1 7.4 2. 3 S: 100/1 7.4 100/1 9.0 7 Compound 2 20/1 .0 11 Examole 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 was prepared; said coupling product is hereinunder named reagent A.

This reagent A may be represented the following statistic formula NH

NH

(CH

2 2 (H 2) 2

NH

2 -CH-CO-(NH-CH-CO) -NH-CH-COOH S-0(CHO

OH

t j- **1W, ~bin which n 327 k A Synthesis of reagent A.

5 mg (1.04 17 moles) of polylysine chlorhydrate (M.W 48000) supplied by Sigma Chemicals were dissolved in 500f of borate buffer 0.01 M pH 8.9;the pH of the resulting solution was thereafter ajusted to 12hith sodium hydroxide(0.5 M) and added -6 to 2.7 g (5.2 10 moles) of above compound 1 previously deposited on the bottom of a test tube by evaporation of a solution of said compound 1 into CH 2 Cl 2 The reaction was continued for one hour at 200 C under agitation.

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

8 Coupling of reagent A with antibody 3D 3 120 l of phosphate buffer 25 mM pH 5 were added to 80 yl of reagent A and thereafter 100 l of a solution. of carbodiimide (2mg/ml of water).

After 2 or 3 minutes, 400j1l of a solution of antibody 303 2 0 0 yl of 3D 3 at 9.6 mg/ml in phosphate buffer 50 mM, pH 7.4 200y1 of phosphate buffer 200 mM pH 8) The incubation was effected for 1 hour at 20" C, then one night at 40 C and 1 hour at 200 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 pg/ml. Said evolution wa;submitted to the fluorescence inhibition test described in example 12.

The fluorescence intqnsities of the different solutions were as follows Reference solution 16 bound solution 134 free solution 150 The efficacity E was 11.9 w roa

R-

A.

1Le

Claims (18)

1. Homogeneous process as hereinbefore defined 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 receptor to the analyte, characterized in that 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 aiialyte and at least one of its receptor; one of the two reagents being coupled with a luminescent compound and the other reagent possessing a heavy atom or units containing a eas heavy atom; 3) incubating the medium after addition of each reagent or after the addition of both 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 as hereinbefore defined.

2. Process according to claim 1, which consists of an excess method, characterized in that it consists in 1) adding to 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 possessing a heavy atom or units containing a 3 heavy atom; 3) incubating the medium after addition of each reagent or after the addition of both reagents; 4) exciting the resulting medium and measuring the signal emitted at equilibrium or during the kinetics. f y 0- ~i~K I ii I~f~ii~iii~ 7 36

3. Process according to claim 1, which consists of a competition method, characterized in that it 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 measuring the signal emitted at equilibrium or dur- ing the kinetics.

4. Process according to claim 1, which consists of a competition method, characterized in that it 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 the resulting medium and measuring the vignal emitted during the kinetics or at equilibrium.

Process according to any one of claims 1 to 4, characterized in that the analyte is a biological or non- biological substance.

6. Process according to any one of claims 1 to 4, characterized in that the analyte is selected among the group consisting of antiboqies, __tigens, toxins, eazymes, proteirs, hormones, steroids, avidin, biotin, micro-orga- nisms and haptens and non-biological substances capable of binding specifically with a ligand such as drugs. V \j i I 37

7. Process according to any one of claims 1 to characterized in that the analyte is prolactin or carcino- embryonic 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.

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 compouud 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.

Process according to any one of claims 1 2 and to 14, for the determination of antigens or haptens S 35 by the excess method, charpaterized in that it consists -38 in incubating the medium containing the target antigen with a first, fluorescein-labelled antibody in the presence of a givern quantity of a second iodinated antibody, or vice versa, the said atibodies having different specificities for the target antigen.

16. A kit for the homogeneous, as hereinbefore defined 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 receptor to the analyte, one of the reagents being coupled with a luminescent compound 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 the diluents or buffers required for the determination.

17. Kit according to claim 16, characterized in that the S* luminescent compound is a chemoluminescent compound and wherein 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. A-7 9 ~N 39 ABSTRACT The present invention relates to a honogeneous 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, process consisting in t 1) adding to said meditl 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 :eceptors 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) incu- bating the medium after 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. J 1 1