DE2545980A1 - METHOD FOR THE IMMUNOENZYMATIC DETERMINATION OF PROGESTERONE - Google Patents

Description

PATENT ADVERTISERS WILCKEN StLAUFER

DR. HUGO WILCKEN · DIPL.-INC. THOMAS WILCKeN · OIPL-CHEM. DR. WOLFGANG LAUFES

LOBECK LOBECK MÖNCHEN

INSTITUT PASTEUR

25, rue du Dueteur Roux

Paris 15eTne (France)

Method for the immunoenzymatic determination of progesterone

The invention relates to a method for quantitative immunoenzymatic determination or dosage of progesterone,

The method according to the invention is based on a determination technique in which a competition between the free Progesterone molecules on the one hand and progesterone molecules on the other, which are attached to an enzyme are coupled to the same antibody sites of the antiprogesterone (Ab), and the precipitation of the Ab insolubilized anti-amma globulins.

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LU »EC K 1 · Brail · Strofle 52-54 ■ Telephone (0451) 758 88 # I MUNICH tO · PrinzregentensfroBe 74 · Telephone (089J 4773« Posfichedc: Hamburg 138119-204 Banks: Commerzbank Lübeck (BLZ 23040022) Account No. 390187 ■ DeuUche Bank MOnchen (BLZ 70070010) account no. 65/05433

The development of the imrnunoer: zyriatis.? He.n Best iTKiunp-en is based on recent research. " ¹ Ie ^p .ixierunfr an enzyme to an antigen or to an antigen has been described by histological examinations CNAKAME PK and PIFRCK Π. ~>. -Jr (1 ^ 7). Enzyme-Iaheled antibodies. Prenaration ani application for the localization of antigens. J. Histochem. 1 · 'ί, 9? 9, AVRAMEAS F. Cl9 ^). Coupling of enzymes to pm ^f .eLns with p; lutaraldehydes. Use of the conjugates for the detection of antigens and antibodies . Immunochemistry 6, M), The. First immunoenzymatic determination concerns the rest of the gonadotronic hormone in the urine Cl'hormone ponadotrope ehorlonique CHCO) "}, whereby HCG is marked with peroxidase * - and the antibody is coupled to cellulose, fVAN MEEMEN BK and PC "U ^T TRS AHWM (1971) Immunoassay usine; antigen-enzyme conjugates. F ¹ ERS Letters 15.2321. The authors use the technique of polymerizing the first antibody or the technique of polymerizing the second antibody. In the first type the technique gave one a feeling opportunity in the same;. The order of magnitude achieved in the inhibition of HMmoagglutination. The second type of technique is ten to twenty times more sensitive, but it does not achieve the sensitivity of radioimmunological determination.

The radioimmunological determination is based on an isotope Dilution in a reaction medium that conforms to the law of mass action follows. There is competition between the radioactive molecules and the unknown comparison or sample .Tolecules opposite the pale antibody seats.

The first immunoenzymatic determination of a 'substance with * a p-eringen molecular weight: DMP Γ ENGVALL E. and PERLMAMN P. Ί97?). Snzyme-1 in>: ed immunosorbet assay (ELISAT.

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25A5980

III Quantitation of sneoi ^ i - ¹ antibodies by enzyme-labeled anti-immunoprlobulin in antifren-coated tubes. The Journal of Immunology IGQ, 129: does not give a satisfactory sensitivity that one has to add 5.10 " ^H ONP * ^f ol / liter in order to achieve a 50 H-lt? E InMM of the enzymatic activity.

Other immunoenzymatic systems make sense for the Mornhin CrUBENSTEIN KE, f-OFNEIDER ^D .S. and TILMATI EF (197?) "Homogeneous ^M enzyr.e immunoassay; a new inrnunochemical technique; BBRC ¹ J", R ¹ JfJ and for the "^ stro ^ ene [VAN WEEMEN BK and SCHUüRS AT ^T .WM (197?) ..?. not the procedure, the sensitivity of radioimmunoassay provisions - described immunoassay using hapten-enzyme conjugates FERS Letters ^ 17J, the sensitivity of these provisions reached even after a ^v erbesserun.

French Patents 73.17.1R1 and 71. ¹ 16,179 relate to methods for the detection and for the determination of hapten. The first patent describing the nature of the hapten coupling states that the increased molecular weight substance used to generate the antibodies is different from that used for the conjugated hapten-enzyme coupling.

The difference in the type of coupling can be based on the following things:

a) a different bond or bridge bond of a chemical nature,

b) Haptens of different chemical types, dip one represent immunological relationship,

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ORIGINAL 1NSPECTÖÖ

c) a connection ar. another 3t ^ l2e of the ^IJ aptenmoleküls.

Thus, the progesterone estinmunr "significantly as ^13" in "the Pe3timmunp; R3yr.tene with which the al'ralis ^ he Progesteronll-succinylnhosnatase with the anti-t ^ ror-estrone-ll-succinyl-Rinderserunialbumin combined is , four to ten times less sensitive are p; epenüher dem "ro ^ estPfoii than the alkaline progesterone-11-sue: a nylphosphatase / anti-nrc rest er on-1 2-succinyl bovine serum albunin systems (page 10, lines 11 to? O).

The basic principle of the method described in this patent specification follows in the Oepsatz to the determination method of the present ^ rfindunn; in which the same coupling of the Haptens and the higher molecular weight substance and the Hapten enzyme is used.

The second patent does not concern the regulation proceed for progesterone according to the procedure of the present invention.

With the present method for immunoenzymatic determination of progesterone according to the invention one can obtain an amount / sensitivity curve, its sensitivity is equivalent to the radioimmunological determination, but with the method according to the invention, the There are no disadvantages associated with radioimmunological examination methods: Expensive examination materials and examination devices (such as radioactive counters, scintillation fluids, radioactive substances), difficulties with the radioactive substances (such as degradation of the antigen and radioactive antibodies, the element period) and sanitary and legal problems.

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Excellent sensitivity of the residual tuning method is obtained in particular due to the use of an antiprogeateron-specific antibody.

A hapten antibody is produced in which the hapten is coupled to a higher molecular weight substance, usually made up of a protein. This coupling product is injected into an animal and the antibodies are isolated in a conventional manner. In the method according to the invention these means are adapted to the particular needs and you get a very sensitive immunoenzymatic Determination procedure.

The invention relates to a method for the immunoenzymatic determination of progesterone on the basis of competitive reactions in a liquid environment of progesterone molecules (referred to as Ag molecules) and of an enzyme such as β-D-galactosidase coupled progesterone molecules (referred to as Ag ^ GZ) against the same antibody seats formed by an antiprogesterone serum (referred to as Ab,.) and a precipitation of soluble complexes obtained by a second insoluble antibody (referred to as Ab-), the reaction leads to a culot containing all antibody populations, and a supernatant or liquid supernatant (surnageänt), which contains all Ag and Ag-ίϊΖ tractions that have not _{reacted with Ab 1} , which is characterized in that the same progesterone derivative as namely the 11-eC -hemisuccinate of progesterone for the coupling with the ß-Oalactosidase and used for the production of the Antiprogesteronserum.

In the present method according to the invention, the coupling of the progesterone is realized with the aid of a

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functional grouping resulting from the hemisuccinate radical exists at carbon 11, whereby an immunogen can be obtained, the injection of which into an animal produces the formation of particular ones and specific responses. This “great specificity enables an immunological determination of the nlasmic Progesterone without prior chromatography of the extracts.

The immunoenzymatic ^ estimmur.Pis method requires mutual influence or V / echs ^ lw? It consists of four types of molecules: an antigen (or skin) (At), which is produced by progesterone; an antiserum (Ah ..) which dispenses with the product made by 11-oC - "emisuccinate of polysterone; an antigen which is marked by an enzyme, that is, the 11- -hemisuccinate of progesterone which is marked by 3-oalactosidase ( Ag-ClZ); and finally an insolubilized antiserum precipitant (Abp). At the end of the reaction, a sediment or precipitate is obtained which contains all antibody ponulations and a supernatant which contains the fractions of Ag and Ag-OZ which do not have reacted with Ab _1.

The measurement of the enzymatic activity becomes more advantageous Way carried out by means of the sediment (culot).

Carrying out the method according to the invention requires the preparation or production of the individual components. The selected enzyme, namely ß-D-ftalactosidase, is first purified using an Eschericia Coll strain. The purification can include, for example, the following process steps: building up the bacteria, taking them up with two volumes of cleaning buffer (tampon de purification) (10 mM Tris-Acetat oH 7.6.10 mM of MgCl ₂ , 5 mM of Mg-TItriplex, 10 mM ft -Mercaptoethanol (ß-SH) and 0.5 % NaCl) grind and sonicate with ultrasound, after centrifugation, dilute the supernatant twice with

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the same buffer, which causes an execution to be executed Proteins is evoked. Pann we; 1 centrifuged uni decanted and the overstani is filled in again with a saturated 33% ammonium sulfate. This precipitate is suspended with the same buffer until complete removal of ammonium sulfate dialyzed un-1 die ß-Oalaetosldase is purified on a DEAE-Senhadex column (Sephadex en gradient de molarite). The enzymatic activity is then measured.

The determination is carried out by ? ml of a f .- 'ialactosidase mixture are poured into a "PM ^ -Pu ff er, which consists of 70 mM Na ₂ HPO ^, 30 mM NaHpPO ^, 1 tnM MgSO ₇ ,, o, 2 mM MnSO ₁₁ and 2 mM Mg- Titriplex consists of adjusting the nH value to 7 and adding 7 ml / 1 ß-SH with 0.5 ml / ONPG (ortho-Nltrophenyl-ß-D-galactosidase) up to Ί Incubate at 2R ⁰ C and the reaction is stopped with 1 ml Na-CO-. (IM). The number of enzymatic units is given by the following formula:

Enzymatic units: = D.O. 4? 0 nm χ dil

£. t (mn)

with £ = 5.1Ο "" ³ .

If the incubation is carried out in "PM ₂ " ft-SH at 15 ° C, the number of enzymatic units is obtained by dividing by the correction factor 2.6. "

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That used to measure enzymatic activity The substrate is ortho-trophenol calactoside. In present ß-iialactoslü3ase becomes ortho-nitrophenol in the reaction medium (with yellow color) and galactose franked. The postage of the ortho-nitrophenol is a function of the existing ones Amount of enzyme in the medium. At a certain concentration of the substrate and at a certain or fixed During the incubation period, the optical density, measured by snectrophotometry, changes accordingly (due to the color intensity) the larger or smaller menjre of the β-galactosldase.

This allows a certain enzymatic ^ unit to be determined such as the amount of ß-galactosidase, dl # hydrolyzes a micromolecule substrate in a unit of time (equal to one minute).

Then the coupling of the II-flC-hemisuccinate des Progesterone carried out with the ß-galactosidase und.es is cleaned as described above using a soluble Garbondiimlds. This reaction is carried out in two stages. During the first stage, known as "Activation", the reaction of the carbodiimide with the II-eC-hdmisuccinate of progesterone and in this way an O-acyl compound is obtained which is very reactive with nucleophiles is: ·

R'N = C = NR ¹

N ¹ R -s- C - NHR ' f R. O
- C = O

\ "

-Q-

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In the second stage of the process, the O-azyl connection is reacted with the ß-cfalactosidase. In this case, a covalent coupling of the hemlsuccinate de :: - ^p rofester ^ ns is carried out in detail, the COOH group of which has been activated by means of the carbodiimide, with the lysine NHp- ^ rupnen of ^ -lala ^ tosiriase.

NO'

I /

R-C-O-C

+ R "NH.

H /

ρ _ C - NHR "+ O = C

NHR ¹

MHR '

The reaction components are located in progesterone-oalactosidase in a ratio Pro / OZ = 130.

In a preferred embodiment of the Konolung in the method according to the invention, 7.10 "mol 11 - ^ - Femisuccinat des
Progesterone (0.1 ml) was added to 10 ^"5 mol of carbodiimide, HCl (0.01 ml), the pH is adjusted to ^, 7 and allowed to incubate for 30 minutes at ambient temperature. Then, 5, ^ 5.10 mol ß -Galactosidase (0.1 ml) was added, which represents an enzymatic activity of ΐβΟ.ΟΟΟ EU / mg. The pH is adjusted to 5.5 and it is allowed to incubate for 12 hours at H ⁰ C. The reaction is then carried out by 1 ml of "PMp" buffer is stopped and the patient is dialyzed with 2 liters of buffer containing 5 g of carbon dextran, with the remaining enzymatic activity

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142,000 EU / mg. The product obtained in this way is passed through a Run Sephadex G 25 column (0 = 1 cm, V = 10 ml) to remove the progesterone bound to β-galactosldase (Pro = Oz) to separate free progesterone.

The procedure then continues with the antiprogesterone serum to obtain. This is a particularly important process stage, since the specificity of the Antiprogesterone antibodies reduce the final precision of the determination method certainly. According to the method according to the invention, one has the ll - ^ - femi-euccinate of progesterone known processes coupled to bovine serum albumin (RSA) (Erlanger B.F., BOREK. F. BETSER S.M. and LIEBERMANN S. (1957). Steroid protein conjugates J. Biol. Chem. 228, 713. VAITUKAITIS J., RABBINS J.B. NO STROKE. E. and ROSS T. (1971). A method for producing specific antisera with small doses of immunogenic J. Clin. Endocr. 33,988).

The coupling of progesterone with the help of a functional group of the hemisuccinate introduced at carbon 11 enables the manufacture of an immunogen which, when injected into an animal, results in the production of antibodies which are particularly specific, as can be seen from Table I below.

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Table I.

Steroids Cross reactions (?) Deoxycorticosterone 3 5 ß-pregnane 3.20-dlon 2, P 5 rt-pregnan 3,20-ion 2 6 ß-hydroxyprogesterone 1.2 17 * C -hydroxyprogesterone 0.5 20A -hydroxyprogesterone o, n Corticosterone 0.3 testosterone 0.0R Cortisol 0.03 5 pregnenolone 0.03 Aldosterone 0.008 estriol <10 " ⁵

These results were obtained according to the following principle:

The antiprogesterone antibodies that bind to progesterone marked with tritium can release radioactive progesterone and spontaneously unlabelled progesterone displace after an exchange with unlabelled progesterone, which has been introduced in the medium.

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The balance between the proportion of unlabeled progesterone antibodies (Ac) and the labeled Progesterone-Ac adjusts is a function of the unlabelled progesterone added to the medium. If you instead of the unlabelled progesterone (progesterone froide) Adding another steroid such as aldosterone, you can only have a very small exchange between the bound radioactive Determine progesterone and the aldosterone present in the medium. The anti-progesterone antibodies are very specific for the progesterone and they only bind to the aldosterone in a very small way.

The lower the exchange between the initial radioactive complex (progesterone antibodies) and that added in the environment Mixture, the lower the percentage of reaction croise "e. This allows great specificity For example, an immunological determination of the plasmatic progesterone without prior chromatography of the extracts, obtained after a simple extraction using an organic solvent.

Finally, the antieerum is produced in two stages is precipitated. In the first place, the proper preparation of the same is essential, in the second place, the insolubilization the obtained antibodies by polymerization. In order to produce rabbit serum anti-globulins, one injects the "procedure described above" 10 mg dissolved in 1 ml physiological serum and 2 ml Freund's complete from animals such as sheep or goats. The injections were with 2.5 mg of antigen carried out by intramuscular and subcutaneous routes and the next day with 2 mg intravenously. The amount of antibodies per millimeter was determined by microprecipitation

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analysis, three antisera were used, with 2 mg / ml (goat), 13 mg / ml (goat) and 13.6 mr / ml (sheep) being titrated. The Insolubles; of the antibody-anti § -Glubolins from rabbits was realized by means of ethylene-chloroformate according to the following procedure: Precipitate the ßammagluboline out by a saturated solution of ammonium sulfate are two volumes of serum fed in and lSsst with stirring for 30 minutes at room temperature and centrifuged for 15 minutes . The precipitate is then taken up and dialyzed against a sodium phosphate buffer (nH 6.3) until the ammonium sulfate has been completely removed (control with 1% BaCl ₃ ). The product made in this way, which consists of gamma globulins and other proteins, is passed over a DEAE cellulose column and the lammagluholins are eluted by a sodium phosphate buffer (pH = 6.3) and are obtained in a very pure state.

In another procedure, the goat or sheep serum is dialyzed directly with sodium phosphate buffer and thrown through given a DEAE cellulose column. The elution becomes the Gammaglobullne is made with the same sodium phosphate buffer carried out. The so obtained Oammaglobullne are very pure. A solution of amma globulins is then prepared at 50 mg / ml and then it is poured in dropwise ethyl chloroformate to in a solution of 0. OiI ml / ml. The pH will be held at 5.0. After three hours of stirring, it is centrifuged and washed three times with physiological water before the absorbs the precipitate with buffer. It is then homogenized with a syringe that has a very fine needle is equipped. To determine the amounts of polymerized antibodies to be used for the determination, the binding force of the polymerized antibodies must be determined

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will. For this purpose, one incubates in each of different solutions (1/1, 1/10, 1/20, 1/50) of antipolymerized antigammaglobulins intersecting dilutions (dilutions croissants) of the antiprogesterone serum ranging from 1 / loo to 1 / 10,000. After centrifugation, the same amount of tritiated progesterone is added to the supernatant in each reaction tube in order to test for the possible presence of anti-progesterone antibodies. The experimental procedure can be carried out in the following way: Mix 0.2 ml of anti-progesterone serum in 0.2 ml of polymerized anti-γ-globulins. It is then allowed to ^{incubate for 30 minutes at U 0} C, centrifuged for 10 minutes at 5000 revolutions per minute and 50,000 com Ή -progesterone (0.1 ml) is added to the supernatant and it is allowed to incubate for 30 minutes at ambient temperature and then for 30 minutes in melting ice . It is then separated with 1 ml of carbon dextran, after minutes of contact it is centrifuged and the radioactivity is then measured in the supernatant. The results obtained are shown graphically in the accompanying drawing (Fier. 1). A sufficient concentration of polymer is used for a given anti-serum dilution! For example, for a dilution of 1/5000 antiprogesterone a dilution of 1/10 polymerize antilamma-frlobulins must be used.

In the present method according to the invention for the immunoenzymatic determination of progesterone becomes a immunological reaction used in which the progesterone sample and the progesterone extract become an antiprogesterone serum is added and the coupling product becomes progesterone-beta-galactosidase admitted. Subsequently, an immunoprecipitation reaction is carried out by means of supposedly insoluble anti-

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body and the enzymatic activity becomes more beneficial Way determined in the sediment.

For carrying out the determination method according to the invention it is preferable to use an analysis case, which is included in the The following main reaction components contain:

1) Antiprogesterone antibodies (in concentrated oier lyophilized form).

2) Progesterone complex bound to ß-halactosidase.

3) Antibody precipitating agents (Ab _? ).

Ό An enzyme substrate such as ONPO.

5) A buffering agent such as "PM" buffer.

6) A range scale for progesterone with different concentrations.

The enzymatic determination is preferably carried out at ^ 5 ° C., but can also be carried out ^{at 2 ° C. in the same way.}

In the following description, for example, the progesterone determination in a biological environment such as can be described in the plasma of pregnant rats · This is a comparative study with the radioimmunological Determination carried out.

The blood taken up in heparin from the carotid artery of the pregnant rats is centrifuged for 20 minutes at 5000 revolutions per minute and the plasma is decanted. A plasma tischee volume of 0.1 ml is added into a conical tube at a normal speed (V = 15 ml) and run for 2 hours at 60 ⁰ C incubation for binding to reduce (proteines vectrices) of progesterone on the proteins. After lasting 2 minutes

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Stirring on vortex was extracted with 10 ml of petroleum ether. The petroleum ether phase is collected and evaporated to dryness in the same tube. The dry extract is taken up in 0.5 nl "PMp" buffer and allowed to these 15 minutes at 45 ⁰ C are. There are compounds of 1/2 and 1/5 prepared and fractions of 0.1 ml are taken to be used for the immunoenzymatic and radioimmunological determinations in duplicate. For the same conditions, 0.1 ml of plasma from a male rat is treated in order to assess the influence of aspecific material such as plasma or solvents. It was found that after the addition of radioactive progesterone or plasma by means of petroleum ether extraction, over 95% could be extracted.

Know the conditions of the immunoenzymatic determination can be taken from the following Table II.

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Table II

From ₁ (dilution 1/5000 Volume (ml) Experiments
conditions 30 minutes
Arbeltstempe
rature Mood extract or
default 0.1 ? h 30
Working temp
rature Ag-GZ (80 enzymatic a
conduct at 28 ° C) 0.1 30 minutes
Working temperature From ₂ (dilution l / lo)
« O ₁ I 0.1

For comparison purposes, a normal rabbit serum was used instead of Ab ..

From the values in Table III it can be seen that the sensitivity of the immunoenzymatic process according to the invention (E.I.E.) is equivalent to the values obtained using radioimmunological methods (E.R.I.).

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Table III

<· .R.I. (ng / -nl) E.I.E, (ng / ml)

Pregnant rats

91 61 11 12

17 31 31 68 63

87 70 11b 91a 11a 11a 20a 20b 16

Mighty swan women

37, 5 ? 9 31 15th 5 40 36 34 47 44 44 30th 32 30th 26th 3 ?, 5 39 η, Ρ 12, R. 16 18th 13.6 14th 27.5 2 * 14.4 12, 4th 12.6 13th 12.5 13th 30th 25th 24 24 15th 17th 10.5 13th 0.4 ο, 6th 0.3 ο, 0.5 1 ο, 3 ι, ? 0.6 0, 5

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Table III (continued

E.R.I. (n.3 / ml) E.I.E.

2, R

Pregnant women

1 5fi

1 5f

3 31

Claims; -? 0 -

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Claims (12)

Claims Method for the immunoenzymatisehen determination of progesterone on the basis of competitive reactions in a liquid environment of propiesterone molecules (referred to as Ag molecules) and progesterone molecules (referred to as Af-ΠΖ) coupled by an enzyme (such as ß-D-galactosidase) to the same antibody seats, formed by a Antiprogesterone serum (referred to as Ab) and a precipitation of soluble comolexes obtained by a second insoluble antibody (referred to as Abp), the reaction leading to a sediment (culot) containing all antibody populations and a supernatant or supernatant. containing supernatant liquid (surnageant), which all Ap-- and Ag-GZ fractions not Ab _-. have reacted, characterized in that the same progesterone derivative such as the 11-C-hemisuccinate of progesterone is used for the coupling with the ß-galactosidase and for the production of the antiprogesterone serum. 2. The method according to claim 1, characterized in that the ß-D-Oalactosidaee prior to coupling is cleaned with the progesterone. 3. The method according to claim 2, characterized in that the coupling by means of a means such as soluble carbodiimide was carried out » 4. The method according to claims 1 to 3, characterized g e k e η η draws that the insoluble antibodies through. Polymerization were made insoluble. 5. The method according to claim Ί, characterized in that g e k e η η that the antibodies prior to polymerization - 21 609819/0797 have been cleaned. 6. The method according to claims 1 to ^π , characterized in that the insoluble antibodies are nolynerisiert.e anti-gamma-olobulins. 7. Reagent set for the best immune; of progesterone characterized by the fact that? «he antipropesterone antibody, a progesterone comolex coupled to ^ -laactosidase is, a filler for antibodies, a buffer and includes a progesterone comparison scale. 8. Reagent set according to claim ⁷ , characterized in that ¹³ , the progesterone is 11- hemisuccinate of progesterone. 9. Reagent set according to claims 7 and R, characterized in that the buffer consists of 70 mM Na ₂ HPO ^, 30 mM NaH ₂ PO ^, 1 mM MgSO ₁ ^, 0.2 mM MnSO ₁ J and 2 mM Mg-Titriplex with the pH adjusted to 7. 10. Reagent set according to claims 7 to 9, characterized in that it is a substrate which enables the determination of the enzyme. 11. Reagent kit according to claim 10, characterized in that the substrate is ortho-nitrophenylß-D-galactoside is. 12. Reäktionsmittelatz according to claims 7 to 10, characterized characterized in that the Antipro ^ esteron antibodies are in concentrated or lyophilized form. 609819/0797 Blank page