CA1071535A - Method for enzyme immunoassay of progesterone - Google Patents
Method for enzyme immunoassay of progesteroneInfo
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- CA1071535A CA1071535A CA237,476A CA237476A CA1071535A CA 1071535 A CA1071535 A CA 1071535A CA 237476 A CA237476 A CA 237476A CA 1071535 A CA1071535 A CA 1071535A
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- progesterone
- antibody
- enzyme
- derivative
- galactosidase
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
- G01N33/743—Steroid hormones
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Abstract
ABSTRACT OF THE DISCLOSURE
" A method for enzyme immunoassay of progesterone "
A method for enzyme immunoassay of progesterone.
The method for enzyme immunoassay of progesterone making use of competitive reactions in a liquid medium of molecules of the said progesterone, molecules referred to as Ag, and molecules of progesterone coupled to an enzyme, namely .beta.-D-galactosidase (3.2.1.23), molecules referred to as Ag-GZ, for the same sites of an antibody consisting of an anti-progesterone serum referred to as Ab1 and of precipitation of soluble complexes obtained by a second insoluble antibody referred to as Ab2, the reaction resulting in a solid phase containing all the antibody populations and of a supernatent containing the Ag and Ag-GZ fractions which have not reacted with Ab1, is characterized by the use of a same progesterone derivative, namely progesterone -11-.alpha.-hemisuccinate, for coupling with .beta.-galactosidase (3.2.1.23) and for the production of anti-progesterone serum.
Determination of progesterone in plasma.
" A method for enzyme immunoassay of progesterone "
A method for enzyme immunoassay of progesterone.
The method for enzyme immunoassay of progesterone making use of competitive reactions in a liquid medium of molecules of the said progesterone, molecules referred to as Ag, and molecules of progesterone coupled to an enzyme, namely .beta.-D-galactosidase (3.2.1.23), molecules referred to as Ag-GZ, for the same sites of an antibody consisting of an anti-progesterone serum referred to as Ab1 and of precipitation of soluble complexes obtained by a second insoluble antibody referred to as Ab2, the reaction resulting in a solid phase containing all the antibody populations and of a supernatent containing the Ag and Ag-GZ fractions which have not reacted with Ab1, is characterized by the use of a same progesterone derivative, namely progesterone -11-.alpha.-hemisuccinate, for coupling with .beta.-galactosidase (3.2.1.23) and for the production of anti-progesterone serum.
Determination of progesterone in plasma.
Description
The present invention relates to a method for enzyme immunoassay of progesterone.
The method of the invention takes advantage of the teaching of the know technique relating to an assay involving a competition between molecules of free progesterone on the one han~ and molecules of progesterone coupled to an enzyme, with respect to the same anti-progesterone antibody (Abl) sites, and the precipitation of abl by insolubilized anti-gammaglobulines ( Ab2).
Enzyme immunoassay is a technique which has only recently been developed. The coupling of an enzyme to an antigen or an antibody was first described for histological studies [NAKANE P.K. and PIERCE G.B. Jr (1967). Enzyme-labelled antibodies. Preparation and application for the localization of antigens, J. Histochem. Cytochem. 14, 929 , AV~AMEAS S. (1969). Coupling of enzymes to proteins with glutaraldehyde. Use of the conjugates for the detection of antigens and antibodies. Immunochemistry 6,43]. The first enzyme immunoassay concerns that of the chorionic gonadotropin enzyme (CGE) in urine, in which CGE is labelled with peroxidase and the antibody coupled with cellulose [VAN WEEMEN
B.K. and SCHUURS A.H.W.M. (1971). Immunoassay using antigen-enzyme conjugates FEBS Letters 15, 232]. The authors used the technique of polymerization of the first antibody, or that of polymerization of the second antibody. The first technique had a sensitivity approximately that obtained by the inhibi-tion of hemagglutination. The second technique was 10 to 20 times more sensitive, but was not so sensitive as radioimmu-nological determination.
Radioi~unoassay is based on isotopic dilution in a reaction medium which obeys the law of mass action. There is ~7~L535 competition between the radioactive molecules and the molecules of the standard or the unknown sample with respect to the same antibody sites.
The first enzyme immunoassay of a low molecular weight substance :DNP [ENGVALL E. and PERLMANN P. (1972). Enzyme-linked immunosorbent assay (ELISA). III - Quantitation of specific antibodies by enzyme-labelled anti-immunoglubulin in antigen-coated -tubes. The Journal of Immunology 109, 129] was not very sensitive because, to obtain 50% inhibition of enzyme activity 5.10 4 mole/liter DMP had to be added.
Other enzyme immunoassay systems have been presented for morphine [ RUBENSTEIN K.E., SCHNEIDER R.S. and ULMAN E.F.
(1972) "Homogeneous" enzyme immunoassay; a new immunochemical technique. B.B.R.C. 47, 846 ], and the oestrogens [ VAN WEEMEN
B~K. and SCHUURS A.H.W.M. (1972). Immunoassay using hapten-enzyme conjugates. FEBS Letters 24,77 ]. Although the sensitivity of these assays is better, it is not so great as that obtained with radioimmunoassay.
French patents 73.17.181 (publication No. 2,184,371 -dated November 26, 1973) and 71.46.179 (publication No.
The method of the invention takes advantage of the teaching of the know technique relating to an assay involving a competition between molecules of free progesterone on the one han~ and molecules of progesterone coupled to an enzyme, with respect to the same anti-progesterone antibody (Abl) sites, and the precipitation of abl by insolubilized anti-gammaglobulines ( Ab2).
Enzyme immunoassay is a technique which has only recently been developed. The coupling of an enzyme to an antigen or an antibody was first described for histological studies [NAKANE P.K. and PIERCE G.B. Jr (1967). Enzyme-labelled antibodies. Preparation and application for the localization of antigens, J. Histochem. Cytochem. 14, 929 , AV~AMEAS S. (1969). Coupling of enzymes to proteins with glutaraldehyde. Use of the conjugates for the detection of antigens and antibodies. Immunochemistry 6,43]. The first enzyme immunoassay concerns that of the chorionic gonadotropin enzyme (CGE) in urine, in which CGE is labelled with peroxidase and the antibody coupled with cellulose [VAN WEEMEN
B.K. and SCHUURS A.H.W.M. (1971). Immunoassay using antigen-enzyme conjugates FEBS Letters 15, 232]. The authors used the technique of polymerization of the first antibody, or that of polymerization of the second antibody. The first technique had a sensitivity approximately that obtained by the inhibi-tion of hemagglutination. The second technique was 10 to 20 times more sensitive, but was not so sensitive as radioimmu-nological determination.
Radioi~unoassay is based on isotopic dilution in a reaction medium which obeys the law of mass action. There is ~7~L535 competition between the radioactive molecules and the molecules of the standard or the unknown sample with respect to the same antibody sites.
The first enzyme immunoassay of a low molecular weight substance :DNP [ENGVALL E. and PERLMANN P. (1972). Enzyme-linked immunosorbent assay (ELISA). III - Quantitation of specific antibodies by enzyme-labelled anti-immunoglubulin in antigen-coated -tubes. The Journal of Immunology 109, 129] was not very sensitive because, to obtain 50% inhibition of enzyme activity 5.10 4 mole/liter DMP had to be added.
Other enzyme immunoassay systems have been presented for morphine [ RUBENSTEIN K.E., SCHNEIDER R.S. and ULMAN E.F.
(1972) "Homogeneous" enzyme immunoassay; a new immunochemical technique. B.B.R.C. 47, 846 ], and the oestrogens [ VAN WEEMEN
B~K. and SCHUURS A.H.W.M. (1972). Immunoassay using hapten-enzyme conjugates. FEBS Letters 24,77 ]. Although the sensitivity of these assays is better, it is not so great as that obtained with radioimmunoassay.
French patents 73.17.181 (publication No. 2,184,371 -dated November 26, 1973) and 71.46.179 (publication No.
2,120,835 - dated July 24, 1972) relate to processes for the detection and determination of hapten; in the first document mentioned the nature of the coupling of hapten to high molecular weight substance, used to form the antibody, differs from that of the conjugated hapten-enzyme coupling.
The difference in nature of the coupling may be due to:
a) a bond or bridge of different chemical nature b) haptens of chemically different nature having an immunological relationship 53~
c) coupling of the hapten molecule in another position.
Thus, in the assay of progesterone, it is sta-ted that the systems of determination where alkaline progesterone~
succinyl-phosphatase is combined with anti-progesterone-ll succinyl- bovine serum-albumine are four to ten-fold less sensitive to progesterone than the alkaline progesterone-ll-succinyl-phosphatase/anti-progesterone 12-succinyl-bovine serum-albumine (page 10, lines 11 to 20).
The basic principle of this patent is incompatible with the determination according to the invention, which uses the same hapten-high molecular weighk and haptenenzyme coupling.
The second patent cited does not teach the assay of progesterone according to the present invention.
The process forenzyme immunoassay of progesterone according to the invention makes it possible to obtain a dose-response curve the sensitivity of which is equal to that of radioimmunoassay while not possessing the drawbacks inherent in this type of determination : high cost of material (radiation counter tube, scintillating liquids, radioactive substances), drawbacks of radioactive substances (breakdown of the radioactive antigen or antibody, period of the element) and problems of health and legislation.
The excellent sensitivity of the assay is notably obtained by the use of a specific anti-progesterone antibody.
It is known how to prepare a hapten antibody by coupling hapten to a high molecular weight substance, which is generally a protein, by injecting said coupling product to an animal and by isolating the antibody in the conventional manner. The invention uses this means, but adapting it to the specific 3G requirements of a very sensitive enzyme immunoassay process.
r~
;535 The process for enzyme immunoassay of progesterone using rival reactions in a liquid medium of the molecules of the said progesterone, molecules referred to as Ag, and the molecules of progesterone coupled to an enzyme, namely ~-D-galactosidase, molecules referred to as Ag-GZ, with respect to the same sites of an antibody consisting of an anti-progesterone serum referred to as Abl, and to a precipitation of the soluble complexes obtained by a second insoluble anti-body referred to as Ab2, the reaction providing a solid phase containing all the populations of antibodies and a supernatent containing the Ag and Ag-GZ fractions which have not reacted with Abl, is characterized in that a same progesterone ~erivative is used, namely progesterone ll-a-hemisuccinate, for coupling with ~-galactosidase and for the production of anti-progesterone serum.
According to the present invention, coupling of progesterone was effected by means of a functional group consisting of the hemisuccinate radical fixed to carbon 11, which made it possible to obtain an immunogen which, when 2 injected to the animal, resulted in the formation of particularly specific antibodies. This very high level of specificity permits immunoassay of the plasmic progesterone without preliminary chromatography of the extracts.
The process of enzyme immunoassay developed necessitates the interaction of 4 types of molecules : an antigen (or hapten) (Ag) consisting of progesterone; a corresponding antiserum produced from progesterone ll-a-hemisuccinate; an enzyme-labelled antigen, that is to say, progesterone ll-a-hemisuccinate labelled with ~-galactosidase (3.2.1.23) (Ag-GZ) and, finally, and insolubilized, precipitating antiserum (Ab2).
At the end of the reaction there is obtained : a solid phase L53~
containing all the antibody populations and a supernatent containing the Ag and Ag-GZ fractions which have not reacted with Abl.
Measurement of enzyme activity is advantageously effected on the solid phase.
The embodiment of the process necessitates preparation of the components. The enzyme selected, ~-D-galactosidase (3.2.1.23), is first purified from a constituent strain of E.Coli; the various purification steps include, for example:
having the bacteria, adding thereto two volumes of purification buffer (10 mM trisacetate pH 7.6, 10 mM of MgC12, 5 mM of titriplex Mg, 10mM of ~-mercaptoethanol (~ SH) and 0.5~
NaCl), grinding and ultrasonic treatment; after centrifugation the supernatent is diluted two-fold with the same buffer, this causing the precipitation of certain proteins. The product is centrifuged and decanted and the supernatent is again precipitated with ammonium sulphate at 33% of saturation.
Said precipitateis put in suspension with the same buffer and dialyzed until complete disappearance of the ammonium sulphate when the ~-galactosldase is purified on a DEAE -*
Sephadex column by molarity gradient. Enzyme activity is then measured.
Assaying is effected by pouring 2 ml of a mixture of ~-galactosidase in a "PM2" buffer consisting of 70 mM of 25 Na2HPO4, 30 mM of NaH2PO4, 1 mM of MgSO4,0.2 mM of MnSO4 and 2 mM titriplex Mg, the pH being adjusted to 7, then 7 ml/l of B SH with 0.5 ml of 4% ONPG (ortho-nitrophenyl-~-D-galactoside). The product is left to incubate in a water-bath at 28C and the reaction is stopped by 1 ml of Na2CO3 lM. The number of enzyme units is given by the formule :
* Trade Mark 5 53~
Enzyme unitS = D.O 420 nm x dil with ~ = 5.10 3 If incubation is effected at 45C in "PM2" ~-SH, the number of enzyme units is obtained by dlvision by a cor-relation factor of 2.6.
Ortho-nitrophenol galactoside is the substrate used to measure enzyme activity. Ortho-nitrophenol (yellow colour) and galactose are released into the reaction medium in the presence of ~-galactosidase. Orthonitrophenol is released as a function of the amount of enzyme present in the medium. For a given concentration of substrate and a given incubation time, the value of optical density (due to the intensity of colouring) measured with a spectrophotometer varies with the amount of ~-galactosidase.
This makes it possible to define an enzyme unit as being the amount of ~-galactosidase which hydrolyzes one micromole of substrate per unit of time ( = one minute).
Coupling of progesterone ll-a-hemisuccinate to ~-galactosidase, purified by the process described hereinabove,is then effected by means of a "soluble" carbodiimide. The reaction takes place in two steps o the first, "activation"
step consisting of the reaction of carbodiimide with progesterone ll-a-hemisuccinate, resulting in an O-acylurea which is very reactive with the nucleophilic product R'N = C = NR' R'N = C - NHR' + ~
~0 0 RC
`OH R - C = O
~7~53~
The second step consists of the reaction of O-acylurea obtained with ~-galactosidase, and more precisely by covalently coupling the progesterone hemisuccinate -the COOH of which has been activated with carbodiimide, with the NH2 of the lysines of the B-galactosidase O NR' o NH~' R-C-O-C + R"NH2 - 3 R-C-MHR" + O = C
MHR' NHR' The progesterone-galactosidase reagents are used in a ratio Pro : GZ = 130.
In a particular embodiment of coupling, 7.10 8 mole of progesterone ll-a-hemisuccinate (O.l ml) was added to 10 5 mole of carbodiimide, HCl (O.Ol ml), the pH being adjusted to 4~7, the mixture was left to incubate for 30 min. at ambient temperature followed by the addition of 5.45 10 10 mole of ~-galactosidase (O.l ml) representing an enzyme activity of 160,000 EU/mg, the p~I was adjusted to 5.5 and the product was left to incubate for 12 hours at 4C, the reaction then being arrested by 1 ml of "P~2" buffer and dialysis was carried out against 2 liters of buf~er containing 5g of dextran carbon.
The remaining enzyme activity was 142.000 EU/mg. The product obtained was then flowed through a Sephadex G 25 column (0 = 1 cm, V = 10 ml) to separate the progesterone coupled to ~-galactosldase(Pro - Gz) from the free pro-gesterone.
The step for obtaining the antiprogesterone serum was then carried out, this being a particularly important step, as the specificity of the anti-progesterone antibodies * Txade Mark - 7 -~7~35i de-termines the final precision of assaying. According to the present invention, progesterone 11 a-hemisuccinate was coupled to bovine serum albumine (BSA) by known techniques.
[ Erlanger B.F., BOREK F., BEISER S.M. and LIEBERMAN S.
(1957j. Steroid protein conjugates JO ~iol. Chem. 228, 713 VAITUKAITIS J., RABBINS J.B., MIESCHL~G E. and ROSS T.
(1971). A method for producing specific antisera with small doses of immunogen J. Clin, Endocr. 33, 988 ].
Coupling progesterone by means of functional group, hemisuccinate, fixed to carbon 11, made it possible to obtain an immunogen which when injected to an animal resulted in the formation of particularly specific antibodies as is seen from the table below:
TABLE I
Steroids Crossed reactions (~) -desoxycorticosterone 3 5 ~-pregnan 3.20-dione 2.8 5 a-pregnan,3.20-dione 2 6 ~-hydroxyprogesterone 1.2 17a-hydroxyprogesterone 0.5 20 a hydroxyprogesterone 0.4 corticosterone 0.3 testosterone 0.08 cortisol 0-03 5-pregnenolone 0.03 aldosterone 0.008 oestriol ~10 5 These results were obtained by reason of the following principle:
' -- ~
~7~L5~
Owing to an exchange with the cold progesterone (not labelled) in-troduced into the medium, anti-progesterone antibodies, coupled to tritium-labelled progesterone, are capable of releasing the radioactive progesterone and spontaneously displace the cold progesterone from its sites.
The equilibrium established between the amount of unlabelled progesterone-antibody (Ac) and labelled progesterone-Ac is a function of the cold progesterone added to the medium. On the other hand, if instead of the cold progesterone another steroid such as aldosterone, for example, is added, there is very little exchange between the coupled radioactive progesterone and the aldosterone of the medium.
The anti-progesterone antibodies being very specific to progesterone, they will only couple very weakly with aldosterone.
Therefore, the weaker the exchange between the initial radioactive complex (progesterone-antibody) and the compound added to the medium, the lower the percentage of the crossed reaction will be.
This extreme specificity permits, for example, immunoassay of plasmatic progesterone without preliminary chromatography of the extracts obtained after a simple extraction with an organic solvent.
The precipitating antiserum is then prepared in two steps : in the first place its production proper and, in the second, insolubilization of the antibodies obtained, by polymerization. To prepare rabbit anti- ~ -globuline serum, 10 mg dissolved in 1 ml physiological salt solution and 2 ml complete Freund's adjuvant per animal (goat or sheep) is injected by the process previously described. Booster doses .
~7~5~i were given every month consisting of 2.5 mg antigen given by intramuscular and subcutaneous injection and 2 mg the following day by intravenous injection. The amount of anti-bodies per ml was determined by microprecipitation analysis.
Three antisera were used, each titering 2 mg/ml (goat);
13 mg/ml (goat), 13.6 mg/ml (sheep). Insolubilization of rabbit anti-gamma-globuline antibodies was carried out with ethyl chloroformiate according to the following process :
the gamma-globulines were precipitated by adding one volume f saturated ammonium sulphate to two volumes of serum, the mixture was stirred for 30 minutes at ambient temperature and centrifuged for 15 minutes.
The precipitate was retrieved and dialyzed against a sodium phosphate buffer (pH 6.3) until the ammonium sulphate had disappeared (checked by 1% BaC12). The product obtained, consisting of gamma-globulines and other proteins, was flowed through a DEAE cellulose column, the gamma globulines, eluted with a sodium phosphate buffer (pH = 6.3), were taken from the column and were obtained in a very pure state.
Another method consisted in using the goat or sheep serum which was dialyzed directly against the sodium phosphate buffer and, after dialysis, flowed through the DEAE column. Elution of the gamma globulines is effected by the same sodium phosphate buffer. The gamma-globulines obtained are very highly purified. A 50 mg/ml ~ -globuline solution was prepared, to which was added drop by drop ethyl chloroformiate at a rate of 0.04 ml/ml of solution. The pH
was maintained at 5Ø After stirring, for 3 hours, the solution was centrifuged and washed three times with physiological salt solution before mixing the precipitate with a buffer. Finally, it was homogenized by means of a ~ 10 --. ~7~53~
syringe with a very fine need]e.
In order to determine the amounts of polymerized antibodies to be used in the assay, it is necessary to determine the coupling power of the polymerized antibodies;
for this, for each of the dilutions (1/1, 1/10, 1/20, 1/50) of polymerized anti-gamma-globulines, increasing dilutions of anti-progesterone serum from 1/100 to 1/10,000 were incubated. After centrifuging, the same amount of tritiated progesterone was added to the supernatent of each reaction tube in order to test the possible presence of anti-progesterone antibodies. As an example, the following experimental procedure can be described : 0.2ml of anti-progasterone serum is mixed with 0.2ml of polymerized anti-gamma-globulines; the mixture is left to incubate for 30 minutes at 4C and is then centrifuged forlO minutes at 5000 r.p.m.; 50,000 cpm of3H-progesterone (O.lml) is added to the supernatent and the mixture is left to incubate for 30 minutes at ambient temperature, then 30 minutes in melting ice;
separation is effected with 1 ml dextran carbon, after contact for 10 minutes the product is centrifuged, the radioactivity in the supernatent is then assessed. The results obtained are given graphically in the drawing which forms a part of this specification; for a given dilution of antiserum a sufficient concentration of polymerized anti-gamma-globulines will be used so that all the first antibodies are coupled thereto; thus, for example, for a dilution of 1/5000 anti-progesterone, a dilution of 1/10 polymerized anti-gamma-globulines will be necessary.
The present invention therefore provides a process for enzyme immunoassay of progesterone comprising an immune \~ -~6~7~L535 reaction proper wherein the said progesterone extract or standard is added to the antiprogesterone serum and to the product of progesterone-~-galactosidase coupling followed by immunoprecipitation with an insolubilized antibody;
enzyme activity is advantageously determined on the solid phase.
In one aspect of khis invention there is provided a methodfor enzyme immunoassay of progesterone making use of the competitive reactions in a liquid medium of the molecules of the said progesterone, molecules referred to as Ag, and progesterone molecules coupled to ~-D-galactosidase enzyme (3.2.1.23), molecules referred to as Ag-GZ, with respect to the same sites of an antibody consisting of an anti-progesterone serum referred to as Abl and to a precipitation of soluble complexes obtained by a second insoluble antibody referred to as Ab2. The reaction results in a solid phase containing all the antibody populations and in a supernatent containing the Ag and Ag-GZ fractions which have not reacted with Abl. Progesterone ll-~-hemisuccinate is used for coupling with ~-galactosidase (3.2.1.23) and for the production of anti-progesterone serum.
In another aspect of this invention there is provided a method for the detection and determination by enzyme immuno-assay of a bindable progesterone. The method comprises the steps of:
- 25 a. providing a given quantity oE a conjugate of a given derivative of the progesterone with ~-D-galactosidase enzyme (3.2.1.23), said given derivative being progesterone 11-~-hemisuccinate;
b. providing a corresponding given quantity of an anti body against a conjugate of said given derivative with a high ~7~53~i molecular weight substance, both of said conjugates involving coupling of a chemically identical derivative of the pro-gesterone by a chemically identical bond or bridge at the same position of the progesterone derivative molecule;
c. admixing a sample of a fluid containing the progesterone to be determined with the reactants of steps (a) and (b) to form a reaction mixture and allowing the reaction to go to completion;
d. separating the resulting mixture into a liquid phase and a solid phase by adding an insolubilized antibody against the antibody of step (b); and e. determining the quantity of the progesterone from the measure of enzyme activity of either separated phase, said method having a determination sensitivity of at least 0.3 nanograms per mill.iliter.
In order to carry out the process of the invention it is preferable to use a box of reagents consisting essentially of :
- anti-progesterone antibodies (in concentrated or freeze-dried form) - a progesterone complex coupled with B-galactosidase, - a precipitating antibody (Ab2) - an enzyme substrate such as ONPG
- a buffer, such as "PM2" buffer - a standard range of progesterone at different concentrations.
Enzyme determination is preferably effected at 45C, but can also take place at 28~C.
As an example, determination of progesterone was carried out in a biological medium - plasma from pregnant rats - and ~7~L535 a comparative study was conducted with radioimmunologic determination.
The blood taken on heparin from the jugular vein of pregnant rats is centrifuged for 20 min. at 5000 r.p.m.
and the plasma is decanted. A volume of 0.1 ml of plasma is put in a standardized ground conical tube (V = 15 ml) and incubated for 2 hours at 60C to reduce the coupling of the progesterone to the vector proteins. Extraction is effected with 10 ml petroleum ether after stirring for *
2 min. in a Vortex apparatus. The petroleum ether phase is recovered and evaporated to dryness in an identical tube.
The dry extract is mixed with 0.5 ml of "PM2" buffer and left to stand for 15 min. at 45C. 1/2 and 1/5 dilutions are made and 0.1 ml fractions are taken twice to be used for enzyme immunoassays and radioimmunoassay. Under the same conditions 0.1 ml of mole rat plasma or "PM2" buffer is treated to assess the influence of a specific * Trade Mark 97~i3~
, -materi.al ~pl~ma ~nd solvent~. A preliminary study sho~ed that ~e~.roleum e~her extrac~ion was consta~tiy superior to 95~ after the additio~ of radioactlve ~rogesterone or plasma.
' The cond-itions o~ enzyme imrrunoassay can be summarized on table II below : -TA~IE II
o : Volume (ml) : Experimental : : conditions Abl(dilution 1/5000) . 0,]. ~ 30 minllt*s ~ Laboratory : èxtract to ~e 0,1 ~ temperature : determined or : ~ :
standard . Ag-GZ ~Q enz me . 0,1 2 h ~0 : wlLts at ,~C~ Laboratory : temperature : A~ (dllu~:Lon 1/10) : 0,1 : ~0 minukes : ~~ : laboratory : ^: temperature . ` :
As experimental control, nor.mal rabbit serum was used instead o~ Abl.
The appended figure and t~ble III clearly show ~'nat ~he sens~tivity o~ the enzyme immunoassay technique (E,I.A.) is equal to that obtained by radioimmunologic determination (R.I.D.).
~ 15 -7~
TA:BIE III
R.I.D. (ng/ml) E,I.A. (n~/ml) PRE GNANT ~A TS
92 30 3~ . 5 gl ~ 31 11 40 ~8 12 36 31~, `~il 30 32 ~' ~50 26 68 ,~,2 . 5 39 67 25 2~
9 - 1.3.~ 12.8 %7 1.6. 18 - 1~ 6 14 1 lb ~7 5 28 91a 14, 4 12 . 4 lla l? . 6 13 lla 1~ . 5 13 20a 30 25 20b - 24 - 24 1~ 5 17 7 10.5 13 ' ' '' :
5~5 _I:IE III
R . I, D ~ (ng,/ml ) :~: . I . A . (~:/n~l NON-PREGNANT IAJOI~
o . 1~ o ~ 6 2 0.~5 0.
;5 . G.5 0.~ 1.2 8 o~6 o.s, g 2.4 2.8 P~ ANT lAiO~
5~ ~iO
The difference in nature of the coupling may be due to:
a) a bond or bridge of different chemical nature b) haptens of chemically different nature having an immunological relationship 53~
c) coupling of the hapten molecule in another position.
Thus, in the assay of progesterone, it is sta-ted that the systems of determination where alkaline progesterone~
succinyl-phosphatase is combined with anti-progesterone-ll succinyl- bovine serum-albumine are four to ten-fold less sensitive to progesterone than the alkaline progesterone-ll-succinyl-phosphatase/anti-progesterone 12-succinyl-bovine serum-albumine (page 10, lines 11 to 20).
The basic principle of this patent is incompatible with the determination according to the invention, which uses the same hapten-high molecular weighk and haptenenzyme coupling.
The second patent cited does not teach the assay of progesterone according to the present invention.
The process forenzyme immunoassay of progesterone according to the invention makes it possible to obtain a dose-response curve the sensitivity of which is equal to that of radioimmunoassay while not possessing the drawbacks inherent in this type of determination : high cost of material (radiation counter tube, scintillating liquids, radioactive substances), drawbacks of radioactive substances (breakdown of the radioactive antigen or antibody, period of the element) and problems of health and legislation.
The excellent sensitivity of the assay is notably obtained by the use of a specific anti-progesterone antibody.
It is known how to prepare a hapten antibody by coupling hapten to a high molecular weight substance, which is generally a protein, by injecting said coupling product to an animal and by isolating the antibody in the conventional manner. The invention uses this means, but adapting it to the specific 3G requirements of a very sensitive enzyme immunoassay process.
r~
;535 The process for enzyme immunoassay of progesterone using rival reactions in a liquid medium of the molecules of the said progesterone, molecules referred to as Ag, and the molecules of progesterone coupled to an enzyme, namely ~-D-galactosidase, molecules referred to as Ag-GZ, with respect to the same sites of an antibody consisting of an anti-progesterone serum referred to as Abl, and to a precipitation of the soluble complexes obtained by a second insoluble anti-body referred to as Ab2, the reaction providing a solid phase containing all the populations of antibodies and a supernatent containing the Ag and Ag-GZ fractions which have not reacted with Abl, is characterized in that a same progesterone ~erivative is used, namely progesterone ll-a-hemisuccinate, for coupling with ~-galactosidase and for the production of anti-progesterone serum.
According to the present invention, coupling of progesterone was effected by means of a functional group consisting of the hemisuccinate radical fixed to carbon 11, which made it possible to obtain an immunogen which, when 2 injected to the animal, resulted in the formation of particularly specific antibodies. This very high level of specificity permits immunoassay of the plasmic progesterone without preliminary chromatography of the extracts.
The process of enzyme immunoassay developed necessitates the interaction of 4 types of molecules : an antigen (or hapten) (Ag) consisting of progesterone; a corresponding antiserum produced from progesterone ll-a-hemisuccinate; an enzyme-labelled antigen, that is to say, progesterone ll-a-hemisuccinate labelled with ~-galactosidase (3.2.1.23) (Ag-GZ) and, finally, and insolubilized, precipitating antiserum (Ab2).
At the end of the reaction there is obtained : a solid phase L53~
containing all the antibody populations and a supernatent containing the Ag and Ag-GZ fractions which have not reacted with Abl.
Measurement of enzyme activity is advantageously effected on the solid phase.
The embodiment of the process necessitates preparation of the components. The enzyme selected, ~-D-galactosidase (3.2.1.23), is first purified from a constituent strain of E.Coli; the various purification steps include, for example:
having the bacteria, adding thereto two volumes of purification buffer (10 mM trisacetate pH 7.6, 10 mM of MgC12, 5 mM of titriplex Mg, 10mM of ~-mercaptoethanol (~ SH) and 0.5~
NaCl), grinding and ultrasonic treatment; after centrifugation the supernatent is diluted two-fold with the same buffer, this causing the precipitation of certain proteins. The product is centrifuged and decanted and the supernatent is again precipitated with ammonium sulphate at 33% of saturation.
Said precipitateis put in suspension with the same buffer and dialyzed until complete disappearance of the ammonium sulphate when the ~-galactosldase is purified on a DEAE -*
Sephadex column by molarity gradient. Enzyme activity is then measured.
Assaying is effected by pouring 2 ml of a mixture of ~-galactosidase in a "PM2" buffer consisting of 70 mM of 25 Na2HPO4, 30 mM of NaH2PO4, 1 mM of MgSO4,0.2 mM of MnSO4 and 2 mM titriplex Mg, the pH being adjusted to 7, then 7 ml/l of B SH with 0.5 ml of 4% ONPG (ortho-nitrophenyl-~-D-galactoside). The product is left to incubate in a water-bath at 28C and the reaction is stopped by 1 ml of Na2CO3 lM. The number of enzyme units is given by the formule :
* Trade Mark 5 53~
Enzyme unitS = D.O 420 nm x dil with ~ = 5.10 3 If incubation is effected at 45C in "PM2" ~-SH, the number of enzyme units is obtained by dlvision by a cor-relation factor of 2.6.
Ortho-nitrophenol galactoside is the substrate used to measure enzyme activity. Ortho-nitrophenol (yellow colour) and galactose are released into the reaction medium in the presence of ~-galactosidase. Orthonitrophenol is released as a function of the amount of enzyme present in the medium. For a given concentration of substrate and a given incubation time, the value of optical density (due to the intensity of colouring) measured with a spectrophotometer varies with the amount of ~-galactosidase.
This makes it possible to define an enzyme unit as being the amount of ~-galactosidase which hydrolyzes one micromole of substrate per unit of time ( = one minute).
Coupling of progesterone ll-a-hemisuccinate to ~-galactosidase, purified by the process described hereinabove,is then effected by means of a "soluble" carbodiimide. The reaction takes place in two steps o the first, "activation"
step consisting of the reaction of carbodiimide with progesterone ll-a-hemisuccinate, resulting in an O-acylurea which is very reactive with the nucleophilic product R'N = C = NR' R'N = C - NHR' + ~
~0 0 RC
`OH R - C = O
~7~53~
The second step consists of the reaction of O-acylurea obtained with ~-galactosidase, and more precisely by covalently coupling the progesterone hemisuccinate -the COOH of which has been activated with carbodiimide, with the NH2 of the lysines of the B-galactosidase O NR' o NH~' R-C-O-C + R"NH2 - 3 R-C-MHR" + O = C
MHR' NHR' The progesterone-galactosidase reagents are used in a ratio Pro : GZ = 130.
In a particular embodiment of coupling, 7.10 8 mole of progesterone ll-a-hemisuccinate (O.l ml) was added to 10 5 mole of carbodiimide, HCl (O.Ol ml), the pH being adjusted to 4~7, the mixture was left to incubate for 30 min. at ambient temperature followed by the addition of 5.45 10 10 mole of ~-galactosidase (O.l ml) representing an enzyme activity of 160,000 EU/mg, the p~I was adjusted to 5.5 and the product was left to incubate for 12 hours at 4C, the reaction then being arrested by 1 ml of "P~2" buffer and dialysis was carried out against 2 liters of buf~er containing 5g of dextran carbon.
The remaining enzyme activity was 142.000 EU/mg. The product obtained was then flowed through a Sephadex G 25 column (0 = 1 cm, V = 10 ml) to separate the progesterone coupled to ~-galactosldase(Pro - Gz) from the free pro-gesterone.
The step for obtaining the antiprogesterone serum was then carried out, this being a particularly important step, as the specificity of the anti-progesterone antibodies * Txade Mark - 7 -~7~35i de-termines the final precision of assaying. According to the present invention, progesterone 11 a-hemisuccinate was coupled to bovine serum albumine (BSA) by known techniques.
[ Erlanger B.F., BOREK F., BEISER S.M. and LIEBERMAN S.
(1957j. Steroid protein conjugates JO ~iol. Chem. 228, 713 VAITUKAITIS J., RABBINS J.B., MIESCHL~G E. and ROSS T.
(1971). A method for producing specific antisera with small doses of immunogen J. Clin, Endocr. 33, 988 ].
Coupling progesterone by means of functional group, hemisuccinate, fixed to carbon 11, made it possible to obtain an immunogen which when injected to an animal resulted in the formation of particularly specific antibodies as is seen from the table below:
TABLE I
Steroids Crossed reactions (~) -desoxycorticosterone 3 5 ~-pregnan 3.20-dione 2.8 5 a-pregnan,3.20-dione 2 6 ~-hydroxyprogesterone 1.2 17a-hydroxyprogesterone 0.5 20 a hydroxyprogesterone 0.4 corticosterone 0.3 testosterone 0.08 cortisol 0-03 5-pregnenolone 0.03 aldosterone 0.008 oestriol ~10 5 These results were obtained by reason of the following principle:
' -- ~
~7~L5~
Owing to an exchange with the cold progesterone (not labelled) in-troduced into the medium, anti-progesterone antibodies, coupled to tritium-labelled progesterone, are capable of releasing the radioactive progesterone and spontaneously displace the cold progesterone from its sites.
The equilibrium established between the amount of unlabelled progesterone-antibody (Ac) and labelled progesterone-Ac is a function of the cold progesterone added to the medium. On the other hand, if instead of the cold progesterone another steroid such as aldosterone, for example, is added, there is very little exchange between the coupled radioactive progesterone and the aldosterone of the medium.
The anti-progesterone antibodies being very specific to progesterone, they will only couple very weakly with aldosterone.
Therefore, the weaker the exchange between the initial radioactive complex (progesterone-antibody) and the compound added to the medium, the lower the percentage of the crossed reaction will be.
This extreme specificity permits, for example, immunoassay of plasmatic progesterone without preliminary chromatography of the extracts obtained after a simple extraction with an organic solvent.
The precipitating antiserum is then prepared in two steps : in the first place its production proper and, in the second, insolubilization of the antibodies obtained, by polymerization. To prepare rabbit anti- ~ -globuline serum, 10 mg dissolved in 1 ml physiological salt solution and 2 ml complete Freund's adjuvant per animal (goat or sheep) is injected by the process previously described. Booster doses .
~7~5~i were given every month consisting of 2.5 mg antigen given by intramuscular and subcutaneous injection and 2 mg the following day by intravenous injection. The amount of anti-bodies per ml was determined by microprecipitation analysis.
Three antisera were used, each titering 2 mg/ml (goat);
13 mg/ml (goat), 13.6 mg/ml (sheep). Insolubilization of rabbit anti-gamma-globuline antibodies was carried out with ethyl chloroformiate according to the following process :
the gamma-globulines were precipitated by adding one volume f saturated ammonium sulphate to two volumes of serum, the mixture was stirred for 30 minutes at ambient temperature and centrifuged for 15 minutes.
The precipitate was retrieved and dialyzed against a sodium phosphate buffer (pH 6.3) until the ammonium sulphate had disappeared (checked by 1% BaC12). The product obtained, consisting of gamma-globulines and other proteins, was flowed through a DEAE cellulose column, the gamma globulines, eluted with a sodium phosphate buffer (pH = 6.3), were taken from the column and were obtained in a very pure state.
Another method consisted in using the goat or sheep serum which was dialyzed directly against the sodium phosphate buffer and, after dialysis, flowed through the DEAE column. Elution of the gamma globulines is effected by the same sodium phosphate buffer. The gamma-globulines obtained are very highly purified. A 50 mg/ml ~ -globuline solution was prepared, to which was added drop by drop ethyl chloroformiate at a rate of 0.04 ml/ml of solution. The pH
was maintained at 5Ø After stirring, for 3 hours, the solution was centrifuged and washed three times with physiological salt solution before mixing the precipitate with a buffer. Finally, it was homogenized by means of a ~ 10 --. ~7~53~
syringe with a very fine need]e.
In order to determine the amounts of polymerized antibodies to be used in the assay, it is necessary to determine the coupling power of the polymerized antibodies;
for this, for each of the dilutions (1/1, 1/10, 1/20, 1/50) of polymerized anti-gamma-globulines, increasing dilutions of anti-progesterone serum from 1/100 to 1/10,000 were incubated. After centrifuging, the same amount of tritiated progesterone was added to the supernatent of each reaction tube in order to test the possible presence of anti-progesterone antibodies. As an example, the following experimental procedure can be described : 0.2ml of anti-progasterone serum is mixed with 0.2ml of polymerized anti-gamma-globulines; the mixture is left to incubate for 30 minutes at 4C and is then centrifuged forlO minutes at 5000 r.p.m.; 50,000 cpm of3H-progesterone (O.lml) is added to the supernatent and the mixture is left to incubate for 30 minutes at ambient temperature, then 30 minutes in melting ice;
separation is effected with 1 ml dextran carbon, after contact for 10 minutes the product is centrifuged, the radioactivity in the supernatent is then assessed. The results obtained are given graphically in the drawing which forms a part of this specification; for a given dilution of antiserum a sufficient concentration of polymerized anti-gamma-globulines will be used so that all the first antibodies are coupled thereto; thus, for example, for a dilution of 1/5000 anti-progesterone, a dilution of 1/10 polymerized anti-gamma-globulines will be necessary.
The present invention therefore provides a process for enzyme immunoassay of progesterone comprising an immune \~ -~6~7~L535 reaction proper wherein the said progesterone extract or standard is added to the antiprogesterone serum and to the product of progesterone-~-galactosidase coupling followed by immunoprecipitation with an insolubilized antibody;
enzyme activity is advantageously determined on the solid phase.
In one aspect of khis invention there is provided a methodfor enzyme immunoassay of progesterone making use of the competitive reactions in a liquid medium of the molecules of the said progesterone, molecules referred to as Ag, and progesterone molecules coupled to ~-D-galactosidase enzyme (3.2.1.23), molecules referred to as Ag-GZ, with respect to the same sites of an antibody consisting of an anti-progesterone serum referred to as Abl and to a precipitation of soluble complexes obtained by a second insoluble antibody referred to as Ab2. The reaction results in a solid phase containing all the antibody populations and in a supernatent containing the Ag and Ag-GZ fractions which have not reacted with Abl. Progesterone ll-~-hemisuccinate is used for coupling with ~-galactosidase (3.2.1.23) and for the production of anti-progesterone serum.
In another aspect of this invention there is provided a method for the detection and determination by enzyme immuno-assay of a bindable progesterone. The method comprises the steps of:
- 25 a. providing a given quantity oE a conjugate of a given derivative of the progesterone with ~-D-galactosidase enzyme (3.2.1.23), said given derivative being progesterone 11-~-hemisuccinate;
b. providing a corresponding given quantity of an anti body against a conjugate of said given derivative with a high ~7~53~i molecular weight substance, both of said conjugates involving coupling of a chemically identical derivative of the pro-gesterone by a chemically identical bond or bridge at the same position of the progesterone derivative molecule;
c. admixing a sample of a fluid containing the progesterone to be determined with the reactants of steps (a) and (b) to form a reaction mixture and allowing the reaction to go to completion;
d. separating the resulting mixture into a liquid phase and a solid phase by adding an insolubilized antibody against the antibody of step (b); and e. determining the quantity of the progesterone from the measure of enzyme activity of either separated phase, said method having a determination sensitivity of at least 0.3 nanograms per mill.iliter.
In order to carry out the process of the invention it is preferable to use a box of reagents consisting essentially of :
- anti-progesterone antibodies (in concentrated or freeze-dried form) - a progesterone complex coupled with B-galactosidase, - a precipitating antibody (Ab2) - an enzyme substrate such as ONPG
- a buffer, such as "PM2" buffer - a standard range of progesterone at different concentrations.
Enzyme determination is preferably effected at 45C, but can also take place at 28~C.
As an example, determination of progesterone was carried out in a biological medium - plasma from pregnant rats - and ~7~L535 a comparative study was conducted with radioimmunologic determination.
The blood taken on heparin from the jugular vein of pregnant rats is centrifuged for 20 min. at 5000 r.p.m.
and the plasma is decanted. A volume of 0.1 ml of plasma is put in a standardized ground conical tube (V = 15 ml) and incubated for 2 hours at 60C to reduce the coupling of the progesterone to the vector proteins. Extraction is effected with 10 ml petroleum ether after stirring for *
2 min. in a Vortex apparatus. The petroleum ether phase is recovered and evaporated to dryness in an identical tube.
The dry extract is mixed with 0.5 ml of "PM2" buffer and left to stand for 15 min. at 45C. 1/2 and 1/5 dilutions are made and 0.1 ml fractions are taken twice to be used for enzyme immunoassays and radioimmunoassay. Under the same conditions 0.1 ml of mole rat plasma or "PM2" buffer is treated to assess the influence of a specific * Trade Mark 97~i3~
, -materi.al ~pl~ma ~nd solvent~. A preliminary study sho~ed that ~e~.roleum e~her extrac~ion was consta~tiy superior to 95~ after the additio~ of radioactlve ~rogesterone or plasma.
' The cond-itions o~ enzyme imrrunoassay can be summarized on table II below : -TA~IE II
o : Volume (ml) : Experimental : : conditions Abl(dilution 1/5000) . 0,]. ~ 30 minllt*s ~ Laboratory : èxtract to ~e 0,1 ~ temperature : determined or : ~ :
standard . Ag-GZ ~Q enz me . 0,1 2 h ~0 : wlLts at ,~C~ Laboratory : temperature : A~ (dllu~:Lon 1/10) : 0,1 : ~0 minukes : ~~ : laboratory : ^: temperature . ` :
As experimental control, nor.mal rabbit serum was used instead o~ Abl.
The appended figure and t~ble III clearly show ~'nat ~he sens~tivity o~ the enzyme immunoassay technique (E,I.A.) is equal to that obtained by radioimmunologic determination (R.I.D.).
~ 15 -7~
TA:BIE III
R.I.D. (ng/ml) E,I.A. (n~/ml) PRE GNANT ~A TS
92 30 3~ . 5 gl ~ 31 11 40 ~8 12 36 31~, `~il 30 32 ~' ~50 26 68 ,~,2 . 5 39 67 25 2~
9 - 1.3.~ 12.8 %7 1.6. 18 - 1~ 6 14 1 lb ~7 5 28 91a 14, 4 12 . 4 lla l? . 6 13 lla 1~ . 5 13 20a 30 25 20b - 24 - 24 1~ 5 17 7 10.5 13 ' ' '' :
5~5 _I:IE III
R . I, D ~ (ng,/ml ) :~: . I . A . (~:/n~l NON-PREGNANT IAJOI~
o . 1~ o ~ 6 2 0.~5 0.
;5 . G.5 0.~ 1.2 8 o~6 o.s, g 2.4 2.8 P~ ANT lAiO~
5~ ~iO
3 ~:L 42 __ 17 ~
~3 :
~3 :
Claims (10)
1. A method for enzyme immunoassay of progesterone making use of the competitive reactions in a liquid medium of the molecules of the said progesterone, molecules referred to as Ag, and progesterone molecules coupled to .beta.-D-glactosidase enzyme (3.2.1.23), molecules referred to as Ag-GZ, with respect to the same sites of an antibody consisting of an anti progesterone serum referred to as Ab1 and to a precipitation of soluble complexes obtained by a second insoluble antibody referred to as Ab2, the reaction resulting in a solid phase containing all the antibody populations and in a supernatent containing the Ag and Ag-GZ fractions which have not reacted with Ab1, wherein progesterone 11-?-hemisuccinate is used for coupling with .beta.-galactosidase (3.2.1.23) and for the production of anti-progesterone serum.
2. A method according to Claim 1, wherein .beta.-D-galactosidase (3.2.1.23) is purified before coupling with the progesterone derivative.
3. A method according to Claim 2, wherein the coupling is effected with an agent such as a soluble carbodiimide.
4. A method according to Claim 1 wherein the insoluble antibody was insolubilized by polymerization.
5. A method according to Claim 4, wherein the anti-bodies were purified prior to polymerization.
6. A method according to Claim 1 wherein the insoluble antibody is polymerized anti-gamma-globuline.
7. A method for the detection and determination by enzyme immunoassay of a bindable progesterone comprising the steps of:
a. providing a given quantity of a conjugate of a given derivative of the progesterone with .beta.-D-galactosidase enzyme (3.2.1.23), said given derivative being progesterone 11-?-hemisuccinate;
b. providing a corresponding given quantity of an antibody against a conjugate of said given derivative with a high molecular weight substance, both of said conjugates involving coupling of a chemically identical derivative of the progesterone by a chemically identical bond or bridge at the same position of the progesterone derivative molecule;
c. admixing a sample of a fluid containing the progesterone to be determined with the reactants of steps (a) and (b) to form a reaction mixture and allowing the reaction to go to completion;
d. separating the resulting mixture into a liquid phase and a solid phase by adding an insolubilized antibody against the antibody of step (b); and e. determining the quantity of the progesterone from the measure of enzyme activity of either separated phase; said method having a determination sensitivity of at least 0.3 nano-grams per milliliter.
a. providing a given quantity of a conjugate of a given derivative of the progesterone with .beta.-D-galactosidase enzyme (3.2.1.23), said given derivative being progesterone 11-?-hemisuccinate;
b. providing a corresponding given quantity of an antibody against a conjugate of said given derivative with a high molecular weight substance, both of said conjugates involving coupling of a chemically identical derivative of the progesterone by a chemically identical bond or bridge at the same position of the progesterone derivative molecule;
c. admixing a sample of a fluid containing the progesterone to be determined with the reactants of steps (a) and (b) to form a reaction mixture and allowing the reaction to go to completion;
d. separating the resulting mixture into a liquid phase and a solid phase by adding an insolubilized antibody against the antibody of step (b); and e. determining the quantity of the progesterone from the measure of enzyme activity of either separated phase; said method having a determination sensitivity of at least 0.3 nano-grams per milliliter.
8. The method of Claim 7 wherein said given derivative is progesterone 11-?-hemisuccinate.
9. The method of Claim 7 wherein said conjugate is formed by activating said given derivative with a soluble carbodiimide to form o-acylurea and reacting the o-acylurea with a lysine of .beta.-galactosidase.
10. The method of Claim 7 wherein said insolubilized antibody is polymerized anti-gamma globuline.
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| FR7434519A FR2288312A1 (en) | 1974-10-14 | 1974-10-14 | PROGESTERONE IMMUNOENZYMATIC ASSAY PROCESS |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1071535A true CA1071535A (en) | 1980-02-12 |
Family
ID=9144070
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA237,476A Expired CA1071535A (en) | 1974-10-14 | 1975-10-10 | Method for enzyme immunoassay of progesterone |
Country Status (8)
| Country | Link |
|---|---|
| JP (1) | JPS5191325A (en) |
| BE (1) | BE834248A (en) |
| CA (1) | CA1071535A (en) |
| DE (1) | DE2545980A1 (en) |
| ES (1) | ES441738A1 (en) |
| FR (1) | FR2288312A1 (en) |
| GB (1) | GB1531974A (en) |
| NL (1) | NL7512025A (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS6111664A (en) | 1984-06-28 | 1986-01-20 | Ono Pharmaceut Co Ltd | Reagent composition for enzyme immunomeasurement of prostaglandin and measuring method of prostaglandin using said composition |
| CH670709A5 (en) * | 1986-04-24 | 1989-06-30 | Univ Moskovsk |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3637711A (en) * | 1968-03-25 | 1972-01-25 | Ashland Oil Inc | Beta-alkenyl substituted 8-hydroxyquinolines |
| NL154599B (en) * | 1970-12-28 | 1977-09-15 | Organon Nv | PROCEDURE FOR DETERMINING AND DETERMINING SPECIFIC BINDING PROTEINS AND THEIR CORRESPONDING BINDABLE SUBSTANCES, AND TEST PACKAGING. |
| NL171930C (en) * | 1972-05-11 | 1983-06-01 | Akzo Nv | METHOD FOR DETERMINING AND DETERMINING BITES AND TEST PACKAGING. |
-
1974
- 1974-10-14 FR FR7434519A patent/FR2288312A1/en active Granted
-
1975
- 1975-10-06 BE BE160740A patent/BE834248A/en not_active IP Right Cessation
- 1975-10-10 CA CA237,476A patent/CA1071535A/en not_active Expired
- 1975-10-13 NL NL7512025A patent/NL7512025A/en not_active Application Discontinuation
- 1975-10-13 ES ES441738A patent/ES441738A1/en not_active Expired
- 1975-10-14 DE DE19752545980 patent/DE2545980A1/en active Pending
- 1975-10-14 GB GB4191275A patent/GB1531974A/en not_active Expired
- 1975-10-14 JP JP50122988A patent/JPS5191325A/en active Pending
Also Published As
| Publication number | Publication date |
|---|---|
| ES441738A1 (en) | 1977-03-16 |
| JPS5191325A (en) | 1976-08-10 |
| BE834248A (en) | 1976-04-06 |
| FR2288312A1 (en) | 1976-05-14 |
| FR2288312B1 (en) | 1977-03-18 |
| NL7512025A (en) | 1976-04-20 |
| GB1531974A (en) | 1978-11-15 |
| DE2545980A1 (en) | 1976-05-06 |
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