Note: Descriptions are shown in the official language in which they were submitted.
535
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.
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
-- 4
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
-- 11 --
\~ -
~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
- 12 -
~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
- 13 -
~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
- 14 -
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
61 35 32J5
11 40 ~8
12 36 31~,
- 1 46 47
17 - 44 44
`~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~
1 56 5
5~ ~iO
3 ~:L 42
__
17 ~
~3 :
