Note: Descriptions are shown in the official language in which they were submitted.
1160~6
RAN 4093/51
The present invention is concerned with an enzyme-
-immune process according to the so-called "sandwich
principle". According to this principle, the substance to
be determined, which can be an antigen, an antibody or a
hapten, is reacted with two immunologically active reaction
partners. Usually, one of these immunologically active
reaction partners is bound to a water-insoluble carrier,
while the other is marked with a suitable enzyme. In
practice, the substance to be determined is firstly reacted
with the reaction partner bound to the carrier, whereupon,
after phase separation and washing, the substance which is
meanwhile immunologically bound to the carrier is reacted
with the second, enzyme-marked reaction partner. After
renewed phase separation, the enzymatic reaction for the
quantitative detection of the substance to be determined is
carried out either in the solid or the liquid phase.
It has hitherto been the opinion that, in the case of
enzyme-immune processes, the immunological reaction of the
substance to be determined had to be effected with the two
corresponding immunologically active reaction partners
successively in a first and in a second reaction step.
566
-- 2
In the scope of the present invention it has now
surprisingly been found that, contrary to the hitherto
existing opinion, it is possible to work in e~fect in a
"one-pot process", with the substance to be determined
being reacted simultaneously with both immunologically
active reaction partners during a single incubation.
The present invention is accordingly concerned
with an enzyme-immune process for the detection and
determination of a substance with an immunologically active
reaction partner, which is marked with an enzyme, as well
as an immunologically active reaction partner, which is
or will be bound to a water-insoluble carrier, by incubation
of the substance to be determined with the immunologically
active reaction partners, subsequent separation of solid and
liquid phases and measurement of the extent of the enzyme
marking either in the solid or in the liquid phase as the
extent of the amount of the substance to be determined, which
is characterised in that for the immunological reaction the
substance to be determined as well as the immunologically
active reaction partners are incubated from the outset and
together and only once.
The substance to be determined can be any constituents
in physiological fluids, cell extracts or tissue extracts
for which there are present or can be formed immunological
reaction partners, and which possess two or more immuno-
logically active sites. Such substances include peptides,
~l~O';~
proteins, lipoproteins, glycoproteins, sterols, steroids,
lipoids, nucleic acids, enzymes, ho:rmones, polysaccharides
and alkaloids, Preferred immunologically active substances
are natural antigens such as hormones, enzymes, organ-
-specific antigens, connective tissue components, blood cell
antigens, plasma proteins and pathological globulins.
Especially preferred substances are carcinoembryonic antigen
(CEA) as well as human choriongonadotropin (HCG).
Thus the present invention provides an immunoassay
process for determining substances having at least two immuno-
logically active sites comprising:
a) preparing a mixture of
(i) a substance having at least two immunologically
active sites or epitopes,
(ii) an immunologically active reaction partner having
an enzyme marker, and
(iii) an immunologically active reaction partner which
is or will be bound to a water insoluble carrier,
b) incubating said mixture,
c) separating the solid and liquid phase,
d) determining the amount of substance by measuring the
extent of the enzyme marking in the solid or liquid phase,
the improvement consisting in
e) using different monoclonal antibodies as the immunologi-
cally active reaction partners, orf) using a monoclonal antibody and an antibody from a dif-
ferent animal species as the immunologically active
~-~ reaction partners.
116~S~j
- 3a -
In the method in accordance with the invention the
immunologically active reaction partner, which is marked with
an enzyme, serves as the indicator of the immunological reac-
tion. Preferred enzymes are alkaline phosphatase (EC 3.1.3.1),
malate dehydrogenase (EC 1.11.37), glucose-6-phosphate de-
hydrogenase (EC 1.1.1.49), glucose oxidase (EC 1.1.3.4), gluco-
amylase (EC 3.2.1.3), galactosldase (EC 3.2.1.22/3.2.1.23) and
acetylcholine esterase (EC 3.1.1.7). Peroxidase from horse-
radish (EC 1.11.1.7) is an especially preferred enzyme label.
The enzyme as the indicator of the immunological
reaction is measured in the liquid phase or, preferably, in
the solid phase according to known methods and is a measurement
for the amount of the substance to be determined. When the
label is peroxidase from horseradish the amount of enzyme is
preferably measured on the basis of the catalytic activity
present, which is determined with the aid of hydrogen
peroxide and o-phenylenediamine as the redox indicator. In
.,
,6
-- 4
this case, after a catalytic reaction lasting for 30 minutes
the colour intensity of the oxid:Lsed redox indicator is
measured photometrically.
The second immunologically active reaction partner
serves for ~he separation of the substance to be determined
from the analytical sample. For this separation step the
second immunologically active reaction partner can be bound
to a water-insoluble carrier from the outset or can be bound
to such a carrier during or after the immunological reaction.
Suitable water-insoluble carriers for the second
immunologicaliy active reaction partner are organic and
inorganic polymers [amylases, dextrans, natural or modified
celluloses, polyacrylamides, agaroses, magnetite, porous
glass powder, polyvinylidene fluoride (Kynar) and latex],
the inner wall of test vessels (test tubes, titre plates or
cuvettes of glass or artificial materials) as well as the
surface of solid bodies (rods of glass and artificial
material, rods with terminal thickening, rods with terminal
lobes or lamellae). Spheres of glass and artificial
material are especially suitable carriers for the method in
accordance with the invention.
~ he second immunologically active reaction partner
can be bound to the water-insoluble carrier physically
(a~sorptively) or chemically or with
-- 5
the aid of a further reaction partner, which, in turn,
is bound to a carrier.
It is essential for the method in accordance with the
invention that the substance to be determined possesses at
least two immunologically active sites (epitopes), which
can be recognised by and react with the two immunologically
active reaction partners, the reaction partner which is
bound to a carrier and the reaction partner which is marked
with an enzyme. As the immunologically active reaction
partners there are preferably used two different components,
which indeed both react with the substance to be determined,
but each cf which is directed to different immunologically
active sites. For the determinatlon of antigens there are
especially suitable two antibodies for the particular antigen,
the antibodies having been produced in two different animal
species and being directed towards different epitopes of
the antigen. The combination of antibodies of different
- clones or of monoclonal antibodies and antibodies from a
different animal species is particularly suitable.
For the immunological reaction the sample with the
substance to be determined can be used directly or can be
pre-diluted or pre-treated in a suitable manner. For the
pre-treatment the substance to be determined can be isolated,
enriched or freed from troublesome ingredients.
-- 6
According to the process in accordance with the
invention, the substance to be determined is rea~ted
simultaneously with the enzyme-marked as well as the carrier-
-bound immunologically active reaction partner. The sequence
in which the reagents are added is governed by the type of
carrier system chosen. When a sensitised plastic sphere is
used, the enzyme-marked reaction partner and the substance
to be determined are initially placed together in a suitable
test tube and subsequently the sphere sensitised with the
other reaction partner is added.
The immunological reaction is carried out in a suitable
buffer system so as to maintain an optimum pH-value, which
can lie between 4 and 9. Preferred buffers are, for example,
acetate buffer, citrate buffer, phosphate buffer, tris
buffer, triethanolamine buffer, borate buffer or glycine
buffer. Buffer mixtures can also be used.
The immunological reaction is preferably carried out
at a temperature between 0C and 55C. Normally, the
immunological reaction velocity increases with higher
temperatures, whereby under otherwise similar test conditions
the equilibrium is achieved more rapidly.
The incubation of the substance to be determined with
the enzyme-marked reaction partner and the carrier-bound
reaction partner can be carried out until the equilibrium
- 25 has been reached. The immunological reaction can, however,
'iÇ;6
-- 7 --
also be stopped at an earlier point in time by separating
the solid and the liquid phase after a defined incubation
period and measuring the extent of the enzyme marking
either in the liquid phase or in the solid phase.
In the immunological reaction precautions can be
taken to stabilize the immunological activity of the reaction
partners and of the substance to be determined as well as of
the enzyme. Further, ingredients such as proteins and
detergents~to eliminate unspecific reactions, to reduce
inhibiting influences or to enhance activation can be
added to the incubation solution.
The novel method in accordance wlth the present
invention is extraordinarily sensitive and is distinguished
by its simplicity in operation.
-- 8 --
The following Examples illustrate the invention.
Example 1
-
Quantitative determination of CEA in plasma of patients
with a monoclonal CEA antibody and a customary CEA antibody
(goat)
Into the requisite number of test tubes (10 x 75 mm)
are in each case pipetted 0.2 ml of test solution (0.2 mol/l
of N2H2P04/Na2HP04, pH 6.5 with 2 g/l of bovine serum
albumin, 20~ of normal goat serum and 0.2 ~g/ml of goat-
-anti-CEA-peroxidase conjugate), 0.050 ml of the patient's
plasma to be analysed or of the CEA standard (0 ng/ml of
CEA, 2.5 ng/ml of CEA, 10 ng/ml of CEA and 20 ng/ml of CEA)
and of the CEA control serum (5.0 ng/ml of CEA + 1.0 ng/ml)
are admixed, in each case there is added a polystyrene sphere
(0 = 6.5 mm) sensitised with monoclonal mouse anti-CEA~ and
the mixture is incubated at 37C for 16 hours. Subsequently,
the polystyrene spheres are washed three times with in each
case 2-5 ml of distilled water, transferred into in each case
0.5 ml of substrate buffer for the determination of the
activity of the peroxidase (0.1 mol/l of potassium citrate
buffer of pH 5.0 with 6 mmol/1 of hydrogen peroxide and
20 mmol/l of o-phenylenediamine) and incubated for 30 minutes
at room temperature (22C). In order to stop the peroxidic
activity and to intensify the colour intensity 2.0 ml of lN
l~()S~
- 9 - ~
hydrochloric acid are admixed and withi~ 30 minutes the
extinction is measured photometrically at the wa-~elength
492 nm. The values of a CEA determination are compiled in
Table I and compared with the values which have been obtained
with the radioimmunoassay of ROCHE.
*The preparation of the monoclonal mouse anti-CEA
is carried out in analogy to the method described in
Journal of Immunological Methods, 32 (1980) 297-304, there
being used as the starting cell line for the fusion the
myeloma line Sp 2/01-AG which is deposited at ATCC under
No. CRL 8006. The fusion is carried out with spleen cells
of mice which ha~e been immunised with CEA. The immunisation
of the mice was carried out in analog~ to Table 1 of the
aforementioned publication, the first two immunisations
being carried out with in each case 50 ~g of CEA, immunisations
3 and 4 being omitted, immunisation 5 being carried out with
50 ~ of CEA and immunisations 6-8 being carried out with in
- each case 200 ~g of CEA.
~1~S~6
-- 10 --
Table I
Sample material ~E492 nm/RT/30 min-
CEA s tandard
0 ng/ml CEA O. 103
2.5 ng/ml CEA 0.330
10.0 ng/ml CEA O . 9 78
20~0 ng/ml CEA 1.850
I
CEA control serum
5.0 ng/ml CEA O . 540
Process in accordance
Patient's plasm ROCHE RIA test with the invention
No. 72120.6 ng/ml 1.2 ng/ml
No. 71882.2 ng/ml 1.0 ng/ml
No. 72201.2 ng/ml 1.4 ng/ml
No. 72181.2 ng/ml 1.3 ng/ml
No. 72342.5 ng/ml 2.7 ng/ml
No. 72492.4 ng/ml 2.0 ng/ml
No. 72032.3 ng/ml 2.0 ng/ml
No. 72233.0 ng/ml 3.3 ng/ml
No. 72473.1 ng/ml 2.8 ng/ml
No. 72584.6 ng/ml 4.3 ng/ml
No. 72154.9 ng/ml 5.5 ng/ml
No. 72195.0 ng/ml 5.9 ng/mg
No. 81808.6 ng/ml 8.5 ng/ml
No. 724811.2 ng/ml 10.7 ng/ml
No. 726214.2 ng/ml 14.3 ng/ml
No. 720115.6 ng/ml 15.3 ng/ml
~1605~;6
Values below 2.5 ng/ml of CEA lie in the normal range,
while values above 2.5 ng/ml lie in the patholo~ical range.
Example 2
Quantitative determination of CEA in plasma of patients
with CE~ antibodies from two different animal s ecies
P
(goat and guinea pig)
Into the requisite number of test tubes (lO x 75 mm)
are in each case pipetted 0.2 ml of test solution (0.2 mol/l
of NaH2P04/Na2HP04, pH 6.5 with 2 g/l of bovine serum
albumin, 20~ of normal goat serum and 0.2 ~g/ml of goat-
-anti-CEA-peroxidase conjugate), 0.050 ml of the patient's
plasma to be analysed or of the CEA standard (0 ng/ml of
CEA, 2.5 ng/ml of CEA, 10 ng/ml of CEA and 20 ng/ml of CEA)
and of the CEA control serum (5.0 ng/ml of CEA + l.0 mg/ml)
are admixed, in each case there is added a polystyrene sphere
(0 = 6.5 mm) sensitised with guinea pig-anti-CEA and the
mixture is incubated at 37C for 16 hours. Subsequently, the
polystyrene spheres are washed three times with in each case
2-5 ml of distilled water, transferred into in each case
0.5 ml of substrate buffer for the determination of the
activity of the peroxidase (0.1 mol/l of potassium citrate
buffer of pH 5.0 with 6 mmol/l of hydrogen peroxide and 20
mmoljl of o-phenylenediamine) and incubated for 30 minutes
US~
at room temperature (22C~. In order to stop the peroxidic
activity and to intensify the colour intensity 2.0 ml of
lN hydrochloric acid are admixed and within 30 minutes the
extinction is measured photometrically at the wavelength
492 nm. The values of a CEA determination are compiled in
Table II and compared with the values which have been
obtained with the radioimmunoassay of ROCHE.
;6
- 13 --
Table II
Sample material _ ~E492 nm/RT/30 min.
CEA standard
O ng/ml CEA 0.058
2.5 ng/ml CEA 0.270
10.0 ng/ml CEA 0.830
20.0 ng/ml CEA 1.515
CEA control serum
5.0 ng/ml CEA 0.470
Process in accordance
Patient's plasma ROCHE RIA test with the invention
No. 72201.2 ng/ml 1.3 ng/ml
No. 72181.2 ng/ml 1.3 ng/ml
No. 72342.5 ng/ml 2.4 ng/ml
No. 72492.4 ng/ml 2.2 ng/ml
No. 72032.3 ng/ml 2.2 ng/ml
No. 72233.0 ng/ml 3.0 ng/ml
No. 72473.1 ng/ml 3.2 ng/ml
No. 72584.6 ng/ml 4.5 ng/ml
No. 72154.9 ng/ml 5.3 ng/ml
No. 72195.0 ng/ml 5.6 ng/ml
No. 81808.6 ng/ml 8.5 ng/ml
No. 724811.2 ng/ml10.9 ng/ml
No. 726214.2 ng/ml14.2 ng/ml
No. 720115.6 ng/ml15.0 ng/ml
- 14 -
Values below 2.5 ng/ml of OE A lie in the normal
range, while values above 2.5 ng/ml lie in the pathological
range.
Example 3
Quantitative determination of CEA in plasma of Patients
with monoclonal CEA antibodies from two different clones
_
Into the requisite number of test tubes (10 x 75 mm)
are in each case pipetted 0.2 ml of test solution
(0.2 mol/l of NaH2P04/Na2HP04 pH 6.5 with 2 g/l of bovine
lo serum albumin, 20% of normal goat serum and 0.15 ~g/ml
of monoclonal mouse-anti-CEA peroxidase conjugate*), 0.050
ml of the patient's plasma to be analysed or of the CEA
standard (0 ng/ml of CEA, 2.5 ng/ml of CEA, 10 ng/ml of
CEA and 20 ng/ml of CEA) and of the CEA control serum
(5.0 ng/ml of CEA + 1.0 ng/ml) are admixed, in each case
there is added a polystyrene sphere (0 = 6.5 mm) sensitised
with monoclonal mouse anti-CEA* and the mixture is incubated
at 37C for 16 hours. Subsequently, the polystyrene
spheres are washed three times with in each case 2-5 ml
of distilled water, transferred into in each case 0.5 ml of
substrate buffer for the determination of the activity
of the peroxidase (0.1 mol/l of potassium citrate buffer
of pH -5.0 with 6 mmol/l of hydrogen peroxide and 20 mmol/l
~6V~i6
- 15 -
of o-phenylenediamine) and incubated for 30 minutes at
room temperature ~22C). In order to stop the peroxidic
activity and to intensify the colour intensity 2.0 ml of lN
hydrochloric acid are admixed and within 30 minutes the
extinction is measured photometrically at the wavelength
492 nm. The values of a CEA determination are compiled
in Table III and compared with the values which have been
obtained with the radioimmunoassay of ROCHE.
*The preparation of the monoclonal mouse anti-CEA is
carried out in analogy to the method described in Journal
of Immunological Methods, 32 (1980) 297-304 ! there being
used as the starting cell line for the fusion the myeloma
line Sp 2/01-Ag which is deposited at ATCC under No CRL 8006.
The fusion is carried 01~t with spleen cells of mice which
have been immunised with CEA. The immunisation of the mice
was carried out in analogy to Table 1 of the aforementioned
publication, the first two immunisations being carried out
with in each case 50 ~g of CEA, immunisations 3 and 4 being
omitted, immunisation S being carried out with 50 ~g of CEA
and immunisations 6-8 being carried out with in each case
200 ~g of CEA. There are used two different, suitable
monoclonal antibodies which are directed towards different
epitopes of the OE A antigen.
i~6~S~6
-- 16 --
Table ~II
Sample material ~E492 nmJRT/30 min.
CEA standard
O ng/ml CEA 0.390
2.5 ng/ml CEA 0.440
10.0 ng/ml CEA 0.620
20.0 ng/ml CEA 0.860
_ _ .
CEA control serum
5.0 ng/ml CEA 0.510
_
atient's plasma ROCHE RIA test Process in accord-
ance with the
invention
No. 72201.2 ng/ml Ø8 ng/ml
No. 72342.5 ng/ml 3.0 ng/ml
No. 72233.0 ng/ml 3.5 ng/ml
No. 72473.1 ng/ml 3.2 ng/ml
No. 72584.6 ng/ml 4.4 ng/ml
No. 72154.9 ng/ml 5.0 ng/ml
No. 72195.0 ng/ml 5.4 ng/ml
No. 81808.6 ng/ml 8.6 ng/ml
No. 724811.2 ng/ml 11.0 ng/ml .
No. 726214.2 ng/ml 14.0 ng/ml
No. 720115.6 ng/ml 16.0 ng/ml
_ _
S~SF~
- 17 -
Values below 2.5 ng/ml of CEA lie in the normal
range, while values above 2.5 ng/ml lie in the
pathological range.
Example 4
Quantitative determination of HCG in serum/plasma/urine:
Into the requisite number of test tubes (10 x 75 mm)
are in each case pipetted 0.2 ml of test solution (0.1 mol/l
of NaH2P04lNa2HP04, pH 7.0 with 2 g/l of bovine serum
albumin, and 1.0 ~g/ml of monoclonal mouse-anti-HCG-
-peroxidase conjugate*), 0~050 ml Of the patient's plasma
to be analysed or of the HCG standard (0, 25, 50, 100 and
250 mIU/ml of HCG) is admixed, in each case there is added
a polystyrene sphere (0 = 6.5 mm) sensitised with rabbit-
-anti-HCG and the mixture is incuba~ed at room temperature
for 16 hours in a water-saturated at~osphere. Subsequently,
the polystyrene spheres are washed three times with in each
case 2-5 ml of distilled water, transferred into in each
case 0.5 ml of substrate buffer for the determination of the
activity of the peroxidase (0.1 mol/l of potassium citrate
buffer of pH 5.0 with 6 mmol/l of hydrogen peroxide and
20 mmol/l of o-phenylenediamine) and incubated for 30
minutes at room temperature tl6-30C). In order to
;S6
- 18 --
stop the peroxidic activity and to intensify the colour
intensity 2.0 ml of lN hydrochloric acid are admixed and
within 30 minutes the extinction is measured photometrically
at the wavelength 492 nm. The values of a HCG determination
in serum and urine are compiled int~e following Tabie.
*The preparation of the monoclonal anti-HCG can be
carried out according to one of the methods described in
Journal of Immunological Methods, 32 (1980) 297-304.
HCG determination in serum and in urine
1. Standard curves
~E /30 min./RT
492 nm
mIU HCG/ml Serum Urine
0 0.185 0.385
0.385 0.525
0.530 0.720
15100 0.830 1.05
250 1.185 1 1.59
- lg -
2. _atient1s samples
Sample No. ~E492nm/3 min./RT = mIU HCG/ml sample
.
Urine N-1032 E 0.375 0
" 58-418 0.575 (x 50) * 1500
" 58414 0.775 (x 100) * 5500
" 58416 0.810 (x 500) * 32000
Serum 2559 0.155 0
" 2560 0.125 0
" 1543 0.195 1.5
" 2673 0.505 44
" 1167 1.085 181
" 1492 0.98 (x 10) * 1370
" 2793 0.77 (x 20) * 1740
" 924 0.69 (x 100) * 7300
* Dilution of the sample
onversion: 1 ng of pure HCG corresponds to ca. 10 mIU
of HCG.
- 20 -
Example 5
Quantitative determination of HCG in serum with monoclonal
HCG antibodies from two different clones
Into the requisite number of test tubes (10 x 75 mm)
are in each case pipetted 0.2 ml of test solution
(0.1 mol/l of NaH2P04/Na2HP04,pH 7.0 with 2 g/l of
bovine serum albumin and 1.0 ~Ig/ml of monoclonal mouse-
-anti-HCG-peroxidase conjugate*), 0.050 ml of the patient's
plasma to be analysed or of the HCG standard (0, 25, 50, 100
and 200 mIU/ml of HCG) are admixed, in each case there is
added a polystyrene sphere (0 = 6.5 mm) sensitised with
monoclonal mouse-anti-~CG* and the mixture is incubated,
for example, at 37C for. 2 hours. Subsequently, the
polystyrene spheres are washed three times with in each
case 2-5 ml of distilled water, transferred into in each
case 0.5 ml of substrate buffer for the determination of
the activity of the peroxidase (0.1 mol/l of potassium
citrate buffer of pH 5.0 with 6 mmol/l of hydrogen peroxide
and 20 mmol/l of o-phenylenediamine) and incubated for 30
minutes at room temperature (16-30C). In oraer to stop
the peroxidic activity and to intensify the colour intensity
1.0 ml of 2N hydrochloric acid are admixed and ~ithin 30 minutes
the extinction is measured photometrically at the wavelength
492 nm. The values of HCG determinations in serum are
compiled in the following Table.
- 21 -
*The preparatisn of the monoclonal mouse HCG antibody
is carried out according to the method described in Journal
of Immunological Methods, 32 (1980) 297-304. There are
used two different, suitable monoclonal antibodies which
are directed towards different epitopes of the HCG antigen.
HCG determination in human serum (average values from
duplicate determinations)
1. Standard curves
GE 492 nm/30 min./RT
10 mIU/ml HCG 1) Serum 2) Plasma 3) Urine 4) Buffer
I
0 0.038 0.054 0.158 0.180
0.226 _ _ _
0.475 o,544 0,557 0.716
100 0.905 0.955 0.970 1.40
200 1.75 1.55 1.90 2044
l~t`~
- 22 -
The HCG values of the examples of patient's sera
compiled below were read-off from standard curve 1). Standard
curves 2), 3) and 4) were established on another day with new HCG
standards.
2. Patient's sera
~E 492 nm/30 min./RT mIU/ml HCG
Serum measured from curve 1)
. ~
Pool 091080 0.041 0
No. 2801 0.025 0
No. 3881 0.450 47
No. 2673 1.13 127
No. 1167 2.12 (1:2) 470
No. 4891 o.975 (1:50)5,450
No. 3240 1.06 (1:20) 2,280
No. 4418 1.475 (1:1000)167,000
