Note: Descriptions are shown in the official language in which they were submitted.
2(~(~8~
-- 1 --
D e s c r i p t i o n
The invention concerns a method for the determination of
immunologically detectable substances according to a
heterogeneous immunoassay principle by incubation with
at least two receptors Rl and R2, of which Rl mediates
the binding to the solid phase and R2 is a conjugate of
a ligand capable of specific binding to the substance to
be determined and a label, in which a solid phase is
used on which a specifically bindable component is
bound, separation of the solid from the liquid phase and
determination of the label in one of the two phases as
well as a suitable reagent therefor.
A multitude of clinically important parameters are
determined with immunological detection methods. There
are a large variety of homogeneous and heterogeneous
variants of methods for immunoassays. Frequently a
method according to the sandwich principle is used or a
variant procedure derived from it. Usually the sample
solution is incubated with a receptor which is capable
of binding to the substance to be determined and which
carries a label, and a receptor capable of specific
binding to the substance to be determined which is bound
to the solid phase or mediates the binding to the solid
phase. In this process, bound complexes of substance to
be detected and labelled receptor are formed on the
solid phase via the receptor which mediàtes the binding
to the solid phase. ~y separation of the solid from the
liquid phase, the bound label can be easily isolated
from the unbound label and can be detected in one of the
two phases. The amount of label is a measure of the
content of substance to be detected.
z~ o
There are different situations in which not only one
test but several different tests have to be carried out
in parallel. This is, for example, the case in the
diagnosis of antibodies directed towards particular
viruses such as e.g. anti-HIV antibodies or antibodies
directed towards hepatitis antigens. In general, a
multitude of different antibodies are formed by the body
to an antigen or hapten i.e. antibodies that are capable
of binding to different epitopes. Some viruses have the
property to continually change their surface so that it
is very difficult either to find antibodies which enable
a reliable detection of the antigen or to find model
substances by which all antibodies formed to a
particular virus can be detected. For this reason one
tries to increase the accuracy of the detection in
parallel tests by using different antibodies which are
capable of binding to different viral antigens or by use
of different antigenic determinants. Furthermore, for
many disease conditions it is important to detect
different parameters in parallel, for example, in tumour
diagnosis in the search for tumour markers or in the
investigation of allergy patients for the detection of
allergens or of anti-allergen antibodies. It is also
necessary to carry out different tests in parallel when
investigating blood for transfusions in order to
determine if the blood contains any risk factors such as
HIV antibodies, hepatitis pathogens etc.
Up to now it has been very complicated to detect
different antigens or antibodies in parallel, since a
separate test has to be carried out for each individual
substance which is time-consuming and laborious. It has
also been suggested that several antibodies be
determined simultaneously by immobilizing different
antigens on the solid phase. However, as a rule, this
0
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method did not yield satisfactory results since, on the
one hand, interfering reactions occurred and, on the
other hand, the binding capacity of the solid phase was
so low for such tests that the sensitivity was lower
than that required in practice.
It was therefore the object of the present invention to
provide a method with which several parameters can be
determined simultaneously and with great accuracy and
speed.
,
This object is achieved by a method for the
determination of immunologically detectable substances
according to a heterogeneous immunoassay principle by
incubation with at least two receptors R1 and R2, of
which Rl mediates the binding to the solid phase and R2
is a conjugate of a ligand capable of specific binding
to the substance to be determined and a label, in which
a solid phase is used on which a specifically bindable
reaction component is bound which is characterized in
that a material is used as the solid phase on the
surface of which specifically bindable partners are
bound and that the sample solution is incubated with
several different receptors Rl whereby each receptor R1
has binding sites for the specifically bindable partners
bound to the solid phase as well as binding sites for
one of the substances to be determined.
Surprisingly, different substances can be successfully
determined simultaneously with the method according to
the present invention. A whole spectrum of desired
parameters can be covered simply and rapidly.
Z0~8~)0
-- 4
The method according to the present invention is
suitable for all variants of the heterogeneous
immunoassay.
;
A solid phase is used for the method according to the
present invention on the surface of which specific
binding partners are bound. The well-known materials can
be used for the solid phase such as plastic, glass,
paper carriers, ceramics, latex, magnetic particles and
others. The solid phase can be present e.g. in the form
of reaction tubes, reagent carrier strips or beads. In a
preferred embodiment the solid phase is present in the
form of a reaction vessel and especially preferred in a
cuvette form whose walls are at least partially coated
on the inner surface with specific binding partners. The
known materials are suitable as materials for the
reaction vessel. For this polystyrene, copolymers of
polystyrene, polycarbonates, polyacrylates and
polymethacrylates are preferred.
The coating of the carrier material with the specific
binding pairs can either be carried out directly or via
a carrier material or a spacer. Binding to a soluble
protein with a molecular weight above 500000 which is
then adsorbed to the inner surface of the reaction
vessel is, for example, suitable. Equally suitable is
binding via a spacer which can be covalently or
adsorptively bound to the surface of the reaction vessel
via a functional group. Methods and agents for this are
known. In a preferred embodiment a carr.ier material is
used as the solid phase which is prepared according to
the method described in DE-A-36 40 412.8. In a further
preferred embodiment the solid phase is a reagent
carrier which was obtained by activating a fibre pad
made of a mixture of cellulose and synthetic fibres with
2(~ 0
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periodate and coating it with the specific binding
partner which had been previously treated with acid. A
method for producing such reagent carriers is described
in DE-A-35 43 749.
A specific binding partner is bound to the surface of
the solid phase. This partner must be capable of binding
to a part of receptor R1. Since the binding between the
partner bound to the solid phase and the receptor R
should not interfere with the ability of the receptor R
to bind to the substance to be determined, then, if
receptor R1 is an antibody, either antibodies which are
directed towards the Fc part of this antibody are used
or protein A is used or partners Pl of a specific
binding pair are imn~obilized. Antigen-antibody, hapten-
antibody, lectin-carbohydrate, biotin-antibiotin-
antibody, biotin-streptavidin as well as biotin-avidin
are, for example, suitable as the specific binding pair.
Partners which are capable of binding to biotin, in
particular streptavidin or avidin, are preferably
immobilized on the solid phase. If protein A is used as
P1 or P2 only receptor Rl should be a complete antibody
in the immunoassay, while a Fab ar F(ab')2 fragment
should be used as the labelled receptor in order not to
cause non-specific binding of the labelled receptor to
the solid phase which would result in a falsification of
the results.
The sample solution is incubated with different
xeceptors Rl. Receptors are used as the receptors Rl
which have binding sites for the partner bound to the
solid phase as well as binding sites for one of the
substances to be detected. Complete antibodies of all
subclasses which are capable of binding to one of the
substances to be detected can be used as receptors Rl.
2~ n
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In this case either antibodies are bound to the solid
phase which are directed towards the Fc part of the
antibody used for Rl or protein A is bound. In the
latter case, the labelled antibody should not be a
complete antibody, in order not to cause non-specific
binding to the solid phase which would result in a
falsification of the result. In a further embodiment
conjugates of partners P2 complementary to partner P1
which is bound to the solid phase as well as ligands
capable of binding to one of the substances to be
detected are used as receptors Rl. The immobilization is
effected via the partner P2 which binds partner P1.
Preferably receptor Rl contains a hapten such as FITC,
p-nitrophenol, saponin, digoxin, or, particularly
preferably, biotin as partner Pl. The ligand binds to
one of the substances to be detected. Depending on the
substance to be detected the ligand can be an antibody,
antibody fragment, antigen, hapten or binding protein.
Whole or purified parts of pathogens such as bacterial
antigens, protozoal antigens, allergens or viral
antigens can be used as antigens. The antigens can
consist of native material, recombinant material or of
chemically synthesized or modified peptides or
carbohydrates. Thus, for example, in the detection of
HIV antibodies, different antigenic determinants such as
p24, gp41 and gp32 are used as ligands for the receptors
Rl. If the type and course of a hepatitis illness is to
be detected, then the different virus antigens HBcAg,
HBsAg, HBeAg, HAV etc. can be used as ligands for
receptor Rl. If the method is to be used for the
detection of allergies, then the different allergens are
used to detect the presence of corresponding antibodies.
For the detection of tumour markers, the corresponding
different antibodies are used for receptor Rl, for
example, the antibodies to CEA, AFP, CA15.3 can be used
in parallel for the receptors Rl. For the determination
2~
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of hormones, antibodies to TSH, LH, FSH, cortisol,
prolactin etc. may be considered. Likewise for the
detection of diseases, antibodies to the different
viral, bacterial or protozoal antigens can be used.
Instead of the antibodies, their fragments such as Fab,
Fab' or F(ab')2 fragments can, of course, also be used.
The antibodies can be polyclonal or monoclonal.
The second receptor R2 used in the method according to
the present invention is a conjugate of a ligand capable
of binding to one of the substances to be determined and
a label. Many possibilities are known for the labelling
and are suitable for the present method. As examples,
radioactive isotopes such as 125I 131I 51Cr 35S and
3H, enzymes such as peroxidase, ~-galactosidase or
alkaline phosphatase, fluorescent, chemiluminescent or
substanc~s detectable by other means can be used. The
ligand used for R2 can be a component capable of binding
to all substances to be detected e.g. in the case in
which different antigens are used for the detection of
definite antibodies such as e.g. HIV. In this case the
label for all receptors R2 is also the same. The
simultaneous determination of IgE antibodies of
different allergens is analogous. If different
parameters are to be determined in parallel, different
ligands can be used for each of the receptors R2 which
are capable of specific binding to the individual
substances to be detected. The determination of the
individual parameters in parallel can be carried out in
two variants. In a first variant the method is carried
out in several phases. In this case the different
receptors R2 which each carry the same label are added
in succession. Since each receptor R2 can only react
with one substance to be detected, the portion of bound
or unbound label can be determined after each addition
2~
of the receptor and is a measure of the respective
substance to be detected. In a second variant, the
method is carried out in a s:ingle phase~ This variant is
preferred because it can also be carried out in
automated analysers. In this case, the different
recept~rs R2 each carry different labels which enable a
simultaneous determination e.y. different isotopes with
distinguishable emissions or fluorescent compounds which
fluoresce at different wavelengths. Equally suitable is
labelling with different enzymes. In this case different
substrates are added for the analysis which form colours
under the action of the respective enzyme that has an
adsorption maximum at different wavelengths. The same
ligands as for receptor R1 are suitable as ligands for
receptor R2. Depending of the determination to be
carried out, the ligands used for receptor R2 can be
identical with those ligands used for receptor Rl or
different from them. For the detection of antigens,
antibodies directed towards the antigens or their
fragments, as well as binding proteins can be used as
ligands.
Antibodies directed towards e.g. IgG, IgA, IgM, IgE or
fragments of these antibodies or antigens capable of
binding to the antibodies to be detected or haptens can
for example be used for the detection of antibodies. The
receptors R2 can be used in the form of mixtures or in
the form of cross-linked products. A cross-linking can
be carried out in a well-known way, for example, by use
of bifunctional or polyfunctional linkers. Methods for
this are known to the expert and do not need to be
described in more detail.
The sample solution can either be incubated
simultaneously with all receptors or first with receptor
20~
g
R1 and then with the different receptors R2. Further
variants of the method are possible and known to the
expert. In this process, the reaction with both
receptors Rl and R2 is carried out in a homogeneous
phase.
:`
Using the method according to the present invention it
is possible to carry out simultaneous determinations for
many different parameters. The simple procedure enables
the simultaneous determination in automated analysers.
`
By using a surface which is coated according to the
present invention with a partner of a specific binding
pair, sufficient binding capacity is available so that
all parameters can be detected accurately and
reproducibly.
The method is also suitable for a rapid diagnosis. By
testing simultaneously for HIV antibodies and HBsAg
blood samples which contain these risk factors can be
eliminated immediately without the necessity for more
accurate diagnostics. On the other hand, a rapid test
can check at the outset of an examination if risk
factors are present and, if this is the case, then a
more detailed diagnosis can follow. It is thus possible
according to the present invention to establish very
quickly if particular indications for a disease are
present which then simplifies the additional
examinations.
20~81~0
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The in~ention is elucidated in more detail by the
following Figures and Examples.
ig. 1 shows a diagram for a simultaneous
determination of HIV antibodies and HBsAg.
ig. 2 shows a diagram for a simultaneous
determination of different tumour markers.
The simultaneous determination of HIV antibodies and
HBsAg is shown diagrammatically in Fig. 1. Antibodies
directed towards fluorescein isothiocyanate (FITC) are
immobilized on a solid phase. The sample solution is
incubated with different conjugates as receptors R1
which all contain FITC and either at least one epitope
capable of binding to HIV antibodies or anti-HBsAg
antibodies. After a wash step the sample is incubated
with labelled anti-human-IgG antibody and anti-HBsAg
antibody as receptors R2. The label is radioactive
iodine. Complexes then form from the receptors Rl, HIV
antibodies or HBsAg and from receptors R2, which are
bound to the solid phase by the binding of FITC to the
immobilized anti-FITC antibodies.
Fig. 2 shows a diagram of a simultaneous determination
of tumour antigens. In this case anti-Fc~ antibodies are
immobilized on the solid phase. Antibodies directed
towards tumour antigens are used for each of the
receptors Rl. Cross-linked peroxidase which carries the
Fab fragment of antibodies directed towards tumour
antigens is used as receptor R2. During the incubation
the anti-tumour-antigen antibodies bind the tumour
antigen present in the sample~ In addition the cross-
linked POD mol~cules also bind to the tumour antigens by
zo~
; means of the corresponding Fab antibodies. The
immobilization results from the binding via the anti-Fc
antibodies.
E x a m p 1 e
Anti-HIV test
`:
HIV antibodies are determined in a two-step sandwich
immunoassay. Reagents with the following compositions
are used for the test.
.
Reaqent 1:
10 7 mol/l each of one or several biotinylated HIV
antigens,
40 mmol/l phosphate buffer, pH 7.0
0.9 % by weight sodium chloride
10 % by volume bovine serum albumin.
The following antigens were used for this: HIV antigens
prepared by genetic engineering which correspond to
HIVl-gp41 (gp41-rek., CentocorTM-pl21) and HIVl-p24
(p24-rek., CentocorTM-pg2), chemically synthesized
peptides from HIV1-gp41 (gp41-pep, Wand et al., PNAS,
83, 6159, 1986) and HIV2-gp32 (gp32-pep, Gnann, J.W. et
al., Science, 237, 1346, 1987). These antigens were
labelled with biotin as described by Leary et al., PNAS,
80, 4045 (1983).
- 12 -
Reaqent 2.
20 mV/ml of a conjugate of sheep antibody to human
immunoglobulin and POD
40 mmol/l phosphate buffer, pH 7.0
0.05 ~ by weight Tween 20
0.2 % bovine serum albumin
0.2 % bovine IgG.
Polystvrene tube
Thermally aggregated BSA which is denoted below as
thermo-BSA is prepared in the following manner: 1 g BSA
was dissolved in 100 ml 50 mmol potassium phosphate
solution at a pH of 7.0, heated to 7CC and maintained
for 4 hours at this temperature with gentle stirring.
The solution was cooled, filtered and adjusted to a
concentration of 50 mg/ml. Subsequently it was dialyzed
against the 30 fold volume of redistilled water.
Preparation of a conjugate of streptavidin with thermo-
BSA: Streptavidin isolated from Streptomyces avidinii
was reacted with maleimido-hexanoyl-N-hydroxy-
succinimide and in this way streptavidin was obtained
which carries ma]eimido groups. Thermo-BSA was reacted
with S-acetylmercaptosuccinic acid-anhydride and
afterwards the protected SH-groups were released by
addition of hydroxylamine. The streptavidin containing
maleimido groups was then mixed with the thermo-BSA
contain.ing SH-groups to form the desired conjugate.
Plastic tubes made of polystyrene were then coated with
streptavidin-thermo-BSA conjugate. The coating of the
tubes was carried out with 1.5 ml of a streptavidin-
thermo-BSA solution in which the molar ratio of the two
components was 1.8:1 (10 ~g/ml) in 40 mmol/l sodium
phosphate buffer, pH 7.4 at 20C during 18 to 24 hours.
2~ )0
- 13 -
After aspirating the tubes they were re-coated for
30 minutes at 20'C with 1.8 ml of a solution of 2 %
saccharose, o.~ % sodium chloride and 0.3 % BSA. After
drying (24 hours at 20C and 40 % relative humidity) the
tubes were ready to use and stable.
,"
1 ml Reagent 1 and 10 ~1 human serum or plasma were
incubated for one hour at room temperature in a
polystyrene tube coated with streptavidin-thermo-BSA
conjugate. Afterwards it was washed three times with tap
water and incubated for one hour at room temperature
with 1 ml Reagent 2. It was washed again three times
with tap water and 1 ml ABTS substrate solution was
added for the test reaction. After 60 minutes the
adsorbance at 422nm was measured photometrically.
The anti-HIV test was carried out using individual HIV
antigens and combinations of antigens. In doing so, it
turned out that the test procedure in polystyrene tubes
coated with streptavidin-thermo-BSA conjugate is
suitable for the simultaneous determination of several
antibodies or populations of antibodies (screening test;
Table 1) irrespective of whether these are directed
towards the same virus or several viruses or antigens of
interest.
2C~81~0
- 14 -
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E x a m p 1 e 2
.,
Hepatitis S antigen and HIV antibodies were determined
in a sample solution using a two-step sandwich
immunoassay. The reagents used had the following
composition:
Reagent 1:
40 mmol/l phosphate buffer, pH 7.0
0.1 % Tween 20
0.9 % NaCl
0.01 % phenol
0.2 % bovine serum albumin
300 ng/ml of a FITC labelled monoclonal antibody to
HBsAg as well as 600 ng/ml of FlTC-labelled p24 and of
FITC-labelled gp41 antigen of HIV.
(FITC = fluorescein isothiocynate)
Reaqent 2:
40 mmol/l phosphate buffer, pH 7.0
0.1 % Tween 20
0.2 mol/l sodium tartrate
0.01 % phenol
0.2 % bovine serum albumin
5000 cpm each of a 125I labelled polyclonal sheep
antibody to human IgG antibody as well as of a
monoclonal antibody to HBsAg.
Polystyrene tubes were coated for 18 hours at room
temperature with 1 ml sheep antibody to FITC (50 ~g/ml
in 0.1 mol/l carbonate buffer, pH 9.6) and coated with
1 ml bovine serum albumin (10 mg/ml in 0.1 mol/l
phosphate buffer) for one hour at room temperature.
:~ 20(~81~(~
: - 16 -
So ~l sample was incubated for 2 hours at room
temperature with 1 ml Reagent l. After washing three
times with water it was incubated for one hour at room
temperature with l ml Reagent 2. After washing three
.~ times the tube was measured in a gamma counter. The
results are shown in Table 2.
T a b l e 2
sample HBsAg Anti-~IVmeasured value
(ng/ml)(Western blot)in the
simultaneous test
1 12 positive2125 cpm
2 <0.2 negative 52 cpm
3 <0.2 positive1159 cpm
4 <0.2 positive3041 cpm
<0.2 negative 47 cpm
6 125 negative 830 cpm
7 203 negative1102 cpm
8 <0.2 negative 69 cpm
9 <0.2 negative 29 cpm
<0.2 negative 58 cpm
11 <0.2 positive2552 cpm
12 <0.2 negative 54 cpm
13 52 negative 472 cpm
14 <0.2 negative 39 cpm
~0.2 negative 43 cpm
- 17 -
E x a m p 1 e 3
The tumour mar~ers CEA and CAlS.3 were determined
simultaneously in a sample using a one-step sandwich
immunoassay. The reagent used had the following
composition:
120 mU/ml of a conjugate of POD and Fab fragments each
of a monoclonal antibody to the tumour marker CEA and
CA15.3
~0 mmol/l phosphate buffer, pH 7.0
TM
0.5 ~ Pluronic F6~
0.2 mol/l sodium tartrate
0.01 % phenol
0.2 % bovine serum albumin
300 ng/ml each of a biotinylated monoclonal antibody to
CEA and CA15.3.
The requirements for the monoclonal antibodies to CEA
and CA15.3 are merely that the Fab fragments in the POD
conjugate are each directed towards another epltope than
the biotinylated antibodies.
Polystyrene tubes were coated for 18 hours at room
temperature with 1 ml sheep anti-mouse-Fc~ antibody
(50 ~g/ml in 0.1 mol/l carbonate buffer, pH 9.6) and
coated with 1 ml bovine serum albumin (10 mg/ml in
0.1 mol/i phosphate buffer) for one hour at room
temperature. 1 ml of the reagent and 100 ~1 sample were
incubated for 2 hours at room temperature. Afterwards
they were washed three times with tap water. Then 1 ml
ABT~ substrate solution was added for the test
reaction. After one hour the absorbance at 405 nm was
f~ `
.~ `
.
2~
~ 18 -
measured photometrically. The results are shown in
Table 3.
T a b l e 3
Simultaneous determination of tumour markers
Content
sample CEA CA15.3 measured value in the
(ng/ml) (U/ml)simultaneous test
1 <0.5 1 76 mA
2 453 4 3580 mA
3 2 6 58 mA
4 212 5 2950 mA
4 7 76 mA
6 9 5 43 mA
793 mA
8 6 3 72 mA
9 50 4 250 mA
8 9 83 mA
11 3 7 59 mA
12 9 192 1483 mA
13 3 4 63 mA
14 5 4 51 mA
