Note: Descriptions are shown in the official language in which they were submitted.
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D e s c r i P t i o n
The invention concerns a process for the determination
of an immunologically detectable substance based on a
heterogeneous immunoassay by use of a solid phase on
which one of the immunologically active reaction
components is bound, a suitable reaction vessel therefor
as well as use of a standard reaction vessel.
Immunoassay procedures are often used to determine
substances which can be specifically bound. In this
process one partner of a binding pair, capable of
specific binding to one another, is reacted with its
specific receptor which is labelled in a known way. The
conjugate of both these substances can then still react
with a receptor which is specific for the conjugate or
for one of the two parts of the conjugate. There are
many variants of these immunological procedures. In such
methods it is advantageous if one of the receptors is
present bound to a solid phase. This facilitates the
separation of the bound and non-bound reaction partners.
For the determination of the substance capable of being
specifically bound, the amount of labelled reaction
partner bound to the solid phase or of labelled reaction
partner present in the solution is measured and related
to the amount of the reaction partner to be determined
according to known procedures.
Plastic tubes or microtitre plates on the inner surface
of which the reaction partner is bound, or spheres on
the outer surface of which the reaction partner is
bound, are usually used as the solid phase for
immunological methods.
1 3367 5~
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In these methods the binding of the specific reaction
partner to the respective surface must be carried out in
such a way that it does not lose its ability to
specifically bind to the substance which is capable of
specific binding to it. For this reason the reaction
partner is usually bound adsorptively to the solid
phase. It was therefore already suggested that the
binding of the reaction partner to the solid phase
should be achieved via a coupling agent which mediates
the binding. In this process one should take care that
the binding of the reaction partner to the binding agent
does not destroy the region of the molecule which
specifically reacts or alternatively that the reaction
partner is bound in such a way that its reactive site is
facing away from the solid phase and towards its binding
partner.
A disadvantage of all these known processes is that a
special solid phase has to be provided for each specific
reaction. This means that for each individual test
another solid phase has to be produced, stored and then
used for the determination which is tedious.
The present invention seeks to
provide a process and a reaction vessel which are
suitable for many different methods of detection. For
routine clinical diagnosis many different parameters
such as hormones, tumour markers, as well as parameters
for infection, allergy or fertility are determined from
a single blood sample. The concentration of the
parameters to be determined covers a wide range.
Parameters in low concentrations such as for example TSH
and prolactin are in the range of 10 10 to 10-12 mol/l,
whereas parameters in high concentrations like T3 and T4
are at a concentration of about 10 7 to 10 8 mol/l. For
B
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this purpose it would be desirable if a different tube
did not have to be used in every case for the individual
determinations but rather if a standard reaction vessel
could be used for all determinations.
According to the invention there is provided a process for the
determination of an immunologically detectable substance
based on a heterogeneous immunoassay by use of a solid
phase on which one of the immunologically active
reaction components is bound ,wherein a reaction vessel
is used as the solid phase on the inner surface of which
streptavidin or avidin is bound in such an amount that
0.1 to 2.5 ~g are present per ml reaction volume. The
amount quoted refers to streptavidin or avidin which is
accessible to the binding partner in the form of a
biotinylated protein or hapten during the course of the
immunological reaction.
The process according to the present invention is
applicable to all methods of determination in which the
receptor, which can be an antibody or an antigen, which
is to be immobilized on the solid phase is conjugated
with biotin. The streptavidin or avidin respectively,
bound to the solid phase according to the present
invention, adhere well to all tube materials and the
binding is stable against detergents. In the process of
establishing calibration curves which are necessary for
the evaluation of many immunological methods, the
streptavidin or avidin bound to the solid phase yield
steep calibration curves with small blank readings which
lead to an increase in the accuracy. A further advantage
is that lot to lot variations in the amount of coated
streptavidin had only negligible influences on the
results of measurements. The particular advantage of the
process according to the present invention is that the
1 336759
amount of streptavidin or avidin bound can be so
adjusted that the solid phase can be used for all known
processes based on heterogeneous immunoassays. A further
advantage is that several substances, especially
antibodies, but also antigens, can be determined
simultaneously with the process according to the present
invention. This will be elucidated in more detail in
Example 7.
In a particularly preferred embodiment the reaction
vessel used as the solid phase is at the same time used
as a cuvette for the photometric determination of the
label. This is particularly advantageous since only a
single vessel is necessary to carry out the entire
reaction. In this case the reaction vessel is so
constructed that it has two optically transparent wall
areas facing one another. The entire wall area can be
coated with streptavidin or avidin.
According to the present invention a reaction vessel is
used as the solid phase on the inner surface of which
streptavidin or avidin is bound. The binding of
streptavidin or avidin is carried out by methods known
to the expert (EP-A 0 122 209~ The streptavidin or avidin
can either be bound directly to the solid phase or
alternatively bound via a spacer or substances which
mediate the binding.
In a preferred embodiment of the present invention
streptavidin or avidin is coupled to a soluble protein
with a molecular weight above about 500 000 and then
this conjugate is adsorbed to a hydrophobic solid phase
as described for example in the patent application
DE-A36 40 412 . It is preferable to use a protein which
has been made hydrophobic.
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This increase in hydrophobicity can result from the
application of heat, treatment with acid, denaturing
agents and/or chaotropic ions and/or by chemical
coupling to a hydrophobic compound. Treatment with these
agents also leads to an increase in the molecular
weight. The increase in molecular weight can be also
achieved by cross-linking with a bi-or polyfunctional
protein reagent.
Acetic acid, propionic acid, lactic acid or hydrochloric
acid can for example be used as the acid. The usual
concentrations are 1 to 100 mmol/l with reaction times
from 10 min to 16 hours.
Suitable chaotropic ions are for example thiocyanates,
iodides, fluorides, bromides, perchlorates and
sulphates. Suitable denaturing agents are for example
guanidine hydrochloride or urea. The concentrations of
these are usually from 10 mmol/l to 6 mol/l.
For the derivatisation with hydrophobic compounds
soluble fatty acids, lipoids in a low or high molecular
weight form as well as synthetic polymers such as
polypropylene glycol or soluble copolymers of
polystyrene are preferably used. The derivatisation is
carried out by methods familiar to the expert.
The cross-linking by means of bi- or polyfunctional
compounds is carried out with known protein binding
reagents. These are compounds which have at least two
functional groups which can be either the same or
different and which can react with functional groups of
proteins by means of these functional groups. It is
preferred to use compounds which consist of an alkyl
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chain at the end of which succinimide, maleimide, and/or
aldehyde groups are located.
The protein is cross-linked with the bi- or
polyfunctional compound in a known way by reacting the
soluble protein and the bi- or polyfunctional compound.
In order to increase the hydrophobicity and/or to cross-
link, it is preferable to use proteins with a molecular
weight from 10 000 to 700 000. Particularly preferred is
bovine serum albumin, lipase or immuno-~-globulin.
Streptavidin or avidin is then coupled to the protein
using a known method. Suitable coupling methods are
described for example in Ishikawa, J., Immunoassay 4
(1983), 209-327.
The conjugate obtained from streptavidin or avidin and
protein is then adsorbed on to the plastic surface which
serves as the solid phase. Before the conjugate of
protein and streptavidin or avidin is adsorbed onto the
hydrophobic solid phase it is also possible to
chemically or physically pretreat the solid phase. Thus
for example a plastic surface can be swollen in advance
or activated in another known manner. The adsorptive
binding to the solid phase results from strong and weak
interactions, hydrophobic forces, dipole-dipole or
ion-dipole interactions.
Reaction vessels made of supporting materials with a
surface tension which is smaller than the surface
tension of the soluble hydrophobic protein i.e. which
are more hydrophobic than protein are particularly
suitable as the hydrophobic solid phase. Use of
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supporting materials with a surface tension of
40 erg/cm2 is preferred. Particularly suitable are
polystyrene, polymethacrylate, ~eflon,* polyamide,
copolymers of styrene and acrylonitrile, glass and
cellulose products.
The solid phase material produced according to the
present invention is used in the determination of many
different parameters. In this process one of the
substances capable of binding to the parameter to be
determined is conjugated with biotin which in turn binds
to the streptavidin or avidin respectively bound to the
solid phase. By this means the specific complexes which
form as a result of the immunoassay process can be
immobilized and then determined. Therefore in accordance
with the present invention as much streptavidin or
avidin respectively is bound to the solid phase that per
ml reaction volume 0.1 to 2.5 ~g are available for
binding to the biotinylated substance capable of
specific binding.
The said reaction volume denotes the sum of sample
volume and test reagent volume.
It is preferred to coat the solid phase material such
that 1 to 2 ~g streptavidin or avidin respectively are
available for binding to the biotinylated substance
capable of specific binding. The amounts of the
biotinylated substance capable of specific binding is in
the range of from 10-16 to 10-8 mol/reaction volume.
A further embodiment of the present invention is a
reaction vessel with optically transparent wall areas
which face one another and with avidin or streptavidin
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coated walls which are at least partially within the
inner wall region intended as a receptacle for liquid,
wherein the inner space of the container intended as a
receptacle for liquid and the respective streptavidin or
avidin content of the coating are so matched that 0.1 to
2.5 ~g streptavidin or avidin are present per ml
reaction volume.
This reaction vessel is particularly suitable for
carrying out the process according to the present
invention. Since it has optically transparent wall areas
which face one another and also a coating of
streptavidin or avidin it can be used simultaneously to
carry out the reaction and for the photometric
determination. As a result of the coating with 0.1 to
2.5 ~g streptavidin or avidin respectively per ml
reaction volume, this reaction vessel can be used for
the determination of different parameters on the basis
of a heterogeneous immunoassay.
The reaction vessel constitutes the solid phase. It
consists of materials which are usually used for this
purpose such as plastics, glass etc. The optically
transparent wall areas also consist of known materials.
Particularly preferred in this case are polystyrene,
copolymers of polystyrene, polycarbonates, polyacrylates
and polymethacrylates.
Streptavidin or avidin can either be coated directly or
via a carrier material or a spacer. For example the
binding of streptavidin or avidin to a soluble protein
with a molecular weight above 500 000 which is then
adsorbed to the inner surface of the reaction vessel is
suitable.
1 336759
g
An embodiment of the invention is also the use of a
standard reaction vessel for the determination of
different parameters using processes based on
immunoassays, which consists of a vessel on the inner
surface of which streptavidin or avidin is bound in such
an amount that for the test reaction 0.1 to 2.5 ~g
streptavidin or avidin are available per ml reaction
volume.
According to the present invention a process and a
reaction vessel are provided in which streptavidin or
avidin are bound permanently and with good adhesion to
the inner surface of a reaction vessel which serves as a
solid phase. The adhesion is so good that even the
addition of detergents does not lead to the detachment
of the substance. The reaction vessel provided according
to the present invention can be universally employed and
is suitable for carrying out methods of determination
for very many parameters and therefore facilitates the
performance of routine determinations.
The invention is elucidated by the following figures and
examples. The figures show :
ig. 1 a family of calibration curves for a TSH
determination using immobilized streptavidin
in various amounts and biotinylated anti-TSH-
antibody;
ig. 2 a calibration curve for a prolactin test;
Fig. 3 a calibration curve for a CEA-test and
ig. 4 a calibration curve for a T4-test.
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Fig. 5 a family of calibration curves for an anti-HBs
determination using immobilized streptavidin
in different amounts.
The monoclonal antibodies against TSH used in the
examples originate from hybridoma cell lines which were
deposited at the European Collection of Animal Cell
Cultures, Porton Down, GB, under the numbers
ECACC 87122201 and ECACC 87122202.
E x a m p 1 e
The binding to the solid phase of the conjugate
according to the present invention and of the
hydrophobic protein and streptavidin was investigated:
- Thermally aggregated BSA, from now on referred to as
thermo-BSA, was prepared as follows: 1 g BSA was
dissolved in 100 ml of 50 mmol/l potassium phosphate
solution with a pH of 7.0, and was heated to 70C and
kept at this temperature for 4 hours during which it was
gently stirred. The solution was cooled down, filtered
and adjusted to a concentration of 50 mg/ml. Afterwards
it was dialyzed against a thirtyfold volume of re-
distilled water.
- Preparation of a conjugate of streptavidin with
thermo-BSA: streptavidin was isolated from Streptomyces
avidinii and was converted by maleimido-hexanoyl-N-
hydroxy-succinimide to a streptavidin containing
maleimido groups. Thermo-BSA was converted with
s-acetyl-mercapto-succinic acid anhydride and the
protected SH-groups were afterwards released by the
addition of hydroxylamine. The desired conjugate was
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then obtained by mixing the streptavidin containing
maleimido groups and the thermo-BSA containing
SH-groups.
- Plastic tubes made of polystyrene were then coated
with the streptavidin-thermo-BSA-conjugate. The coating
of the tubes was carried out with 1.5 ml of a
streptavidin-thermo-BSA-solution for 18 to ~4 hours,
whereby the molar ratio of both components was 1.8 : 1
(10~g/ml) in 40 mmol/l sodium phosphate buffer, pH 7.4
at 20C.
After aspiration of the tubes they were recoated with
1.8 ml of a solution containing 2% sucrose, 0.9% sodium
chloride and 0.3% BSA for 30 min at 20OC. After drying
(24 hours at 20OC and 40% relative humidity) the tubes
were ready for use and stable.
E x a m p 1 e 2
One-step test for TSH
Biotinylated monoclonal anti-TSH-antibody was prepared.
For this purpose the anti-TSH-antibody,
ECACC 87122201, was converted with biotin in the usual
way, whereby the coupling too~ place via amino groups.
Antibody and biotin were used in the proportion of
1 : 16. ~00 ng of the biotinylated antibody thus
obtained were dissolved in 1 ml buffer (40 mmol/l sodium
phosphate, 0.5% Pluronic F68, 0.2 mol/l sodium tartrate
as well as 0.01% phenol) and then incubated for two
hours in a coated tube as described in Example 1 with
200 ~1 sample or standard solution respectively
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containing a known amount of TSH and 75 mU of an anti-
TSH-antibody conjugated with peroxidase
(ECACC 87122202) Afterwards it was washed three times
with tap water and 1 ml ABTS-substrate solution was
added. After one hour the absorption was measured at
405 nm. The results are shown in Fig. 1. It turns out
that a satisfactory result is obtained with immobilized
streptavidin when an amount of more than O.l ~g/ml test
volume is used.
The streptavidin required for binding of the
biotinylated antibody used in the test was determined.
For this the amount of freely available biotin was
determined according to the method of Bayer, Edward A.,
Meir-Wilchek, Analytical Biochemistry 154 (1986), 367-
370. Assuming that 1 ~g antibody was biotinylated in an
antibody to biotin ratio of 1 : 15 (Guesdon, I. L., J.
Histochem. Cytochem. 27 (1979) 1131-1139), it follows
that 1 to 2 mol biotin per mol antibody are available
for binding, i.e. 1.5 to 3 ng biotin are freely
accessible per ~g antibody.
A tube coated with 10 ~g/ml streptavidin-thermo-BSA-
conjugate (Example 1) has a binding capacity for biotin
of 15 to 20 ng per ml test volume, determined with
radioactive 14C-biotin.
The binding capacity of the streptavidin tubes for
biotinylated immuno-~-globulin is 1.7 to 2 ~g/ml test
volume. The determination was carried out with
125I-labelled ~-globulin (labelled according to the
method of McConahey and Dixon 1966, Int. Arch. Allergy
29/185).
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E x a m ~ 1 e 3
Prolactin test
Prolactin is determined by a one-step-sandwich-
immunoassay. A reagent of the following composition is
used for the detection:
- 50 mU/ml of a conjugate of POD and a monoclonal
antibody specific for prolactin,
- 40 mmol/l phosphate buffer, pH 7.0
0.5% Pluronic F65 (polyoxyethylene polypropylene)
0.2 mol/l sodium tartrate
0.01% phenol
0.2% bovine serum albumin
400 ng/ml of a biotinylated monoclonal antibody
against prolactin.
Both monoclonal antibodies are only required to detect
prolactin and to be directed against the various
epitopes of prolactin. 1 ml of this reagent and 50 ~1
sample were incubated for 30 min at room temperature in
a polystyrene tube coated with streptavidin-thermo-BSA-
conjugate, obtained according to Example 1. Afterwards
it was washed three times with tap water. 1 ml ABT ~-
substrate solution was added for the test reaction. The
absorbance was measured photometrically after 30 min at
405 nm. The results are shown in Fig.2.
ABT ~ : 2.2'-azino-di-[3-ethylbenzthiazoline-6-
sulphonate]-di-ammonium salt
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E x a m p 1 e 4
CEA test
CEA was determined in a sample solution by a one-step-
sandwich-immunoassay. The reagent used contained the
following:
120 mU/ml of a conjugate of POD and a monoclonal
antibody against CEA,
- 40 mmol/l phosphate buffer, pH 7.0
0.5% Pluronic F68
0.2 mol/l sodium tartrate
0.01% phenol
0.2% bovine serum albumin
500 ng/ml of a biotinylated monoclonal
antibody against CEA.
The only requirements for both monoclonal antibodies
against CEA are that they detect CEA and that each is
directed against a different epitope of CEA.
1 ml reagent and 100 ~1 sample were incubated for 2
hours at room temperature in a polystyrene tube coated
with streptavidin-thermo-BSA, obtained according to
Example 1. Afterwards it was washed three times with tap
water. 1 ml ABTS~ substrate solution was added for the
test reaction. After one hour the absorbance was
measured photometrically at 405 nm. The results are
shown in Fig. 3.
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E x a m p l e 5
T4 test
T4 was determined in a competitive immunoassay using
biotinylated anti-T4-antibodies. For this purpose 500 ~l
of a reagent consisting of:
00 mU/ml thyroxine-POD-conjugate (prepared
according to EP 209 155)
120 mmol/l barbiturate
18.2 mmol/l phosphate buffer, pH 8.6
0.04% by weight ANS (8-anilino-1-naphthaline
-sulphonic acid)
0.2% by weight bovine serum albumin,
as well as 20 ~l sample were incubated for 10 min at
room temperature in a polystyrene tube coated with
streptavidin-thermo-BSA-conjugate, obtained according to
Example 1. Afterwards 500 ~l of a second reagent
consisting of:
~g/ml biotinylated polyclonal antibody against
T4,
120 mmol/l phosphate buffer, pH 8.6,
0.04% by weight ANS (8-anilino-1-naphthaline
-sulphonic acid),
0.2% by weight bovine serum albumin,
were added and incubated for a further 30 min at room
temperature. After washing it three times with tap water
1 ml ABT ~ substrate solution was then added and
incubated for 30 min at room temperature. Afterwards the
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absorbance was measured photometrically at 405 nm. The
results are shown in Fig. 4.
E x a m p 1 e 6
Anti-HBs test
HBs-antibodies were determined in a one-step-sandwich-
immunoassay. A reagent with the following composition
was used for the determination:
60 mU/ml of a conjugate of POD and HBs-antigen
40 mmol/l phosphate buffer, pH 7.0
200 mmol/l sodium tartrate
0.5% by weight Pluronic F68
0.01% by weight phenol
0.2% bovine serum albumin
0.1% by weight bovine-IgG
150 ng/ml biotinylated HBsAg (prepared according
to Immunol. Letters 8 (1984), 273).
1 ml of this reagent and 200 ~1 sample were incubated
for 4 hours at room temperature in a polystyrene tube
coated with a conjugate of streptavidin-thermo-BSA.
After washing it three times with tap water 1 ml of
ABTS-substrate solution was added for the test reaction.
After 60 min the absorbance at 422 nm was measured
photometrically.
The anti-HBs-test was performed in tubes using different
amounts of immobilized streptavidin. The results are
shown in Fig. 5. It can be seen that when streptavidin
is used in amounts which are more than those recommended
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in the present invention then the test becomes
significantly less sensitive.
E x a m p 1 e 7
Anti-HIV test
HIV-antibodies are determined in a two-step-sandwich-
immunoassay. A reagent with the following composition
was used for the determination.
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
The following were used as antigens: HIVl-antigen
produced by genetic engineering corresponding to HIVl-
gp41 (gp41-rek., CentocorTM-pl21) and HIVl-p24 (p24-
rek., CentocorTM-pg2), chemically synthesized peptide
from HIVl-gp41 (gp41-pep, Wang 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).
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Reagent 2:
20 mU/ml of a conjugate of sheep antibodies against
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
1 ml of reagent 1 and 10 ~1 human serum or plasma were
incubated for 1 hour at room temperature in a
polystyrene tube coated with a conjugate of
streptavidin-thermo-BSA. Afterwards the tube was washed
three times with tap water and incubated for 1 hour at
room temperature with 1 ml reagent 2. The tube was again
washed three times with tap water and 1 ml ABTS
substrate solution was added for the test reaction.
After 60 min the absorbance at 422 nm was measured
photometrically.
The anti-HIV-test was carried out using individual HIV-
antigens and combinations of antigens. As a result it
was demonstrated that the test procedure in polystyrene
tubes coated with a conjugate of streptavidin-thermo-BSA
is suitable for the determination of individual antigen-
specific antibodies as well as for the simultaneous
determination of several antibodies or antibody
populations (screening test ; table 1), irrespective of
whether these antibodies are directed against the same
virus or against several viruses or against the antigens
of interest.
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T a b 1 e
Human serum sample
Anti- Anti- Anti- Anti- Anti- Anti-
HIV-Antigens Neg. HIV I HIV I HIV I HIV II HIV I HIV II
Amount of
HIV-Antigen
gp41-rek. 521250 812 521 102 223 75
p24-rek. 43786 1515 212 491 1531 263
gp41-pep 71831 301 295 63 52 59
gp32-pep 38 41 50 62 1819 45 810
gp41-,p24-rek. 451402 1818 618 550 1559 266
gp41-,p24-rek.+ 551380 1753 599 1723 1529 878
gp32-pep
