Note: Descriptions are shown in the official language in which they were submitted.
2195752
WWibshmy
11051P WO
Oligomeric carrier molecules with defined incorporated
marker groups and haptens
DESCRIPTION
The present invention concerns new conjugates, processes
for their production as well as the use of these
conjugates as antigens in immunological methods of
detection or for DNA diagnostics.
The detection of immunoglobulins in body fluids, in
particular in human sera, is used to diagnose infections
with microorganisms, in particular viruses, such as HIV,
hepatitis viruses etc. The presence of specific
immunoglobulins in the examined sample is usually
detected by reaction with one or'several antigens that
react with the specific immunoglobulins. Methods for the
determination of specific immunoglobulins in the sample
liquid must be sensitive, reliable, simple and rapid.
A further immunological method is a competitive
immunoassay in which an analyte is detected
qualitatively and quantitatively in such a way that a
hapten that is immunologically analogous to the analyte
and the analyte compete for binding sites on a receptor
e.g. an antibody. The analyte analogue is in this case
usually used in a labelled form or in a form capable of
binding to a solid phase.
In recent years more and more detection systems based on
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2195752
non-radioactive marker groups have been developed in
which the presence of an analyte, e.g. a specific
antibody, in the examined sample can be determined with
the aid of optical (e.g. luminescent or fluorescent),
NMR-active or metal-precipitating detection systems.
EP-A-0 307 149 discloses an immunological test for an
antibody in which two recombinant polypeptides are used
as antigens one of which is immobilized on a solid phase
and the other carries a marker group whereby both
recombinant antigens are expressed in different
organisms in order to increase the specificity of the
test.
EP-A-0 366 673 discloses a method for the detection of
antibodies in a sample in which an antibody is detected
by reaction with a purMied labelled antigen and with
the same purified antigen in a solid phase-bound form.
Human IgG is for example disclosed as an antigen.
EP-A-0 386 713 describes a method for the detection of
antibodies against HIV using two solid supports in which
various HIV antigens are immobilized on the two solid
supports each of which is brought into contact with an
aliquot of a sample and with a labelled HIV antigen
wherein the presence of antibodies is detected by a
positive reaction in at least one of the tests.
Recombinantly produced polypeptides are disclosed as HIV
antigens.
EP-A-0 507 586 describes a method for carrying out an
immunological test for a specific immunoglobulin in
which a sample is brought into contact with two antigens
capable of binding the immunoglobulin, wherein the first
Ate.:{
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antigen carries a group suitable for binding to a solid
support and the second antigen carries a marker group.
The marker group can be a direct marker group e.g. an
enzyme, a chromogen, a metal particle, or also an
indirect marker group i.e. the marker group attached to
the antigen can react with a receptor for the marker
group which in turn carries a signal-generating group. A
fluorescein derivative is mentioned as an example of .
such an indirect marker group, the receptor of which is
an antibody which in turn is coupled to an enzyme.
Polypeptides such as the hepatitis B surface antigen are
disclosed as antigens. SH groups are introduced into
this antigen by derivatization which are used to couple
the fluorescein.
EP-A-0 507 587 discloses a method which is specifically
suitable for the detection of IgM antibodies in which
the sample is incubated with a labelled antigen which is
directed against the antibody to be detected and with a
second antibody which is also directed against the
antibody to be detected and is capable of binding to a
solid phase.
EP-A-O 199 804 and EP-A-0 580 979 disclose an
immunological method of detection using antigens which
are labelled with luminescent metal chelate groups and
in particular with ruthenium and osmium chelate groups.
Immunoglobulins are used as antigens which are
statistically labelled by reaction with activated metal
complexes.
EP-A-0 178 450 discloses metal chelates in particular
ruthenium complexes to which an immunologically active
material for example an antibody can be coupled.
4 2195752
Coupling is achieved by statistical reaction of the
immunologically reactive material with the metal
chelate.
EP-A-O 255 534 discloses a luminescence, immunoassay
using a metal chelate-coupled antigen or antibody.
Coupling is for example achieved by statistical reaction
of a metal chelate active ester derivative with an
antibody.
WO 90/05301 discloses a method for the detection and for
the quantitative determination of analytes by
electrochemiluminescencd using luminescent metal
chelates which are coupled to (i) an added analyte, (ii)
a binding partner of the analyte or (iii) a reactive
component that can bind. to (i) or (ii). Luminescence is
measured after binding lEhe metal chelates to activated
and optionally magnetic microparticles.
In the immunological methods for detecting antibodies
known from the state of the art polypeptide antigens are
usually used which are normally produced by recombinant
DNA methods. However, problems may occur when using such
polypeptide antigens. Thus recombinant polypeptides can
often only be produced in the form of fusion
polypeptides in which case the fused part can lead to
false positive results in the test. In addition
polypeptides produced by recombinant expression often
only have a very low stability in the sample solution
and tend to aggregate. A further disadvantage is that it
is often not possible to selectively and reproducibly
introduce marker groups into such polypeptides.
Moreover the production of recombinant polypeptide
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antigens involves high costs and large variations in the'
immunological reactivity in different lots of the
recombinant polypeptides can occur.
Even in competitive immunoassays which have very high
requirements for sensitivity and precision it is often
very difficult to achieve the required lower detection
limits using known antigens when detecting analytes that
are only present in very low concentrations such as
estradiol or testosterone in particular with detection
systems based on electrochemiluminescence.
The object of the present invention was therefore to
provide a process with which antigens for immunological
tests can be produced in a simple and efficient manner
wherein the disadvantages of the antigens known from the
state of the art are ate least partially eliminated. In
addition the process should enable a selective and
reproducible introduction of marker groups into the
antigens.
This problem is resolved by conjugates comprising a
polymeric carrier with a maximum of 100 monomeric units
which contains 1 - 10 hapten molecules and 1 - 10 marker
or solid phase binding groups coupled to reactive side
groups wherein the monomeric units are selected from
amino acids, nucleotides and peptidic nucleic acids.
When using the conjugates according to the invention
that contain 1 - 10 hapten molecules and a defined
number of marker or solid phase binding groups as
antigens in an immunological method of detection it is
surprisingly possible to achieve a considerable higher
sensitivity and precision and at the same time at a
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reduced lower detection limit compared to known
monomeric and multimeric antigens. Moreover the
conjugates according to the invention can be constructed
in a simple manner by solid phase synthesis e.g. a
peptide solid phase synthesis. For this monomeric units,
e.g. amino acid derivatives, that are derivatized by a
hapten molecule or a marker or solid phase binding group
can be incorporated at predetermined positions. In
addition it is possible to selectively incorporate
additional haptens or marker or solid phase binding
groups after completion of the solid phase synthesis at
positions of the carrier chain at which monomers are
located having free functional groups. This enables a
defined and reproducible incorporation of hapten
molecules and marker or solid phase binding groups into
the conjugate. The distances between individual groups
on the conjugate can be'exactly defined and varied if
necessary. The signal,q enching can be kept low by
selecting the distance of the marker groups on the
conjugate so that the signal strength increases
proportionaly to the number of marker groups. A defined
spatial orientation of marker groups also contributes to
the improvement of the signal strength e.g. in the case
of helical carriers. The distances between marker groups
are therefore preferably 3-6 or/and 13-16 monomeric
units in the case of helical carriers e.g. single-
stranded or double-stranded nucleic acids.
The polymeric carrier molecule which forms the backbone
of the conjugate has a maximum length of 100 monomeric
units preferably of 3 - 80 monomeric units and
especially preferably of 5 - 60 monomeric units.
The monomeric units are selected from amino acids,
nucleotides and peptidic nucleic acids. The polymeric
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carrier preferably comprises a peptide chain, preferably
a linear peptide chain, composed of amino acids.
However, the carrier can also be an oligonucleotide to
the reactive side groups of which hapten molecules and
marker or solid phase binding groups are coupled.
In addition the polymeric carrier can be composed of
peptidic nucleic acids. Peptidic nucleic acids comprise
a polyamide backbone made of the same or different
monomeric units of the formula
(CH2)k-CHR'-N[CO-(CH2)i-L]-CH2-(CH2)m-NH-CO-, in which L
is selected from the group comprising hydrogen, phenyl,
naturally-occurring nucleobases and non-naturally-
occurring nucleobases,'R' is selected from the group
comprising hydrogen and-the side chains of amino acids,
preferably a amino acids, that occur naturally or non-
naturally, k and m are each independently 0 or 1 and i
is independently 0 to'5. The hapten molecules and marker
or solid phase binding groups can be coupled to the
nucleobases or/and amino acid side chains of the
peptidic nucleic acids. Peptidic nucleic acids and their
production are described in W092/20703. Reference is
herewith made to this disclosure.
Even in the case of peptidic nucleic acids the carrier
can be present as a single or double strand. Double-
stranded carriers with at least one PNA strand e.g. a
PNA strand and a nucleic acid strand e.g. a DNA strand
are particularly preferred.
The conjugate contains 1 - 10 hapten molecules
preferably 1 - 6 hapten molecules and especially
preferably 1 or 2 hapten molecules. The hapten is
preferably an immunological reactive molecule having a
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molecular mass of 100 - 2000 Da. Such haptens can for
example be selected from pharmacological active
substances such as antibiotics, opiates, amphetamines,
barbiturates, cytostatic agents (e.g. gentamicin,
tobramycin, vancomycin etc.) paracetamol, salicylates,
phenytoin, quinine and quinine derivatives, theophyllin
etc., hormones and metabolites such as sterols, bile
acids, sexual hormones (e.g. estradiol, estriol,
testosterone, progesterone, pregnenolone and derivatives
thereof), corticoids (e.g. cortisol, corticosterone,
cortisone and derivatives thereof), cardenolides and
cardenolide-glycosides (e.g. digoxin, digoxigenin,
strophanthin, bufadienolides etc.) steroid-sapogenines,
steroid alkaloids, peptide hormones, creatinine, thyroid
hormones (e.g T3, T4), neurotransmitters (e.g.
serotonin, choline, y-aminobutyric acid), vitamins and
mediators such as prosthglandins, leucotrienes, leuco-
endiines and thromboxanes.
On the other hand the hapten can also be selected from
immunologically reactive peptide epitopes preferably
having a length of up to 30 amino acids. Such peptide
epitopes can for example be derived from pathogenic
organisms e.g. bacteria, viruses and protozoa or from
autoimmune antigens. The immunologically reactive
peptide epitopes can for example be derived from viral
antigens e.g. the amino acid sequences of HIV I, HIV II
or hepatitis C virus (HCV).
In addition the hapten can also be selected from nucleic
acids with a length of preferably up to 50 nucleotides
that are complementary to a nucleic acid sequence which
is to be detected in the sample. Finally the hapten can
also be selected from peptidic nucleic acids with a
length of up to 50 monomeric units.
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Moreover the conjugate according to the invention
contains 1 - 10 preferably 2 - 8 marker or solid phase
binding groups. Preferred examples of marker groups are
luminescent metal chelates and fluorescent labels.
Preferred examples of solid phase binding groups are
biotin and biotin analogues such as desthiobiotin and
iminobiotin which can specifically react with
streptavidin or avidin.
The hapten molecules and marker or solid phase binding
groups are preferably coupled to the carrier chain via
reactive amino or/and thiol side groups particularly
preferably via reactive primary amino side groups. Such
side groups can be produced by incorporating appropriate
monomers e.g. amino acids such as lysine, ornithine,
hydroxylysine or cysteine into the carrier chain.
In certain embodiments of the present invention it may
be preferable to incorporate a spacer between the hapten
and the marker or solid phase binding group and the
carrier chain. The spacer is preferably flexible and has
a chain length of preferably 3-30 atoms. The spacer
particularly preferably contains hydrophilic groups such
as oxyalkylene or/and hydroxy side groups.
Preferred marker groups are luminescent metal chelates
i.e. metal chelates which can generate a detectable
luminescence reaction. This luminescence reaction can
for example be detected by fluorescence or by
electrochemiluminescence measurement. The metal of these
metal chelates is for example a transition metal or a
rare earth metal. The metal is preferably ruthenium,
osmium, rhenium, iridium, rhodium, platinum, indium,
palladium, molybdenum, technetium, copper, chromium or
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2195752
tungsten. Ruthenium, iridium, rhenium, chromium and
osmium are particularly preferred. Ruthenium is most
preferred.
The ligands which form the metal chel~_te together with
the metal are usually polydentate ligands i.e. ligands
with several co-ordination sites. Polydentate ligands
for example include aromatic and aliphatic ligands.
Suitable aromatic polydentate ligands include aromatic
heterocyclic ligands. Preferred aromatic heterocyclic
ligands are polyheterocycles containing nitrogen such as
for example bipyridyl, bipyrazyl, terpyridyl and
phenanthrolyl. These ligands can for example contain
substituents such as alkyl, substituted alkyl, aryl,
substituted aryl, aralkyl, carboxylate, carboxyaldehyde,
carboxamide, cyano, amino, hydroxy, imino,
hydroxycarbonyl, aminoorbonyl, amidine, guanidinium,
ureide, groups containing sulphur, groups containing
phosphorus and the carboxylate ester of N-hydroxy-
succinimide. Preferred ligands contain C2-C3 alkylenoxy,
C2-C3 alkylenethio and C2-C3 alkylene amino groups, in
particular ethylene-oxy groups. The chelate can also
contain one or several monodentate ligands. Examples of
monodentate ligands comprise carbon monoxide, cyanides,
isocyanides, halogenides and aliphatic, aromatic and
heterocyclic phosphines, amines, stilbenes and arsines.
The luminescent metal chelate is particularly preferably
selected from metal chelates with bipyridyl or
phenanthrolyl ligands. Examples of suitable metal
chelates and the production thereof are described in EP-
A-0 178 450, EP-A-0 255 534, EP-A-0 580 979 and WO
90/05301. Reference is herewith made to these
disclosures. Ruthenium-(bipyridyl)3 chelates are the
most preferred metal chelates. These chelates are
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commercially available in the form of active ester
derivatives for example from the Igen Inc. Company
(Rockville, MD, USA).
When using a luminescent metal complex which is
detectable by an electrochemiluminescence reaction as
the marker group, the incorporation of at least. one
positive or/and negative charge carrier e.g. amino or
carboxylate groups into the carrier chain or/and the
spacer between metal complex and carrier chain has
proven to be advantageous. The carrier chain
particularly preferably contains one or several negative
charges which can for example be generated by
incorporation of glutamic acid or asparaginic acid
during the synthesis. Also in the case of other marker
or solid phase binding'groups e.g. fluorescent groups or
biotin it may be advantageous to incorporate charge
carriers into the carrier chain or/and into the spacer.
A further example of preferred marker groups are
fluorescent labels such as fluorescein, coumarin,
rhodamine, resorufin, cyanine and derivatives thereof.
When using fluorescent marker groups it has proven to be
advantageous to use a helical structure of the carrier
backbone which immobilizes the fluorescent marker groups
with regard to spatial orientation and spacing in order
to prevent fluorescence quenching by energy transfer.
Examples of monomers which result in a suitable helical
structure are proline or a peptidic nucleic acid
derivative with a proline side group.
The conjugates according to the invention are produced
by a process in which a polymeric carrier, preferably a
peptide carrier, composed of monomeric units is
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synthesized on a solid phase in which (a) during the
synthesis monomer derivatives are introduced at
predetermined positions on the carrier which are
covalently coupled to hapten molecules or/and marker or
solid phase binding groups or/and (b) after the
synthesis activated hapten molecules or/and marker or
solid phase binding groups are coupled to reactive side
groups of the carrier.
In variant (a) of the method according to the invention
a monomer derivative is introduced during the solid
phase synthesis which is covalently coupled to a hapten
molecule or/and a marker, or solid phase binding group
preferably via a primary amino side group of a basic
amino acid such as lysine or ornithine or via a thiol
side group of an amino acid such as cysteine. The
corresponding monomer c rivatives are for example
synthesized by coupling an activated hapten molecule or
an activated marker or solid phase binding group e.g. an
active ester derivative to a free primary amino group or
a maleimide derivative to a free thiol group of
optionally partially protected monomer derivatives e.g.
amino acid derivatives. A preferred metal chelate-
coupled lysine derivative is shown in Fig. 1. Fig. 2
shows a biotinylated lysine derivative.
The term "active ester" within the sense of the present
invention encompasses activated ester groups which can
react with free amino groups of peptides under such
conditions that no interfering side reactions with other
reactive groups of the peptide can occur. An N-hydroxy-
succinimide ester is preferably used as the active ester
derivative. Analogous p-nitrophenyl, pentafluorophenyl,
imidazolyl or N-hydoxybenzotriazolyl esters can also be
used in addition to N-hydroxysuccinimide esters.
21 752
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The synthesis of derivatives of hapten, marker groups
and solid phase binding groups that are suitable for
incorporation into oligonucleotide carriers is described
in Theisen et al. (Tetrahedron Letters 33 (1992), 5033-
5036) using the fluorescent dye 5-carboxyfluorescein
which is converted into a phosphoramidite derivative as
an example. This phosphoramidite derivative can be
incorporated at the 5' end or/and the 3' end of
oligonucleotides or within the oligonucleotide sequence.
According to variant (b) of the process according to the
invention the introduced group is coupled after cleaving
protecting groups of the, monomer derivatives used for
the solid phase synthesis preferably to amino or/and
thiol side groups especially preferably to primary amino
side groups of the carrier.
Ss
The haptens and marker or solid phase binding groups are
preferably introduced according to variant (a) i.e. by
using monomer derivatives during the solid phase
synthesis that are coupled to the group that is to be
introduced in each case. According to this variant
luminescent metal chelates, biotin or peptide haptens
can for example be introduced without problems. However,
in the case of sensitive fluorescent dyes or labels or
other haptens such as steroids this procedure is
unsuited since these substances can be destroyed under
the conditions of the solid phase syntheses. In this
case the conjugates are synthesized according to process
variant (b) i.e. by subsequent coupling to the completed
carrier molecule. Of course it is also possible to use a
combination of process variants (a) and (b).
It is also possible to introduce two different groups
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according to variant (b),i.e. after completion of the
synthesis e.g. a hapten and a marker group or a hapten
and a solid phase binding group. In this connection the
process according to variant (b) can for example be
carried out in such a way that the first group to be
introduced is coupled to amino side groups and the
second group to be introduced is coupled to thiol side
groups of the carrier molecule. On the other hand both
groups to be introduced can each be coupled selectively
to predetermined primary amino side groups of the
carrier in which a monomer derivative with a first
protecting group is used for the amino side group at
positions of the carrier at which hapten molecules are
to be coupled and a monomer derivative with a second
protecting group for the amino side group is used at
positions of the carrier at which marker or solid phase
binding groups are to be coupled and the first and the
second protecting group are selected in such a way that
it enables a selective cleavage of the protecting groups
and thus a selective coupling in two reaction steps. For
this purpose the first and second protecting group can
be selected from acid-labile amino protecting groups
such as Boc or acid-stable protecting groups such as
phenylacetyl.
In the process according to the invention the carrier
molecule having the desired monomer sequence is
synthesized on a solid phase. The peptide carriers are
preferably produced using a commercial peptide
synthesizer (e.g. the instruments A 431 or A 433 from
Applied Biosystems). The synthesis is carried out
according to known methods preferably starting at the
carboxyl terminus of the peptide using amino acid
derivatives. Amino acid derivatives are preferably used
whose amino terminal group required for coupling is
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derivatized with a fluorenylmethyloxycarbonyl (Fmoc)
residue. Reactive side groups of the amino acids used
contain protecting groups that can be readily cleaved
off after completion of the peptide synthesis. Preferred
examples of this are protecting groups such as
triphenylmethyl (Trt), t-butyl ether (tBu), t-butyl
ester (OtBu), tert.-butoxycarbonyl (Boc), 2,2,5,7,8-
penta-methylchroman-6-sulfonyl (Pmc) or phenylacetyl.
The amino side chains of lysine residues or of other
amino acid derivatives with primary amino side groups
that are located at positions of the peptide at which it
is intended to introduce, a hapten or label are
covalently coupled to the group to be introduced
according to variant (a).
In addition to the 20 natural amino acids the peptide
can also contain artificial amino acids such as J-
alanine, y-amino-butyric acid, c-amino-caproic acid,
norleucine or ornithine. These artificial amino acids
are used for the synthesis in a protected form
analogously to the natural amino acids.
According to variant (b) of the process according to the
invention the hapten or the label is introduced after
completion of the synthesis by reacting the peptide
after cleavage of protecting groups with the activated
group desired in each case which reacts with free
primary amino groups of the peptide. 1.5 to 4
equivalents of active ester are preferably used per free
primary amino group. Subsequently the reaction product
is purified, preferably by HPLC. The introduction of two
different activated groups according to variant (b) is
achieved by using two selectively cleavable protecting
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groups as elucidated above.
The peptide backbone of the conjugate has a non-
immunologically reactive amino acid sequence i.e. an
amino acid sequence which does not interfere with the
test procedure in the intended application of the
conjugate as an antigen in an immunological method of
detection.
On the other hand the backbone of the carrier molecule
can also be composed of nucleotides or peptidic nucleic
acids. The synthesis of an oligonucleotide carrier
molecule can be carried bout in a commercial DNA
synthesizer. The hapten molecules and the marker or
solid phase binding groups are preferably introduced as
phosphoramidite derivatives (Theisen et al., supra;
Applied Biosystems, User Bulletin 67, FAM Amidite, May
1992) or/and they are subsequently coupled to free
reactive side chains. Carrier molecules based on
peptidic nucleic acids are synthesized analogously to a
solid phase peptide synthesis e.g. according to the
method described in W092/20703. The hapten molecules or
marker or solid phase binding groups can be introduced
according to the methods described for peptide and
oligonucleotide carriers.
The present invention also concerns the use of the
conjugates as antigens in an immunological method if the
hapten molecule is an immunologically reactive molecule
or for DNA diagnostics if the hapten is a nucleic acid.
Conjugates which contain more than one hapten molecule
can be used in immunological detection methods as
polyhaptens.
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A preferred embodiment of the invention concerns the use
of the conjugates in an immunological method for the
determination of specific antibodies in a sample liquid.
Such antibodies are preferably determined which indicate
an infection by microorganisms such as bacteria, viruses
or protozoa. Antibodies directed against viruses e.g.
antibodies directed against HIV or hepatitis viruses are
particularly preferably determined. The sample liquid is
preferably serum and particularly preferably human
serum. In addition it is preferred that the conjugates
according to the invention are used in an immunological
method in a bridge test format.
Thus the present invention concerns a method for the
immunological determination of a specific antibody in a
sample liquid which is*characterized in that (a) the
sample liquid is incubated with at least one conjugate
according to the invention which is directed against the
antibody to be determined and (b) the antibody is
detected via binding to the peptide.
The immunological method of determination according to
the invention can in fact be carried out according to
any known test format e.g. in a homogeneous immunoassay
with a single reaction phase or in a heterogeneous
immunoassay with more than one reaction phase. A
heterogeneous test format is preferably used in which
the presence of the antibody is detected in the presence
of a solid phase. one embodiment of this test format is
the so-called double antigen bridge test design. In this
case the sample liquid is incubated in the presence of a
reactive solid phase with two antigens directed against
the antibody to be determined of which the first antigen
carries a marker group and the second antigen is bound
to the solid phase or is present in a form capable of
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2195752
binding to the solid phase. The first or/and the second
antigen is a conjugate according to the invention. The
antibody to be determined in the sample liquid is
detected after optionally separating the solid phase
from the incubation liquid by determining the label in
the solid phase or/and in the liquid phase. The first
antigen is preferably a 'conjugate labelled with a
luminescent metal chelate or a fluorescent group. The
second antigen is preferably labelled with biotin and'is
capable of binding to a solid phase which is coated with
streptavidin or avidin.
The test procedure preferably comprises mixing the
sample liquid with the'first labelled antigen and the
second antigen on the solid phase in order to obtain a
labelled immobilized complex of first antigen, antibody
and solid phase-bound second antigen. Compared to other
test formats for detecting antibodies, the bridge test
format leads to an improvement in sensitivity i.e. all
immunoglobulin classes such as IgG, IgM, IgA and IgE are
detected as well as in specificity i.e. the unspecific
reactivity is reduced.
A second preferred embodiment of the invention concerns
the use of the conjugates in a competitive immunoassay.
Competitive immunoassays are generally used to detect
lower molecular analytes and can in principle be carried
out in two test formats the "labelled antibody" format
and the "labelled analogue" format. In the "labelled
antibody" format the sample liquid containing the
analyte to be. determined is incubated with a hapten
capable of binding to a solid phase which competes
immunologically with the analyte and a labelled
receptor, e.g. an antibody, directed against the hapten
and the analyte. In this test format the label bound to
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the solid phase is inversely proportional to the
concentration of the analyte. In the "labelled analogue"
format the sample liquid containing the analyte to be
determined is incubated with a labelled hapten that
immunologically competes with the analyte and a receptor
directed against the analyte and the hapten which is
capable of binding to a solid phase. The amount of solid
phase-bound labelled hapten is then inversely
proportional to the concentration of free analyte to be
determined.
Thus the present invention concerns a method for the
detection of an analyte,in a sample liquid based on the
principle of a competitive immunoassay in a "labelled
analogue" format which is characterized in that (a) the
sample liquid is incubated in the presence of a reactive
solid phase with a conji gate according to the invention
which contains a marker group and with a receptor which
is bound to the solid phase or is capable of binding to
the solid phase and which can enter into a specific
immunological reaction with the analyte and the hapten
component of the conjugate, (b) the solid phase is
optionally separated from the incubation liquid and (c)
the presence or/and the amount of analyte in the sample
liquid is determined by measuring the marker component
of the conjugate in the solid phase or/and in the
incubation liquid. A biotinylated antibody or a
biotinylated antibody fragment is preferably used as the
immobilizable receptor and a solid'phase coated with
streptavidin or avidin is preferably used as the
reactive solid phase.
Yet a further subject matter of the present invention is
a method for detecting an analyte in a sample liquid
based on the principle of a competitive immunoassay in a
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"labelled antibody" format which is characterized in
that (a) the sample liquid is incubated in the presence
of a reactive solid phase with a conjugate according to
the invention which contains a solid phase binding group
and with a receptor which carries a marker group and
which can enter into a specific immunological reaction
with the analyte and the hapten component of the
conjugate, (b) the solid phase is optionally separated
from the incubation liquid and (c) the presence or/and
the amount of analyte in the sample liquid is determined
by measuring the marker component of the receptor in the
solid phase or/and in the incubation liquid. A
biotinylated conjugate and a solid phase coated with
streptavidin or avidinds preferably used. An antibody
or an antibody fragment is preferably used as the
labelled receptor.
Ss
The luminescent metalichelate groups are preferably
detected by means of electrochemiluminescence in which
luminescent species are generated electrochemically on
the surface of an electrode. The luminescence can be
detected qualitatively or/and quantitatively. Examples
for carrying out luminescence assays can be found in EP-
A-0 580 979, WO 90/05301, WO 90/11511 and WO 92/14138.
Reference is herewith made to the processes and devices
for luminescence assays disclosed therein. The solid
phase in electrochemiluminescence assays is preferably
composed of microparticles and particularly preferably
of magnetic microparticles that are provided with a
coating that interacts with the second antigen on the
solid phase. The microparticles are preferably coated
with streptavidin.
Electrochemiluminescence is preferably measured in the
presence of a reducing agent for the metal complex e.g.
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an amine. Aliphatic amines are preferred, in particular
primary, secondary and tertiary alkylamines whose alkyl
groups each have one to three carbon atoms.
Tripropylamine is particularly preferred. However, the
amine can also be an aromatic amine such as aniline or a
heterocyclic amine.
Furthermore a non-ionic surface active agent e.g. an
ethoxylated phenol can optionally be present as an
amplifier. Such substances are for.example commercially
available under the names Triton X100*or Triton N-401*.
On the other hand the lthninescent metal chelate group
can also be detected by fluorescence in which the metal
chelate is excited by irradiation with light of a
suitable wavelength and, the fluorescence radiation
resulting therefrom is measured. Examples for carrying
out fluorescence assays are given in EP-A-0 178 450 and
EP-A-O 255 534. Reference is herewith made to this
disclosure.
Detection of fluorescent groups which like the
luminescent metal chelates are also preferred marker
groups of the conjugates according to the invention can
- as stated above - be carried out by excitation and
measurement of the fluorescence in a known manner.
Yet a further subject of the present invention is an
immunological reagent which contains at least one
labelled or solid phase-bindable conjugate according to
the invention. A reagent for the immunological
determination of a specific antibody based on the
principle of a double antigen bridge test contains (a) a
labelled conjugate according to the invention or/and (b)
* Trademarks
22 -
2195752
a further conjugate according to the invention which is
bound to a solid phase or is present in a form capable
of binding to a solid phase.
A reagent for determining an analyte, preferably a lower
molecular analyte based, on the principle of a
competitive immunoassay contains a labelled conjugate
("labelled analogue" format) or a solid phase bindable
conjugate ("labelled antibody" format) which competes
immunologically with the analyte to be determined for
binding to a receptor. The reagent for a competitive
immunoassay preferably contains spatially separated from
the conjugate according.to the invention either a
labelled receptor ("labelled antibody" format) or a
solid phase bindable receptor ("labelled analogue"
format) which can react immunologically with the analyte
to be determined and wash the conjugate according to the
invention.
The invention is further elucidated by the following
examples and figures. They show:
Fig. 1 a metal chelate-lysine derivative
Fig. 2 a biotin-lysine derivative
Fig. 3 a conjugate according to the invention
Fig. 4 a reference conjugate
Example 1
Production of a metal chelate-lysine derivative
6 mmol of the ruthenium complex Ru(bipyridine)2
(bipyridine-CO-N-hydroxysuccinimide ester) according to
EP-A-O 580 979 was dissolved in 50 ml dimethylformamide
and a solution of a-Fmoc lysine was added dropwise.
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After removing the solvent, the residue was dissolved in
a small amount of acetone, admixed with 300 ml
chloroform and briefly heated to boiling. After
separating the solvent, the compound shown in Fig. 1 was
obtained as a solid.
Example 2
Production of metal chelate-labelled and biotinylated.
peptides
The metal chelate-labelled and biotinylated peptides
were produced by means of fluorenylmethyloxycarbonyl-
(Fmoc) solid phase peptide synthesis on a batch peptide
synthesizer e.g. from Applied Biosystems A431 or A433.
For this 4.0 equivalent's of the amino acid derivatives
shown in Table 1 were'used in each case:
St
Table 1:
A Fmoc-Ala-OH
C Fmoc-Cys(Trt)-OH
D Fmoc-Asp(OtBu)-OH
E Fmoc-Glu(OtBu)-OH
F Fmoc-Phe-OH
G Fmoc-Gly-OH
H Fmoc-His(Trt)-OH
I Fmoc-Ile-OH
K Fmoc-Lys(Boc)-OH
L Fmoc-Leu-OH
M Fmoc-Met-OH
N Fmoc-Asn(Trt)-OH
P Fmoc-Pro-OH
Q Fmoc-Gln(Trt)-OH
R Fmoc-Arg(Pmc)-OH
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S Fmoc-Ser(tBu)-OH
T Fmoc-Thr(tBu)-OH
U Fmoc-l3Alanine-OH
V Fmoc-Val-OH
W Fmoc-Trp-OH
Y Fmoc-Tyr(tBu)-OH
Z Fmoc-E-aminocaproic acid-OH
Nle Fmoc-E-norleucine-OH
Abu Fmoc-y-aminobutyric acid-OH
Introduction of metal chelate and biotin groups into the
peptide sequence was carried out by direct incorporation
of metal chelate-coupled or biotin-coupled amino acid
derivatives e.g. within the sequence via a lysine
residue E-derivatized 4,i.th a metal chelate active ester
(Fig. 1) or via a biotin-derivatized lysine residue
(Fig. 2) or N-terminally by using a corresponding a-
derivatized amino acid residue.
The amino acids or amino acid derivatives were dissolved
in N-methylpyrrolidone. The peptide was synthesized on
400-500 mg 4-(2',41-dimethoxyphenyl-Fmoc-aminomethyl)-
phenoxy resin (Tetrahedron Letters 28 (1987), 2107) with
a loading of 0.4-0.7 mmol/g (JACS 95 (1973), 1328). The
coupling reactions were carried out for 20 minutes in
dimethylformamide as a reaction medium with 4
equivalents dicyclohexylcarbodiimide and 4 equivalents
of N-hydroxybenzotriazole relative to the Fmoc-amino
acid derivative. The Fmoc group was cleaved in 20
minutes after each synthesis step using 20 % piperidine
in dimethylformamide.
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The release of the peptide from the support and the
cleavage of the.acid-labile protecting groups was
achieved in 40 min at room temperature with 20 ml
trifluoro acetic acid, 0.5 ml ethanedithiol, 1 ml
thioanisole, 1.5 g phenol and 1 ml water. The reaction
solution was subsequently admixed with 300 ml cooled
diisopropyl ether and kept at 0 C for 40 min to
completely precipitate the peptide. The precipitate was
filtered, washed again with diisopropyl ether, dissolved
in a small amount of 50 % acetic acid and lyophilized.
The crude material obtained was purified in ca. 120 min.
by means of preparative HPLC on delta-PAK*RP C18
material (column 50 x 300 mm, 100 A, 15 p) using an
appropriate gradient (eluant A: water, 0.1 % trifluoro-
acetic acid, eluant B: acetonitrile, 0.1 % trifluoro-
acetic acid). The identity of the eluted material was
checked by means of ion-spray mass spectrometry.
Acid-stable phenylacetyl protecting groups were removed
at room temperature enzymatically using immobilized or
soluble penicillin G-amidase in aqueous solution with an
organic solvent component.
Example 3
Coupling to haptens
The metal chelate-labelled or biotinylated peptide was
dissolved in dimethylformamide and a slight excess (ca.
30 %) of the activated hapten (e.g. N-hydroxysuccinimide
ester) was added with regard to the positions on the
peptide capable of coupling (e.g. amino side groups of
lysine). It was stirred for 1 hour at room temperature,
the solvent was removed in a high vacuum and the peptide
was purified by means of preparative HPLC.
*TradpSnarks
- 26 - 2195752
Conjugates having the following structure were prepared
using the metal chelate-lysine derivative shown in Fig.
1 as the marker group and estradiol (E2) as the hapten:
I: AcK(BPRu)UEUEUK(E2)UEUEUK(BPRu)UEUK(E2)-NH2
II: AcK(BPRu)UEUEUK(E2)UEUEUK(BPRu)U-NH2
The conjugate I is shown in Fig. 3. The amino terminus
of the peptide chains is protected by acetyl (Ac). The
carboxyl terminus is present as an acid amide group.
The metal chelate and hapten molecules are in each case
coupled to the peptide chain via the E-amino side group
of the lysines.
A conjugate having the sequence of conjugate I is
synthesized in an analogous manner using testosterone as
a hapten molecule.
The structure of the conjugates produced was examined
and confirmed by means of 1H-NMR (500 MHz).
Example 4
Determination of estradiol in serum
A competitive two-step assay based on the "labelled
analogue" format was carried out to determine estradiol
in human serum. For this 90 l solution 1 (0.69 nmol/1
conjugate I according to the invention (Fig. 3) or
1.68 nmol/1 reference conjugate (Fig. 4) each in
50 mmol/1 4-morpholine ethane sulfonic acid (MES), pH
6.8, 0.1 % bovine serum albumin, 0.1 % Thesit, 0.01 %
methylisothiazolone, 0.1 % oxypyrion, 30 ng/ml
* Trademarks
IB
2195752
27 -
detachment reagent dihydrotestosterone) was incubated
for 10 min at 37 C in a polystyrene vessel together with
50 Al sample (serum sample or estradiol standard).
Subsequently 90 Al solution 2 (0.7 gg/ml or 1.4 gg/ml
conjugate of biotin and polyclonal anti-estradiol rabbit
Fab' in 50 mmol/l MES buffer, pH 6.0) and 50 Al bead
suspension (720 g/ml streptavidin-coated magnetic
particles, Dynal Company) were added in succession.
After incubating for a further 10 minutes at 37 C,
150 Al of the mixture was transferred into a measuring
cell. There the bead particles and the ruthenium label
adhering thereto was magnetically concentrated on the
electrode surface and the chemiluminescence signal
generated electrochemically was detected at 28 C.
The result of this expeSriment shown in Table 2 shows
that the conjugate according to the invention has a
considerably improved-test performance with regard to
sensitivity and to lower detection limit compared to the
reference conjugate.
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Table 2
Antigen Conjugate I Conjugate
(invention) (reference)
Antigen concentration (nmol/1) 0.69 1.68
Ruthenium complex concentration 1.68 1.68
(nmol/1)
Concentration antiserum 0.7 1.4
(Mg/ml)
Counts Standard F 80412 87709
Counts Standard B 653420 1223219
Counts Standard A 861140 1445270
Ratio B/A 0.759 0.847
Ratio F/A 0.093 0.061
Ratio F/E 0.546 0.442
lower detection limit 15.9 25.1
pg/ml (3 % CV)
