Note: Descriptions are shown in the official language in which they were submitted.
''' 4~ BEHRINGWERKE AKTIENGESELLSCHAFT 1995/B 033 - Ma 1016
Dr. Pfe/Zi
Immunological determination method
The present invention relates to a competitive immuno-
chemical determination method, in which the concen-
tration of the analyte is determined in a 1-step
process and the analyte in this case competes with a
first and a second specific binding component for
binding to a third specific binding component, the
first specific binding component being bound to a
water-insoluble support and the second specific binding
component being provided with a signal-generating
label.
Immunological determination methods have gained
outstanding importance in - many areas of clinical
diagnosis since the first description of a radioimmuno-
assay (1959) and the first enzyme immunoassay.
In an enzyme immunoassay for the determination of an
analyte, immunological binding and reaction components
are used, such as, for example, haptens, antigens,
antibodies or fragments of antibodies. The binding and
reaction components used can in this case on the one
hand be present bound to a solid phase or conjugated to
a signal-generating label, e.g. a labeling enzyme via,
for example, a covalent bond. Solid phases used are
concave shaped articles, such as, for example, tubes or
hollows in the form of microtiter plates, but also
convex shaped articles, such as, for example, spheres.
Planar solid phases, such as, for example, test strips,
are also used. Alkaline phosphatase, (3-galactosidase
and horseradish peroxidase are frequently employed as
labeling enzymes, chromogenic, fluorogenic or lumi-
nescent compounds being used as substrates. The
composition of sample, incubation and washing buffers,
as well as the various substrate/chromogen reagents,
are known to the person skilled in the art.
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2
In contrast to homogeneous enzyme immunoassays, in
heterogeneous enzyme immunoassays unbound reaction
components are removed from bound reaction components
by a phase separation and subsequent washing steps. In
this case, a differentiation is made between the 1-step
and the 2-step process. Whereas in the 1-step process
altogether only one separation step ("bound/free"
separation) is carried out, this takes place in the 2
step process after each individual incubation or
reaction step.
Generally, enzyme immunoassays according to the immuno-
logical reaction principles are divided into non-
competitive and competitive techniques. The noncompeti-
tive techniques ("sandwich" tests) are distinguished in
that solid phase-bound and signal-generating reaction
components are present in a large molar excess in
comparison with the analyte to be determined. For the
formation of the "sandwich" complex, at least two
binding sites of the analyte are necessary, which in
each case are recognized by the solid phase-bound or
signal-generating reaction components. The measured
signal activity of the "sandwich" complex formed is in
this case directly proportional to the analyte concen-
tration.
In the competitive techniques, on the other hand, one
of the reaction components is limiting for immune
complex formation due to its low concentration, so that
a competition takes place between reaction component
and analyte around at least one binding site of the
mutual binding component. Within the competitive
techniques, these, for their part, can be subdivided
into two groups. In the first group the number of
insolubilized binding components is lower than the
number of signal-generating reaction components and the
analyte molecules to be determined, while in the second
group the concentration of the signal-generating
reaction component is lower than the number of
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insolubilized binding components and free analyte
molecules. In both cases, the signal activity of the
complex formed is inversely proportional to the
measured analyte concentration.
On comparison of the two immunological reaction
principles, it can be determined that the known
competitive techniques are inferior to noncompetitive
techniques with regard to sensitivity, measuring range,
specificity, robustness and incubation time (EKINS R.
(1 9 8 5) CURRENT CONCEPTS AND FUTURE DEVELOPMENTS: IN ALTERNATIVE
IMMUNOASSAYS).
The differing affinity of the serum antibodies to be
detected therefore very significantly determines
whether the 1-step or 2-step process is employed. In
the detection of low-affinity antibodies, the 2-step
process has proven advantageous compared with the
1-step process, in particular if the serum incubation
time is carried out overnight as the first step.
However, care is to be taken that in the second
incubation step an equilibrium is not established
between the bound and unbound signal-generating
reaction components, which would lead to a displacement
of low-affinity analyte antibodies. The detection of
low-affinity antibodies is thus in principle better
feasible using the 2-step process than with the 1-step
process, which is easier to carry out and shorter for
the user.
The purity of the solid phase-bound antigen, for
binding of which both the antibodies to be detected and
the signal-generating reaction components compete in a
competitive enzyme immunoassay, plays an important
part. The epitope density of the insolubilized antigen
is in this case crucial for the sensitivity of
detection (KENNY G. et al. (1983) J. CLIN. MICROBIOL. 17,
655-665). If a protein mixture is employed, as is the
case, for example, with a purified HAV virus antigen,
21933~4~
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the sensitivity of detection can be considerably
adversely affected if the virus-specific protein
fractions represent only a small fraction of the total
protein employed. In order to avoid this, in the known
processes an appropriately highly purified antigen must
therefore be used, the production of the necessary
degree of purity being associated with a considerable
expense and correspondingly turning out to be
difficult, expensive and time-consuming (PURCELL R. et
al. (1976) JOURNAL of IMMUNOLOGY 116, 349-356) . In parti-
cular in the detection of low-affinity anti-HAV anti-
bodies which are formed immediately after inoculation
has taken place, the various commercially available
diagnostic tests show a poor sensitivity. Only by the
use of purified HAV antigen or the vaccine antigen was
it possible to improve the sensitivity in the detection
of low-affinity antibodies (DELEM A. (1992) BIOLOGICALS 20,
289-291).
The object on which the present invention is based
therefore consisted in developing a process which
allows the detection of low-affinity antibodies with
the aid of the 1-step process and in this case an only
minimally purified antigen can be used.
This object was essentially achieved by the embodiments
provided in the patent claims.
The immunochemical process according to the invention
for the determination of an analyte by means of a
heterogeneous competitive determination process
includes the following steps:
a) incubation of the analyte with a first, a second
and a third specific binding component, the first
specific binding component being bound to a water-
insoluble solid phase (solid phase-bound reaction
component) and the second specific binding compo-
nent being provided with a signal-generating label
_, ,~ - 5 _
(signal-generating reaction component), and the
analyte and the first and the second specific
binding components competing for binding to the
third specific binding component (common binding
component),
b) separation of the signal-generating label bound to
the solid phase via the third specific binding
component from the unbound fraction,
c) measurement of the signal which can be generated
by the bound fraction of the label, and
d) determination of the analyte concentration by
comparison of the values found in step c) with a
standard curve plotted under identical conditions
or calculated theoretically.
Analytes which can be determined by means of the
process according to the invention are known per se to
the person skilled in the art. Advantageously, the
analyte is an antibody, in particular an antibody of
human or animal origin, which is relevant in micro-
biological diagnosis, in particular in the field of
infection diagnosis such as is carried out in blood
banks, such as, for example, anti-HAV antibodies, anti-
HIV antibodies, anti-HCV antibodies or antibodies in
the diagnosis of hepatitis B.
The first and the second specific binding components
must be able to react specifically with the third
specific binding component. Advantageously, the first
and the second specific binding components are
antibodies, monoclonal or polyclonal, or antibody
fragments. To carry out the process according to the
invention, however, lectins or synthetic/recombinant
antibodies can also be employed.
Fundamentally, the process according to the invention
21~33~4
can also be used for the detection of antigens which
are not antibodies, it then being possible for the
first and the second specific binding components to be
an antigen and the third specific binding component to
be an antibody.
Solid phases as such as known per se to the person
skilled in the art. Advantageously, water-insoluble
solid phases are employed, such as, for example, latex
particles, magnetically attractable particles or micro-
titer plates.
Signal-generating labels as such are known per se to
the person skilled in the art. Such a label can either
be conjugated directly to the specific binding compo-
nent concerned or by means of a reversible coupling
known per se to the person skilled in the art, such as,
for example, biotin-streptavidin, fos-jun or antibody-
antigen bonds.
Signal-generating labels which are employed are
preferably components which are capable. of chemo-
luminescence or fluorescence or enzymes which can
convert the luminogenic, fluorogenic or chromogenic
substrates. In the case of; the enzymes, horseradish
peroxidase is particularly preferred. In the case of
the chemoluminescent labels, the compounds described in
European Patent Applications EP-A-0 257 541 and EP-A-
0 330 050 are particularly preferred.
Customarily, in the immunochemical processes described,
after the separation of the solid and the liquid phase
the signal is measured either on the solid phase or in
the supernatant separated off. From the measured
signal, the analyte concentration is determined in a
manner known per se to the person skilled in the art by
means of a so-called standard curve. To plot the
standard curve, the signals are measured using the
respective determination process for known analyte
_ - 7 -
concentrations and converted into a curve form either
graphically or mathematically. Such a standard curve
can also be calculated theoretically with a certain
accuracy on account of the known physical and chemical
properties of the specific binding components.
The process according to the invention can be used in
immunochemical methods in which competition takes place
between the analyte to be determined and the signal-
generating reaction components and/or insolubilized
reaction components for binding to a common binding
component.
The process according to the invention is furthermore
distinguished in that the common binding component only
has to be minimally purified. It has turned out to be
significant that the common binding component should
have at least two different binding sites, one binding
site being recognized by the insolubilized reaction
components, while the signal-generating reaction compo-
nents bind to the second binding site. In the presence
of the analyte to be detected, specific competition of
the analyte molecules with the signal-generating and/or
the insolubilized reaction components then takes place
for binding to the common binding component. In this
case, it has proven to be essential for the process
according to the invention that in the absence of an
analyte the formation of a signal-forming "sandwich"
complex is not prevented by the competition between
insolubilized and signal-generating reaction compo-
nents.
Surprisingly, it was found in the process according to
the invention for the detection of antibodies against
the hepatitis A virus that in the absence of an analyte
(= negative control) no competition between the signal-
generating reaction components (= monoclonal anti-HAV-
specific antibody conjugate) and the insolubilized
reaction components (= polyclonal anti-HAV-specific
- _ ~I93344
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antibodies) occurred for binding to the common binding
component (= HAV antigen) and as a result the formation
of a signal-generating "sandwich" complex is made
possible. This was all the more surprising because the
known monoclonal antibodies against HAV can almost
completely block the binding of polyclonal antibodies
to the virus in various competitive immunoassays (LEMON
S. et al. (1993) VIROLOGY 4, 285-295; HUGHES J. et al.
(1984) J. VIROL. 52, 465-473; STAPLETON J. et al. (1987)
J. VIROL. 61, 491-498). However, in the presence of
analyte molecules (= anti-HAV-specific antibodies -
positive control) it was just as surprisingly possible
to find that specific competition for the common
binding component can take place between the analyte
and the insolubilized reaction components and/or
signal-generating reaction components.
The finding was also surprising that the common binding
component only had to be purified minimally or not at
all in order to be able to detect, even using the
1-step process, low-affinity antibodies which can occur
in the early phase of an infection or after inoculation
has taken place. In the process according to the
invention, possibly due to the presence of solid phase-
bound reaction components, conditions are created which
allow particularly effective and specific competition
between the analyte and the signal-generating and/or
solid phase-bound reaction components for the binding
sites of the common binding component. Minimally
purified in the sense of the present invention in thi s
case means that the proportion of specific protein is
less than 80, preferably less than 50 %, very prefer-
ably less than 20 0.
Reaction components preferably used in the context of
the invention are antibodies or defined fragments of
antibodies. The preparation of polyclonal or monoclonal
aritlbOdleS (KOHLER G. arid MILSTEIN C. (1975) NATURE 256,
495-497) is carried out by a method known per se to the
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person skilled in the art. Besides polyclonal
antibodies, monoclonal antibodies or fragments thereof
(F(ab')2 or Fab') can also be employed. According to
the process according to the invention, in this case
one reaction component is bound to the water-insoluble
solid phase, while the second reaction component is
employed as a signal-generating component. Preferably,
in the process according to the invention a polyclonal
antibody is bound to the solid phase and a monoclonal
antibody which is conjugated to a labeling enzyme is
used as a signal-generating reaction component. The
preparation of the monoclonal conjugate used in the
invention is likewise known to the person skilled in
the art (review article: ISHIKAWA E. et al. (1983) J.
IMMUNOASSAY 4 , 2 0 9 - 3 2 7 ) .
The suitability of the antibodies used can be deter-
mined, for example, by experiments known per se to the
person skilled in the art.
A binding component which can be employed in the
context of the invention is any macromolecule which has
at least two separate binding sites for the
insolubilized and the signal-generating reaction
components. Suitable macromolecules in this case are
proteins - optionally modified by carbohydrates and/or
lipids-, carbohydrates, lipids, synthetic polymers and
nucleic acids. The molecular weight of the binding
component is preferably between 50,000 and 2 million.
The process according to the invention can be used in
all immunological detection methods in which, in
heterologous immunoassays, specific competition can
take place between the analyte and solid phase-bound
reaction component and/or signal-generating reaction
component for at least two binding sites of a common
binding component.
CA 02193344 2004-08-18
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Description of the figures
The detection limit of the process according to the
invention was determined with the aid of defined
dilutions of an anti-HAV standard (WHO standard). In
Fig. 1, the measured extinctions of various antibody
concentrations are shown in a semilogarithmic presen-
tation. Owing to the competitive assay construction,
the extinctions decrease with increasing antibody
concentration.
The calculated analytical sensitivity is 13.4 IU/1, the
extinction of the negative control as a cut-off having
been halved.
It was possible to show with the aid of inoculation
seroconversions that low-affinity antibodies against
HAV are detectable at a very early stage using the
process according to the invention. In Fig. 2 is shown
the time course of a typical anti-HAV seroconversion
(0193). Serum samples were taken from inoculation
subjects at various times and the latter investigated
using the process according to the invention. The
ratios shown in this case correspond to quotients from
the measured extinctions and the cut-off. Ratios of < 1
indicate that, with the detectable occurrence of anti-
HAV antibodies, a seroconversion has taken place. As
shown in Fig. 2, it was even possible after inoculation
had taken place using the process according to the
invention to detect a seroconversion from the second
week.
In Fig. 3, the measured extinctions of the sero-
conversion 0193 investigated were quantified with the
aid of the calibration curve of Fig. 1. Even from the
2nd week, an antibody concentration was determined
which clearly exceeds the inoculation protection
threshold value of 20 IU/l.
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The following examples are intended to illustrate the
invention:
Abbreviations:
HAV: hepatitis A virus
POD: peroxidase
SH: ~ulfhvdryl groups
ATCC'~"': American tissue cell cultureTM
Examples
Example 1
a): Conjugation of antibodies
Monoclonal antibodies against HAV are reacted with a
heterobifunctional reagent (TARRIMORE et al. (1983) J.
IMM. METH. 62, 123-131), then incubated with SH-
activated peroxidase (KING et al. (1978) BIOCHEMISTRY 17,
1499-1506) and subsequently purified by gel chromato-
graphy.
b): Preparation of HAV antigen
To prepare the HAV antigen, commercially available
cells, such as, for example, human diploid embryonic
lung fibroblasts, are infected with a characteristic
hepatitis A virus strain, such as, for example, ATCCTM
HM-175. After several days, the removed cell super-
natant is centrifuged, the cell pellet obtained is
taken up in a customary storage buffer and the antigen
is inactivated according to a process known to the
person skilled in the art . The inactivated antigen can
be used further without additional purification.
The process according to the invention is in this case
not restricted to the abovementioned method for the
preparation of HAV antigen, but other preparation
processes for antigen isolation known to the person
skilled in the art can also be used. Moreover,
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commercially available HAV antigen preparations can
also be employed in the process according to the inven-
tion.
c): Coating of the hollows of microtiter plates
A determined amount of human polyclonal anti-HAV
antibody (16 ~,g/ml) is added with gentle stirring to a
coating solution (sodium carbonate 0.01 moll pH 9.6)
and the mixture is homogenized for about 30 minutes.
The coating of the individual hollows of a microtiter
plate was carried out subsequently using a coating
volume of 150 ~.1. After an incubation overnight at room
temperature, the coating solution is aspirated and the
individual hollows of the microtiter plate are washed
twice with a wash solution (0.25 mol/1 of citric
acid/0.05 mol/1 of tris pH 7.4). Following the last
washing step, the individual hollows of the microtiter
plate are sucked empty and it is sealed into aluminum
films together with packed drying agent (e. g. silica
gel). The coated microtiter plates are stored at 4°C
until they are used.
d) : Enzyme iamnunoassay for the determination of anti-
HAV antibodies
The process according to the invention is a competitive
enzyme immunoassay by the 1-step process. Sample to be
investigated (25 ~,l) , conjugate (= POD-conjugated anti-
HAV-specific monoclonal antibody, 50 ~,1) and HAV
antigen (50 ~.l) are added successively to the hollows
of a microtiter plate which are coated with human
polyclonal anti-HAV-specific antibodies. The coated
microtiter plate, conjugate and HAV antigens were taken
from the Enzygnost~ anti-HAV test kit (order code OQEC
Behringwerke AG, Marburg). If the sample to be
investigated in this case contains the anti-HAV
antibodies to be determined, these compete with the
conjugate molecules and/or insolubilized polyclonal
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anti-HAV-specific antibodies for binding to the HAV
antigen. After an incubation time of 2 hours at 37°C,
excess conjugate and unbound reactants are removed by
aspiration and washing four times, e.g. with the
Behring ELISA Processor II or Behring ELISA Processor
III (Behringwerke AG, Marburg), and the amount of the
bound conjugate is determined (30 min, room tempera-
ture, protected from light) by the addition of 100 ~.1
of substrate/chromogen solution (Behringwerke AG, order
code OUVP). The enzymatic reaction of the chromogen
tetramethylbenzidine dihydrochloride is interrupted by
addition of 100 ~.1 of 0.5 N sulfuric acid and the
extinction at 450 nm is determined photometrically. The
measured extinction is in this case indirectly propor-
tional to the anti-HAV-antibody concentration contained
in the sample.
e): Comparison of the process according to the inven-
tion with an alternative, fully monoclonal process.
The process according to the invention is an enzyme
immunoassay by the 1-step process which contains a
polyclonal anti-HAV-specific antibody of human origin
bound to the solid phase and, as conjugate antibody, a
monoclonal anti-HAV-specific mouse antibody. The
comparison process differs from the process according
to the invention in that the polyclonal solid-phase
antibody is replaced by a monoclonal antibody.
In Table 1, the process according to the invention is
shown with regard to signal intensity of the negative
control and measured detection limit with the alterna-
tive, fully monoclonal process.
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Table 1: Comparison of the process according to the
invention with a fully monoclonal process
Extinction of the Detection
negative control limit
Process according
to the invention 1427 mE 13 IU/1
Fully monoclonal
process 543 mE 28 IU/1
The higher signal intensity of the negative control and
the improved detection limit of the process according
to the invention developed' from a polyclonal and a
monoclonal anti-HAV-antibody clearly show the
superiority of the process according to the invention
compared with an alternative, fully monoclonal process.
