Note: Descriptions are shown in the official language in which they were submitted.
WO 92/21973 2 ~ ~L 07 0 5 PCI'/SE92/00386
METHOD AND SEN80R ~5AN8 FOE~ DETE:RMINING MYOCARDIAL
INFARCTION ~IARI~ERS
S FIELD OF THE INVENTION
This invention relates to a method of simultaneously
assessing from blood samples the variation with time of at
least two different and independent markers indicative of
myocardial damage to provide for early and reliable
diagnosis of myocardial infarction as well as to improve
the monitoring of the treatment and recovery after a
myocardial event. The invention also relates to a sensor
unit to be used in the method.
BACl~GROIJNl) OF T}{E: INVENTION AND PRIOR ART
Acute myocardial infarction (AMI) is a major cause of
deaths in the developed countries, the medical term
describing the development of ischemia and necrosis of a
portion of the myocardium. The ischemia is caused by
occlusion of the artery by a thrombus preventing normal
blood flow to the affected area of the myocardium and
eventually resulting in tissue necrosis.
A number of serious complications like pulmonary
edema, hypertension, thromboembolism, and ruptures of the
heart can occur in AMI. Correct diagnosis and appropriate
treatment at an early stage increases the chance of
survival and diminishes the risk of complications. It is
therefore essential that patients with suspected AMI are
immediately sent to hospital for diagnosis and treatment.
~ The current clinical diagnosis of AMI is based on
chest pain (sudden onset of pain which sustains for more
than 15 minutes?, electrocardiogram (ECG) and levels and
kinetics of heart muscle specific enzymes and proteins.
While chest pain and observed ECG changes may be sufficient
for a safe diagnosis in some cases, they are, however,
often of a rather nonspecific nature, and the final
confirmation of AMI and differentiation from other cardiac
or non-cardiac events can for a large group of patients
only be made by monitoring the mentioned biochemical
parameters by blood sampling at regular intervals, usually
W092/2197~ PCT/SE92/~386
6 to 18 hours between sampling, and analysis at centralized
laboratories. Today a patient with suspected AMI therefore
may spend from a few hours to more than 48 hours at the
coronary or intensive care unit before a reliable diagnosis
can be made, depending on the individual patient and the
readiness of the physician.
It is, of course, desirable that the correct diagnosis
could be made in all cases at an earlier stage. On one
hand, in case of an AMI, the early intervention with
appropriate treatment with thro~bolytics would reduce the
mortality and diminish the risk of complications. In fact,
thrombolytics have to be used within the first six hours
from the onset of pain to be beneficial, the mortality
being substantially reduced the earlier the treatment is
established (around 10% per hour). On the other hand, the
relatively larger group of patients (about two thirds) not
suffering from AMI could be excluded from expensive,
inappropriate and sometimes fatal therapy, other causes of
the AMI-like symptoms being e.g. angina pectoris or pain
from the gastro-enterological tract. Thus, the treatment
with thrombolytics has severe side effects such as stroke
and haemorrhagic bleedings. Today up to around 2% of the
patients treated with the current thrombolytics, tissue -
plasminogen activator (tPA) and streptokinase, are hit with
25 these side effects. Accurate diagnosis is thus fundamental `
in these cases.-Also, the great economic importance of such
early identification of the patients not requiring the AMI
treatment is readily appreciated, considering the
substantial cost reductions obtained when such patients
after a much shorter time than before may be transferred to
an ordinary medical ward or in the most favourable cases
can be sent home.
In the treatment of AMI by thrombolytics the objective
of the treatment is to achieve reperfusion of the blocked
coronary artery before the tissue has got an irreversible
damage. It is known that the cardiac enzymes show different
patterns if a reperfusion is achieved or not. It is a
matter of course that a real-time analysis permitting
.
WO92/21973 2 1 1 f~ 7 ~ S PCT/SE~2/~3~
bedside monitoring of the patients would be desired. so far
this has not been feasible with today's assay methods and
technology.
Similarly, it would, of course, also be desirable to
be able to continuously monitor cardiac enzymes for
detecting a possible AMI initiated during thorax surgery
before closing the thorax.
Current serum assays are based on the leakage of
intra-cellular enzymes or other proteins from the damaged
or necrotic cells which result from the myocardial
infarction. The recognized standard assay for the detection
of a heart attack has for a long time been the creatine
kinase (CK) test. This test involves the determination of
CX and its isoenzyme CX-MB which exhibit detectable peak
concentratior.s around twelve hours after the myocardial
event has occurred. Another conventional protein tested is
lactate dehydrogenase (LD) and its isoenzymes, and in
recent times the determination of noncatalytic proteins
like myoglobin and troponin T has been rendered possible as
a result of sensitive immunological determination methods.
Still another protein known to be released from cardiac ;~
muscle following myocardial infarction and forming the
basis of a test is myosin light chain. Each one of the
mentioned marker proteins exhibits specific serum
concentration changes corresponding to the kinetics and
release of the respective protein in an acute infarction.
The combined analysis of two markers has been
suggested to improve the recognition of myocardial
infarction. Thus, combined analysis of CK and LD is
described in e.g. Limbird, Lee, Diss. Abstr. Int. B 1974,
34(ll), 5322-3.
W09l/01498 discloses the simultaneous or sequential
testing for creatine kinase and myosin light chain for the
early detection of a myocardial infarction. While the
combination of the two assays is said to provide a very
reliable diagnostic test for the early phase of a heart
attack, it also permits differentiation betwèen a
myocardial infarction and other ischemic events causing
,
W092/21973 ~ 0~ PCT/SE92/~38
cardiac pain, such as angina pectoris. The latter feature
is due to the fact that myosin light chain is released not
only in case of an acute myocardial infarction, but also
for other cardiac injuries, ~hereas creatine kinase is
substantially only released in the instance of an acute
myocardial infarction.
While the dual combination assays of the type
described above offer a more accurate and reliable -
diagnosis, they suffer from the basic disadvantage of being
l~ based on conventional type assays, usually solid-phase
enzyme immunoassays, which have to be performed at a
laboratory and usually cause a considerable time to pass
before the final diagnosis can be made, as mentioned above.
There is therefore a need of an assay procedure which
permits a reliable diagnosis of a possible myocardial
infarction to be obtained in a shorter time, which in its
entirety may be performed at or in the close vicinity of
the patient, and which may easily be automated.
8~MMARY OF TRE INV~NTION
One object of the present in~ention is therefore to
provide an assay method by which it is possible to reliably
diagnose AMI in such a short time that a treatment with
thrombolytics will be beneficial.
Another object of the invention is to provide an assay
method by which it is possible to rapidly and reliably
exclude AMI to thereby reduce care costs in that such
patients within a short time may--be transferred to an -~
ordinary (s~bstantially less costly) ward or even be sent
home.
Still another object of the invention is to provide an
assay method by which it is possible to efficiently monitor
the treatment of an AMI with thrombolytics such that the
treatment may be i D ediately interrupted as soon as
reperfusion has been obtained, or the patient may be
subjected to an alternative treatment in case reperfusion
is not obtained in a reaæonable time.
Another object of the invention is to provide an assay
method for the diagnosis of AMI or monitoring of treatment
,
W092/21973 C~ ? A~ J ~ PCT/SE92/00386
with thrombolytics that may be performed at or close to the
patient's bedside.
Still another object of the invention is to provide an
assay method for monitoring cardiac enzymes to detect the
possible initiation of an AMI during thorax surgery, and
which may be performed in the operating room.
A further object of the invention is to provide a
sensor means for use in the assay methods mentioned above.
The above mentioned objects are achieved with the
method and sensor means of the present invention. A basic
concept of the invention resides in determining with short
intervals the variation with time of at least two,
preferably at least three different markers, i.e. enzymes
or proteins, of myocardial infarction in real-time
lS measurements in a flow cell system, which measurements are -~
based upon interactions of the analytes with ligands bound ;-
to a sensor surface, the interactions causing a detectable ~-
change of the physico-chemical characteristics of the
surface. While the study of at least two different cardiac
analytes or markers will provide sufficient information for
insuring an early diagnosis, the use of sensor surface ~-
technology to detect analyte-ligand interactions provides
for real-time analytical procedures which are sufficiently
rapid for permitting the analysis of blood samples taken at
short intervals and which also are apt to automation and
need not be performed at a laboratory, but may readily be
performed at the patient's bedside or in an operating room.
It is to be noted that the terms "different markers"
or "different analytes" as used herein mean markers or
analytes originating from different and discrete genomes.
Therefore, any post-translational modifications like
glycosylation or fragmentation of macromolecules do not
qualify them as being regarded as "different" analytes. For
example, CX-MMA and CK-MMB are not different analytes in
the context of the present invention.
Thus, in accordance with one aspect of the invention
there is provided a method of AMI marker determination or
monitoring, comprising the steps of
WO92/21973 ~rl ~5 6 PCT/SE92/00386
(i) simultaneously determining from a first blood,
serum or plasma sample from a patient at least two,
preferably at least three different analytes indicative of
myocardial infarction by contacting in a flow cell or cells
the sample with one or more sensor surface areas each
supporting a different ligand or mixture of different
ligands capable of specifically binding to a respective
analyte, and, optionally after additionally binding an
analyte specific reagent or reagent complex to the bound ;
analytes, detecting any binding interaction of each analyte
with its ligand as a consequential change of the physico-
chemical characteristics of the sensor surface;
(ii) removing each bound analyte from its sensor
surface bound ligand by passing a regenerating liquid
lS through the flow cell;
(iii) repeating step (i) for at least a second blood,
serum or plasma sample taken from the patient at a -~
determined time interval from said first sample; and
(iv) determining from the results of steps (i) to
(iii) the variation with time of said cardiac analytes.
By the term "reagent complex" as used herein is meant
that the reagent is bound to one ore more other species.
Such a reagent complex may be added as an assembly or be
formed successively after the reagent has bound to the
analyte as will be further elucidated below.
In accordance with another aspect of the invention
there is provided a sensor means comprising, immobilized to
one or more sensing areas thereof, either individually or
in combination, at least two, preferably at least three
different ligands, each ligand being capable of
specifically binding to a respective analyte indicative of
myocardial infarction, said sensor means being adapted for
the detection of any analyte-ligand interaction as a
consequential change of the physico-chemical
characteristics of the sensing surface, and said ligand
supporting surface areas being regeneratable after the
coupling of analytes thereto.
W092~21973 21 1 D 7 ~.3 5 PCT/SE92/~386
The different ligands may thus be either individually
immobilized to respective sensing areas, ~r co-immobilized
to a single sensing area. Combinations of these embodiments
are, of course, also possible, i.e. that there are two or ~-
more sensing areas, each area supporting co-immobilized
ligands.
In a preferred embodiment the sensor surface is a
surface capable of exhibiting surface plasmon resonance -
(SPR) and the detection of the cardiac analytes or markers
is carried out by surface plasmon resonance spectrometry.
BRIEF D~8CRIPTION OF TH~ PRA~ING8
Other objects and advantages of the invention will be
apparent to those skilled in the art from the following
detailed description thereof taken in conjunction with the
accompanying drawings. In the drawings:
Fig. l is a schematic illustration of a per se known
SPR-based flow cell measurement system;
Pig. 2 is an exploded sectional partial view of a flow
cell unit useful for the purposes of the present invention;
Fig. 3 is an SPR-sensor diagram showing the co-
immobili2ation of a monoclonal antibody specific for CR-M~
and a monoclonal antibody specific for myoglobin to a
sensing surface;
Fig. 4 is a corresponding diagram as in Fig. 3 showing
the analysis of a sample containin~ elevated levels of CK-
MB and myoglobin using the sensing surface with immobilized
CR-MB and myoglobin monoclonals in Fig. 3; and
Fig. 5 is a corresponding diagram as in Fig. 4 showing
the analysis of a sample not containing CK-MB and only a
normal level of myoglobin.
DETAI~ED ~ESCRIPTION OF THE INVENTION
As mentioned above one basic feature of the invention
resides in the simultaneous measurement of several, viz. at
least two, preferably, however, at least three, or even
e.g. four or five, different myocardial infarction markers
or analytes. While the diagnostic sensitivity and
specificity of such markers will vary, the appropriate
selection of analytes and the determination thereof ~-
~092/21973 q ~O l~ PCT/SE~2/~ ~6
simultaneously from blood samples taken at short intervals
will provide for an analytical picture or pattern which
will be very useful for diagnosing or excluding AMI. The
relationship between the various analytes during a certain ~-
time interval will also give an indication of how long an
infarction condition has been going on, for example based
upon the rise of the level of one analyte and the decline
of another. Likewise, the areas under the respective
analyte graphs will indicate the extent of the damage to
10 the cardiac muscle tissue. -
The ter~ "simultaneously" as used herein is to be
understood in a rather broad sense meaning that the ~;
respective determinations of the analytes need not be
started exactly simultaneously but that certain delays may
be permitted as long as a comparison between the different
analyte levels determined may be considered as meaningful.
Another basic feature of the invention lies in the
- already mentioned repeated analyses at, preferably, regular
short intervals permitting the variation of time of the
cardiac markers to be determined. Such information will
considerably improve the possibilities of making a reliable
AMI di:~gnosis or exclusion.
In combination the above basic features will permit a
very reliable diagnosis or exclusion of AMI to be
established at a desirably short time after the patient has
been brought to hospital, or in the alternative, efficient
monitoring of the treatment with thrombolytics as well as
of thorax surgery.
An apparatus system enabling the desired type of
analytical procedures to be performed should (i) rely on a
measuring principle based upon the interaction of the
analytes with ligands immobilized to a sensor surface and
detecting complex formation as a consequential change of
the physico-chemical properties of the sensor surface; (ii)
have a flow cell system permitting the desired simultaneous
detection of several analytes; and (iii) have a sensor
surface or surfaces which can be regenerated in situ in the
sense that bound analytes may be removed from the
WO92/21973 2 1 I (~ r~ O L PCT/SE92/~386
respective ligands to permit consecutive analyses on one
and the same sensor surface(s) after a regenerating step.
All the requirements (i) to (iii) may be met by analytical
technology which is known per se in the art.
In order to increase the above mentioned change of
the physico-chemical properties of the sensor surface,
further complexing of the bound analytes, and thereby an
increase of the thickness of the bound substance layer, may
be accomplished by reacting the ligand bound analytes with
a secondary reagent specific to the analyte, i.e. a
sandwich assay, as is per se known in the art. Such bound
secondary reagent may then, if desired, be further
complexed by reacting it with a tertiary reagent, etc. to
still more increase the surface change to be detected.
In the case of optically based methods, and especially
those based upon SPR spectroscopy, it is possible to
increase the detected surface change further by labelling
of the secondary and/or tertiary reaqent, etc by an
optically dense species, such as a particulate label, for
example, glass, latex, colloidal silver or gold, metal
oxide or ferritin; see e.g. EP-A-276 142.
By using a secondary reagent to perform a sandwich
type assay on the sensor surface as outlined above, not
only the sensitivity and in some cases the specificity will
be increased, but also the dynamic measuring range may be
extended in that the primary response may be used rather
than the secondary response if the ran~e of the latter
would be insufficient.
The measuring principle as defined above will enable
real-time measurements to be conducted and thereby permit
the desired performance of consecutive analyses of blood
samples taken with short intervals. Such a measuring
principle, and the sensor surface associated therewith, is
also readily adaptable to flow cell systems. Among methods
relying on the said measuring principle may be mentioned
internal reflectance methods, particularly evanescent wave
spectroscopy (EWS), such as attenuated internal reflection
(ATR) spectroscopy, total internal reflectance fluorescence
WO92/21973 ~ PCT/SE92/~386
(TIRF) spectroscopy, and surface plasmon resonance (SPR)
spectroscopy, or wave guide spectroscopy. All these methods
are based upon the examination of an optical property of a
solution bordering a surface where total internal
reflection has occurred. Other types of sensors that may be
used are those based upon photoacoustic piezoelectric,
surface acoustic wave (SAW), or electrochemical
measurements, etc. Particularly suitable for the purposes
of the invention is SPR spectroscopy as will be described
lQ in more detail below.
A flow cell system is essential for providing a rapid
and easy to handle system apt to automation. By the term
"flow cell" (which is to be interpreted in a broad sense)
is meant that the sample and other analytical fluids will
flow past the sensing surface or surfaces at a constant
rate. Such a system may, as mentioned above, in one
embodiment thereof comprise a plurality of sensing surface
areas, i.e. one for each cardiac analyte as well as a -
control area, and optionally one or more areas for other
purposes as will be described below. The flow may be either
in parallel or in series with respect to the different
sensing surface areas. A parallel flow cell sy,tem may
comprise several separate flow cells arranged in parallel,
whereas in a series system several separate flow cells may
be connected in series. A parallel or serial flow system,
respectively, may also be formed by several defined sensing
surface areas contained within a single flow cell. For an
example of a flow cell system comprising several flow
cells, it is referred to our published PCT-application WO
90/05295 (the full disclosure of which is incorporated by
reference herein) disclosing a fluid handling block unit
comprising a plurality of open channels for defining
multiple flow cells together with a sensor plate applied
thereto.
In another and novel embodiment of flow cell system,
the ligands for all the cardiac analytes may, as already
mentioned above, be co-immobilized in a single surface
are~. Thus, the specific response (Stenberg et al., J.
WO92/21973 11 2 1 1 ~ 7 ~I 5 PCT/SE92/00386
Colloid Interface Sci. 143 (1991) 513) for surface
concentration detecting devices, such as surface plasmon
resonance detection, is in principle (at least down to the
diffraction limitations ~f the optical system) independent
of the size of the detecting area. This is in contrast to
absolute measuring devices such as or~inary solid phase
assays where the specific response is area dependent.
Normally, the different immobilized capturing
molecules are immobilized to different detection areas as
described above. The independence of area size for the
surface concentration detecting device will, however, in
accordance with this novel and inventive concept, allow
reduction in the number of detection areas by co-
immobilization of two or more different capturing molecules
15 onto the same area, the specificity then being revealed by -~
sequential injections of second reagents (such as
antibodies) specific for each analyte bound by the
respective capturing molecules. As is readily understood, --
such a procedure will greatly reduce the complexity of the --
optical and mechanical design of the system for the
concentration determination of a panel of analytes.
Sensor surfaces which may be regenerated as defined
above a substantial number of times, e.~. 50 to 100 times,
are known per se in the art and are, for example, described ~-
in our published PCT-application Wo 90/05305 (the full
disclosure of which is incorporated by reference herein).
These sensor surfaces comprise a substrate coated with a
metal film to which has been attached a layer of an organic -
polymer or a hydrogel forming a so-called basal surface
which may contain functional groups for selectively binding
the desired ligands. For further details on the production
of such surfaces it is referred to our published PCT-
application W0 90/OS303 (the full disclosure of which is
incorporated by reference herein). Such a surface is easily
regeneratable in situ in a flow cell. Thus, after the
determination of one sample is completed, the bound
analytes can be removed from the respective ligands to
prepare the sensing surface(s) for a subsequent assay of
W092t21973 ~ I2 PCT/SE92/~386
another sample. This is made possible by the binding of the
analyte to the ligand being broken by the regenerating
fluid whereas the binding of the ligand to the sensor
surface is not.
Exemplary of myocardial infarction analytes or markers
from which the test panel may be selected are myoglobin,
creatine kinase (CK) (also known as creatine phosphokinase)
and its isoenzymes and isomers thereof, especially CK-MB,
tropomyosin, lactate dehydrogenase (LD), troponin C, T and
~O I, aspartate aminotransferase, and myosin, especially the
light chain thereof.
Presently preferred infarction markers for the
purposes of the present invention are CK-MB, myoglobin and
troponin I.
However, none of the above mentioned markers is
absolutely specific for acute cardiac muscle damage. For
example, both CK-MB and myoglobin may also be present in
skeletal muscle damages. It is therefore, as already
mentioned above, preferable to simultaneously test for one
or more control analytes. One example of such a control
analyte is carbonic anhydrase III (CA III) which has been
shown to be present in substantial amounts in skeletal
muscle, in minor amounts in i.a. smooth muscle cells, but
not at all in the myocardium. CA III can therefore be used
to distinguish whether increased concentrations of e.g.
myoglobin originate from cardiac or skeletal muscle (see
e.g. Vaananen H.K. et al., Clin. Chem. 36/4, 635-638
( 1990 ) .
The ligands for the specifically binding cardiac
analytes, such as those mentioned above, are usually
antibodies, particularly monoclonal antibodies. By the term
"antibodies" as used herein is also to be understood active
antibody fragments, antibodies and fragments thereof
produced by genetic engineering, etc.
The functionalizing of the basal sensor surface with
the ligands is simplified if the ligands are chimeric
molecules, i.e. comprise a common part for binding to the
; basal surface and a variable part for binding to the
WO92/21973 13 2 I i 0 7 0 ~ PCT/SEg2/00386
different cardiac analytes. In the case that the sensing
surface comprises a basal dextran layer, the chimeric
molecule may consist of an antibody to dextran which is
conjugated to a monoclonal directed against the desired
cardiac analyte.
Antibodies that may be used as ligands are described
in the prior art, and may also be produced by methods known
per se, e.g. by hybridoma or recombinant DNA technology. -
Thus, antibodies against creatine kinase and creatine
kinase MB are, for example, described in EP-A-288 179, EP-
A-339 814, EP-A-261 781, and US-A-4,912,033.
Antibodies against myosin light chain are, for
instance, disclosed by W0 91/01498, W0 90/15329, and US-A-
4,879,216.
Antibodies directed against troponin T are described ~
in, for example, EP-A-394,819, and antibodies against ;
troponin I are described by Cummins B. et al., Biochem.
Soc. Trans. 15 (1987) 1060-61.
Myoglobin antibodies are, for instance, disclosed in
JP-A-54011231.
Antibodies directed against lactate dehydrogenase are
described in, for example, DE-A-2 350 711.
The preparation of antibodies directed against CA III -~
are described by Vaananen H.K. et al., Histochemistry 1985;
83: 231-5, and by Kato K. et al., Clin. Chim. Acta 141
(1984) 169-177.-
Several antibodies are also commercially available,
such as monoclonals against CK-MB and myoglobin. -
For the functionalization of the sensor surfaces with
the ligands for the respective cardiac analytes it is
referred to the aforementioned WO 90/05a305.
The blood samples taken from the patient may be
analyzed directly as whole blood, but it may be preferable
to use plasma or serum prepared from the blood sample, e.g.
as obtained by an initial centrifugation or filtration
step.
With the above described method and sensor means of
-the present invention, using the mentioned analytical
W092/21973 ~ 14 PCT/SE92/~386
apparatus, it is thus possible to in a short time, by
taking blood samples at short intervals and using bedside
apparatus and thereby dispensing with central laboratory
determinations, diagnose or exclude, respectively, an AMI.
Similarly, the treatment with a thrombolytic, as well as
the state of the myocardium during heart surgery may
readily be monitored, as already mentioned above.
A further beneficial feature of the method of the
present invention is that it may also provide information
about the presence in the blood samples of antibodies
against streptokinase. Thus, whereas streptokinase is àbout
tenfold cheaper than tissue plasminogen activator (tPA),
tPA is necessary if the patient has a high level of
antibodies against streptokinase or is allergic thereto.
About 90% of the patients experiencing a reinfarcation
within a year have neutralizing antibodies. Neutralizing
antibodies can also occur after a flu, if the infection is
caused by streptococcal bacteria. Therefore, the method of
the invention preferably also comprises testing for such
neutralizing streptokinase antibodies by including a
streptokinase ligand (or an antibody against the anti-
streptokinase antibody) in the or one of the sensing areas.
Thereby such crucial information about the level of
neutralizing streptokinase antibodies in a patient can be
determined at the same time as the AMI diagnosis is
performed.
In a preferred embodiment the present invention thus
comprises determining (i) at least two, and preferably at
least three, different infarction analytes; (II) one or
optionally more control analytes; and (iii) streptokinase
antibodies. `
Hereinafter the invention will be described, by way of
example only, with regard to a particular SPR-based
embodiment, reference being made to Figs. 1 and 2 mentioned
above. First, however, the surface plasmon resonance (SPR)
phenomenon will be briefly explained.
If the angle of incidence of light directed towards an
interface between two transparent media of different
~ :
WO92/21973 2 t ~l ~ 7 0 ~ PCT/SE92/~3~
refractive indices exceeds a critical angle, the light is
reflected back into the medium having the higher refractive
index, so-called total internal reflection. However,
despite the total reflection an electromagnetic field
component of the light called the "evanescent wave"
penetrates a short distance (of the order of a wave length)
into the medium of lower refractive index. If the interface
~etween the media is coated with a thin metal film, such as
silver or gold, and the light is plane-polarized and
monochromatic, the evanescent wave will at a certain angle
of incidence interact with collective electron
oscillations, called plasmons, in the metal. This
phenomenon - called surface plasmon resonance (SPR) - will
be observed as an intensity dip in the reflected light. To
couple the light to the intèrface such that SPR arises, two
alternative arrangements are used, either a metallized
diffraction grating (Woods effect) or a metallized glass
prism or a prism in optical contact with a metallized glass
substrate (Kretschmann effect). The specific angle of the
occurrence of SPR is sensitive to refractive index changes
close to the interface of the medium of the lower
refractive index. Thus, if the high refractive index medium
is glass and the medium of lower refractive index is an
aqueous solution in contact with the glass, changes of the
solution close to the interface, e.g. by the adsorption of
a protein layer, will cause a corresponding shift of the
resonance angle. SPR may therefore be used for detecting
e.g. immunoassay reactions as is well known in the art.
A schematic illustration of an SPR based biosensor
system of the flow cell type which is known in the art and
may be used for the purposes of the invention is shown in
- Fig. l. In the figure a flow channel l has an open top
portion covered by a sensor plate or chip 2 of glass coated
with a metal film 3, more specifically of gold, to define a
3S flow cell. A prism 4 contacts the other side of the glass
plate 2 to couple a wedge-shaped beam 5 of p-polarized
light from a monochromatic light source 6 thereto. The
reflected light is directed against a detection unit 7
wog2/2l973 ~ Q ~ 16 PCT/SE92/~386
comprising a matrix (i.e. rows and columns) of
photodetectors. The gold film surface exposed to the fluid
passing through flow cell 1 has ligands 8 immobilized
thereto as will be further described below.
Since the beam of light 5 reflected at the glass-metal
interface represents a continuous range of incident angles,
a shift in the resonance angle caused by a change in the
concentration of biomolecules at the metal surface may be
detected by the detector unit 7. ~or a more comprehensive
description of the above described biosensor system,
including optical system, sensor unit, and liquid handling
unit, it is referred to our aforementioned W0 90/05295.
The use of a biosensor system of the above outlined
type for the purposes of the present invention will now be
described. Fig. 2 schematically illustrates a part of a
liquid handling block unit 9 comprising four flow cells 10,
corresponding to flow cell 1 in Fig. 1. The flow cells 10
- are, as in Fig. 1, defined by upwardly open channel parts
11 covered by a sensor plate 12, corresponding to sensor
plate 2 in Fig. 1. In the figure is also illustrated an
optointerface 13 for effectina optical contact between the
sensor plate 12 and a prism co-responding to prism 4 in
Fig. 1. The optointerface consists of a thin glass plate
having elastic material pieces 14 on both sides with a
refractive index matching that of the sensor plate 12. When
the assembly of liquid handling block unit 9, sensor plate
12 and optointerface 13 is arranged in the biosensor system
schematically illustrated in Fig. 1 (replacing flow channel
1 and sensor plate 2 therein), the wedge-shaped beam 5 will
extend transversely across the flow cells, each flow cell
corresponding to, e.g., one column of photodetectors in the
detector matrix 7.
The gold-coated glass plate 12 has, as a specific
example known per se in the art, a hydrophilic matrix of
non-crosslinked carboxymethylated dextran covalently bonded
to the gold film through an optically and biologically
inert linker layer of long chain hydrocarbon. Ligands can
be covalently immobilized to the dextran layer after
WO92/21973 17 2 1 ~ O / ~ ~ PCT~SEg2/00386
activation, e.g. by derivatization with N-hydroxy-
succinimide (NHS), mediated by N-ethyl-N'-~dimethyl-
aminopropyl)carbodiimide (EDC). The NHS-ester formed
readily reacts with uncharged primary amino groups of the
ligands to be coupled thereto.
In the flow cells l0 shown in Fig. 2 the sensing areas
forming the top part of each flow cell may, for the
purposes of the invention, in three of the flow cells l0
support ligands reactive with a respective cardiac analyte
l0 to be determined whereas the fourth flow cell preferably is ~-
used as a control or reference. For example, one flow cell
may support a monoclonal antibody against creatine kinase
MB (CK-MB), a second flow cell a monoclonal antibody
against myoglobin, a third flow cell a monoclonal antibody
against troponin I, and a fourth flow cell a monoclonal
antibody against carbonic anhydrase III (CA III) as a
control. Of course, more than four flow cells may be used
if desired. Thus, for instance, a fifth flow cell may
support streptokinase ligands for determining streptokinase -
antibodies in a sample. Optionally, a sixth flow cell may
support a ligand for an additional cardiac infarction
analyte.
For further details on the immobilization of ligands
and reaction conditions for the coupling of the analytes to
be determined, it is referred, in addition to the above
mentioned WO 90/05303 and WO 90/05305, to the manual "Real-
time Biospecific Interaction Analysis, A Guide to Methods
and Applications" (l990), Pharmacia Biosensor AB, Uppsala,
Sweden (the full disclosure of which is incorporated by
reference herein).
A test of a blood plasma sample for the cardiac
enzymes creatine kinase MB, myoglobin, and troponin I using
the analytical system illustrated in Figs. l and 2 may be
performed as follows. After removal of the blood cells from
a blood sample, a defined volume of the plasma is
introduced into the liquid handling unit 9 and evenly
distributed among the four flow cells l0. When flowing
through the flow cells the mentioned cardiac enzymes, if
Wo92/21973 ~Q~ 18 PCT/SE9~/~3~
present, will bind to the respective sensing surface
supporting the proper ligand. This will cause a shift of
the resonance angle, which shift is proportional to the
amount of cardiac enzyme bound to the surface, as described
S above. Thereby the level of each analyte in the sample may
be determined. To increase the resonance angle shift
detected, the ligand bound analytes are preferably reacted
with a secondary reagent in sandwich assay fashion. As
already mentioned above, such a secondary reagent may
optionally be labelled with an optically dense species to
still more increase the shift. Alternatively, a tertiary
reagent may be used. After the detection step, the sensor
surface is regenerated by passing a regenerating agent,
e.g. glycine buffer, or phosphoric, formic or hydrochloric
acid (lO-lO0 mM), through the cell. A whole such analytical
procedure will take about 15 minutes.
Instead of using several flow cells as described
above, a single flow cell may be used which has ligands for
all the analytes of interest co-immobilized on the same
sensing surface area. This embodiment will also be
illustrated in the Example below.
When a patient with suspected AMI has been brought to
the coronary care unit in possession of the biosensor
apparatus described above at the patient's bedside, and it
has not been possible to establish a safe diagnosis based
upon symptoms and ECG, blood samples are taken regularly,
say every 15 to 30 minutes, and successively analyzed in
the described fashion. While already the first sample
tested may exhibit such levels of one or more of the -
analytes tested for that an AMI may directly be diagnosed,
a safe and reliable AMI diagnosis or exclusion, based upon
the development with time of the analyte levels, will in
the majority of cases be obtained within about no more than
about two to three hours. In the case of a diagnosed AMI,
the patient can then be treated with thrombolytics, i.e.
streptokinase or tPA. Preferably, the test panel of the ~;
sensor unit also contains a sensing surface area for
testing for streptokinase antibodies, and information about
WO92/21973 2 ~ 1 0 f ~ ~ PCT/SEg2/~386
the proper thrombolytic and dosage thereof to select may
thereby be obtained at the same time, i.e. resulting in
that the dose of streptokinase is adjusted or that tpA is
selected rather than streptokinase.
The same bedside apparatus and test panel may then be
used for monitoring the thrombolytic treatment to observe a
reperfusion as soon as possible after it has taken place,
so that the per se risky treatment may be stopped when no -~
longer necessary or an alternative treatment may be
introduced if reperfusion is not obtained in a reasonable
time. Especially in such monitoring of thrombolytic
treatment, relatively high levels of an analyte of
interest, e.g. CK-MB, may be obtained. Thus, if a secondary
reagent is used as described above, the dynamic range of
the secondary response may not be sufficient for permitting
the development of the peak value of a specific analyte to
be monitored. The occurrence and exact monitoring of such a
peak indicative of reperfusion may then be performed by
instead measuring the primary response, i.e. the complex ~-
formation between the analyte and the immobilized ligand.
Naturally, the above de,cribed procedure and method may
also be used for monitoring the myocardium state during
thorax surgery such that an AMI initiated during the
surgery may be detected and treated before t~e thorax is
closed.
In the following non-limiting example, the detection
of CX-MB and myoglobin in a plasma sample in accordance
with the present invention is described, using a commercial
SPR-based biosensor instrument (BIAcoreTM) and sensing
30 surface (Sensor ChipTM CM5) (both marketed by Pharmacia ~-;
Biosensor AB, Uppsala, Sweden).
~XAMPLE
A. ~o-immobilization of monoclonal antibodies on sensing
surface
Immobilization of a monoclonal antibody specific for
CK-MB and a monoclonal antibody specific for myoglobin was
performed in the biosensor instrument in t~e following
manner:
WO92/21973 ~ 20 PCT/SE92/~ ~
A continuous flow of HBS (lO mM Hepes buffer, O.l5 M
NaCl, 3.4 mM EDTA, 0.05 ~ Tween), pH 7.4, over the sensing
surface was maintained at 5 ~l/min. A fraction of the
carboxyl groups on the;sensing surface was activated to
form reactive N-hydroxysuccinimide esters by injecting into
the instrument 35 ~l of a solution containing 0.2 M l-
ethyl-3-(dimethylaminopropyl)carbodiimide hydrochloride
(~DC) and 0.05 M N-hydroxysuccinimide (NHS) in water. 35 ~l
of the antibody solution containing 50 ~g/ml of a
monoclonal antibody specific for CK-MB (obtained from
BiosPacific, Emeryville, California, U.S.A.) and 50 ~g/ml
of a monoclonal antibody specific for myoglobin (obtained
from the Institute of General and Molecular Pathology,
Tartu State University, Tartu, Estonia) in lO mM sodium
acetate, pH 5.0, were then injected. A buffer with a pH
below the pI of the antibody will give a positive net
charge of the protein, and at low ionic strength the
antibodies will preconcentrate to the remaining negatively
charged carboxyl groups on the surface via electrostatic
20 attraction giving a high antibody concentration in the -
matrix. The preconcentration allows fast immobilization
with low amount of antibodies. Remaining reactive ester
groups were deactivated by injection of 35 ~l of l M
ethanolamine hydrochloride, pH 8.5. Tbe sensorgram obtained
25 is shown in Fig. 3 (response in resonance units, RU, ~-
plotted versus time in seconds). The response signal was
evaluated at two levels: 20 seconds before the injection of
EDC/NHS (A) and 9 minutes after the injection of
ethanolamine (8). B minus A thus defines the immobilized
amount of the two antibodies.
B. Analvsis of Plasma sam~les
The analysis of CK-MB and myoglobin at elevated levels
in a plasma sample, using the sensing surface with co-
immobilized antibodies prepared in section A above, was
performed in the following manner:
A continuous flow of HBS (lO mM Hepes buffer, 0.15 M
NaCl, 3.4 mM EDTA, 0.05 % Tween), pH 7.4, over the sensing
surface was maintained at 5 ~l/min. 35 ~l of a plasma
WO92/21973 2 1 1 0 7 0 ~ PCT/SE92/00386
21
sample containing CK-MB and myoglobin were injected into
the instrument. 4 ~1 each of second antibodies specific for
CK-MB and myoglobin, respectively, at a concentration of
100 ~g/ml were then injected in sequence followed by 4 ~1
of 10 mM glycine-HCl, pH 2.5. The sensorgram obtained is
shown in Fig. 4 (response in resonance units, RU, plotted
versus time in seconds). The response signal was evaluated
at four levels: 20 seconds before the injection of the
sample (A), 20 seconds before the injection of the second
antibody specific for CK-MB (B), 20 seconds before the
injection of the second antibody specific for myoglobin
(C), and 20 seconds before the injection of glycine-HCl -~
(D). Thus, A defines the baseline, B minus A defines the
plasma response, C minus B defines the specific response
for CK-MB, and D minus C defines the specific response for
myoglobin. The analysis time was 18 minutes. -
The same procedure as described above was then ~-~
performed for a plasma sample not containing CK-MB and a
normal myoglobin level. The sensorgram obtained is shown in
20 Fig. 5. As appears therefrom, no response was obtained when -~
injecting the second antibody specific for CK-MB, whereas a
weak response was obtained for the second antibody specific
for myoglobin.
The invention is, of course, not restricted to the -
above specifically described embodiments, but many
modifications and changes may be made without departing
from the scope of the general inventive concept as defined
in the subsequent claims.
