JPH06508204A - Measuring method and sensor device for myocardial infarction marker - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Field of the invention: Method and sensor device for measuring myocardial infarction marker The present invention provides a blood sample with at least two different and independent results indicative of myocardial damage. Provides early and reliable diagnosis of myocardial infarction by simultaneously evaluating changes in markers over time and, at the same time, to methods for improving treatment and monitoring of recovery after a myocardial accident. The present invention It also relates to a sensor device for use in this method.

Background of the invention and prior art Acute myocardial infarction (A MI) is the leading cause of death in developed countries. Ischemia was affected by myocardium Caused by blockage of an artery by a blood clot that prevents normal blood flow to the area and eventually leading to tissue necrosis.

Some serious complications such as pulmonary edema, hypertension, thrombosis and cardiac rupture can occur with AMI. It can happen in Early correct diagnosis and appropriate treatment increase chances of survival. and reduce the risk of complications. Therefore, patients suspected of having AMI should be diagnosed immediately. It is essential that the patient be sent to a hospital for treatment.

The current clinical diagnosis of AMI is chest pain (pain that starts suddenly and lasts for more than 15 minutes). ), electrocardiogram (ECG) and levels and dynamics of cardiac muscle-specific enzymes and proteins. Chest pain and observed ECG changes may not be safe for diagnosis in some cases. are sometimes sufficient, however they are often rather non-specific. As it is of a different nature, final confirmation of AMI and other cardiac and non-cardiac matters are required. For most patients, identification of the cause occurs at regular time intervals, usually between 6 and 18 hours. Blood was collected at intervals and analyzed in a central laboratory to monitor the biochemical parameters mentioned above. This is possible only by looking at it. Therefore, patients currently suspected of having AMI are Depending on the preparedness of the individual patient and physician, the cardiac Patients may spend several hours to more than 48 hours in treatment or intensive care.

Of course, it is desirable in all cases to be able to make the correct diagnosis at an early stage. one On the other hand, in the case of 8Ml, early appropriate treatment with thrombolytic agents can prevent death. rate and reduce the risk of complications. In fact, because thrombolytic agents are beneficial should be used within the first 6 hours of the onset of pain, and sooner (when treatment is certain). If done in fact, the mortality rate is significantly reduced (about 10% per hour). On the other hand, AMI A relatively large group of patients (approximately two-thirds) not affected by and can be excluded from treatment, which is sometimes fatal, in which case other A Causes of MI-like symptoms are, for example, angina pectoris or pain originating from the gastrointestinal tract. this As such, treatment with thrombolytic agents can reduce strokes and hemorrhage (haeIllorrhagic). It is accompanied by severe side effects such as bleeding. Thrombolysis currently used by the agents tissue plasminogen activator (tPA) and streptokinase Up to approximately 2% of treated patients experience these side effects. Therefore, accurate A thorough diagnosis is important in these cases. ^MI treatment is necessary at such an early stage. The great economic importance of identifying patients who do not Can be transferred to a regular medical ward after a shorter time or is most preferred. This is acceptable given the substantial cost savings that would be achieved if the patient could be sent home or sent home. This is easily understood.・In the treatment of AMI with thrombolytic agents, The goal is to achieve reperfusion of occluded coronary arteries before tissue is irreversibly damaged. It is to accomplish. When reperfusion is achieved or absent, cardiac enzymes differ. It is known that this pattern shows a certain pattern. A remote site that allows for monitoring at the patient's bedside. Naturally, real-time analysis is desired. So far, the current This was not possible using the assay methods and techniques available.

Similarly, detecting AMI that may begin before closing the chest during thoracic surgery Of course, it would be desirable to be able to constantly monitor cardiac enzymes in order to

Current serum assays detect intracellular enzymes from damaged or necrotic cells resulting from myocardial infarction. or based on leakage of other proteins. Accepted for heart attack detection The standard assay for this has long been the creatine kinase (CK) test. this exam includes the measurement of CK and its isoenzyme CK-MB, which indicates that a myocardial accident has occurred. It shows a detectable peak concentration after about 12 hours. Other conventional test proteins are Lactate dehydrogenase (LD) and its isozymes, and recently mi Measurement of non-catalytic proteins such as oglobin and troponin T is a highly sensitive immunoassay. This became possible as a result of scientific measurement methods. Following myocardial infarction, sulcus detachment from the heart muscle and Yet another protein known to form the basis of the test is the myosin chain. It is. Each one of the marker proteins mentioned above has a specific serum concentration depending on the kinetics. Changes and release of the respective proteins in acute infarction are shown.

It has been proposed that a combined analysis of the two markers improves the recognition of myocardial infarction. Ta. For example, analysis combining CK and LD is performed by Limbird Lee. , Diss, Abstr, Int.

B 1974.34 (11), 5322-3.

1091101498 uses creatine kinase and myocardial infarction for early detection of myocardial infarction. Discloses simultaneous or sequential testing of thin chains. The combination of these two assays is While it is said to provide an extremely reliable diagnostic test for early stage heart attacks, it It also makes it possible to differentiate between myocardial infarction and other ischemic events that cause heart disease, such as angina pectoris. Ru. The latter characteristic is that myosin chains are present not only in cases of acute myocardial infarction, but also in other cardiac disorders. Creatine kinase is also released in cases of acute myocardial infarction, whereas creatine kinase is essentially released in acute myocardial infarction. Due to the fact that it is released only when .

Dual combination assays of the type described above provide more accurate and reliable diagnosis. However, these are conventional format assays, usually solid-phase enzyme immunoassays. based on the above-mentioned final The disadvantage is that a considerable amount of time may elapse before a diagnosis can be made.

Therefore, an approach that allows a reliable diagnosis of a possible myocardial infarction to be made in a short time. procedures that are performed entirely next to or in the vicinity of the patient. Therefore, one of the features of the present invention is to The aim is to provide reliable 8M treatment within a short period of time such that treatment with thrombolytic agents is beneficial. The object of the present invention is to provide an assay method that allows the diagnosis of I.

Another object of the invention is to quickly and reliably diagnose AMI and thereby exclude it. transfer such patients to regular wards (substantially less expensive) within a short period of time? Or assay methods that can even be sent home, reducing treatment costs. The goal is to provide the following.

Yet another object of the invention is to effectively monitor the treatment of AMI with thrombolytic agents. treatment is discontinued as soon as possible and reperfusion is achieved. or treat the patient with another treatment if reperfusion is not achieved within a reasonable time. The purpose of the present invention is to provide an assay method that enables

Another object of the invention is that it can be carried out at or near the patient's bedside. Provides an assay method for diagnosing AMI or monitoring treatment with thrombolytic agents. Is Rukoto.

Yet another object of the invention is to monitor cardiac enzymes that may occur during thoracic surgery. We present an assay method that detects AMI that cannot be detected and that can be performed in the operating room. It is to provide.

Another object of the present invention is to provide a sensor device for use in the above assay method. Is Rukoto.

The above objects are achieved by the method and sensor device of the invention. Basics of the invention The concept is that at least two, preferably at least three different markers of myocardial infarction , that is, real-time changes in enzymes or proteins in a flow cell system. The purpose of this measurement is to measure the analyte at short time intervals. -based on interactions with surface-bound ligands, and this interaction is causing detectable changes in the physicochemical characteristics of the surface. at least two different minds Studies of visceral analytes or markers provide sufficient information to warrant early diagnosis. can be shortened using sensor surface technology that detects analyte-ligand interactions. Fast enough and automated to allow analysis of blood samples taken at close time intervals It is also easy to perform and does not need to be performed in the laboratory and can be performed at the patient's pedside or hand. A real-time analysis procedure is provided that can be easily performed in the operating room.

As used in the present invention, the terms "different markers" or "different analytes" refer to different and It should be noted that this refers to markers or analytes derived from two separate genomes. It is. Therefore, post-translational modifications such as glycosylation or fragmentation of macromolecules are does not qualify it to be considered as an analyte. For example, CK-MMA and CK MMa is not a distinct analyte in the context of the present invention.

According to one embodiment of the invention, therefore, (i) the sample is placed in a flow cell or within which different ligands or in contact with one or more sensor surface areas that support a mixture of different ligands. and optionally additionally analyte-specific reagents or reagent complexes. After binding to the bound analyte, each analyte has all Detect binding interactions as changes in the resulting physicochemical characteristics of the sensor surface. The first sample of blood, serum, or plasma from the patient is tested for myocardial infarction. At least two, preferably at least three different analytes shown are measured simultaneously. (if) each bound analyte is linked to the ligand bound to its sensor surface. by passing the regeneration liquid through a flow cell, (ffl) Step (i) is performed by collecting samples from the patient at determined time intervals from said first sample. repeat for at least a second blood, serum or plasma sample taken; and (tv) Determine the temporal changes in the cardiac analyte from the results of steps (i) to (Sat) A method of measuring or monitoring an MI marker is provided which comprises the steps of:

As used herein, the term "reagent complex" means that a reagent is associated with one or more other substances. It means that it is combined with quality. Such reagent complexes should be added as aggregates. or after the reagent has combined with the analyte as described further below. It can be formed continuously.

In other embodiments of the invention, the one or more sensing regions may be individually or at least two, preferably at least three different immobilized in combination A sensor device is provided comprising ligands, each ligand being indicative of myocardial infarction. The sensor device has the ability to specifically bind to each analyte. Misaligned analyte-ligand interactions also result in physicochemical changes in the sensing surface. is adapted to detect as a change in the characteristic and the ligand supports The surface area can be regenerated after the analyte has bound thereto.

That is, different ligands are immobilized individually on their respective sensing regions, or They can be simultaneously immobilized on a single sensing area. Combinations of these embodiments , i.e. there are two or more sensing regions and each region is co-immobilized. It is of course also possible to support the ligand.

In a preferred embodiment, the sensor surface exhibits surface plasmon resonance (SPR). and the detection of cardiac analytes or markers is Performed by SMON resonance spectroscopy.

Brief description of the drawing Other objects and advantages of the invention will be found in the detailed description thereof below, together with the accompanying drawings. It will be clearer to those skilled in the art.

In the drawings, FIG. 1 is a schematic diagram of a flow cell measuring device based on SPR, which is known per se. can be, FIG. 2 is an exploded partial cross-sectional view of a flow cell unit useful for purposes of the present invention. , Figure 3 shows a monoclonal antibody specific to CK-MB and a antibody specific to myoglobin. 5PR-sensor diagram showing simultaneous immobilization of noclonal antibodies on a sensing surface. Figure 4 shows the immobilized CK-MB and myoglobin monoclonal cells in Figure 3. increased levels of CK-MB and myoglobin using a sensing surface with FIG. 5 is a diagram corresponding to FIG. 3 showing the analysis of samples containing CK-MB and FIG. Corresponding to Figure 4, which shows the analysis of a sample containing only normal levels of myoglobin, without FIG.

Detailed description of the invention As stated above, the basic features of the present invention are several, namely at least two. of, preferably at least three, but also possible for example four or even five It consists in the simultaneous measurement of different myocardial infarction markers or analytes.

Although the diagnostic sensitivity and specificity of such markers may vary, short Appropriate selection of analytes and their simultaneous measurement for blood samples taken at time intervals can provide analytical diagrams or patterns that are extremely useful for diagnosing or excluding AMI. Probably. The relationship between various analytes during a certain time interval may be e.g. How long the infarct condition has progressed based on the rise in levels of analytes and other declines in It will also give you an indication of what you are doing. Similarly, below each analyte graph The area will indicate the extent of damage to the heart muscle tissue.

The term "simultaneously" used in the present invention should be interpreted in a rather broad sense. Yes, i.e. each measurement of the analyte does not have to start exactly at the same time. , insofar as comparisons between measured levels of different analytes are considered meaningful. A certain amount of delay is acceptable.

Another essential feature of the invention is a preferred or in the repetition of the described analysis at regular short time intervals. Such feelings information significantly alters the likelihood of making a reliable or unreliable diagnosis of AMI. will do good.

Combined with the basic characteristics mentioned above, the outlook after a patient is brought to the hospital is to ensure highly reliable diagnosis or exclusion of AIIIT within a short period of time. or, in another case, treatment with thrombolytic agents and also thoracic surgical procedures. effective monitoring of the technique would be possible.

Analytical devices that allow the execution of the desired type of analytical procedure are: (i) a sensor for the analyte; – immobilized on the surface or based on interaction with Gantt and the resulting sensor The measurement principle detects complex formation as a change in the physicochemical properties of the surface. (if) a flow cell device capable of the desired simultaneous detection of several analytes; and (ffl) after the regeneration process, the bound analyte is continuously on one and the same sensor surface(s) removed from the One that can be played back in its original position in the sense that it can be analyzed. or have multiple sensor surfaces. This requirement (i) All of ~(ffl) shall be satisfied by analytical techniques known in this technical field. Can be done.

In order to increase the changes in the physicochemical properties of the sensor surface described above, additional binding molecules can be added. Complexing the analyte and thereby increasing the thickness of the binding material layer will improve the ability of the ligand to bind. Reacting the analyte with a second reagent specific for the analyte, i.e. This is achieved by sandwich assays known in the art. like this The bound second reagent is then further reacted with a third reagent, if desired, to form a complex. The number of surface changes to be detected can be further increased.

Optical methods, especially those based on SPR spectroscopy, increase detection surface changes. Additionally, optically dense materials such as particulate labels, such as second and/or third reagents, can be added to For example, labeled with glass, latex, colloidal silver or gold, metal oxides or ferritin. For example, reference is made to EP-A-276142.

Sandwich mold onto the sensor surface using the second reagent as outlined above. By performing the assay, it not only increases the sensitivity and in some cases the specificity. , the dynamic measurement range can be expanded, and if the quadratic response range is insufficient, - A quadratic response can be used rather than a next response.

The measurement principle identified above makes it possible to carry out real-time measurements, thereby reducing It allows performing the desired continuous analysis of blood samples taken at long time intervals. Like that The measurement principle and associated sensor surface are easily adapted to flow cell systems. You can also do that. Based on the measurement principle mentioned above (EWS), e.g. attenuated internal reflection (ATR) ) spectroscopy, total internal reflection fluorescence (TIRF) spectroscopy, and surface plasmon Mention may be made of sound (SPR) spectroscopy or waveguide spectroscopy. these All methods are based on the examination of the optical properties of a solution in contact with a surface where total internal reflection occurs. Other types of sensors that can be used are photoacoustic piezoelectric, surface acoustic wave (S AW) or based on electrochemical measurements. Particularly suitable for the purposes of the invention The method used is SPR spectroscopy, which is described in more detail below.

Flow cell systems are easy to automate and are necessary to create devices that are quick and easy to handle. It is essential. The term "flow cell" (which should be interpreted broadly) refers to the and other analytical liquids flow past one or more sensing surfaces at a constant velocity. means. As mentioned above, such a system in one embodiment has multiple a sensing surface area, i.e. an area for each cardiac analyte as well as a control area; and optionally one or more areas for other purposes as described below. Ru. This flow may be in parallel or in series with respect to different sensing surface areas. good. A parallel flow cell system consists of several separate flow cells arranged in parallel, On the other hand, in a series device several separate flow cells can be connected in series. .

Parallel or serial flow systems each contain several confines contained within a single flow cell. It may also be formed from a textured sensing surface. Flow consisting of several flow cells A cell-based example is our published PCT application WO90105295 (which ), the entire disclosure of which is incorporated herein by reference, includes a number of flow Consists of a number of open channels to form cells and a sensor plate applied to them A liquid handling block unit is disclosed.

In another novel embodiment of the flow cell system, for all cardiac analytes co-immobilization of the ligands on a single surface area as already mentioned above. Can be done. Therefore, surface concentration detection devices such as surface plasmon resonance detection heterogeneous responses (Steinberg et al., J.

Co11oid Interface Sci, 143 (1991), 51 3) is basically the size of the detection area (at least below the diffraction limit of the optical device) is unrelated. This is typically used for absolute measurement devices where the specific response is area dependent, e.g. This contrasts with solid-phase assays.

Typically, different immobilized capture molecules are immobilized in different detection regions as described above. It will be done. However, this new and original concept makes it possible for surface concentration detection devices to It is independent of the size of the area that there are two or more different capturing parts. Simultaneous immobilization of children in the same area makes it possible to reduce the number of detection areas, and then The specificity of the second assay is determined by the specificity of each analyte bound to each capture molecule. revealed by subsequent injection of a drug (eg antibody). easily understood However, such a procedure may require would significantly reduce the complexity of the optical and mechanical design.

As clearly explained above, it can be played quite a number of times, e.g. 50-100 times. Sensor surfaces that can be used are per se known in the art and are e.g. as described in published PCT application W090105305 of (incorporated herein by reference). These sensor surfaces are coated with a metal film. The so-called basic surface contains functional groups that selectively bind the desired ligand to the attached substrate. consisting of a layer of organic polymer or hydrogel attached that forms a Further details on the fabrication of such surfaces can be found in our published PCT publication. Application No. 1090105303 (the entire disclosure of which is incorporated herein by reference) is referenced. Such surfaces can be easily regenerated in situ in the flow cell. I can do it. Therefore, after the measurement of one sample is completed, the bound analyte is Sensing surfaces (one or more than one).

This occurs because the bond between the analyte and the ligand is broken by the regenerating solution, causing the ligand to become active. This is possible because the bond to the surface is not broken.

An example of a myocardial infarction analyte or marker from which a test panel is selected is myoglobin. , creatine kinase (CK) (also known as creatine phosphokinase) and its isozymes and isomers, especially CK-MB, tropomyosin, lactic acid Dehydrogenase (LD), ) Roponin C, T and ■, Aspartate Aminotran spherase, and myosin, especially its light chain.

Presently preferred myocardial infarction markers for the purposes of the present invention are CK-MB, myoglobin and Trobonin I.

However, none of the above markers are unconditionally specific for acute myocardial infarction injury. It's not unusual. For example, both CK-MB and myoglobin are present in skeletal muscle injury. There may be. Therefore, as already mentioned above, one or more contrasting subdivisions It is preferable to simultaneously test for analyte substances. One such control analyte An example of this is carbonic anhydrase II [(C^■), which has a significant amount in skeletal muscle, It exists in small amounts in muscle, but it is known that it does not exist in cardiac muscle. Therefore, CAIII may be caused by increased concentrations of e.g. myoglobin originating from cardiac or skeletal muscle. (e.g. VaananenH, K, et al., Cl1n, Chell, 36/4.635-638 (1990 ) is referred to).

Gants that specifically bind cardiac analytes, such as those mentioned above, are usually especially monoclonal antibodies. As used herein, the term "antibody" refers to an active These should be understood as body fragments, antibodies and their fragments produced by genetic engineering.

Functionalization of the basic sensor surface with a ligand involves the ligand being a chimeric molecule, i.e. Consisting of a common portion that binds to the underlying surface and variable portions that bind to various cardiac analytes. It becomes easier if If the sensing surface consists of a basic dextran layer, the chimeric molecule for dextran coupled with a monoclonal directed toward the desired myocardial analyte. It consists of antibodies that

Antibodies that can be used as ligands are described in the prior art and by techniques known per se, e.g. by the hybridoma method or by recombinant DNA. It can also be created using technology.

Thus, antibodies against creatine kinase and creatine kinase MB are For example, EP-^-288179, EP-A-339814, EP-^-2617 81 and US-A-4,912,033.

Antibodies against myosin chains are disclosed in, for example, Wo 91101498, To 90/1 5329 and 115-A-4,879,21,6.

Antibodies against troponin T are described, for example, in EP-A-394,819. and antibodies against trobonin I such as Cull1m1ns B, etc., Bi ochem, Sac, Trans, 15 (1987), 1060-6 1.

Myoglobin antibodies are disclosed, for example, in JP-^-54011231.

Antibodies against lactate dehydrogenase are described, for example, in DE-^-2350711. There is.

Preparation of antibodies against CAm was described by Vaananen Ho et al.

Histochemistry, 1985; 83: 231-5 , and Kat.

K9 et al., C11n, ChiIl, Acta, 141 (1984), 16 9-177.

Several antibodies, such as monoclonals against CK-IIIB and myoglobin, are commercially available. You can also get items for sale.

Functionalization of sensing surfaces with ligands for respective cardiac analytes The above-mentioned 10901050305 is referred to.

Although blood samples taken from patients can be analyzed directly as whole blood, blood samples using plasma or serum obtained from e.g. by an initial centrifugation or filtration process. is preferable.

Thus, the above-described method and sensor device of the invention allows the use of the previously described analytical device. Blood samples are taken at short intervals within a short period of time using a bedside device. and thereby permit the respective measurement, diagnosis or exclusion of AMI by a central laboratory. It can be made unnecessary. Similarly, treatment with thrombolytic agents as already mentioned above The condition of the myocardium during medical treatment and cardiac surgery can be easily monitored.

Another beneficial feature of the method of the invention is that it It also provides information regarding the presence of antibodies against the virus. In other words, strep Tokinase is approximately 10 times cheaper than tissue plasminogen activator (TP^). streptokinase, but the patient has high levels of antibodies to streptokinase or tP^ is required if you are allergic to. Experienced reinfarction within 1 year Approximately 90% of patients with this disease have neutralizing antibodies. Neutralizing antibodies are present after influenza infection. However, this can occur if the infection is caused by streptococci. Therefore, the book The method of the invention preferably includes streptokinase ligation in the sensing region or in one of the sensing regions. (or antibodies against anti-streptokinase antibodies). This includes testing for streptokinase-neutralizing antibodies such as . The patient's Critical information regarding the level of streptokinase-neutralizing antibodies can be used to perform an AMI diagnosis. You can read and write at the same time.

In a preferred embodiment, the invention thus provides (i) fewer (both two, and preferably Preferably at least three different infarct analytes, (ti) one or optionally If more control analytes and (ffi) streptokinase antibodies are measured, It consists of things.

Hereinafter, the invention will be described based on the specific SPH, to which reference is made to FIGS. 1 and 2 above by way of example. Only aspects will be explained. However, first the surface plasmon The sound (SPR) phenomenon will be briefly explained.

When the angle of incidence of light towards the interface between two transparent media of different refractive index exceeds the critical angle When the light is reflected into a medium with a high refractive index, this is called total internal reflection. be.

However, despite total reflection, the magnetic field of light, which is called "enokunesent wave" The components enter the low index medium over a short distance (on the order of the wavelength). media interface is coated with a thin metal film, e.g. silver or gold, and the light is plane polarized. If the metal is monochromatic, the evanescent wave is a collective electron oscillation in the metal at a certain angle of incidence. They interact and are called plasmons. This phenomenon is called surface plasmon resonance (SPR). The phenomenon is observed as a temporary dip in the intensity of reflected light. Ru. To couple light to an interface so that SPR occurs, there are two possible configurations: metallized diffraction gratings (Woods effect) or metallized glass plates. Prisms in optical contact with rhythm or metal-coated glass substrates (Kretschmann effect). The specific angle of SPR generation is at the interface of a medium with a low refractive index. Sensitive to adjacent refractive index changes. Therefore, if the high refractive index medium is glass If the low refractive index medium is an aqueous solution in contact with the glass, the interface Close solution changes, e.g. due to adsorption of protein layers, result in corresponding changes in the resonance angle. will occur. Therefore, SPR is, for example, a well-known technique in this technical field. It can be used to detect immunoassay reactions.

Flows known in the art and which can be used for the purposes of the present invention A schematic diagram of a cellular SPH-based biosensor device is shown in FIG. In the figure, The flow channel 1 has an open tip, which is coated with a metal membrane 3, more specifically gold. Covered with a coated glass sensor plate or chip to form a flow cell. Ru. The prism 4 contacts the other side of the glass plate 2 and receives polarized light from the monochromatic light source 6 thereon. The wedge-shaped beams 5 are tied together. The reflected light passes through a matrix of photodetectors (i.e. ie, to a detection device 7 consisting of rows and columns). Exposure to liquid passing through flow cell 1 The gold-coated surface has ligand 8 immobilized on it and is described in more detail below. do.

Since the light beam 5 reflected at the glass-metal interface represents a continuous range of angles of incidence, The detection device 7 detects changes in the resonance angle caused by changes in the concentration of biomolecules on the surface. can be detected. A more comprehensive description of the biosensor devices mentioned above including chemical equipment, sensor units, and liquid handling units, but our aforementioned 1. 090105295 is referenced.

The use of a biosensor device of the type outlined above for the purposes of the present invention will now be described. Ru. FIG. 2 shows a part of a liquid handling block unit 9 consisting of four flow cells 10. 1, the flow cell corresponds to the flow cell 1 of FIG. In Figure 1 As shown in FIG. - It is covered with a sensor plate 12 corresponding to the plate 2. Sensor in the drawing - to achieve optical contact between the plate 12 and the prism corresponding to prism 4 in FIG. A second optical interface 13 is also shown. The optical interface matches the refractive index of the sensor plate 12 on both sides. It consists of a thin glass plate with a piece of elastic material 14 having a refractive index of .

Figure 1 shows the assembly of the liquid handling block unit 9, the sensor plate 12, and the optical interface 13. When placed in the biosensor device shown schematically in the flow channel 1 and sensor plate 2), the wedge-shaped beam 5 extends across the flow cell. and each flow cell corresponds to, for example, one row of photodetectors of the detector matrix 7.

Gold-coated glass plate 12, which is a special example known per se in the art, is an optical Covalently bonded to the gold coating through a layer of a long-chain hydrocarbon linker that is both biologically and inert. with a hydrophilic matrix of uncrosslinked carboxymethylated dextran . The ligand can be activated, e.g. N-ethyl-N'-(dimethylaminopropyl) N-hydroxysuccinimide (NH5) via carbodiimide (EDC) l:, J, L is covalently immobilized on the dextran layer after activation by derivatization. can do. The NH3-ester formed is the unligand-loaded first It can easily react with and bond to monoamino groups.

In the flow cell 10 shown in FIG. 2, a sensor for forming the tip portion of each flow cell For purposes of the present invention, three of the flow cells 10 correspond to each heart area being measured. supports the analyte and reactive ligand, while a fourth flow cell preferably supports the control Or it can be used as a reference.

For example, one flow cell has a monomer for creatine kinase MB (CK-MB). A second flow cell contains a monoclonal antibody against myoglobin. a third flow cell contains a monoclonal antibody against troponin, and The fourth flow cell was a monochrome cell for carbonic anhydrase m (C^■) as a control. can support null antibodies. Of course, four or more flow cells can be used if desired. can be used. Thus, for example, the fifth flow cell can support streptokinase ligand to measure streptokinase antibodies. Wear.

Optionally, a sixth flow cell supports a ligand for another myocardial infarction analyte. You can also hold it.

Further reaction conditions for immobilization of the ligand and binding of the analyte to be measured For further details, please refer to W090105303 and Wo90105305 mentioned above. As a guidebook, ``Guide to Real-Time Biospecific Interaction Analysis, Methods, and Applications'' ( 1990), Pharmacia Biosensor AB, Uppsa la, Sweden, the entire disclosure of which is incorporated herein by reference. ).

Using the analyzer shown in Figures 1 and 2, we analyzed the cardiac enzymes creatine kinase MB, myoglycan Testing of plasma samples for Robin and Troponin I can be performed as follows. . After removing blood cells from the blood sample, a defined amount of plasma is transferred to a liquid handling unit. 9 and evenly distributed to the four flow cells 10. Cardiac enzymes mentioned above are present When the cardiac enzymes flow through the flow cell, each sensor supports the appropriate ligand. will bind to known surfaces. This causes a change in the resonance angle, and this change will be proportional to the amount of cardiac enzyme bound to the surface as explained in . This results in The amount of each analyte in the sample can be determined.

To increase the resonance angle changes detected, the ligand-bound analyte is placed in a sandwich. It is preferable to react with the second reagent in a second assay method. As already mentioned above, Such second reagent may optionally be optically added to further increase the change. It may be preferable to label with a concentrated substance. Or use a third reagent. You can also do that. After the detection step, the sensor surface is treated with a regenerant such as glycine buffer, Or regenerate by passing phosphoric acid, formic acid or hydrochloric acid (10-100mM). all of Such an analytical procedure requires approximately 15 minutes.

Instead of using several flow cells as described above, all analytes of interest Using a single flow cell with both substance ligands co-immobilized on the same sensing surface area. can be used. This embodiment is also illustrated in the examples below.

If a patient suspected of having AMI has the above-mentioned biosensor device at the patient's bedside, was admitted to a coronary intensive care unit and based on symptoms and an electrocardiogram (ECG). If a safe diagnosis cannot be made, blood samples should be collected regularly, i.e. Samples are taken every 30 minutes and analyzed sequentially as described above. First sample tested has already been immediately diagnosed as A11l as a level of one or more of the analytes. Safe and reliable diagnosis or exclusion of AMI, even in cases where it is possible to Within about 2-3 hours in most cases based on the evolution of analyte levels over time. will be obtained. 8) If diagnosed as II, the patient should subsequently receive thrombolytic agents, etc. Thus, treatment with streptokinase or tPA can be performed. preferably This sensor unit test panel is suitable for testing streptokinase antibodies. Information on selecting appropriate thrombolytic agents and their dosages, including known surface areas. can be obtained at the same time, i.e. as a result the dose of streptokinase tP^ is selected rather than regulated or streptokinase.

Then, using the same bedside equipment and test panel, examine where reperfusion has occurred. treatment with thrombolytic agents should be monitored as soon as possible after the so that this inherently dangerous treatment is no longer needed, discontinued or repeated. Other treatments can be employed if perfusion cannot be obtained within a reasonable time.

Examples of analytes of interest, especially when monitoring treatment with such thrombolytic agents For example, a relatively high level of CK-MB may be obtained. Therefore, as mentioned above When using a second reagent, the kinetic range of the second response is determined by the specific analyte peak being monitored. may not be sufficient to allow expansion of the network value. That then shows reperfusion. The occurrence and accurate monitoring of peaks such as This can be carried out by measuring the formation of a complex with a regulated ligand.

Of course, the procedures and methods described above can be used to monitor the condition of muscles of interest during thoracic surgery. can be used to detect AMI that begins during surgery and to close the chest. It can be treated before it occurs.

The following non-limiting examples include commercially available SPH-based biosensor devices (BIA core　”’　) and sensing surface (5sensorChip　''　CM5　 ) (Both are Pharmacia Biosensor AB, Uppsal a.

CK- in plasma samples according to the invention using Detection of MB and myoglobin is described and a monoclonal specific to CK-MB Biosensor immobilization of antibodies and monoclonal antibodies specific to myoglobin The following was executed on the device.

HBS at pH 7.4 (10 mM Hepes buffer, 0.15 M NaCl 1,3,401 M EDTA, 0.05% Tween) constant The flow was maintained at 5 μm/min. Both carboxyl groups on the sensing surface are submerged in water. 0.2M 1-ethyl-3-(dimethylaminopropyl)carbodiimide hydrochloride< EDC> and a solution containing 0.05M N-hydroxysuccinimide (NFIS). Activate by injecting 35 μm of the solution into the device to form a reactive N-hydroxyscreen. forming a cinimide ester. Then 10mM sodium acetate at pH 5.0 CK-MB-specific monoclonal antibody (BiosP acific.

Emeryville, California, U.S.A.), and 50 Mg/111 monoclonal antibodies specific for myoglobin (Inst Itute of General and Molecular Patho Logy.

Tartu 5tate University, Tartu, Eston 35 μm of antibody solution (obtained from IA) was injected. p[ A buffer of I creates a positive net charge on the protein and at low ionic strengths The antibody is attracted to the remaining negative surface by electrostatic attraction giving a high antibody concentration in the matrix. will be preconcentrated into charged carboxyl groups. This preconcentration allows for small amounts of Allows fast immobilization in the body. The remaining reactive ester groups are 35 μm pi (g, Deactivated by injection of 5 IM of ethanolamine hydrochloric acid. Sen obtained The Sardaram is shown in Figure 3 (response in resonance units, RU plotted against time in seconds). lot). There are two levels of response signals: 20 seconds for EDC/NFIS Evaluations were made before (A) and 9 seconds after ethanolamine injection (B). Therefore B minor S A represents the immobilized amount of the two antibodies.

B. Analysis of plasma samples Using the sensing surface with co-immobilized antibodies prepared in section A above Analysis of elevated concentrations of CK-MB and myoglobin in plasma samples was performed as follows. Executed.

p) 17.4 HBS (1 hM Hepes buffer, 0, 15 Constant flow of M NaCl + nM EDTA, 0.05% Tree) The speed was maintained at 5 μm/min. Plasma sample 35 containing CK-11B and myoglobin μm was injected into the device. specific for CK-MB and myoglobin of 4 μm each. A different second antibody was added at a concentration of 100 u g/val to 4 μm at pH 2.5. QmM glycine-HCl was injected sequentially. The resulting sensor duram is shown in the figure. 4 (response in resonance units, Ru, plotted against time in seconds).

The response signal has four levels: 20 seconds before sample injection (A), specific to CK-MB; (B) 20 seconds before injection of second antibody specific for myoglobin. Evaluations were made 0 seconds before (C) and 20 seconds before glycine-HCl injection (D). Therefore A is baseline, B minus A is plasma response, and C minus B is CK - It is a response specific to MB, and D minus C is a response specific to myoglobin. That's the answer. Analysis time was 18 minutes.

The same procedure as above was then repeated without CK-MB and with normal myoglobin levels. The test was carried out on plasma samples. The obtained sensor duram is shown in FIG. From the drawing As can be seen, there was no response when a second antibody specific for CK-MB was injected. However, a weak response was observed with a second antibody specific to myoglobin. It was.

The invention is, of course, not limited to the embodiments specifically described above; Without departing from the scope of the general inventive concept as defined by the claims below. Many modifications and changes are possible.

FIG.2 Q Masako Niko Submission of translation of written amendment (Article 184-8 of the Patent Act) December 6, 1993

Claims (20)

[Claims] 1. (i) place the sample in a flow cell or cell, each specifically binding to each analyte; one or more supporting different ligands or mixtures of different ligands capable of combining. contacts more sensor surface area and, optionally, additional analyte After binding the substance-specific reagent or reagent complex to the bound apportioning substance, each analyte is A sensor surface that results in all binding interactions of a substance with its ligand First blood from the patient, blood by detecting as changes in the physicochemical characteristics of At least two different analytes indicative of myocardial infarction are present in the serum or plasma sample. measured at the time, (ii) regenerating each bound analyte from the ligand bound to its sensor surface; The liquid is removed by passing it through a flow cell, (iii) collecting step (i) from the patient at determined time intervals from said first sample; repeat for at least a second blood, serum or plasma sample, and (iv) Determine changes over time in the cardiac analyte from the results of steps (i) to (iii). Melt A method for measuring myocardial infarction markers consisting of stages. 2. Changes in the physicochemical characteristics of the sensor surface are measured using evanescent wave spectroscopy. 2. The method of claim 1, wherein the change in refractive index is determined. 3. Claim 2, wherein the evanescent wave spectroscopy is surface plasmon resonance spectroscopy. Method described. 4. The sample is in contact with multiple sensor surface areas, each of which has a different readout. 4. A method as claimed in claim 1, 2 or 3, in which the gun is supported. 5. The sample is in contact with a single sensor surface area and said areas are co-immobilized thereon. 4. The method according to claim 1, 2 or 3, wherein the method has different ligands. 6. Any one of claims 1 to 5, wherein at least three different cardiac analytes are measured. The method described in paragraph 1. 7. Additionally, streptokinase antibodies in the sample can be added to the sensor surface area or measured by binding to the corresponding ligand immobilized on the sensor surface area of 7. A method according to any one of claims 1 to 6, comprising: 8. Cardiac analytes include myoglobin, creatine kinase and its isoenzymes and its isomers, especially creatine kinase MB, myosin, especially its L chain, Pomyosin, lactate dehydrogenase (LD), aspartate aminotransferer and troponin C, T and I. or the method described in paragraph 1. 9. Cardiac analytes include creatine kinase MB, myoglobin and troponin I 9. The method according to claim 8, wherein the method is selected from: 10. The method according to claims 1 to 9, comprising measuring at least one control analyte. The method described in one or more of the above. 11. A claim in which myoglobin is measured and carbonic anhydrase is the control analyte. 10. The method described in 10. 12. Any one of claims 1 to 11 for diagnosing and/or excluding myocardial infarction. Use of the methods described. 13. Any one of claims 1 to 11 for monitoring treatment using thrombolytic agents. Use of the methods described. 14. Immobilized on one or more sensing regions individually or in combination A sensor consisting of at least two, preferably at least three different ligands a device in which each ligand specifically binds to a respective analyte indicative of myocardial infarction; The sensor device is capable of detecting any analyte-ligand interaction. Also adapted to detect as a result changes in the physicochemical characteristics of the sensing surface and the ligand-supporting surface area is exposed to the analyte after binding thereto. A sensor device that is capable of being regenerated. 15. Consisting of a number of sensing regions, each sensing region supporting a different ligand. The sensor device according to claim 14. 16. A claim in which a single sensing region has different ligands co-immobilized thereon. 15. The sensor device according to 14. 17. When the ligand is myoglobin, creatine kinase and its isoenzymes and Its isomers, especially creatine kinase MB, myosin, especially its L chain, tropomi Osin, lactate dehydrogenase (LD), aspartate aminotransferase, and cardiac enzymes or proteins selected from the group consisting of troponin C, T and I. the ligand is preferably selected from monoclonal antibodies against creatine Claim 14 selected from the group consisting of Naze MB, Myoglobin and Troponin I. 17. The sensor device according to any one of items 1 to 16. 18. Additionally, a ligand capable of binding streptokinase antibodies can be added to it. The sensor according to any one of claims 14 to 17, which is immobilized on the sensing region of Device. 19. Additionally, one or more ligands for at least one control analyte can be used. The cell according to any one of claims 14 to 18, wherein the cell is immobilized in a plurality of sensing regions. sensor device. 20. 20. A method according to claims 14 to 19 adapted for surface plasmon resonance spectroscopy. The sensor device according to any one of the above.