Enzyme immunosensor for diagnosis of myocardial infarction

ELSEVIER

Sensors and Actuators B 30 (1996) 71-76

Enzyme immunosensor for diagnosis of myocardial infarction Cordula Siegmann-Thoss a,b, Reinhard Renneberg ", Jan F.C. Glatz ~, Friedrich Spener ~.b.. i Institute of Chemical and Biochemical Sensor Research, Mendelstrafle 7, D-48149 Miinster, Germany b Department of Biochemistry, University ofMiinster, Wilhelra-Klemm-Strasse 2, D-48149 MOnster, Germany ¢ Cardiovascular Research Institute Maastricht, University ofLimburg, Universiteitsinge150, NL-6229 ER, Maastricht, Netherlands Received 8 February 1995; in revised form 20 April 1995; accepted 21 April 1995

Abstract

Amperometric enzyme immunosensors have been developed for the detection of the early myocardial infarction marker heart fatty acid binding protein (H-FABP) in blood plasma. They are based on anti-H-FABP antibodies immobilized on nitrocellulose or activated nylon membranes covering a modified Clark-type oxygen electrode. Two approaches have been tested using bovine H-FABP as a model: (i) the competition principle, where defined amounts of glucose oxidase (GOD)-labelled H-FABP compete with free H-FABP for binding to the immobilized capture antibodies; (ii) the sandwich principle, where the free H-FABP binds to the immobilized capture antibody and to a secondary GOD-labelled antibody, forming a sandwich. In both approaches, after addition of glucose, the activity of the enzyme label is determined with the amperometric electrode within minutes. The immunosensor using the sandwich principle on activated nylon membranes can be used repeatedly for at least 10 measurements after regeneration of the antibody membranes without loss of binding capacity of the capture antibodies. For the determination of human H-FABP, the sandwich principle with monoclonal anti-bovine-H-FABP antibodies as capture and GOD-labelled goat-anti-human-H-FABP antibodies as secondary antibodies is chosen. Initial experiments demonstrate that the sensitivity of the immunosensor is sufficient for the determination of clinical levels of H-FABP in plasma after myocardial infarction (100300 ng ml- 1). Keywords: Fatty acid binding protein; Amperometric enzyme immunosensor; Myocardial infarction diagnosis; Clark-type oxygen electrodes; Glucose oxidase; Immobilization

1. Introduction

1.1. Myocardial infarction diagnosis At present, routine clinical diagnosis of acute myocardial infarction (AMI) is based on three classical findings: (i) the clinical symptoms of A/VII; (ii) typical electrocardiographic changes; and (iii) elevation and decrease of serum enzyme activities. Unfortunately, these findings are not present or easily discerned in every AMI patient [ I ]. In a significant minority of patients acute myocardial infarction cannot be accurately diagnosed by clinical history and electrocardiographic changes alone [ 1 ]. Thus the release of proteins into plasma due to myocardial muscle damage can be used to assist in myocardial infarction diagnosis. Due to the damage of the myocytes, the cell membrane loses its integrity and the intracellular macromolecules diffuse first into the interstitium and subsequently into the intravascular space and lymphatic * Corresponding author. 0925-4005/96/$15.00 © 1996 Elsevier Science S.A. All rights reserved SSDIO925-4005(95)O1750-P

system. Their appearance in blood depends on their intracellular location, their molecular weight, the local blood and lymph flow and the rate of elimination through the kidneys [ 1]. Nowadays the enzymes creatine kinase (CK), lactate dehydrogenase (LDH) and their isoforms are used as potential AMI markers, but their increase in activity occurs only 4-8 h after the onset of myocardial infarction. Due to the development of sensitive and selective immunological determination techniques, non-enzymic proteins, such as troponin T, myoglobin or heart fatty acid binding protein (H-FABP) have gained in importance as A/VIImarkers. In contrast to CK and LDH, the levels of these proteins in plasma rise significantly above their threshold value only 2.5-3.5 h after the onset of the first clinical symptoms of AMI [2-5]. The concentration of troponin T, a structural protein (37 kDa) of the contractile apparatus, starts to increase within 3.5-4 h after the onset of the first symptoms [ 3 ]. Myoglobin, the oxygen-transporting protein of muscle cells, shows an increase in plasma concentration within the first 3 h after

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AMI [6]. Troponin T is of special interest because of the existence of a cardiospecific isoform, while myoglobin appears to be especially useful for the early diagnosis of AMI.

1.2. Fatty acid binding protein Heart-type fatty acid binding protein (H-FABP) belongs to a family ofintracellular 14-15 kDa proteins, which interact with hydrophobic ligands [7]. At least 10 members of this family have been identified and structurally characterized, each showing a specific pattern of tissue expression [ 8]. For example, H-FABP cannot only be found in the heart muscle, but also in skeletal muscle, kidney, mammary gland and brain. In human heart muscle the concentration of H-FABP is high, being 0.5 mg per g wet weight, which is equivalent to 10-20% of the cytoplasmic proteins [ 5 ]. After myocardial infarction H-FABP is released into the plasma of cardiac patients. The level is significantly elevated above its threshold level within 1.5-3 h after the first clinical symptoms of AMI. Glatz et al. showed that H-FABP, like myoglobin, is a sensitive marker, especially for the early assessment or exclusion of AMI and, because of its rapid excretion rate, a suitable marker for monitoring recurrent infarction [5]. Possible interferences due to skeletal muscle damage, from the presence of H-FABP in skeletal muscle tissue, can be assessed from the ratio of the plasma concentrations of myoglobin and H-FABP. Research data on the suitability of H-FABP as a plasma marker for AMI were obtained by Kleine et al. by using a non-competitive enzyme-linked immunosorbent assay of the antigen capture type (sandwich ELISA); however, this method required at least 4 h to be carried out [4]. The application of H-FABP as an early plasma marker in routine clinical practice ideally requires the availability of a rapid assay system. Such a potential system is outlined in this paper.

2.2. Buffers The following buffers were used. PBS: 0.01 M sodium phosphate, 0.154 M NaCI, pH 7.4. PBS-Tween: PBS with 0.05% (v/v) Tween 20. Coating buffer: 0.05 M sodium carbonate, pH 9.6. Blocking buffer: PBS with 4% (w/v) bovine serum albumin. Sample buffer: PBS-Tween with 1% (w/v) bovine serum albumin. Measurement buffer: PBS, pH 5.5. Regenerating buffer: 0.1 M glycine/HCl, pH 2.7.

2.3. Immobilization In this work the antibodies were coupled either by adsorption or covalently via their amino groups. To obtain information about the amount of membrane-bound antibodies, the BCA test (BCA = bicinchoninic acid) described by Smith et al. as modified by Redinbaugh and Turley was used [ 10,11 ].

2.3.1. Immobilization on nitrocellulose (0.45 txm pore size) Membrane discs with a diameter of 6 mm were incubated with 100/zl of a 5 /zg ml- ~ antibody solution (diluted in coating buffer) for 1 h at 37 °C. Afterwards the membranes were washed with PBS-Tween and remaining free binding sites on the membranes were blocked using BSA (200/zl blocking buffer). After washing with PBS-Tween, the membranes were ready to be mounted in front of the electrode or were stored in PBS at 4 °C. These membranes bound the antibodies by adsorption.

2.3.2. Immobilization on lmmunodyne (preactivated nylon) and filter papers (blue ribbon) The immobilization of the antibodies on these membranes followed the same protocol as that used for the adsorptive immobilization on nitrocellulose. The filter papers were preactivated with carbonyldiimidazol as described by Cha and Meyerhoff [ 12]. Both membrane types bound the antibodies covalently.

2. Experimental 2.4. Conjugation protocols 2.1. Materials Glucose oxidase from Aspergillus niger (EC 1.1.3.4) was obtained from Merck (Darmstadt, Germany). Nitrocellulose (0.45/.~m) from Schleicher and Schiill (Dassel, Germany), Immunodyne from Pall Biosupport (USA) and filter papers type 5893 (blue ribbon) from Schleicher and Schiill (Dassel, Germany) were used as membrane materials. Monoclonal anti-bovine-H-FABP antibodies were produced, and hybridomas were culture in the Institute of Chemical and Biochemical Sensor Research, Miinster, as described by Liddell and Cryer [9]. Polyclonal goat-anti-human-HFABP antibodies were produced by the Cardiovascular Research Institute, Maastricht, University of Limburg, and polyclonal rabbit-anti-bovine-H-FABP antibodies by the Institute of Chemical and Biochemical Sensor Research, Miinster.

The preparation of anti-H-FABP antibody-GOD conjugate follows the modified protocol described by Tijssen and Wilson and Nakane [ 13,14]. GOD was conjugated to bovine H-FABP according to the one-step glutaraldehyde method of Tijssen [ 13].

2.5. Assay procedures 2.5.1. Measurement with disposable membranes For single-use measurements nitrocellulose or Immunodyne membranes with immobilized antibodies were used. All incubation steps were carried out for 10 min at 37 °C with gentle shaking. In a first step after immobilization of the antibodies, any remaining free binding sites on the membrane were blocked with 200 /xl per membrane of the blocking buffer. Afterwards, the membranes were incubated with 100

C Siegmann-Thosset al./Sensors andActuatorsB 30 (1996) 71-76 /~1 human H-FABP standard solutions diluted in sample buffer over the concentration range 0 to 100 ng ml-~ or with 100 /~1 plasma sample diluted in sample buffer (dilution 1:10). Finally the membranes were incubated with 100/d of antibody-GOD conjugate (dilution 1:20, 1:30 or 1:50). After each incubation step the membranes were washed five times with 200 pl PBS-Tween, pH 7.4. 2.5.2. Electrode preparation and electrochemical procedures The electrode reaction is based on the reduction of oxygen at a platinum cathode. The platinum working electrode and Ag/AgCI anode were separated from the measuring solution by a gas-permeable Teflon membrane. Using a screw cap, the prepared membrane with the immuno-sandwich complex was placed in front of the electrode. The oxygen, consumed in the reaction of GOD, diffused through both membranes and was reduced at the working electrode at a potential of - 800 inV. The resulting current was proportional to the oxygen concentration, which in turn was proportional to the activity of the marker enzyme. Therefore in the case of the competition format the resulting current was inversely proportional to the antigen concentration, and in the case of the sandwich format directly proportional. After preparing the electrode, the measuring head was immersed into the measurement buffer (2 ml ). The measurement was carried out at room temperature with gentle stirring. The final substrate concentration was 150 mM, obtained by the addition of 100/~13 M/3-D-glucose in distilled H20. The substrate was added after measuring the blank (oxygen concentration of the measuring solution). Due to subsequent oxygen depletion at the electrode the current decreased to a saturation level, where the rate of oxygen diffusion from air balanced the rate of oxygen consumption. The curves were evaluated by measuring the initial slope. 2.5.3. Measurement with renewable membrane For these measurements all incubation steps took place directly on the membrane, which was fixed in front of the electrode. The whole procedure was exactly as described above. After one analysis, the measurement buffer was replaced by the regenerating buffer (0.1 M glycine/HCl, pH 2.7) for 1 min. A subsequent analysis could be carried out after washing the membrane once, followed by the incubation steps described above.

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on nitrocellulose or activated nylon membranes covering a modified Clark-type oxygen electrode. Using bovine H-FABP as a model, two approaches based On the classical enzyme immunoassay were tested: (i) the competition type, where defined amounts of GOD-labelled H-FABP compete with free H-FABP for binding to the immobilized capture antibodies; (ii) the sandwich type, where the free H-FABP binds to the immobilized capture antibodies and forms a sandwich with secondary GOD-labelled antibodies. In both approaches the resulting decrease in oxygen concentration after addition of glucose was determined using the amperometric electrode. To control the antigen-antibody interaction and to compare the enzyme conjugates with established methods, spectrophotometric ELISAs were carried out in parallel with all sensor measurements.

3.1. Selection of membrane for antibody immobilization

To compare immobilization or activation techniques with regard to the amount of bound protein, we chose the BCA test [ 10,11 ]. This assay is based on the reduction of Cu 2+ to Cu + by the protein (biuret reaction) in an alkaline environment. The Cu + formed builds an intense purple complex with the bicinchoninic acid reagent, which can be measured spectrophotometrically at 550 nm; thus the resolubilization of proteins bound to the membrane can be avoided. This comparison showed that nitrocellulose with 20% of the applied antibody concentration immobilized had the highest binding capacity, followed by Immunodyne with 9%. CDI-activated filter papers bound only 4% of the available antibodies (Fig. 1). The test, however, does not give any information on the binding capacity of the immobilized antibodies themselves. Also the test cannot be used for membranes containing groups that are able to react with the BCA reagent. 1,2

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3. Results and discussion Immunosensors which have not yet been applied for routine clinical analysis generally show the same high sensitivity and specificity as ELISA, but allow a rapid analysis (minutes rather than hours) while using relatively simple and userfriendly apparatus. In this work amperometric enzyme immunosensors have been developed for the detection of H-FABP in the blood plasma of cardiac patients. These sensors are based on monoclonal anti-H-FABP antibodies immobilized

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Fig. 1. Bindingof antibodiesby differentmembranes.Proteinwas determinedusingthe BCA test. Filledcircles,membraneincubatedwith 100/tg monoclonalanti-bovine-H-FABPantibodies(mAb) for 1 h at 37 °C; empty circles,calibrationcurveusingthe monoclonalantibodiesin solution.

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3.2. Testing the enzyme immunosensors

11

To find the optimal configuration and measuring technique with respect to the application of the immunosensor in clinical routine analysis, different parameters using bovine H-FABP as analyte were tested. A sensor calibration curve for bovine H-FABP using the competitive principle, nitrocellulose as membrane and GOD as the marker was found to be strictly linear within the range 0 to 100 ng H-FABP/nml buffer (Fig. 2). In principle, this curve reveals the possibility of attaining a measuring range for H-FABP relevant to clinical diagnosis. The dotted line in Fig. 2 shows the non-specific binding of the conjugate to the blocked membrane, an extremely important value for the proper functioning of immunosensors, because it limits the ultimate sensitivity of the measurement. For this reason and because of the difficult optimization of assays of the competitive type, we turned to the sandwich principle. Moreover the nitrocellulose membranes displayed a significant amount of leaching out of the non-covalently immobilized antibodies. Also taking into consideration the low binding capacity of filter papers for antibodies (Fig. 1 ), we selected the Immunodyne membrane for all further experiments. The sensor calibration for bovine H-FABP using the sandwich principle, the Immunodyne membrane and GOD as the marker is shown in Fig. 3. The curve is linear within the range 0-50 ng H-FABP/ml buffer. Due to the covalent coupling of antibodies to Immunodyne, regeneration of the antibody membrane became possible, indeed the use of glycine/HCl buffer, pH 2.7 allowed the rapid dissociation of the antibody / antigen/antibody-enzyme conjugate complex. To assess this regeneration and to examine leaching out, partial loss of binding capacity of the capture antibodies and accumulation of antigen or antibody-enzyme conjugate, we determined a given H-FABP concentration repeatedly. Fig. 4 shows the results of such determinations with 100 ng bovine H-FABP/ ml buffer (hatched bars). After each regeneration step and prior to the determination of the next H-FABP concentration, the membrane was measured without incubation with antigen I

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Fig. 4. Reproducibilityof bovine H-FABP determination with renewable sensormembrane.Immunodynemembraneincubatedwith 35/zgpolyclonal anti-bovine-H-FABPantibodies,blockingreagent4% BSA,dilution of polyclonalanti-bovine-H-FABPantibody--GODconjugate1:20in samplebuffer. Hatched bars, repeatedmeasurementof 100 ng bovineH-FABP/mlbuffer; black bars, control. and conjugate (black bars). The values obtained reflect the non-specific binding of the antibody-enzyme conjugate plus the incomplete regeneration of the antibody membrane. As can be seen in Fig. 4, the overall signal obtained initially increases with the number of regenerations; however, after the fourth regeneration, it scatters within a small mean. This initial increase can be ascribed to the increase in non-specific interactions, which are represented by the black bars. The mean of determinations 2-11 is 14.3 nA with a standard deviation of 0.67. After 10 determinations a decrease in the overall signal is observed, due to increasing denaturation of the capture antibodies brought about by the regeneration process (data not shown). Taking into account an acceptable relative error of + 10%, this renewable immunosensor can thus be used for 10 measurements. The sensitivity of this immunosensor permitted the determination of H-FABP in the concentration range 5-100 ng m l - ~. This allows the increase in human H-FABP concentration to be monitored in blood plasma of cardiac patients, for

C. Siegmann-Thoss et al. /Sensors and Actuators B 30 (1996) 71-76

which maximum levels of the order of 300 ng H-FABP/ml have been reported [4].

3.3. Application of the enzyme immunosensor in the diagnosis of acute myocardial infarction Based on the results reported above, we preferred the sandwich principle for the determination of human H-FABP by using monoclonal anti-bovine-H-FABP antibodies as capture and GOD-labelled polyclonal goat-anti-human-H-FABP antibodies as detector. The monoclonal antibodies were immobilized on the Immunodyne membrane, which was mounted in front of the Clark-type oxygen electrode. Thus the sensor was operated with single use of membranes. In clinical application the regeneration technique cannot be used anyway, due to the requirement for hourly blood measurements over a period of at least 24 h. To demonstrate the reproducibility and to determine the relative error of each sensor measurement using disposable membranes, we measured a given H-FABP concentration ( 10

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ng ml- i ) repeatedly (Fig. 5). The mean of 10 determinations is 2.66 nA rain- 1 with a standard deviation of 0.31. Thus the relative error is + 11%, which should be taken into account in the interpretation of the results shown below. The calibration curve was linear (r=0.98) within the range 5-80 ng H-FABP/ml buffer (Fig. 6) with a standard deviation of about 10%. Recovery experiments using normal plasma spiked with purified human H-FABP yielded an average recovery of 93 + 5%, which compared well with the recovery of the sandwich ELISA (93.5%) [ 15 ]. Thus, with the enzyme immunosensor developed here the measurement of human H-FABP in plasma samples of cardiac patients becomes possible. The sensor allows the use of pre-prepared membranes that are obtained after immobilization of the capture antibodies and saturation of free binding sites on the Immunodyne membrane. The use of this disposable membrane requires 27 rain per sample (Fig. 7), a considerable improvement in comparison to ELISA measurements. Fig. 8 shows data for the release of H-FABP into the plasma of a cardiac patient with diagnosed anteroseptal myocardial infarction and a normal kidney function measured with the immunosensor. The results of the sandwich ELISA for HAb-GOD- glucose conjugate [ measurement H-FABP b'uffer~z~

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Fig. 8. Individual time-content curve of human H-FABP in plasma after acute myocardial infarction, sensor measurement. Plasma of acardiac patient with diagnosed anteroseptal myocardial infarction. The insert shows the results of the sandwich ELISA. Immunodyne membranes each incubated with 300 ng monocional anti-bovine-H-PABPantibodies, blocking reagent 4% BSA, plasma dilution 1:20 in sample buffer, dilution of anti-human-HFABP antibody-GOD conjugate 1:30 in sample buffer.

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FABP in the same plasma samples carded out two years earlier using polyclonal capture antibodies and polyclonal antibody--peroxidase conjugate are given in the insert of Fig. 8 for qualitative comparison. Due to the detection limit of the sensor measurement (5 ng ml - t ) and the relative error ( + 11%) the background is higher, but taking the changes occurring during long-term storage of the plasma into account, the results of the sensor and the ELISA compare well. Moreover, the comparison of both techniques reveals that the sensor allows a five times faster determination of HFABP and opens the possibility of bed-side determinations of human H-FABP for the early diagnosis of AMI.

References [ 1] A.K. Ellis, Serum protein measurements and the diagnosis of acute myocardial infarction, Circulation, 83 ( 1991) 1107-1109. [2 ] H.A. Katus, A. Remppis,F.J. Neumann,T. Scheffold,K.W.Diederich, G. Vinar, A. Noe, G. Matern and W. Kuebler, Diagnosticefficiencyof troponin T measurements in acute myocardialinfarction, Circulation, 83 (1991) 902-912. [3] J. Malr, E. Artner-Dworzak, P. I.eehleitner, J. Smidt, I. Wagner, F. Dienstl and B. Puschendorf,Cardiac troponin T in diagnosis of acute myocardialinfarction, Clin. Chem., 37 ( 1991) 845-852. [4] A.H. Kleine, J.F.C. Glatz, F.A. Van Nieuwenhoven and G.J. Van der Vusse, Release of heart fatty acid-binding protein into plasma after acute myocardialinfarction in man, Mol. Cell. Biochem., 116 (1992) 155-162. [5 ] J;F.C.Glatz, A.H. Kleine, F.A. Van Nieuwenhoven,M.P. Van DieijenVisser, W.Th. Hermens and G.J. Van der Vusse, Fatty acid-binding protein and myoglobinas plasma markers for the early assessment of acute myocardial infarction in man, Tijdschr. Ned. Ver. Klin. Chem., 18 (1993) 144-150. [6] W.S. Kilpatrick, D. Wosorn, J.B. McGuinnessand A.C.A.Glen, Early diagnosis of acute myocardial infarction: CK-MB and myoglobin compared,Ann. Clin. Biochert, 30 (1993) 435--438. [7] C. Unterberg, T. Btirchers, P. Hcjrup, P. Roepstorff, J. Knudsen and F. Spener, Cardiac fatty acid-binding proteins, l Biol. Chert, 256 (1990) 16255-16261. [8] T. B~rehers and F. Spener, Fatty acid binding proteins, Curr. Top. Membr., 40 (1994) 261-294. [ 9 ] J.B.Liddell and A. Cryer,A Practical Guide to MonoclonalAn tibodies, John Wiley, Chicester, UK, 1991. [ 10] P.K. Smith, R.I. Krohn, G.T. Hermanson, A.K. Malla, F.H. Gartner, M.D. Provenzano,N.M. Fujimoto, N.M. Goeke, B.J. Olson and D.C. Klenk, Measurement of protein using bicinchoninic acid, Anal, Biochem., 150 (1985) 76-85. [ 11] M.G. Redinbaugh and R.B. Turley, Adaptation of bicinchoninic acid protein assay for use with microtiter plates and sucrose gradient fractions, Anal, Biochem., 153 (1986) 267-271.

[ 12] G.S. Cha and M.E. Meyerhoff, Potentiornetricion- and bio-selective electrodesbased on asymmetriccelluloseacetate membranes, Talanta, 36 (1989) 271-278. [13] P. Tijssen, Practice and Theory of Enzyme lmmunoassays, Elsevier, Amsterdam, 1985, [ 14] M.B. Wilson and P.K. Nakane, lmmunofluorescence and Related Techniques, Elsevier, Amsterdam, 1978. [ 15] A.P. Kleine, Fatty acid-binding protein as diagnostic marker of acute myocardialinfarctionin man, Ph.D. Thesis, Universityof Maastricht, 1993.

Biographies Cordula Siegmann-Thoss studied chemistry at the University of Mtinster, Germany, and received her Ph.D. there under the supervision of F. Spener. In 1993 she joined the Institute of Chemical and Biochemical Sensor Research (ICB), where she is also responsible for public relations. This paper is part of her Ph.D, thesis. Reinhard Renneberg received his Ph.D. in biochemistry in 1979 from the Central Institute of Molecular Biology (biosensor group) in Berlin-Buch, Germany. He was involved in the development of enzyme sensors for glucose, of microbial sensors and immunosensors. In 1991 he moved to the ICB, where he became head of the Department of Immunosensors. Jan F.C. Glatz received his Ph.D. in biochemistry in 1983 from the University of Nijmegen, Netherlands, on the basis of a thesis on fatty acid oxidation in skeletal and cardiac muscle. At the Cardiovascular Research Institute Maastricht he is currently involved in research on lipid metabolism in the nomoxic and ischaemic heart, with special reference to the role of cytoplasmic fatty acid binding proteins, and in research on the application of fatty acid binding protein as a diagnostic marker of cardiac muscle injury. In 1990 he became an Established Investigator for the Netherlands Heart Foundation. Friedrich Spener received his Ph.D. in chemistry and biology from the University of Graz, Austria. After postdoctoral research at the University of Minnesota, Austin, USA, he worked as section leader at the Federal Centre for Lipid Research in Mtinster, Germany. In 1974 he moved to the Department of Biochemistry, University of MUnster, Germany, where he became professor of biochemistry. In 1991 he additionally became affiliated with the ICB and is director of the Division Biotechnology and Biomedical Technology. His current research interest is mainly in the fields of lipid protein interaction, enzyme technology and biosensor development.