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CLIN. CHEM.

38/3,

386-393 (1992)

Development and In Vitro Characterization of a New Immunoassay of Cardiac Troponin T Hugo A. Katus,’ Siegfried Looser,2 Klaus Hallermayer,2 Borgya,2 Ulrich Essig,2 and Ursula GeuB2 We describe a new one-step enzyme immunoassay of troponin T that uses two specific monoclonal antibodies and streptavidin-coated tubes as the solid phase. The monoclonal antibodies were obtained by conventional hybridoma technology, with human troponin T as antigen. The identity of the cardiac troponin T antigen was confirmed by analysis of the amino acid composition and by partial sequence analysis. The specificityof the monoclonal antibodies for cardiac troponin T was proved by immunoblot analysis and displacement curves. The capture antibody is labeled with biotin. The second antibody is conjugated to horseradish peroxidase (EC 1.11.1.7). The assay [EnzymunTestR system (Boehringer Mannheim GmbH)] is performed in only 90 mm at room temperature; the measuring range for troponin T is 0.1 to 15 g/L. The assay shows excellent between-run precision (CV = 3.3-4.9%). AdditionalKeyphrases:enzyme immunoassay dial infarction forms

biotin-streptavidin

interaction

acute myocarprotein iso-

Measurements of above-normal concentrations of creatine kinase (CK; EC 2.7.3.2) or isoenzyme CK-MB and lactate dehydrogenase (EC 1.1.1.27) in serum are wellestablished indicators for the diagnosis of acute myocardial infarction (AMI).3 Frequently, however, these enzymes are not sufficiently specific and sensitive for accurate diagnosis (1-5). Because of these limitations, an easily measured biochemical marker that is found only in heart muscle and that is consistently released

into serum from severely damaged myocardial cells would be of great value for the detection of myocardial infarction.

Troponin T is the tropomyosin-binding protein of the troponin regulatory complex located on the thin filament of the contractile apparatus. Troponin T exists in three isotype forms-in fast and slow skeletal muscle and in cardiac muscle. These muscle-specific troponin T isoforms are the products of different genes (6, 7). The antigens encoded by these genes reveal residues having high homology of sequences (90%) but also significantly

‘Innere Medizin III, University of Heidelberg, Bergheimer 58, 6900 Heidelberg, F.R.G. 2Boehringer Mannheim GmbH, D-8132 Tutzing, F.R.G.

Str.

3Nonstandard abbreviations: CK, creatine kinase; CK-MB, creatine kinase isoenzyme MB; AMI, acute myocardial infarction; ELISA, enzyme-linked immunosorbent assay; SDS, sodium dodecyl sulfate; and ABTS, 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonate). Received September 17, 1990; accepted January 14, 1992.

386 CLINICALCHEMISTRY,Vol.38, No.3, 1992

Andrew

Remppis,’

Thomas

Scheffold,’

Anneliese

different [homology only 11% (8)1. Further diversity of troponin T structure results from differential RNA processing in the respective muscle type (9-12). These isoforms of fast or slow skeletal muscle troponin T or cardiac muscle troponin T may differ by six to 11 amino acid residues (8, 13). In cardiac muscle of adult rat or chicken, only one troponin T isoform is expressed, whereas two isoforms are found in bovine cardiac muscle (9, 14-16). Wilkinson and Taylor (14) reported the expression of only one troponin T isoform in rabbit myocardium, whereas Anderson and Oakeley (17) identified at least four isoforms. In immunological analysis of human myocardium, Anderson et al. (18) reported

multiple

isoforms of cardiac troponin

T; our group,

however,

using the same techniques, found only one isoform in myocardial tissue of normal and diseased myocardium (19,20). Despite the unresolved issue of the presence or absence of polymorphic isoforms of troponin T in human myocardium, the pronounced divergence of amino acid composition between cardiac and skeletal troponin T allows the differentiation of these molecules by immunological techniques and the development of a cardio-specific test system. Recently, we described an enzyme immunoassay (ELISA) to detect troponin Tin human serum (21). In this one-step sandwich assay, the troponin T antigen was bound to the solid phase by an affinity-purified polyclonal antibody from sheep and detected by peroxidaselabeled monoclonal antibody. Now we have developed a

much more sensitive tropomn is based

T-specific

enzyme immunoassay,

monoclonal

antibodies.

using two The method

on the one-step sandwich assay principle, with streptavidin-coated polystyrene tubes as the solid phase (Figure

1).

Materials and Methods Materials Dianuno-benzidine, Tween 20, and dithiothreitol were from Serva, Heidelberg, F.R.G.; (3-mercaptoethanol and sodium perborate were from Merck, Darmstadt, F.R.G.; and all other chemicals,

immunoreagents,

and streptavidin-coated tubes were from Boehringer Mannheim GmbH, Mannheim, F.R.G. Reagents for troponin T assay. The troponin T assay kit contains 100 streptavidin-coated plastic tubes (binding capacity >14 ng of biotin), incubation buffer (per liter, 10 mmol of citrate and 47 mmol of phosphate, pH 6.3), biotinylated anti-troponin T antibody (1.25 mg/L), anti-troponin T antibody labeled with horseradish peroxidase (>40 U/L), substrate buffer (100 mmol/L phos-

Washkig

solulion

solution

I

___

1)-EJ

-)-E

[

Teste#{174}-systemES 22 and ES 600 analyzers Mannheim GmbH).

]

Preparatioh of Troponin T

>-uI

t) s_

Both human

cardiac and skeletal muscle troponin T from and bovine tissue were prepared by general procedures (24-26). The molecular homogeneity of the purified antigens was assessed by SDS-polyacrylamide gel electrophoresis (27,28).

-

4 thro.ncgen

To determine Absottanc.

solution

{ ABTS [

ofcOlouc

Me.ewe,n.nh/evaluatlon

3

lluponln-T b1olln4atsd

wnpls .ntl-1cgonln-T

antibodies

)-E

1o1,onin-T-antIbody-POO

]

Streptavldm

conjugate

Fig. 1. Test principle for troponin T ELISAone-step sandwich assay with biotin-streptavidin technology POD,peroxidase

phate-citrate buffer, pH 4.4, containing sodium perborate, 3.2 mmollL), substrate [2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonate), ABTS#{174}; 1.9 mmol/L], and six standards of bovine cardiac troponin T (0-15 g/L) in human serum matrix. The purity of the troponin T preparation used as standard material was assessed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (Figure 2). The capture antibody (M7) was biotinylated according to the method of Peters and Baumgarten (22) by using biotin cross-linked with N-hydroxysuccinimide. Horseradish peroxidase was coupled to the second antibody as reported by Wilson and Nakane (23). Apparatus.

The assay was performed

with Enzymun-

170,000 97,400

-

55,400

-

20,100

-

the tropomn

T-specific absorptivity

at

280 nm, we dialyzed purified bovine cardiac troponin T against three changes of 1 mmol/L acetic acid for 27 h at room temperature and then against four changes of doubly distilled water for 48 h. After lyophilization, we dissolved 1 mg of powdered tropomn T in 1 mL of blank buffer (30 mmolfL phosphate, pH 7.3, containing 2 mol of urea, 0.6 mol of KC1, 0.25 mmol of dithiothreitol, and 2 g of sodium azide per liter). The specific absorptivity at 280 nm was 0.706 L g1 cm1.

-

>-:i

(Boehringer

12 Fig. 2. SDS-gel electrophoresis of the bovine cardiac troponin T preparation used as standard material Lane 2:40 rig of bovine cardiac troponin T was analyzed in 8-25% gradient SDS-Phastgel’ under reducing conditions according to the Phastsystemti instruction manual (26). Lane 1: Horse a-macroglobulin (170 kDa), rabbit muscle phosphorylase b (97 kDa), bovine liver glutamate dehydrogenase (55 kDa), porcine muscle lactate dehydrogenase (36 kDa),and soybean trypsin inhibitor(20 kDa) were used as molecular mass markers. The gel was silver-stainedas described in the developmentaltechniqueFileNo. 210 of the Phastsystem instruction manual (26)

Identificationof Cardiac TroponinT Analysis of amino acid composition: Gas-phase hydrolysis was performed with an Applied Biosystems (Ramsey, NJ) amino acid analyzer, Model 420A, with on-line analysis of phenylthiocarbamyl-derivatized amino acids as described in the user bulletin (29). The hydrolyzed samples were loaded into the analyzer and processed, and the tryptophane residues were determined by fluorescence measurements according to Pajot (30). Analysis of amino-terminal sequence: N-terminal sequence analysis was performed by automated Edman degradation, performed with an Applied Biosystems protein sequencer, Model 477A, with on-line analysis of phenylthiohydantoin-derivatized amino acids. CNBr-digestion: Bovine cardiac troponin T solution dialyzed against acetic acid (100 mL/L) was dried by centrifugation under reduced pressure and redissolved in 700 mL’L acetic acid. After pretreatment with 13-mercaptoethanol, CNBr was added to the troponin T solution to a final 500-fold molar excess over methionine

residues. The sample was flushed with nitrogen, sealed, and incubated in the dark at 25 #{176}C for 24 h. The digestion was stopped by dilution with water and stored at 4#{176}C. To remove the cleavage reagent and to separate the cleavage products, we fractionated the digest by using linear gradient elution from a 4.6 mm x 100 Aquapore#{174} RP 300 reversed-phase column (Applied Biosystems, Ramsey, NJ). The following solvent system was used: Solvent A, 1 mL of trifluoroacetic acid per liter of water; Solvent B, 1 mL of trifluoroacetic acid per liter of acetomtrile/water (70/30, by vol). The flow rate was 0.2 mL/min. Absorbance was monitored at 220 nm. Developmentof Monoclonal Antibodies Monoclonal antibodies reactive with human cardiac T were produced by hybridoma formed from P3X 63-Ag8-653 myeloma cells and splenocytes of Balb-C mice (31) immunized with four intraperitoneal injections (100, 50, 50, and 50 .tg) of human cardiac troponin T in Freund’s adjuvant. We screened the cell troponin

CLINICALCHEMISTRY,Vol.38, No. 3, 1992 387

supernates with the ELISA and cloned the positive cells, using a cell sorter (FACS; Becton Dickinson, Heidelberg, F.R.G.); the resulting clone cultures were screened again with the ELISA. Antigen-specific clones were grown in culture, cryopreserved, or injected into the peritoneal cavity of pristane-primed Balb-C mice to produce

ascites fluid. for screening hybridoma ture supernate: In this screening ELISA

antibodies in cell culassay, several washes

with phosphate-buffered saline are performed after each incubation step. First, add affinity-purified cardio-specific polyclonal troponin T antibodies (10 mgfL) to microtiter plates (Nunc, Wiesbaden, F.R.G.). Next, block the remaining protein-binding sites on the microtiter plate by incubation with bovine serum albumin, 10 g/L. Thereafter, incubate human cardiac troponin T for 1 h at room temperature so that it binds to the solid-phase polyclonal antibodies. Add cell culture supernate, incubate

for 1 h, then

check

for binding

of hybridoma

antibodies to the immobilized antibody-troponin T complex by adding a peroxidase-conjugated anti-mouse immunoglobulin Fab fragment (Boehringer Mannheim, 25 UIL). Incubate for another hour at 25 #{176}C, then measure the peroxidase bound to the microtiter plate by adding 100 pL of ABTS substrate (pH 5.0). Measure the resulting color change at 405 nm with an ELISA reader (e.g., Dynatec) after 30 mm at room temperature. Characterization of antibodies by ELISA: The reactivity of selected monoclonal antibodies diac troponin T and the cross-reactivity nal antibodies with human skeletal

with human

car-

of the monoclotropomn

T were

assessed by using the same general test principle as outlined above. To determine the affinity of the hybridoma antibody in cell culture supernate, we modified the test system as follows. First, we incubated less cardiac troponin T (0.4 mg/L) in microtiter wells coated with cardio-specific sheep anti-troponin T antibody. Next, the antibody under investigation was added, with increasing concentrations of cardiac troponin T (50 L, 0 to 3.2 mg/L), to the coated wells. Cardiac tropomn T added in solution competes with the cardiac troponin T immobilized by the polyclonal sheep antibodies for binding to the liquid-phase monoclonal antibody. The amount of free antigen necessary for half-maximal displacement of the monoclonal antibody under investigation is a measure for the affinity of this antibody. To determine the cross-reactivity of the hybridoma antibody in cell supernate, instead of adding increasing amounts of cardiac troponin T we added the skeletal troponin T isoform in increasing concentrations from 7.8 to 30 &g/L.Cross-reactivities of the monoclonal antibodies were calculated by comparing the amounts of human cardiac (C) and skeletal muscle (SM) troponin T necessary for half-maximal displacement of the monoclonal antibody: Cross-reactivity = [concn C (50% intercept)! concn SM (50% intercept)] x 100%. Characterization of antibodies by Western blot: Crude myofibrillar proteins and purified troponin T from dif388 CLINICALCHEMISTRY,Vol.38, No.3, 1992

ferent muscles

and animals

were analyzed

by using 12%

SDS-polyacrylamide gel electrophoresis according to Laemmli (27) or the Phastsystem (Pharmacia, Uppsala, Sweden) (28). To assess the reactivity of monoclonal antibodies with multiple troponin T isoforms ent muscles, we used a 9% polyacrylamide gel. were stained by incubation with AgNO3 (1 formaldehyde (15 mmol/L) for 15 mm, followed

in differ-

The gels gIL) and

by incubation with Na2CO3, 25 gIL, in distilled water. Immunoblot analysis

was performed

essentially

as outlined

by

Towbin et al. (32). Electrotransfer of the proteins from the SDS-gel to cellulose membranes (Immunobilon-Pmembrane; Millipore Corp., Waltham, MA) was achieved by use of 100 mA and 50 V for 25 mm at 4#{176}C. The troponin T antigen was identified by incubating the cellulose membranes for 30 mm at room temperature in phosphate-buffered saline, pH 7.4, containing 2 pg of anti-troponin T antibody and 50 g of bovine serum albumin per liter after the membranes had been blocked with 10 g/L albumin solution. After several washings of the cellulose membrane with phosphate-buffered saline containing 10 g of bovine serum albumin and 10 mL of Tween 20 per liter, the bound anti-troponin T antibody was reacted for 30 mm at room temperature with 4000-fold-diluted sheep anti-mouse IgG-Fc antibody labeled with horseradish peroxidase. The unbound antibody-peroxidase complex was then removed by washing, the immobilized peroxidase was identified by mea-

swing the conversion of substrate (diaminobenzidine, 50 gIL) in the presence of H202 (3 mL/L) for 3 miii at room temperature. Purified human cardiac troponin T and proteins of known molecular mass were used as controls. Assay Procedure Serum samples and standards (200 pL) in duplicate were incubated with conjugate solution (1000 zL) in the streptavidin-coated tubes for 1 h at room temperature.

The tube contents were aspirated and the tubes were rinsed three times with tap water. The substrate solution (1000 iL) was added to the tubes and incubated for 0.5 h at room temperature. Absorbances were read at 405 nm, and troponin T values were calculated from the calibration curve. A new calibration curve was constructed for each assay. Specificity of the Troponin T Assay The assay specificity was tested against known amounts of tropomn T purified from human cardiac and skeletal muscle. The antibodies were used in two arrangements: (a) cardio-specific antibody M7 as capture antibody and antibody 1B1O as labeled antibody (as in the assay kit); (b) antibody 1B1O as capture antibody and cardio-specific antibody M7 as labeled antibody (switched

antibodies).

The nonspecific

binding

of puri-

fied troponin T from human cardiac and skeletal muscle to the streptavidin-coated test tubes was assessed by adding to the test tube only labeled antibody but no anti-troponin To assess

T capture

antibody.

the nonspecific binding of troponin T in serum, we added 200 pL of serum from patients with

AM! or skeletal muscle damage to the test tubes conlabeled antibody but no capture antibody.

taining

Patients Serum specimens were obtained from 15 patients with AM! and 14 patients with skeletal muscle damage. Diagnostic criteria for AM! were typical electrocardiographic changes (development of new Q-waves and reduction of R-waves) and anginal pain. Patients were classified as having skeletal muscle damage if (a) there was clinical evidence of skeletal muscle trauma, (b) there were no or only minor ST-segment or T-wave changes on the electrocardiogram, and (c) the CKMB/CK ratio was