0161-5890/92 $5.00 + 0.00 Pergamon Press plc

Molecular Immunology, Vol. 29, No. 2, pp. 271-218, 1992 Printed in Great Britain.

NEW MONOCLONAL ANTIBODIES AS PROBES FOR HUMAN CARDIAC TROPONIN I: EPITOPIC ANALYSIS WITH SYNTHETIC PEPTIDES CATHERINE LARUE,* HBL~~ DEFACQUE-LACQUEMENT,*CHARLES CALZOLARI,* DUNG LE Ncmmt and BERNARD PAU$ *Sanofi

371 rue du Pr. J. Blayac, 34184 Montpellier Cedex 04, France; C.N.R.S.-I.N.S.E.R.M. de Pharmacologic, Montpellier, France and $FacultC de Pharmacie, Montpellier, France

Recherche,

jCentre

(First received 23 March

1991; accepted in revised form 24 April 1991)

monoclonal antibodies (MAbs) specific for human cardiac troponin I (TnI) were selected to develop a new alternative for specific biological diagnosis of acute myocardial infarction. Using an immunoenzymatic sandwich assay, these MAbs were employed in the mapping of human cardiac TnI and showed six different epitopes. Parts of the TnI peptide sequences were synthesised; the sequences were chosen from the published sequences of mammalian TnI. Immunological assays showed that 8 out of 40 MAbs recognised a RAYATEPHAK (P2) N-terminus cardiac-specific sequence of human TnI. The information obtained from epitopic mapping of TnI and the properties of the peptides allowed pairs of MAbs to be selected for the development of a future specific TnI assay. Abstract-Forty

INTRODUCTION

Because of the high incidence and severity of acute myocardial infarction (AMI), diagnosis must be rapid and accurate. In 1 case out of 4, an electrocardiogram is not sufficient for the diagnosis, particularly in the case of small infarcts (Levy et al. 1987). Assays of plasma parameters are thus essential to confirm clinical hypothesis. The time to appearance of these molecules in the bloodstream seems to be correlated with their mol. wt, half-life and site of storage. Assays of serum enzymes (LDH, C&Z-Mb, GOT) and sometimes of myoglobin are widely performed in the early phase of AM1 (Rotenberg et al., 1989; Vaidya et al., 1988; Thompson et al., 1988). In spite of the relatively high diagnostic specificity of certain enzyme assays (Lee and Goldman, 1986), the frequent serial measurements required in the very early hours after the onset of chest pain and nonspecific pathological variations (Levy, 1987; Cummins et al., 1987a) could present some inconveniences for an absolute specific diagnosis. Our aim was to identify an accurate cardiac-specific biological parameter detectable in serum very early after AMI. Different contractile proteins were possible candidates as they are not normally found in the blood. Different immunoassays using polyclonal or monoclonal antibodies (MAbs), prepared from various cardiac contractile proteins [tropomyosin (Cummins et al., 1981), troponin I (Cummins et al., 19876), light (Katus et al., 1988) and heavy (Leger et al., 1990; Larue et al., 1991) chains of myosin] have been employed in the past. Troponin I (TnI) was chosen for its cardiac specificity. TnI is the inhibitory subunit of troponin, the thin

filament regulatory complex which confers calcium sensitivity to striated muscle actomyosin ATPase activity. Three isoforms of TnI have been identified (Wilkinson and Grand, 1978): two skeletal TnI (fast and slow) isoforms (MW = 19,800 daltons) and a cardiac TnI isoform with an extra 30 residues on the N-terminus resulting in a mol. wt of 22,500 daltons. This isoform has been shown to be released after acute myocardial infarction (AMI) (Cummins et al., 1987b; Cummins and Cummins, 1987). Their assay was based upon the use of polyclonal antibodies against human TnI, but because of the method’s poor sensitivity, could not differentiate between small quantities of TnI (observed in small infarcts) and normal concentrations. In addition, a slight cross-reactivity with skeletal forms was observed. In order to improve both the sensitivity and specificity of tests using this new parameter, we obtained high affinity monoclonal antibodies against human TnI. Several TnI cardiac sequences were synthesised in order to identify cardiac-specific peptide sequences of human TnI and to localise the epitopes recognised by MAbs. Our strategy was mainly supported by the sequence of bovine cardiac TnI (Leszyk et al., 1988) and on the following main criteria: the differences in sequence between bovine cardiac and skeletal forms already known (Fig. 1); sequence homology between rabbit and bovine cardiac forms; an antigenicity index above 1.4 (according to Chou and Fasman); the hypothesis that the human and bovine sequences could be identical. Five peptides were synthesised on the basis of this strategy. The addition of an amide function at the C-terminus of all peptides rendered them peptidic moiety-like. Five analogues of these peptides were also 271

212

CATHERINELARUEet

al.

Avian Fast Sk

MSDEEKKR

Rabbit Fast Sk

GDEEKRN

Rabbit Slow Sk

PEVERK

8 7 6

Rabbit Card

ADESRDA-AGEARPAPA-VRR-S--D-RAYATEPHAKSK

33

Bovine Card

ADRSGGSTAGDTVIPAPPPVRRRSSAINYjRAYATEPHAKlKK

39

_Pl

-NY-P2‘

_PlU

--P2-

Avian Fast Sk Rabbit Fast Sk Rabbit Slow Sk Rabbit Card Bovine Card

G RA C S

76..N..

-p3Avian Fast Sk

I I

..I40

Rabbit Fast Sk . 139 Rabbit Slow Sk ..138

E

166..//..

E

162..//..

V E

163..ll..

Rabbit Card

..167

I D

191..//..

Bovine Card

..173

I

197Jl..

4

P4

D

)

Fig. 1. Alignment of TnI amino acid sequences and synthetic peptides (P, , P,, P,, P, and P, NYP, A dash indicates a deletion introduced to maximise sequence similarity. Residues which are identical in all five sequences are boxed. (Sk: skeletal form; card: cardiac form). The three amino acids shown by bold characters in P, bovine sequence were different in human sequence: P-A; V+I; A+-.

synthesised with the addition of a cystein (Cys) residue and a aminohexanoyl (Ahx) spacer at the N-terminus of each. This “Cys-Ahx” spacer allowed linkage, through this terminus (via the SH group), to a carrier protein for “oriented” binding to enzymes or to solid phases. Immunological recognition (or interaction) of these peptides and their analogues with our MAbs was investigated through immunological assays. MATERIALS Preparation

AND METHODS

of TnI

Human and bovine cardiac and skeletal tissue was frozen immediately after excision in liquid nitrogen and stored at -80°C until used. Cardiac and skeletal TnI were prepared according to the affinity chromatographic method using immobilised troponin C (TnC) (Syska et al., 1974), relying upon the physiological properties of TnC-TnI binding in the presence of calcium. TnC was obtained from fresh or deep-frozen bovine cardiac muscle, purified according to Thulin and Vogel (1988), and coupled to sepharose-4B by means of cyanogen bromide (Cummins et al., 1987b). Human TnI was eluted as a single peak on addition of 10 mmol/l EGTA (Cummins et al., 1987b). After dialysis against 0.5 mol/l NaCl, 20 mmol/l Tris-HCl, pH 7.5,60 mmol/l2-mercaptoethanol, TnI was stored at -20°C until needed. Protein concentrations were determined at 280 nm with the use of E 1%/l cm and gave values of 4.2 and 4.0 for cardiac and skeletal TnI, respectively. Polyacrylamide

gel-electrophoresis

One dimensional polyacrylamide gel electrophoresis in the presence of 0.1% (w/v) sodium dodecyl sulfate

(SDS), 0.1 mol/l K/K, PO, buffer, pH 7.0 was carried out by Phastsystem (Pharmacia) using 4-l 5% (w/v) gradient polyacrylamide gels. Preparation

of monoclonal

antibodies

Zmmunisation. High-responder Biozzi mice (Boumsell and Bernard, 1980) received a subcutaneous injection of 50 pg purified human cardiac TnI in complete Freund’s adjuvant (v/v). Two further injections were given under the same conditions at 6-week intervals. The final 50 pg injection of TnI was given intraperitoneally 3 days before fusion. Fusion and cellproduction. Spleen cells were fused with a myeloma P3-X63-Ag8 653 line using a method adapted from Di Pauli and Raschke (1978). Anti-troponin antibodies in the mouse serum, culture supernatants, ascites or purified IgG fractions were detected by their binding to pure human cardiac TnI, in parallel with skeletal TnI. Antibody-producing hybridomas were subcloned, then frozen in liquid nitrogen. Ascites were produced following intraperitoneal injection of cloned hybridoma cells into Gamma-irradiated (350 rads) and pristan-treated Bulb/c mice. Monoclonal antibodies were then purified from ascites by affinity chromatography on protein A-sepharose (Ey et al., 1978). Peptide synthesis Peptides corresponding to sequences PAPPPVRRRSSA (P,), RAYATEPHAK (P2), KQELEREAEERRG (P3), VKKEDTEKENREVGDWRKN (P4) and PAPPPVRRRSSA-NY-RAYATEPHAK (P,-NYP2) were synthesised, as were the corresponding Pi, Pi, Pi, Pi and P;-NY-P, analogues bearing the additional

New monoclonal antibodies for cardiac troponin I cysteyl-aminohexanoyl sequence at the N-terminus to permit coupling to carrier protein. The peptides were synthesised manually by the solid phase method (Merrifield, 1963) using p-methylbenzyhydrylamine resin (0.5 mmol NH,/g, Novabiochem, Laufelingen, Switzerland) and following the procedure previously described (Le Nguyen et al., 1989). The reactive amino acid side chains were protected as follows: tosyl for Arg; o-chlorobenzyloxycarbonyl for Lys; p-methylbenzyl for Cys; cyclohexyl ester for Asp and Glu; benzyl for Ser and Thr; 2,6-dichlorobenzyl for Tyr, Boc for His. Asn and Gln were used unprotected. The peptides were purified using preparative HPLC under conditions previously described (Le Nguyen et al., 1989). Analytical HPLC runs were carried out on a Lichrospher RP18 column (5 pm endcapped, 4.6 ID x 125 mm, Merck) and preparative runs on a Partisil Cl8 0DS3 column (10 pm, 22 ID x 500 mm, Whatman, Clifton, New Jersey) with the following solvent gradient system: A = 0.1% TFA in H,O; B = 60% CH, CN in H,O + 0.1% TFA. Peptides were dissolved in a minimum volume of solvent B, injected onto the column and eluted in a gradient mode. Several fractions were collected from the main peaks and controlled by analytical HPLC. Those of satisfactory purity (> 99%) were collected and freeze-dried. The preliminary step was to synthesise Pi-NY-P, and P, (and their cysteyl-aminohexanoyl analogues). Subsequently, P, and P, (and analogues) were then synthesised, to define epitopes. Finally, P, (and analogues) was synthesised. Radioimmunoassays (RIA) Indirect solid phase radioimmunoassay. TnI antibodies were detected in mouse serum, culture supernatants, ascites and purified IgG fractions by an indirect solid phase radioimmunoassay (RIA). Cardiac or skeletal TnI (10 pg/ml) in phosphate-buffered solution (PBS) pH 7.4, was adsorbed onto 200 ~1 wells in polyvinyl 96-well microtitre plates (Falcon 3911, Becton Dickinson) by incubating 50 p 1antigen overnight at + 4°C. After washing (PBS-Tween 20, 0.1%) antigen-coated wells were saturated with 4% fetal calf serum (FCS) in PBS for 45 min at 37°C. Test antibody was incubated (50 pi/well) for 2 h at 37°C in PBS-4% FCS in the antigen-coated wells. After washing, lz5I-labeled goat anti-mouse immunoglobulins were added (200,000 cpm/well) in the same buffer and incubated for 2 h at 37°C. After washing, the radioactivity bound in each well was counted. Antibody binding to peptide was also assayed with this direct RIA, using a peptide concentration of 20 pgg/ml for adsorption. Competitive radioimmunoassay. Binding of the different soluble peptides to different MAbs was assayed with the following competitive radioimmunoassay using adsorbed TnI. Dilutions of antibody solution were incubated in the cardiac TnI-coated wells and antibodyantigen binding was determined by addition of radioiodinated anti-mouse immunoglobulins as in the indirect solid-phase radioimmunoassay. The concentration of

273

the antibody used in the technique described below was chosen in the lower third of the linear part of the antibody titration binding curve obtained by plotting radioactivity versus logarithm of antibody dilutions. Cardiac troponin I standards (1000; 100; 10 and 0 ng/ml) or the peptide to be studied (10,000; 1000; 100; 10 and 0 ng/ml) were serially diluted in the PBS-4% FCS solution. Diluted TnI or peptide solutions were then mixed with an equal volume of antibody solution at the previously determined dilution. Aliquots of the antigen antibody mixtures were transferred in duplicate to TnIcoated plates and incubated for 2 h at 37°C. Antibody bound to coated TnI plates was detected using labeled goat anti-mouse immunoglobin. Maximum bound radioactivity was routinely 12,000-20,000 cpm with a background of 500-800 cpm. Peptide recognition by a particular Mab was given in percentage of binding to TnI-coated plates: Percentage B/B, = [(cpm-nsb)/(cpm max-nsb)] x 100, where cpm = signal obtained for a given peptide concentration, cpm max = signal obtained with the MAb alone (without peptide), and nsb = nonspecific binding without MAb and without peptide. 100% MAb binding to coated TnI reflected either a complete lack of recognition of the tested soluble peptide by this MAb, or the blank reference sample (MAb solution). Liquid-phase radioimmunoassay. Pi-NY-P, peptide was iodinated using the chloramine T technique (Greenwood et al., 1963). For each iodination, 1 mCi of “‘1 sodium iodide (Amersham, Buckinghamshire, UK) was allowed to react with 1 pg peptide. Free iodine was separated from the iodinated peptide on an Amprep column (phenyl pH, RPN 1914, Amersham). The specific activity of iodinated peptides was approximately 0.3 mCi/pg, which is equivalent to 0.5 mole of iodine per mole of peptide. MAb binding to iodinated peptides was determined after separation of bound and free peptide, using a double antibody method (Zola and Brook, 1982) and precipitation with a 4% polyethylene glycol 6000 solution (Merck, Darmstadt, Germany). The P; NYP, peptide-antibody association constant for each antibody was determined in the culture supernatant, according to Scatchard et al. (1949). The concentration of cold peptide varied between 1.6 x lop8 and 2.6 x lo-rOmol/l. Two-step immunoenzymometric assay (IEMA) 200 ~1 of purified antibody solution (10 pgg/ml) were adsorbed onto Maxisorp immunotubes (Startube, Nunc, Denmark) overnight at +4”C, in a 0.1 mol/l K/K, PO,, pH 6.8 buffer. After rinsing and saturation for 1 h at room temperature with buffer A (1% (w/v) casein (Sigma), in 0.1 mol/l K/K, PO, buffer pH 6.8), 200 ~1 of cardiac or skeletal TnI (500, 50, 5, 0 ng/ml) diluted in buffer A containing 0.1% Tween 20, were added for 2 h at room temperature. After rinsing, the second purified antibody conjugated with peroxydase enzyme using the glutaraldehyde technique (Avrameas et al., 1969) was

214

CATHERINE

LARUE et al

skeletal isoforms. No MAb recognised only the skeletal isoform. All 40 MAbs were available as culture supernatants, while only 28 MAbs were purified from ascitic fluids. The specificities of all MAbs were also investigated using competitive radioimmunoassay in order to verify their recognition of TnI in solution. Relative antigenic mapping bodies in IEMA Fig. 2. SDS electrophoresis of cardiac and skeletal Tnl. A = cardiac TnI, B = skeletal TnI, C = cardiac + skeletal TnI.

added in the same buffer at a final concentration of 0.5 pg/ml, and incubated for 2 h at room temperature. All intermediate washings were carried out with 0.1 mol K/K, PO, pH 6.8 containing 0.1% Tween 20. After the last rinsing, orthophenylene diamine (10 mg in 20 ml 0.1 mol/l citrate buffer, pH 5.0, containing 40 ~1 H, 0,) was added as a substrate of reaction. After 20 min at room temperature, optical density was read at 490-650 nm. RESULTS Purljkation

of Troponin

I

Cardiac and skeletal muscle yielded similar quantities of TnI: 4.35 f 1.36 mg/g. When characterised by SDS gel electrophoresis, cardiac TnI preparations migrated as a single band (Fig. 2a). Skeletal TnI preparations also migrated as a single component (Fig. 2b), although slightly more rapidly than the cardiac form in keeping with the smaller mol. wt of 19,800 compared to 22,500 daltons. No signs of proteolytic degradation as would be evidenced by smaller peptides were apparent in preparations from fresh cardiac or skeletal muscle. Characterisation

of monoclonal

antibodies

Forty MAbs were selected after indirect radioimmunoassay on absorbed cardiac or skeletal TnI. 25 MAbs (culture supernatants) recognised only human cardiac TnI, while 15 others recognised both cardiac and

of TnI with monoclonal

IEMA assays were employed to build a relative antigenie map of TnI using 28 MAbs out of a total of 40. These 28 MAbs were conjugated to peroxydase-enzyme and allowed to try all the possible pairs of MAbs (784 MAb-pair possibilities) with the 3 standards of cardiac TnI (500, 50, 5 ng/ml) and the nonspecific binding. Zero absorbance with the 500ng/ml TnI standard indicated that the MAb-peroxidase was directed against the same epitope as the coated MAb (or close to it). Conversely, high absorbance indicated complementarity of the two MAbs. Figure 3 shows the antigenic mapping of the 28 purified MAbs studied: 10 MAbs recognised both cardiac and skeletal TnI while 17 recognised only the cardiac isoform. It is of interest to point out all the different possible pairs of cardiac-specific MAbs which could be used to realise a very specific assay. Epitopic analysis with cardiac-specljic P,, P, , P,, P3 and analogues):

peptides

(P,-NY-

Indirect RIA on adsorbed peptides. Eight MAbs (culture supernatants) of the 40 tested recognised adsorbed P,-NY-P, peptide well and Cys-Ahx-P,-NY-P, peptide even better: 3B8, 3C6, 5D12, 5G4, 6Fl2, 6F10, 8Hll and 1 lE12 (Fig. 4 shows an example of three MAbs and a control). Adsorbed Pi peptide was only recognised by 6Fl2. No antibody recognised adsorbed P, or P, (nor the corresponding analogues). Competitive radioimmunoassay on adsorbed TnI. The 40 MAbs were first tested against soluble peptides at pH 7.4. Of the 40 MAbs, the same eight previously-selected MAbs recognised soluble P,-NY-P, and its analogue

c

c+ Cardiac-specific Mabs

anti-

Non cardiac-specific Mabs

Fig. 3. Antigenic mapping of human cardiac troponin I from N terminus to C terminus with MAbs by IEMA method (*; MAbs which recognised P,NYP, peptide). The different designs of boxes represented different epitopes of group of MAbs.

New monoclonal CPM

antibodies for cardiac troponin

275

I

- NSB Adsorbed peptides

:

15000 -

q q q

PI Pi P2

10000 -

H

P’INYPP

01 P-3 El non specific binding

5000 -

0 Mab

8Hll

6F12

llE12

7Dl

Fig. 4. Indirect radioimmunoassay on adsorbed cardiac peptides; example of three MAbs (6F12, 8Hll and 1lE12) and a MA\, control (7Dl was directed against cardiac TnI but did not recognise P,NYP, peptide). Culture supernatants were all diluted 1 in 10. These MAbs recognised adsorbed P,NYP, (and analogue); 7Dl MAb control did not recognise any peptide. of three MAbs and a control shown in Fig. 5). Binding pH-dependency was tested using P, and CysAhx-P, peptides (Pi) against one of the eight MAbs (5G4), and then for the other seven; pH 5.5 was found to be optimal for the peptide-MAb recognition (binding signal was maximal at this pH), and was thus chosen to assay all the cardiac peptides (Table 1 and Fig. 6). It is of interest to note that P,-NY-P,, Pj-NY-P,, P, and Pi were recognised by all eight MAbs (including 11E12); 11El 2 MAb also weakly recognised P, and Pi. On the other hand, P, peptide was not recognised by any antibody. The 8 MAbs demonstrated better recognition of cardiac TnI than P,-NY-P, peptide (and its analogue) or smaller peptides like P, or its analogue (results not shown). Liquid-phase radioimmunoassay of ‘25I-P’, NPY,. As our peptides carry a charge and are thus sensitive to pH, competitive radioimmunoassay was carried out using a scale of pH between 3.5 and 7.4. Maximal immunological peptide recognition was observed for all MAbs at pH 5.0. MAb association constants were determined by Scatchard plot, using Pi-NY-P, peptide (Table 2). The association constants varied from 0.3 to 4.0 x lo-“/ mol/l depending on the MAb tested. (example

Epitopic analysis with noncardiac-spec$c peptides ( P4 and its analogue). Of the 40 tested MAbs, only llE2 MAb slightly recognised adsorbed Pi peptide. 1 lE2

MAb binding on adsorbed TnI was inhibited by P, (90% inhibition for 10 pg/ml and 50% inhibition for 100 pgg/ml) and relatively less inhibited by Pi (94% inhibition for 10 pgg/ml and 65% inhibition for lOOpg/ml), while a control MAb was not inhibited by P, or Pi at these concentrations. Zmmunoenzymometric assay (IEMA)

of TnI

IEMA was carried out with cardiac or skeletal TnI. Different test conditions (pH, buffers, incubation courses) were studied and resulted in the choice of 0.1 mol/l K/K, PO, pH 6.8 buffer containing 1% casein (buffer A) and two-step IEMA, as optimal conditions. All tested MAb pairs were used to construct the relative antigenic map (Fig. 3) as previously described, and also to identify some interesting pairs capable of measuring TnI concentrations by IEMA. As an example, Fig. 7 shows a pair (adsorbed 3B9 versus 8El-peroxydase) which can quantify cardiac TnI concentrations from 0.5 ng/ml to 500 ng/ml without crossreactivity with skeletal TnI over the same concentration range. DISCUSSION The choice of cardiac TnI as a new biological parameter for the diagnosis of acute myocardial infarction, the quality of TnI purification and the optimisation of

% BIB0

80 Peptide concentration

60

:

0 0 ng/ml 100 q/ml

Mab

308

’

5D12

534

8Hll

q

1000 nglml

n

10000 ng/ml

6C4

Fig. 5. Competitive RIA of MAbs binding on adsorbed cardiac TnI, by P, NYPz peptide in solution, at pH 7.4; 3B8, 5D12, 5G4 and 8Hll MAbs recognised P,NYP, peptide, while control MAb (6C4) did not recognise this pcptide.

216

CATHERINELARUEet al. Table 1. Competitive RIA of MAbs binding on adsorbed TnI by a peptide concentration 10,000 ng/ml MAb 3B8 3C6 5D12 5G4 6F10 6F12 8Hll 1lE12

Pl

P’l

++

++

P2

p’2

+++ ++ +++ ++++ +++ +++ +++ ++

PlNYP2 ++ + ++++ +++ ++++ ++ ++ ++++

P’NYP2 ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++

P3

of

P’3

++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++

~ -~ ~ --

~ ~ ~

MAbs which recognised peptides: -no recognition of peptide by the MAb, + 5-39% + + 40-59%, + + + 60-79% and + + + + 80-100% of competition.

a screening method have allowed an important number of MAbs to be produced. Although there is probably a high level of sequence homology between skeletal and cardiac TnI isotypes (Wilkinson and Grand, 1978) we noted a considerable number of MAbs raised against cardiac TnI isotype. A specific immunoenzymometric assay of human cardiac TnI was developed thanks to an accurate selection of MAbs. These high affinity MAbs (n = 28), specific for human TnI, led to the construction of an epitopic map of TnI with at least 6 different epitopes. The sensitivity of our assay (0.5 ng/ml) was higher than that described in literature by Cummins et al. (1987a, b). The specificity of our assay, for an absolute biological diagnosis, was excellent since skeletal TnI was not detected, even at high concentrations. This has great significance in terms of wider diagnostic assay development and suggests that cardiac immunochemical specificity is easily achieved with TnI. Examination of other contractile protein isotypes employed in diagnostic immunoassays does not indicate the same potential for high cardiac specificity. The approach of selecting peptides by comparing known mammalian sequences and seeking for high antigenicity indexes, permitted us to validate our three sequences (Pz, P, , P4) as correct human sequences, since the amino acid sequence of human cardiac TnI has now been published (Vallins et al., 1990).

Three amino acid residues differ in the P, peptide (PAPPPVRRRSSA) compared to the actual human sequence (PAPAPIRRRSS). Of 40 MAbs directed against the whole molecule of human TnI, epitopes corresponding to eight of these could be located. It is of interest to note that these MAbs recognised rather small peptides (some of them have only 10 AA), even if immunological recognition of small amino acid sequences by polyclonal antibodies, or more rarely by monoclonal antibodies, has already been published (Gupta et al., 1988; Gleed et al., 1986; Schulze-Gahmen and Wilson, 1989). The P, sequence (KQELEREAEERRG), although human- and cardiac-specific, was recognised by none of Table 2. Affinity constants of 8 MAbs with ‘25I-P; NYP, peptide, by Scatchard measurement MAb

Affinity constants (1O’“/mol/l)

3B8 3C6 5D12 5G4 6FlO 6F12 8Hll llE12

1.4 1.5 0.6 4.0 0.6 0.8 0.3 2.0

% binding 100

Peptides : H

Pi

0-n

P-1

0-0

P2

o-o

p’2

A-A

PlNYPP

f&A

P’lNYPP

0 0

100

1000

10000

100000

nglml

Fig. 6. Competitive RIA of 1lE12 MAb binding on adsorbed cardiac TnI by different peptides, at pH 5.5.

New monoclonal

antibodies for cardiac troponin

277

carry out a sandwich with P,-NY-P, and two anti-peptide MAbs (results not shown), even if the sensitivity of this assay was not excellent. In conclusion, this work resulted in the production of an important number of MAbs against human TnI, in a relative epitopic map of TnI, and in the localisation of some specific MAbs by peptidic analysis. Suitable combinations of MAbs will be now chosen as accurate tools for a specific immunoenzymometric assay of human cardiac TnI.

Absorbance (490-650 nm) 2.5 7

0 TN I card 0 TN I sk.

0.5

I

5

50

500

nglml

Fig. 7. Two-step immunoenzymometric-assay of cardiac TnI with a pair of MAbs: 3B9 was adsorbed on tubes, 8El was peroxydase-labeled. (sk: skeletal form; card: cardiac form.)

the 40 anti-human TnI MAbs. This sequence was identical in rabbit, bovine and human species (Leszyk et al., 1988; Vallins et al., 1990). Indirect radioimmunoassay was used to select eight MAbs which recognised adsorbed P,-NY-P, and CysAhx P,-NY-P, . These results were confirmed by competition radioimmunoassays showing that all the MAbs recognised not only absorbed P,-NY-P,, but also soluble P,-NY-P, (and its Cys-Ahx-P,-NY-P, analogue) and soluble P, (and its analogue). The coating of small peptides (10 AA for PJ induces considerable conformational constraints, to the detriment of their recognition by specific antibodies. This could explain why seven out of the eight MAbs which recognised P, peptide (and its analogue) in solution, could not recognise adsorbed P, peptide. P,-NY-P, peptide (and its analogue) was better recognised by the eight MAbs than P, (and its analogue) whereas seven out of eight MAbs did not recognise P, sequence at all. The global conformation of P,-NY-P, peptide (24 AA) may more resemble TnI than P, peptide alone, even if P, sequence was not exactly as in humans. All eight MAbs are directed against P, peptide. The ability of 11E 12 MAb to recognise P, sequence and more weakly P, sequence possibly reflects an epitope located near the centre of P,-NY-P, peptide. Moreover, three MAbs out of 8 (5D12, 6F10, llE12) recognised Cys-Ahx-P, peptide better than natural P, peptide. The Cys-Ahx spacer possibly made the peptide conformation more flexible and suitable for antibody accessibility. Liquid-phase RIA with 1251-labeled P,-NY-P, was used to evaluate the affinity constants of MAbs against this radiolabeled peptide. These constants were measured using soluble radiolabeled peptide. Thus the molecular size of the “‘1 atom added to such a small peptide could constrict peptide-antibody recognition. The P, peptide, identical in both human cardiac and skeletal forms, was only recognised by the 1lE2 MAb. Finally, the chosen peptides demonstrated that the MAbs were complementary, since it was possible to

Acknowledgements-The authors would like to thank Drs A. Thevenet, R. Gartner and D. Putman-Cremer for tissue sources and Drs P. Gros and J. P. Liautard for helpful discussion.

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