WO1990015993A1

WO1990015993A1 - Assay for human ventricular myosin lc1 and monoclonal antibody thereto

Info

Publication number: WO1990015993A1
Application number: PCT/US1990/003434
Authority: WO
Inventors: Ban-An Khaw; Philip D. Nicol; Gary R. Matsueda
Original assignee: The General Hospital Corporation
Priority date: 1989-06-14
Filing date: 1990-06-14
Publication date: 1990-12-27
Also published as: AU5853590A

Abstract

A monoclonal antibody capable of forming an immune complex with human ventricular myosin LC1, and having substantially no capability of forming an immune complex with human fast skeletal myosin LC1.

Description

ASSAY FOR HUMAN VENTRICULAR MYOSIN LC1 AND MONOCLONAL

ANTIBCDY THERETO

Background of the Invention The work described herein was made with the aid of government funding and the government therefore has certain rights in the invention.

This invention relates to monoclonal antibodies reactive with human ventricular myosin light chain 1 (LCI).

Ventricular myosin LCI has been detected in the serum of patients following myocardial infarction and patients with cardio yopathy associated with myocyte necrosis. It is known that a highly sensitive assay system for these light chains is desirable. Monoclonal antibodies have been derived which react with, i.e., form immune complexes with, human ventricular myosin LCI. However, because of the high degree of ho ology between human ventricular and both fast (red^ and slow (white) skeletal myosin light chains, these antibodies were not specific to human ventricular myosin light chains, and were capable of forming immune complexes with both fast and slow human skeletal myosin light chains. Following injuries, patients are likely to have fast skeletal myosin light chains in their serum, but are less likely to have slow skeletal myosin light chains in their serum. The amino acid sequences of human ventricular myosin LCI, and of human fast skeletal myosin light chain 1 (fast human skeletal myosin LCI) are known. Hoffman et al. (1988) 16 Nucleic Acids Research 5, 2353; Seidel et al. (1987) 15 Nucleic Acids Research .12, 4989. It appears, from cDNA probe studies, that human slow skeletal myosin LCI is identical to human ventricular myosin LCI. Kurabayashi et al. (1988) 263 J. Biological Chemistry 22. 13930. The sequences of human ventricular myosic LCI and human fast skeletal myosin LCI, while similar, differ in approximately 28% of their corresponding positions.

Summary of the Invention In general, the invention features, in one aspect, a monoclonal antibody capable of forming an immune complex with human ventricular myosin LCI, and having substantially no capability of forming an immune complex with human fast skeletal myosin LCI. The monoclonal antibodies of the invention have high enough affinity for human ventricular myosin LCI to render them useful in specific im unoassays of body fluids (blood, serum, urine or plasma) for ventricular myosin LCI, which is present in elevated amounts in the blood of patients who have recently suffered myocardial infarction (MI) or suffer from other cardiomyopathy associated with myocyte necrosis, and which is thus diagnos ic of MI or other cardiomyopathies. The invention is in part based on our discovery that monoclonal antibodies made using, as imunogens, synthetic peptides unique to ventricular myosin LCI, as compared to fast skeletal myosin LCl,ldespite exhibiting, in some experiments, an apparent affinity for ventricular myosin LCI too low to render them useful in a practical assay, actually do possess the requisite affinity. We discovered this phenomenon when we compared the results of two types of experiments, the first employing radio-iodinated human ventricular LCI in a conventional binding assa , and the second using a radio-labeled ventricular myosin LCI peptide in a competitive assay with unlabeled ventricular myosin

LCI. The first set of experiments, using radiolabled ( 125I) LCI, seemed to indicate very poor affinity of the antibodies for ventricular myosin LCI, too low to be of practical value. The competitive binding experiments, however, gave much better results, leading us to conclude that it was not low affinity, but an artifact of the experimental conditions, which produced the poor results of the first set of experiments. We postulate that the explanation is as follows. Labeling with 125I occurs at the Tyr residues of human ventricular myosin LCI. In conventional binding experiments, the labeled LCI molecule is apparently degraded in a way which produces a substantial quantity of 125I-labeled fragments from the carboxy terminal region of the molecule, which do not bind to the antibody and are thus not counted, producing a low value which does not accurately reflect the true affinity of the antibodies for the ventricular myosin LCI molecule. It was only upon using a competitive assay using unlabeled LCI and labeled ventricular myosin LCI peptide that we discovered that the antibodies in fact have sufficiently high affinity for use in a commercial assay, in preferred embodiments, the antibody of the invention is also capable of forming an immune complex with an antigen containing a peptide sequence containing 5 or more (more preferably, 6 or more, and more preferably 20 or fewer) amino acids and being at least 75% homologous, and more preferably 100% homologous, with a region of human ventricular myosin LCI which differs from the corresponding region of human fast skeletal myosin LCI. In more preferred embodiments, the antibody is capable of forming an immune complex with an antigen containing one of the peptide sequences LYS PRO GLU PRO LYS LYS ASP ASP ALA LYS, MET ALA PRO LYS LYS PRO GLU PRO LYS, PRO GLU PRO PRO PRO GLU PRO GLU ARG PRO, ALA SER LYS ILE LYS ILE GLU PHE THR PRO GLU GLN ILE GLU GLU, THR PRO LYS CYS GLU MET LYS ILE THR TYR GLY GLN CYS, ASN THR LYS MET MET ASP PHE GLU THR PHE, LEU GLN HIS ILE SER LYS ASN LYS ASP THR GLY THR, or GLU ARG LEU THR GLU ASP. The invention also features a hybrid cell (hybridoma) capable of producing an antibody of the invention.

In preferred embodiments, the synthetic peptide with which the antibody is reactive is homologous with a region of human ventricular myosin LCI which differs from the corresponding region of human fast skeletal LCI in at least 1/3 of its amino acid sequence. The synthetic peptide may be bound to a carrier molecule, preferably by a linkage to the C-terminal end, for use as an immunogen to produce antibodies of the invention. The antibodies of the invention can be used in a kit to assay human ventricular myosin LCI; the kit preferably also includes a synthetic peptide of the invention which, like the antibody, can be provided with a label, depending on the format of the assay (any standard immunoassay format can be used, e.g., ELISA, competitive RIA, etc.).

The invention provides monoclonal antibodies which possess both selectivity and high affinity for human ventricular myosin LCI. The invention also provides an assay for human ventricular myosin LCI which does not require the use, as standards, of either human or animal ventricular myosin light chains, which are not readily available.

All monoclonal antibodies having the characteristics described above and being specific for human ventricular myosin LCI are encompassed by the present invention. These monoclonal antibodies are produced by hybrid cells made using conventional hybridization techniques such as are described in Reinherz et al. (1979) J. Immunol. 123, 1312. As is well-known in the monoclonal antibody field, each independently produced hybrid cell line which produces a monoclonal antibody specific to the same particular antigenic determinant is nonetheless different from all others, as is each of the monoclonal antibodies so produced. Thus, while repetition of the procedure described below for producing the monoclonal antibody of the invention will result in the production of a hybrid cell line which produces useful monoclonal antibody specific to human ventricular myosin LCI, it is highly unlikely that it will produce a cell line which produces a monoclonal antibody which is chemically an exact copy of the monoclonal antibody described below.

Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims. Description of the Preferred Embodiments We now turn to a description of the preferred embodiments of the invention, after first describing the drawings. Drawings

Fig. 1 sets forth the amino acid sequences of human ventricular myosin LCI and human fast skeletal myosin LCI, aligned beginning at their amino terminal ends.

Fig. 2 sets forth the amino acid sequence of the first 14 amino acids at the amino terminal end of ventricular myosin LCI, as well as the amino acid sequences of two fragments of this sequence, designated PI and P348.

Fig. 3 is a graph depicting the antibody response over time of P348-immunized mice to human ventricular light chains. Figs. 4a-4c are graphs depicting the antibody response of P348-immunized mice to human ventricular light chains, KLH, and P348.

Fig. 5 is a graph depicting the antibody response of Pl-immunized mice to human ventricular light chains, KLH, and PI. ^**

Fig. 6 is a graph depicting the antibody response of 5 hybridoma cell lines to human ventricular LCI. Fig. 7 is a graph of ^"the results of an 8E3 antibody liquid phase competition assay depicting the absence of inhibition of binding of 1"25-'i P348 to the antibody by human fast myosin light chains. Production of Synthetic Peptides Referring to Figs. 1 and 2, two peptide sequences from the amino terminal end of human ventricular LCI were designated PI and P348. PI was synthesized using the Dupont RAMPS system employing amino acids and a cysteine resin made by Dupont. All chemicals used were amino acid synthesis grade unless otherwise stated. The completeness of coupling was assessed following each amino acid addition by the Kaiser test as described in the RAMPS manual. Following cleavage of the peptides from the resin, employing 95% Trifluoroacetic acid 5% phenol, the finished peptide was precipitated from anhydrous ether three times, dissolved in distilled water, and lyophilized. The crude peptide was subjected to amino acid analysis for confirmation of the amino acid content, and purity was assessed by HPLC analysis on a Vydac C18 column employing a mobile phase of CH3CN/.05% TFA and H20/.05% TFA. P348 was synthesized on an automated solid phase peptide synthesizer using TBOC amino acids, according to standard techniques. The crude peptide was cleaved from the resin with HF, and the purity and amino acid content were assessed as described above for PI. HPLC profiles confirmed a single major peak for each synthetic peptide and no further purification was deemed necessary. All peptides were positive when tested with the Ellman reagent, which detects free sulfhydral groups. Production of Synthetic Peptide Immunoqens

For use as immunogens, both PI and P348 were coupled to the hapten carrier keyhole limpet haemocyanin (KLH) (Calbioche ) via a thiolether bond, as described in Bernatowicz et al . (1986) Analytical Biochemistry 155, 95. The efficiency of coupling and the conjugation of peptide to KLH was assessed by amino acid analysis of the conjugate. Incorporation of peptide into bromacetylated KLH results in the formation of a thiolether bond between the cysteine residue of the peptide and lysines of the KLH. When subjected to acid hyc lysis and subsequent derivitization, this cysteine appears as carboxymethyl cysteine on an HPLC profile. The molar ratio of this CM-cysteine to the average of 3 amino acids which were not present in the peptide thus approximates quite closely the molar incorporation of peptide into KLH. The molar incorporation of carboxymethyl cysteine into the conjugate was used as the measure of the concentration of peptide for subsequent immunizations of mice. Isolation of Human Ventricular Myosin

Human left ventricular tissue was obtained from autopsy material within 48 hours of death. Tissue was frozen in liquid nitrogen and stored at 70 degrees

Celcius until the time of isolation. Cardiac myosin was isolated by the procedure of Katz et al. (1966) Circulation Res. 19, 611-621, and human ventricular light chains were isolated by the method of Perry and Perry (1970) Bioche . J. 119, 31-38. The purification of human ventricular myosin light chain 1 from crude light chains was performed using affinity chromatography on a CNBR activated sepharose 4B column (Pharmacia) which had been coupled to a monoclonal antimyosin light chain 1 antibody (8E3B6D10). Crude light chains at a concentration of 1 mg/ml were applied to the column in .01M Tris Cl pH 7.4, lOmM EDTA and 1 mM PMSF. After incubation for 1 hour at 21 degrees Celcius the column was washed with the same buffer until the absorbence at 280 nm of the eluate fell to zero. The column was then washed to 1 M NaCl in PBSA until the absorbence returned to zero. Myosin light chain 1 was eluted from the column with 0.1 M glycine-HCl, pH 2.5, and eluted fractions containing protein were neutralized immediately with solid Tris. The fractions were then dialyzed overnight against 6 L of 10 mM EDTA, ImM PMSF. Purity of the preparation was assessed by SDS-PAGE on 12% polyacrylamide gels. Synthesis of P348 Bromacetyl Tyrosine

In order to attach a C-terminal Tyr residue for radio-iodination, lyophilized peptide P348 (7mg) was dissolved in 1 ml of 0.1M phosphate buffer, pH 7.4, containing ImM EDTA. This solution was cooled on ice and then 3.4 mg of N-Succinimidyl Bromacetyl Tyrosine was added while the solution was stirring. The solution was allowed to warm to room temperature over the course of 30 minutes. This was followed by HPLC purification of the modified peptide as described above. Aliquots were collected of the pure modified peak which corresponded to 50 ug of modified original peptide, and lyophilized. Iodination of LCI

Radio-iodination of LCI was performed using the chloramine T method, followed by separation on Sephadex G-25. Specific activity of LCI was determined prior to separation as the TCA precipitable counts per minute following precipitation with carrier BSA. Synthesis and iodination of P348-Tyr

HPLC was used to follow the reaction of P348 with Bromo acetyl Tyrosine. HPLC profiles before and after the reaction revealed a shift in elution of P348

10 following the addition of a tyrosine residue. The reaction resulted in 85% of P348 incorporating a tyrosine residue. The peak corresponding to P348TYR was collected and the purified product iodinated using the chloramine T method. Specific activity of the iodinated

^••-5 product was determined by adding 2 mg of carrier peptide to a 10 ul sample from the iodination followed by pre. itation in anhydrous ether at -70 C. The specific activity of the peptide using this technique was 90 Ci/mMole. ⁰ Production of Polyclonal Antisera in A/J Mice

In order to select a mouse which produced adequate antibodies to the synthetic peptides Pi and P348 used for immunization, the following steps were taken. Female A/J mice 6-8 weeks old were purchased 5 from Jackson Laboratories. Groups of 6 mice each were immunized with 50 ug of P348 (incorporated into KLH) and 25ug of PI (incorporated into KLH) in complete Freund's adjuvant (Sigma chemical HR 37a strain) . Mice were boosted with the same dose of peptide KLH conjugate at 0 monthly intervals in an equal volume of Freund's incomplete adjuvent. Mice were subjected to retro orbital bleeds at monthly intervals and titers were determined for polyclonal sera. Solid phase titering was performed employing antigen coated microtiter plates (Falcon) coated with 50 microliters of antigen at a concentration of 50 ug/ml which had been incubated overnight with 10% gamma globulin-free horse sera. Titers were assayed for peptide KLH conjugate, peptide alone, and human ventricular myosin light chains by serially diluting the sera in 1% gamma globulin-free horse sera. Samples of sera were titered against control plates coated with 50 microliters of 1% BSA. Antisera was incubated for 1 hour, washed and then 50 microliters of I Goat antimouse F(ab')₂ (Cappel) (50,000 cpm) was added and plates were incubated for another hour. Following washing with distilled water, individual wells were counted in a Micromedics gamma counter.

Fig. 3 shows the antibody response over time of P348-immunized mice to human ventricular light chains. All mice responded to both the carrier protein _.^**._,H and to the respective peptide equally well in solid phase titering, as shown in Figs. 4a-c. In other experiments, when human ventricular myosin light chains were used as the antigen, animals immunized with P348 were found to have cross reactive polyclonal sera. Titers of cross reactive sera rose by an order of magnitude each month following primary immunization. In contrast, the polyclonal sera obtained from mice immunized with PI did not cross react with human ventricular myosin light chains in solid phase assays, as shown in Fig. 5. Production of Hybridomas and Purified Monoclonal Antibodies

From a panel of mice immunized with P348, the mouse with the highest solid phase titer against human ventricular myosin LCI, as measured as described above, was chosen for fusion. This mouse demonstrated 50% binding of polyclonal sera to antigen coated plates at a serum dilution of 1/10,000.

Myeloma cells SP OA/2 (ATCC) were grown in 20% fetal calf serum (M.A. Bioproducts) DMEM media, as previously described. Three days prior to fusion the mouse was hyperimmunized via a tail vein injection with lOug of P348 conjugated to KLH. Fusion was carried out as described by Kohler and Milstein. Cells were plated into 96 well culture plates in HAT media and fed every three days with HAT media until day seven, at which time the media was changed to HT media. Culture supernatants from wells with hybridoma growth were screened for the presence of anti human ventricular LCI antibody employing both a conventional solid phase plate assay in microtiter plates (results given in Fig. 6) and by liquid phase screening using iodinated human ventricular light chains in an indirect solid phase assay.

Briefly, the solid phase assay was carried out as follows. LCI was coated on wells of a microtiter plate and supernatant from cultured hybridoma cells was added to the wells, incubated, and then the wells washed, leaving LCI-bound monoclonal antibody in several of the wells. Iodinated goat antimouse F(ab')₂ was then added to the wells, and labeled wells detected as an indication of the presence of monoclonal antibody to LCI. In the second liquid phase screening method, goat antimouse F(ab')₂ was coated on the wells of a microtiter plate and incubated with culture supernatants for one hour, followed by a washing step, and then incubation with iodinated LCI; labelled wells indicated monoclonal antibody to LCI. The hybridoma growth frequency in the fusion was 95% and 5 clones were identified which bound ventricular myosin LCI and P348 in both solid and liquid phase screening. These 5 clones were subcloned twice to assure monoclonality. Solid phase screening of these 5 cell lines was undertaken to assess cross reactivity with human ventricular and fast skeletal myosin light chains. All 5 clones recognized ventricular myosin light chains but failed to cross react with fast skeletal myosin LCI.

Isotyping of culture media of the above 5 clones gave the results shown in the table below.

MURINE MONOCLONALS DIRECTED AGAINST P3 8 AND VLC1

ANTIBODY ISOTYPE SPECIFICITY

HC L< PI P348 VLC1 FSLC

5A3 IgG 2a K + +

5B11 IgG 2b K + +

8E3 IgG 2a K + +

9D5 IgG 2a K + +

9G8 IgG 2a K + +

*VLC1 = ventricular LCI

*FSLC - fast skeletal light chains

Four of the five cell lines were propagated in pristane-primed Balb C mice and ascites collected.

Two weeks following priming, 1 x 10 cells were injected into each mouse. Ascites fluid was harvested at the time of maximum ascites production and cells were removed by centrifugation at 1500 x g. Ascites from these four cell lines were subjected to microzone electrophoresis to confirm the presence of antibody. One antibody, designated 8E3B6D10, was purified on a protein A column and its purity assessed in the same fashion.

Monoclonal antibodies were purified employing standard techniques by chromatography on Protein A Sepharose obtained from Sigma Chemical. Antibodies thus purified were subjected to electrophoresis in a Beckman Microzone chamber and stained to asses purity of the preparation. Liquid Binding Studies Liquid phase competitive binding studies were carried out as follows. A conventional triple antibody precipitation method was used to study the binding of antibodies in liquid phase. Rabbit antimouse polyclonal sera as well as goat antirabbit polyclonal sera, which are publicly available from a number of sources, were supplied by Dr. Robert Graham. Purified antibody, ascites, and culture supernatants were titered in liquid phase against 125I P348 to determine, the antibody concentration at which binding was 50% of maximal. This concentration of antibody was then used in the following liquid phase assay.

Each tube contained the following: One hundred microliters of either normal human sera or patient sera, 50 microliters of monoclonal antibody, 330 microliters of 1% BSA/H 0, 10 microliters (16,000 cpm)

125 of radio-iodinated ( I) P348, 10 microliters of unlabeled LCI (affinity purified) or unlabeled P348 at known concentrations. Control tubes were the same except that 50 microliters of a monoclonal IgG 2a Kappa chain (designated 1B9G4) which is specific for human atrial cardiac myosin heavy chain was substituted for the light chain monoclonal antibody. In addition, 6 control tubes contained, substituted for unlabled peptide or protein, 10 microliters of 1% BSA. After addition of all reagents, tubes were incubated overnight at 4°C.

The following day, 50 microliters of rabbit antimouse polyclonal sera was added to each tube and incubated at 4°C. for 30 minutes. Following this, 50 microliters of goat antirabbit sera was added and the tubes were incubated for a further 30 minutes at 4°C. One ml of normal saline was then added and the tubes were spun at 5000 x g for 10 minutes. The supernatants were decanted and the pellets washed twice more in similar fashion. The three supernatants were pooled and both the pellets and supernatants were counted. Results were plotted as normalized percent binding. Concentrations of unknowns were read from a standard curve generated with each assay. All measurements were performed in duplicate and standard deviation and standard error of the mean were calculated from six

-ubes which contained no unlabled antigen and hence represented maximal binding values to 125I peptide P348Tyr in the assay.

The specific ability of antibody 8E3B6D10 to bind 125I-labeled peptide in liquid phase was demonstrated as follows. Using rabbit antimouse sera and goat antirabbit sera the antibody bound 50% of

125I P348Tyr at an antibody concentration of 10 ng/rnl. This concentration of antibody was then used in liquid phase competition studies. When unlabeled P348 was used competitively against 125I P348Tyr, 50% of inhibition of binding of the labeled antigen was seen at a concentration of 0.19 picomoles (2.8pg) of peptide.

When affinity purified ventricular LCI, rather than unlabled peptide, was used to compete with the labeled peptide, the curve was identical to that of the peptide. The 50% inhibition point was 0.19 picomoles (5 ng) , indicating the apparent affinity of the antibody to the peptide was the same as to the native protein.

When affinity purified 125I was used as the probe, binding occurred in liquid phase but the specific activity of the probe as judged by TCA precipitation was consistently low. When labeled LCI probes were used to optimize conditions for liquid phase binding, a much higher antibody concentration was required. The difficulty in iodinating LCI resulted in much larger amounts of the probe and antibody being used, which decreased the sensitivity of the system. When different methods of iodination were used the results were no better. Ventricular LCI Specificity In order to demonstrate the absolute specificity of 8E3B6D10 for human ventricular LCI, fast skeletal light chains were used in the liquid phase competition assay in place of human ventricular LCI. Fast skeletal light chains at a concentration of up to 7.5 ug/ml fail to inhibit the binding of ¹²⁵I P348TYR to the antibody (Fig. 7).

The antibodies and peptides of the invention can be used to assay ventricular myosin LCI in body fluids using any conventional immunoassay format, e.g., a competitive immunoassay in which any ventricular myosin LCI in the sample competes with labeled peptide for binding to the antibody of the invention, and is compared to data for the competition of unlabeled peptide and labeled peptide for binding to the same antibody.

Other embodiments are within the following claims.

Claims

Cl aims

1. A pure monoclonal antibody capable of forming an immune complex with human ventricular myosin LCI, and having substantially no capability of forming an immune complex with human fast skeletal myosin LCI.

2. The pure monoclonal antibody of claim 1, said antibody being capable of forming an immune complex with an antigen containing a peptide sequence comprising 5 or more amino acids homologous with a region of human ventricular myosin LCI which differs from the corresponding region of human fast skeletal myosin LCI.

3. The pure monoclonal antibody of claim 2, said antigen containing between 5 and 20 amino acids, inclusive.

4. The pure monoclonal antibody of claim 1, capable of forming an immune complex with an antigen containing the peptide sequence LYS PRO GLU PRO LYS LYS ASP ASP ALA LYS.

5. The pure monoclonal antibody of claim 1, capable of forming an immune complex with an antigen containing the peptide sequence MET ALA PRO LYS LYS PRO GLU PRO LYS.

6. The pure monoclonal antibody of claim 1, capable of forming an immune complex with an antigen containing the peptide sequence PRO GLU PRO PRO PRO GLU PRO GLU ARG PRO.

7. The pure monoclonal antibody of claim 1, capable of forming an immune complex with an antigen containing the peptide sequence ALA SER LYS ILE LYS ILE GLU PHE THR PRO GLU GLN ILE GLU GLU.

8. The pure monoclonal antibody of claim 1, capable of forming an immune complex with an antigen containing the peptide sequence THR PRO LYS CYS GLU MET LYS ILE THR TYR GLY GLN CYS.

9. The pure monoclonal antibody of claim 1, capable of forming an immune complex with an antigen containing the peptide sequence ASN THR LYS MET MET ASP PHE GLU THR PHE.

10. The pure monoclonal antibody of claim 1, capable of forming an immune complex with an antigen containing the peptide sequence LEU GLN HIS ILE SER LYS ASN LYS ASP THR GLY THR.

11. The pure monoclonal antibody of claim l, capable of forming an immune complex with an antigen containing the peptide sequence GLU ARG LEU THR GLU ASP.

12. A hybrid cell capable of producing the monoclonal antibody of claim 1.

13, A synthetic peptide comprising 5 or more amino acids homologous with a region of human ventricular myosin LCI which differs from the corresponding region of human fast skeletal myosin LCI.

14. The synthetic peptide of claim 13 in which said region of human ventricular myosin LCI differs from the corresponding region of human fast skeletal LCI in at least 1/3 of its amino acid sequence.

15. A synthetic peptide comprising 5 or more amino acids with at least 75% homology to a region of human ventricular myosin LCI which differs from the corresponding region of human fast skeletal myosin LCI, said synthetic peptide differing from said corresponding region of human fast skeletal myosin LCI.

16. The synthetic peptide of claim 13 or 15 comprising six or more amino acids.

17. The synthetic peptide of claim 13 or 15 bound to a carrier molecule.

18. The synthetic peptide of claim 17 bound to said carrier molecule by a linkage to the C-terminal or the A-terminal end of said synthetic peptide.

19. The synthetic peptide of claim 17 bound to said carrier molecule by a thiolether linkage to a cysteine residue added to the C-terminal or the A-terminal end of the synthetic peptide.

20. An immunoassay kit containing (1) a monoclonal antibody capable of forming an immune complex with human ventricular myosin LCI, and having substantially no capability of forming an immune complex with human fast skeletal myosin LCI, said monoclonal antibody further being capable of forming an immune complex with a synthetic peptide comprising 5 or more amino acids homologous with a region of human ventricular myosin LCI which differs from the corresponding region of human fast skeletal myosin LCI, (2) said synthetic peptide, and (3) said synthetic peptide bearing a label.

21. An immunoassay kit containing (1) a monoclonal antibody capable of forming an immune complex with human ventricular myosin LCI, and having substantially no capability of forming an immune complex with human fast skeletal myosin LCI, said monoclonal antibody further being capable of forming an immune complex with a synthetic peptide comprising 5 or more amino acids with at least 75% homology to a region of human ventricular myosin LCI which differs from the corresponding region of human fast skeletal myosin LCI, said synthetic peptide differing from said corresponding region of human fast skeletal myosin LCI, (2) said synthetic peptide, and (3) said synthetic peptide bearing a label.

22. An immunoassay kit containing (1) a monoclonal antibody capable of forming an immune complex with human ventricular myosin LCI, and having substantially no capability of forming an immune complex with human fast skeletal myosin LCI, said monoclonal antibody further being capable of forming an immune complex with a synthetic peptide comprising 5 or more amino acids homologous with a region of human ventricular myosin LCI which differs from the corresponding region of human fast skeletal myosin LCI, (2) said synthetic peptide bearing a label, and (3) a standard inhibition curve of the binding of said synthetic peptide bearing said label to said monoclonal antibody as a function of the concentration of said synthetic peptide.

23. An immunoassay kit containing (1) a monoclonal antibody capable of forming an immune complex with human ventricular myosin LCI, and having substantially no capability of forming an immune complex with human fast skeletal myosin LCI, said monoclonal antibody further being capable of forming an immune complex with a synthetic peptide comprising 5 or more amino acids with at least 75% homology to a region of human ventricular myosin LCI which differs from the corresponding region of human fast skeletal myosin LCI, said synthetic peptide differing from said corresponding region of human fast skeletal myosin LCI, (2) said synthetic peptide bearing a label, and (3) a standard inhibition curve of the binding of said synthetic peptide bearing said label to said monoclonal antibody as a function of the concentration of said synthetic peptide.

24. A method of producing a monoclonal antibody capable of forming an immune complex with human ventricular myosin LCI, and having substantially no capability of forming an immune complex with human fast skeletal myosin LCI, comprising immunizing a mammel with a synthetic peptide comprising 5 or more amino acids homologous with a region of human ventricular myosin LCI which differs from the corresponding region of human fast skeletal myosin LCI, fusing lymphocytes obtained from such mammal with an immortal cell line to produce hybridoma cells, culturing said hybridoma cells to produce supernatant, screening said supernatants for monoclonal antibody capable of forming an immune complex with human ventricular myosin LCI and having substantially no capability of forming an immune complex with human fast skeletal myosin LCI, and purifiying said monoclonal antibody.

25. A method of assaying a sample for human ventricular myosin LCI comprising comparing (1) the inhibition by any said ventricular myosin LCI in said sample of the binding of a pure monoclonal antibody capable of forming an immune complex with human ventricular myosin LCI, and having substantially no capability of forming an immune complex with human fast skeletal myosin LCI, said monoclonal antibody further being capable of forming an immune complex with a synthetic peptide comprising 5 or more amino acids homologous with a region of human ventricular myosin LCI which differs from the corresponding region of human £a_*t skeletal myosin LCI, to said synthetic peptide bearing a label, and (2) the inhibition of the binding of said pure monoclonal antibody to said labeled synthetic peptide by said synthetic peptide in an unlabeled condition.