CA2056407A1 - Monoclonal antibody specific to ventricular myosin light chain1 and enzyme immunoassay for its detection in cardiac patients - Google Patents

Abstract

A competitive method of measuring human ventricular myosin light chain in cardiac patients which comprises incubating at least a monoclonal or polyclonal anti-HVLCS antibody, an enzyme, a VLCS antigen and an unknown amount of HVLCS analyte present in patient's serum. The anti-HVLCS antibody or the VLCS antigen, is directly or indirectly detected by means of enzyme-bound label, whereby the amount of HVLCS analyte, when initially present in patient's serum, is determined by comparing the extent to which the said VLCS antigen is bound to the said anti-HVLCS antibody with a calibration curve obtained from a known amount of said antigen. The anti-HVLCS antibody or the VLCS antigen is solid-phase bound. The anti-HVLCS monoclonal antibody has specificity to at least one of HVLC1 and HVLC2. There is prepared a monoclonal antibody which specifically binds to VLC1 and which is produced by the hybridoma cell line having the ATCC accession number HB 10471.

Description

TITI~E OF THE INVENTION

Monoclonal antibody specific to ventricular myosin light chainl and enzyme immunoassay for its detection in cardiac patients.

5 BACKGROUND OF THE lNVENTlON

Coronary occlusion is a major cause of death and disability in the developed countries. In order to identify the extent of the resulting myocardial infarction, to define an appropriate therapy, and to test its efficacy, clinicians require a precise detection and localization of 10 necrosis, and a retrograde evaluation of the extent of damage.

At present, the diagnosis of myocardial infraction is based on the patient's medical history, electrocardiographic (ECG), ultrasonographic or echocardiographic changes, chest x-ray, serum enzyme determinations, radiologic investigations, radionuclide imaging (thallium 201, technetium 9 9 mT c-pyrophosph ate, radionuclide angiography), positron emission tomography and magnetic resonance imaging. They all represent noninvasive investigations. Invasive tests are carried out in hospitalized patients for valid reasons only and include catheterization and 20 angiocardiography. All of these diagnostic techniques provide the physician with information on both the structure and function of the heart.

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WO 90/15329 PCI/CA91)/OD]77 2~564Q7 Most physicians now rely heavily on serum cardiac marker measurement to help define or exclude acute myocardial infarction.
The biochemical methods most commonly used measure the levels of certain cytoplasmic components which are rapidly released into the 5 serum of patients with acute myocardial infarction from damaged necrotic cardiac cells (l-3). These serum markers are usually measured by their enzymatic activity and those include: lactic dehydrogenase (LDH) and its isoenzymes LDH-1 and LDH-2, creatine phosphokinase (CPK or CK) and its cardiac-specific isoenzymatic form (CK-MB), 10 glutamic exalacetic transaminase (SGO-T), and aspartate transferase (AST) (1-5).

These isoenzymes are not, however, uniquely located in cardiac muscle, which leads to serum elevations in the clinical range in noncardiac related conditions (6-25). For example, creatine-kinase is 15 elevated in patients with acute myocardial infarction as well as in association with chronic alcoholism, cardioversion, skeletal muscle trauma, and intramuscular injection. While CK-MB is a more specific and sensitive indicator of myocardial damage, its usefulness is limited to those patients seen early in infarction. In addition, a 6% false-20 negative incidence has been reported (26) and Roe et al. (27) observedthat $wo dogs induced to infarction failed to release CK-MB into $he serum. LDH isoenzyme analysis lacks the sensitivity and specificity ~;
enjoyed by CK MB (10æ false-negative and 5% false-positive) and elevated elvels of LDH are observed in hemolyzed samples, 25 necessitating care in sarnple collection. Additionally, these enzymes do not correlate directly with the extent of necrosis.

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WO 90/1532~ 2 0 ~ 6 ~ o ~Cr/cA9o/ool77 These assays often fail to indicate the cardiac infarction (false negative) and are also sensitive to background level of enzymes (false positive).

Cardiac muscle specific serum markers Cardiac-muscl~specific markers would be, on the other hand, potentially valuable diagnostic aids in myocardial infarction. The contractile proteins of the myofibril exist in different isotype forms which vary in their tissue distribution, particularly between cardiac and skeletal muscles. These proteins include myosin heavy and light chains, actin, tropomyosin, troponin-I, troponin-T, and troponin-C.
Leakage of those contractile proteins from cardiac cells would illdicate damaged or necro~ic myocardium.

Structu~e of ca~diac and skeletal muscle myosin Myosin is the most abundant contractile protein present in various muscle cells. Structurally, the molecu!e consists of two globular heads attached to a r~d-like tail shaped into ~-helix. On the molecular level, it consists of two hea iy chains of molecular weight 200,000 which are non-covalently associated with four light chains of approximate molecular weight 20,000. The rod is composed of two heavy chains while heads of two pairs of light chains and two heavy chains.

With the advent of such techniques as polyacrylamide ~
electrophoresis, peptide mapping and high performance liquid chromatography, it became apparent that myosin exists in isoenzymatic forms that differ in light and hea~ ~ chain subunit composition.

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wo 90/15329 PC~/CA90/00177 205~41)7 Myosin heavy and light chains are different with respect to DNA sequence, amino acid sequence, molecular weight and ATPase activity, in all the species and muscle types so far investigated.

The light chains from all fast muscles are clearly different in 5 number and mobility (e.e. molecular weight) from those of slow muscles. Fast-twitch muscles have three light chains (LClF, LC2F, LC3), whereas the slow-twitch and cardiac muscles have two light chains (LClS and LC2S). Each myosin head contains one LC2 type light chain, and either a LCI or LC3 type of light chain. The LCI /LC3 light chains 10 were originally called the "alkali light chains" because they could be dissociated from heavy chains only under denaturing conditions such as highly alkaline pH. The LC2 light chains are called "phosphorylatable' or "regulatory" light chains, as they are easier to remove from the heavy chain and, in certain muscles (e.g. smooth 15 muscle), they play an essential role in regulation of contraction Studies have shown that skeletal and cardiac muscle light chains are chemically (28) and immunologically (29) distinct, and each is characterized by a unique amino acid sequence (30), yet many structural features are shared.

In mammalian and human cardiac muscles LCI and LC2 differ in the atria and ventricles (31). Human ventricular LCI has molecular weight of 27,000 daltons while LC2 20,000 daltons. Alkali light chains from all species have higher molecular weight, in the range of 25-28,000 dalton, than light chain 2 class (1~21,000).

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20~6407 The mechanism of cardiac light chain release upon myocardial cell death Previous published clinical and experimental studies have shown that cardiac myosin light chains are liberated from myocardial -5 fibers and can be detected in sera of patients after myocardial infarction (32-4~). In many patients, the release of light chains is biphasic. The appearance and disappearance of these structural proteins in the serum following irreversible ischemic cellular injury differ from that of cytosolic eatine kinase MB and myoglobin in that serum levels of 10 both cardiac myosin light chain 1 and light chain 2 rise early after the onset of infarction and remain elevated for several days after injury (34, 37, 43). The cellular mechanisms responsible for both the early and sustained release of myosin light chains from irreversibly injured myocardium are not known with certainty.

Several investigators (32, 3~38, 46) ha~Je suggested that myosin light chain subunits are initially released after irreversible ischemic iniury from an unassembled intracellular light chain pool. The earlier observations of Horvath and Gaetjens (44), Morkin et al. (4S) and Zak et -~
al. (47) lend support to the existence of a sarcoplasmic pool of myosin light chains in both rabbit skeletal muscle and rat heart myocytes.
Presumably, sarcolemmal damage produced early in the course of ~-ischemic injury leads to the release into the circulation of these unassembled light chains, along with other cytosolic components of the myocyte (CK-MB, ~DH, myoglobin, etc.).

The additional proof for the existence of a soluble pool of unassembled LC1 within the sarcoplasm comes from the studies of Samarel et al. (46). Using antibodies specific for both light and heavy : . , , - ., -. : ~
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WO 90/15329 PCr/CA90~00177 2~64~7 6 chains of myosin, in a well-characterized model for the rapid production of global myocytic cellular injury (the calcium paradox in isolated perfused rat heart), they demonstrated that a soluble pool of unassembled cytoplalsmic light chains exist and is rapidly released 5 from perfused rat heart following sarcole~unal disruption produced by the calcium paradox.

To define an appropriate therapy and to test its efficacy, clinicians require a precise detection and localization of myocardial infarction and a retrograde evaluation of the extent of damage.

The EP-205177 patent application discloses a monoclonal antibody which recognizes the common antigenic determinant of myvsin light chain in human serum of a patient with muscle disease.
This is a radioimmunoassay and radioisotopes such as 125I, 13lI, 3H and -~
l4C, etc. are used.
15Isoto~e Half-life 3H 12.3 years 4C 5,760 years -25I 59.7 days `-~3lI 8.1 da,vs The half-life of a radioisotope is the time it takes for half a mole of that radioisotope to be eliminated.

In spite of the success of the radioimmunoassay technology, the growing concern of the hazards and dangers of radioactive substances, problems of radioactive waste disposal and legislation, which restricts the use of radioisotopes, have fueled the search for alternative label.

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Further, for this EP~205177 radioimmunoassay, an overnight incubation period is also needed, which might be too long for a cardiac patii~at.

As stated in the Merck Manual (Fifteen edition (1987) pp. 489-5 490), in typical MI (myocardial infarctisn), the di.;gnosis is evident from the history, confirmed by the initial ECG and its subsequent evolution, and supported by the serial enzyme changes. In other instances, a definitive diagnosis may not be possible, and patients must be classified as having had a "possible" or "probable" MI; usually the clinical 10 findings are typical or strongly suggestive, but objective confirmation from the ECG and enzyme assay is lacking. If clinical suspicion was strongly based on a characteristic history, most will have suffered a small infarction. -It is wise to c~)nsider MI in all men over age 35 and all women 15 over age 50 when the major complaint is chest pain. It must be differentiated from the pain of pneumonia, pulmonary embolis~
pericarditis, rib fracture, cos~ chondral separation, or chest muscle tenderness after trauma or exertion. Patients often interpret the pain of infarct as indigestion, and evaluation may be difficult, since many have 20 coexisting hiatus hernia, peptic ulcer, or gallbladder disease. Although some relief of the pain of infact-on by belching or follo~ , antacid ~j~
therapy is common, such relief is usually brief or incomplete. Other conditions to b~ considered include acute aortic dissection, renal stone, sp.enic infarction, and a wise variety of abdominal disorders.

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WO 90/15329 PCl/CA90/OOt77 20~6407 Treatment Treatment is designed to: (1) relieve distress, (2) reduce cardiac work, and (3) prevent and treat complications (which are discussed separately, below).

5 Prehospihl treatment: Since 50% of deaths from acute MI occur within 3 to 4 h of onset of the clinical syndrome, the first few hours of management are critical. The major factor causing delay of treatment is the patient's denial that the symptoms represent a serious, potentially life-threatening illness. The immediate threat to life IS primary 10 ventricular fibrillation (fibrillation with previous ventricular premature beats) or, occasionally, heart block or profound bradycardia with consequent hypotension that initiates cardiac arrest. Optimal early management includes rapid diagnosis, alleviation of pain and apprehension, stabilization of heart rhythm and BP (blood pressure), 15 and ~ransportation to a hospital with a monitoring unit.

Most of the time, myocardial infarction are lethal and if there could be a method to detect it at its early stage, many lives could be saved.
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It would thus be highly desirable if there could be provided a 20 mean to rapidly detect early myocardial infarction in a patient.

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WO 90/15329 PCr/CA90/00177 9 20~6~07 SUMMAl~Y OF THE IN~rENTION

Surprisingly and in accordance with the present invention, there is provided rapid enzyme immunoassays for the detection of myocardial infarction.

There is provided in accordance w~th the present invention a sensitive and specific enzyme linked immunosorbent assay (ELISA) for the rapid measurement of human cardiac myosin light chains in serum and urine of patients with myocardial cell death, for diagnosis of myocardial infarction. -The precise detection and localization of myocardial infarction and a retrograde evaluation of the extent of damage is so achieved through the m~asurement of human cardiac myosin light chain in patients.

This method enables the clinicians to know the diagnosis withln a 3 hour period. This method comprises the incubation of a mixture comprising at least an anti-HVLCs monoclonal antibody, an -:
enzyme, a ventricular light chain (VLCS) antigen and an unlcnown amount of HVLCs ~nalyte.

The HVLCs analyte is obtained from the patient's serum or urine. The anti-HVLCs monoclonal antibody or the VLCS antigen is either direc~ly or indirectly detected using an enzym~bound label.
Thereafter, the amount of serum HVLCs is calculated from a standard curve.

wo 90/l5329 pcrlcA9o/oo177 2Q~ Q~ lo The anti-HVLCs monoclonal antibody has specificity to at least HVLCI or HVLC2. Preferably, a competitive method of the present invention is either a solid-phase or a liquid-phase assay. The solid-phase method mainly consist of a solid-phase bound anti-HVLCs 5 antibody or a VLCs antigen whereas the liquid-phase method comprises incubating the abo~ed mentioned components also with a precipitating agent in order to precipitate down the immune complex.

There is provided within the scope of the present invention a monoclonal antibody which specifically binds to VLC1 and which is 10 produced by the hybridoma cell line having the ATCC accession number HB10471.

Also within the scope of the present invention is a sandwich method for measuring HVLCs in cardiac patients. This method comprises the steps of:

a) providing a first anti-H~lLCs monoclonal antibody attached to a solid surface;

b) contacting the antibody-coated solid surface with the sarnple to be tested for a sufficient time to allow an immunologic reaction to occur; and c) contacting the coated-surface according to step b) with a second polyclonal or monoclonal anti-HVLCs antibody.

Also within the scope of the present invention is a diagnostic kit for measuring HVLCS in a sample, adapted to be used according to the method of the present invention.

SU~STITUTE S~EE~

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5 6 ~ 0 7 Surprisingly, the method ol .e present invention will allow, through its specific antibody against HVLCI and HVLC2 and its enzyme-bound label, for a rapid and precise measurement of HVLCS.

Th ~ unexpected early detection of myocardial infarction enables many lives tobe saved.

Other advantages ~ the present invention will be readily illustrated by referring to the following description.

IN THE DR~WINGS

Figure 1 shows a schematic representation of direct ~Fig la) and indirect (Fig lb) competitive ELISA described Ln assay 1.

Figure 2 shows an antibody dilution curve in ELISA.

Figure 3 shows an ELISA calibration curve for DVLC~.
:
Figure 4 shows levels of HVLC~ in sera of patients with myocardial `
infarction and in a rat with _xperimentally induced myocardial 15 infarction.

Figure 5 shows a double sandwich ELISA for DVLCs .

Figure 6 shows a direct competitive solid phase ELISA with immobilized antibody.

Figure 7 shows an indirect competitive solid phase ELISA with immobilized antibody and anti-idiotypic antibody as serrogate of ~ `
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wo so/~s~2s Pcr/t:A9o/ool77 2Q~fi~7 12 I~ETAILED DESCRIrTION OF THE INVENTIQN

The present invention relates to a novel method of rapidly detecting myocardial infarction. This method comprises using cardiac myosin light chains as cardio-diagnostic markers since myosin is the 5 most abundant contractile protein of a myofibril, and cardiac myosin light chains are structurally biochemically and anti-genetically different from skeletal and smooth muscle myosin chains.

These cardiac muscle-specific contractile proteins are ideal markers as their elevations in serum indicate damaged or necrotic 10 myocardium only.

There were raised rabbit polyclonal and mouse monoclonal antibodies to cardiac myosin light chain I and in turn mouse monoclonal anti-idiotypic antibodies to rabbit and mouse anti-myosin light chain I antibodies that represented an internal image of the 15 myosin light chain. Purified, characterized and labelled with enzymes, these antibodies were used to construct solid phase competitive or double-sandwich ELISAs to measure serum levels of cardiac myosin light chains.

There is prepared a monoclonal antibody which specifically 20 binds to VLC1 and which is produced by the hybridoma cell line having the ATCC accession mlmber HBI0471.

In the first one, patient serum light chains compete with known amount of standard light chain preparation for binding to a limited number of sites on anti-light chain antibodies. The same 25 sample light chains, if present, will lessen the amount of bound SUBSTITUTE SHEET

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-WO 90/15329 PCl/CA90/00177 13 ~ . ,, 20~;L07 standard light chains. In the second assay, myosin light chains in the sample compete with anti-idiotypic antibody for the binding to anti-myosin light chain antibody, provided anti-idiotype is the internal image of the antigen.

Standard light chain (VLCs) may be selected from the group consisting of: HVLC~, HVLC2, bovinel, bovine2, porcinel, porcine2, caninel, canine2, ratl, rat2, mousel, mouse2, rabbitl, rabbit2, or from any other philogenetically related species. It can also be a peptide representing the epitope on ventricular myosin light chain 1 or 2, complementary to the antigen binding site on the anti-HVLCs antibody.

The anti-HVLCs antibody or the VLCs antigen is either directly or indirectly detected by means of enzyme-bound label.

At least 25 different enzymes have been employed as labels in enzyme immunoassay (Table I). Enzymes have a number of advantages over other types of label (Engvall, 1980; O'Sullivan et al., 1 379a; Wisdom, 1976):

1. They are relatively cheap, are readily available in a purified forrn, and have a long shelf life.

2. A range of assays are available for measuring enzyme activity, and these can be perfossned on readily available equipment. Many of these assays can also be automated.

3. A single enzyme label can transform many molecules of substrate into product. This amplification effect provides the basis of very ~5 sensitive assays for enzymes. Catalase has a particularly high substrate-.. . . .
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20~6407 14 turnover rate: 30,000 umol hydrogen peroxide/min/mg of enzyme (Haining and Legan, 1972; Zidoni and Kremer, 1974). Unfortunately, it is difficult to label other molecules with this enzyme without loss of activity, and substrates are not available that give readily measurable 5 produc~s (Yolken, 1982).

4. Enzyme activity can be modulated (e.g., by antibody); thus nonseparation enzyme immunoassays are possible.

The preferred enzyme used as label are: horseradish peroxidase, alkaline phosphatase, $-galactosidase, glucose oxidase and urinase.

The solid phase competitive method of the present invention has the anti-HVLCs antibody or the VLCs antigen solid-phase bound.
The solid support of ~is solid phase assay can be selected from any one of the following: a rnicr~titration plate or brealcable strips (poly~inyl, polychloride, polystryrene, etc.), plastic beads, plastic tubes, latex 15 particles, magnetic particles, nitrocellulose or acetate membranes and dipstick.

The liquid phase method of the present invention comprises incubating also with a precipitating agent in order to precipitate down the immune complex. The preferred precipitating agent is selected 20 from the following: anti-mouse Ig anti-serum, immunobeads, immunolatex, pansorbin A, matrix bound protein A and G, polyethylene glycol, and immuno-magnetic particles.

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WO 90/l5329 . ? O S 6 ~ o 7 pcr/cA9o~ool77 TABLE I E~Z~:
Oxidoreductases Malate dehydrogenase Glucose~phosphatase dehydrogenase ~, Glucose oxidase `
Catalase Horse~adish peroxidase Microperoxidase Firefly luciferase Bacterial luciferase Transferases Hexokinase ~Phosphofructokinase Pyruvate kinase Phospho~lucomutase -Ribonuclease A
Hydrolases Acetylcholineesterase Alkaline phosphatase 23 Phospholipase C
~-Amylase ;:
Giucoamylase Lysozyme Glucosidase g Galaosidase Invertase Urease Adenosine dearninase Lyases Carbonic anhytrase Isomerases 5.3-Ketosteroid isomerase _ wo 90Jl5329 , Pcr/cAso/oot77 ~2 0 ~ 16 Also within the scope of the present invention is a kit for measuring HVLC in a sample which comprises three different components:
1) a solid surfaoe having bound thereto an an~-HVLCs antibody;
5 2) a known amount of VLCs antigen;

3) an other HVLCs antibody which recognizes a different epitope on said HVLCS or an anti-idiotypic antibody which has specificity to the first anti-HVLCs antibody and which is detected by means of an enzyme-bound label.

The patient's sam~le is equivalent to the second component and is analyzed in parallele with the standard VLCS antigen.
1) Patient's HVLC Standard VLC solid surface coated with + + anti-HVLCs antibody V adding patient's adding component 2 15sample 3) incubating 4) adding component 3;
5) adding enzymic substratei 6) reading the absorbance.

The standard VLC (known arnount) reading gives a calibration curve, whereas the unknown amount of HVLCs ~patient's sample) is read on the calibration curve.

~ ' '~ ' ' WO 90~1~329 17 2 0 ~ ~ 4 o 7 PCr/CA90/00l77 .. . .
DESIGNINC; ENZYME LINKE~ IMMUNOSORBENT AS~Y FOR VL C
=1 Four different approaches are possible.
Assay 1: Direct or indirect competitive ELISA, where serum or HVLC2 compete with solid phase bound VLC1 or VLC2 accordingly, for binding to the corresponding antibody in solution. The amount of replaced HVLCs, corresponding to the serum HVLCS, is calculated directly from the standard curve.

Assay 2: Double sandwich ELISA, where serum H~LCs bind to an antibody coated solid phase and is then detected with the second antibody directed to a different epitope on HVLCs.

Assay 3: Direct or indirect competitive ELISA where serum HYLCs compete with enzyme labelled VLCs in solution for binding to a solid phase bound anti-HVLCs antibody. The amount of serum HVLCs is calculated from the standard VLCs curve.

Assay 4: Direct or indirect competitive ELISA, where serum HVLC ~;
competes with anti-HVLCs an~i-idiotypic antibody (anti-Id) for binding to a solid phase anti-HVLCs idiotype (Id) and the amount of replaced anti-idiotype corresponds to the serum HVLCs level.

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: .' ' .' ~, WO 90/1~329 PCI`/CA90/00177 20S6~7 ~cam~le I
I. ISOLAllON ~ND PURIFICAllON OF ~ C LIGHT CH~I~ E~2kI
~N~NE. PO~C-NE OR HIJMAN LEFr VENT~lCLE

All steps are performed at 4C. Porcine, canine or human left ventricular muscles are stored in 50% glycerol in 0.06 M KCl, 0.02 M KP
pH 6.5, 0.005 M g-ME, 0.005 M Mgcl2~ 0.0002 M PMSF. The tissues are minced in a cold meat grinder previously washed wi~h 0.2 M EDTA pH

7.0 and a res~ting mince homogenized in 5 volumes of 0.05 M KH2P04 pH 6.8, 0.001 M EDTA, 0.01 M NaPPi pH 7.5, 0.001 Drr, 0.03 M KCl, 0.01 M
MgC12, 0.0001 M PMSF (solution 1) for 1 min. at 60 revolutions (27000 rpm) in Virtis 45~9 homogenizer.

The homogenate is centrifuged for 10 min. at 8000 rpm (Sorvall GS-3~9) and the pellet washed once in 3 volumes of solution 1 and then once in 1% Triton~ in solution 1, followed by five washes in solution 1 to remove traces of Triton~9. Each time, the pellet is resuspended by brief (3 sec.) homogenization at 27000 rpm. The final pellet is homogenized in 400 ml of 0.1 M KH2PO4, 0.03 M KCI, 0.01 M NaPPi, 0.001 M Dl r, 0.001 M EDTA, 0.05 M }~H2W4, 0.05 M MgCI2, 0.00035 M AlP, 0.0002 M PMSF, 1 ug/ml pepstatin A, pH 7.5 and extraction continued for 15 min. while stirring.

Actomyosin containing supernatant obtained after centrifugation for 15 min. at 8000 rpm (Sorvall GS-3~) is filtered through a cheeæ cloth and spun again for 20 min. at 9000 rpm (Sorvall GS-3~). The actomyosin is precipitated from the supernatant with 9 volumes of H20, 0.001 M EDTA and collected by centrifugation at 9000 rpm (Sorvall GS-3~) for 20 min. The pellet is resuspended by , . .

.

6 ~ 0 7 homogenization in a glass Poter homogenizer~) in 87 ml of 0.05 M
NaPPi, pH 7.~, 0.001 M Drr, 0.001 M E~TA, 0.005 M MgC12, 0.002 M All', and protein concentration determined by extinction co-efficient (E280 nm for myosin = 5.6). The myosin solution is made 2 mg~ml and contamLinants (actin and tropomyosin) precipitated with 34% (NH4)2S04, 0.001 M EDTA, pH 6.8 for 30 ~in. s'drring.

The supernatant, containing myosin, is collected by centrifugation for 25 min. at 10,000 rpm and precipitated again with . 42% (NH4)25O4 for 30 min. stirring. This time the myosin containing precipitate is collected by centrifugation at 9000 rpm (Sorvall ~3~)) for 30 min. I~e pellet is resuspended in small volume of 0.05 M Tris/HcL
pH 7.5, 0.5 M KCl, O.OOl M DTT and dialyzed against 200 volumes of the same solvent O/N at 4C, with three buffer changes. The dialysate is then spun at 10000 rpm (~ ~rvall SS-34~9) for 10 min. and myosin containing supernatant precipitated with 9 volumes of d.H2o, 0.001 M
EDTA. The precipitate is spun at 2000( ?m (Sorvall S~34~)) for 15 ~un. ;
To dissociate light from heavy ch~ins of myosin, the pellet is resuspended in small volume of 8 M urea, 0.001 M Drr~ 0.01 M EDTA, 0.05 M Tris, pH 7.5 and stirred for 2 hrs at RT. Heavy chains are precipitated wi~ 10 volumes of cold d.H2o, 1 mM EDT~, pH 7.5 O/N at 4C and collected by centrifugation at 20000 rpm (Sor~all ~34~D) for 10 min.

The light chains containing supernatant is collected by centrifugatior. and is concentrated by membrane ultrafiltration (PMIO~19, Arnicon'~). Froteins are determined by the well known Lowry assay.
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~: : . -- -wo sa/ts329 pcr~cA9o/oo177 ~ -, 20 20~6407 Myosin light chain 1 is separated from light chain 2 on SD~
PAGE and then is electroeluted. 12.5% acrylamide sodium dodecyl sulfate polyacrylarnide gel electrophoresis ~SDS PAGE) is prepared with one large well across the stacking gel. Elelctrophoresis is run at 14 mA
5 overnight. Five mg myosin light chain preparation is loaded per gel.
After the run has been completed, a strip of gel is cut off and stained in Coomassie Blue as a reference. Both light chains are cut out separately and placed into 50 mm dialysis tubing containing 4 ml of O.Z M
Tris/acetate at pH 7.4,1% SDS and 0.001 M Dl~. The tubing is then placed into a horizontal electrophoresis system (Fisher-FB-LHU~I9 1390) filled with running buffer containing 50 mM Tris/acetate at pH 7.4, 0.1% SDS. The system is run at 60V overnight at room temperature.
Gel is removed from the stack and stained with Coomassie to verify the completion of transfer. The content of bags is then dialyzed against 1~ several changes of Tris/acetate buffer.

~xample II
Preparation of antigens for irnmunization Coupling of ventIicular light chains to KLH

To increase the antigenecity of LCI, canine (DVLCl) myosin 20 light chain 1 is conjugated to a carrier protein, keyhole limpet haemocyanine (K~H) according to the instructions of the manufacturer (Cambridge Research Biochemicals Inc.) Briefly, to one vial of KLH (8 mg) 500 ~ll of 0.1 M sodium hydrogen carbonate buffer (pH 8.4) is added and mixed gently. Ten mg 25 of DVICI are dissolved in 1 ml of 0.1 M sodium bicarbonate buffer at ' ~ .-.. .. ., . ~ . . .

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. . . -WO 90/153~9 PCI`/CA90/00177 21 20~6~07 pH 8.4 and added to a cleaned vial containing KLH, 2.5 ml 0.1 M
carbonate buffer, 10 ~g glutaraldehyde (Sigma Chemical Co.). The vial is sealed and stirred for ~4 hours at room temperature. The conjugate is then dialyzed in 30 an tubing against saline for 2~48 hours at 4C (2 5 X 41), while stirring.

~am~ III
Enzyme-linlced immunosorbent a~say (ELISA) Standard for anti-VLCl antibodies Titration of rabbit or mouse immune sera as well as the 10 detection of anti-DVLCI /PVLCI (porcine VLCI)monoclonal antibodies in tissue culture supernatants is performed in ELISA. Antigens (DVLCS, PVLCs or PSkMLCS (porcine slceletal muscle LCs)) are first conjugated to ovalbumin to increase the binding of LCS to plastic Briefly, 200 ~g VLCl in 200 ~ll of PBS are mixed wlth 600 ~lg of ovalbumin (200 ~ll), 200 )lg of EDC (Ethyl-Dimethylaminopropyl-Carbodimide) (100 ~l) and 200 ~lg sulfo-NHS (N-hydroxysulfo, succinimide) (100 ~1) in PBS and are incubated overnight at 4C. The reaction is stopped by the addition of 3 ml of PBS at pH 7.0 and the conjugate dialyzed against several changes of PBS. The volume is 20 reduced by speed-vacuum centrifugation.

Antigen-ovalbumin conjugates are then immobilized on polystyrene microtiter plates (Immulon 1~ sold by Dynatech Labs) at a concentration of 10 ~g/ml in 0.1 M sodium carbonate/bicarbonate buffer at pH 9.6 overnight at 4C. The excess of antigen is removed by 25 washing with Tris-saline (TS)-Tween69 and the remaining binding sites are saturated by a one hour incubation with 1% milk in TS. Immune - . ~ - 1 . -:
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- . - . - . -.- , . - - , , ,,, , ~, . .~ , .. .;. . -WO 90/15329 PCI'/CA90/00177 213~6~Q~ 22 sera or tissue culture supernatants are diluted in T~ulk and reacted with antigen for 1 hour at 37C. After four washes, the plates are incubated with peroxidase-conjugated anti-mouse IgC; (H+L) (E~ioCan Inc.) diluted in T~milk for 1 hour at 37C. The non-bound antibody is 5 washed off and enzymatic ac~vity of peroxidase is revealed with 0.03%
H22 in a chromogen solution ~phenylenediamine at 1 mg/ml of 0.1 M Na citrate buffer, pH 5). The colour reaction is stopped after a 3û
minutes incubation by the additon of 3 N H2SO4 and the absorbance is read in an automatic reader at a wavelength of 492 nm.
~xam~21e IV

PREPA~ATlON OF RA8BlT ANTI{~ARDIAC MYOSIN LIGHT CHAI~I 1 ANTrBODlES

Immunization protocol Antisera are raised in New Zealand White rabbits by injection 15 of 100 ug dog ven~ricular light chains 1 (DVLCI) coupled to KLH and emulsified in Freund's complete adjuvant. The first injection is equally divided between one intra-muscular (i.m.~ site and three intradermal (i.d.) sites. Each booster of 100 ug DVLCI-KLH emulsiffed in incomplete Freund's adjuvant is given i.m. at 2 week intervals. Serum 20 is collected 1 week after the second and each subsequent booster and titered against DVLCI and human ventricular light chains 1 (HVLCI) in ELISA.
'~ .'.' Isolation of immunoglobulins from rabbit antiserum Blood, collected from an ear vein, is left at RT for 2 hrs to clot.
25 The walls of the tube are rimmed with a wooden stick to loosen the - --, -,.- . .. . ..
- ... - . . ..
-. - ,., .. , . - -, ,.. .- , .. . .

wo 91)/15329 PCI/CA90/0017~
23 2056~07 fibrin clot and the clot is cut in~o pieces and left O/N at 4C. To obtain serum, the blood is spun at 3000 rpm (Sorvall SS-34~) for 30 min., serum pipetted out and spun again. Serum is dialyzed against 0.1 M
sodium phosphate buffer, pH 7.2, ~.02% NaN3 an ~.iltered sterile. It is 5 applied to Staphylococcal protein A-Sepharose 4B~ ~SpA) column previously equilibrated with phosphate buffer, at 10 ml/hr according to the method of Hjelm et al. (PEBS Letters 1972;28;73-75). Unbound proteins are washed off with the column buffer and bound immunoglobulins with 0.1 M glycine/HCl, pH 3.0, 0.15 M NaCl into tubes containing 1 M Tris/HCl pH 9Ø IgG is immediately dialyzed ~ -overnight against PBS at 4C, concentrated and stored at -2ûC
. . ".

~am~le V
~ ~$012Y F~

1. Immunization protocol The Balb/c mouse used for this fusion is injected with 100 llg DVLC1-KEH conjugate emulsified with CFA (Complete Freund's Adjuvant) for the primer and with IFA (Incomplete Preund's Adjuvant) for 4 booster in,ections. Inoculatio}~s are administered i.p. at 2-3 week intervals. Three days before fusion, the animal is boosted i.v.
with the conjugate alone.

2. Fusion and antibody production 11.5 x 106 spleen cells are fused with 1.15 x 106 myeloma cells at a ratio of 10:1 with 50% PEG 4000 containing 5% DMSO at pH 7.4 according to the method of St. Groth (4). The viability of cells after fusion is 33%. The fused cells are plated into 10 96-weli plates in HAT
medium supplemented with ECGS at 5 x 104 cells/well and over a ' ~ ~ '. -- ' : ~
... . . .

WO 90~15329 PCI-/CA90/00177 20~6~07 2~ .
spleen cell feeder layer. Seven days later and then every 5 days, cells are fed with HAT medium. Twenty five days after fusion clone FsPgAs is tested in ELISA against PVLCS and PSkMLCS and are found to be strongly positive for PVLCl and weakly positive for PSkMLCS. It is 5 further minicloned in HT medium into 5 wells of a 96-well plate and then one of the wells into a further 10 wells of a 96-well plate. Cells ~:
from one of these 10 wells is expanded gradually into wells of a 24-well plate, 6-well plate, 25 cm2 T-flask, 75 cm2 T-flalsk and finally into 150 cm2 T-flask in DMEM, 10% FBS. Fs IgG is isolated from either tissue 10 culture supernatant or ascitic fluid. For scaling up the production, a few Lifecell(E) bags (Baxter Inc.) are inoculated with 0.1 x 106 cell/ml and grown batchwise for 10 days. Bags are emptied at the end of the culture, cells are collected by centrifugation and supernatant concentrated by membrane ultrafiltration for subsequent purification. ~ -For ascites production, 10 days prior to the injection of cells, 0.5 ml of pristane is inoculated i.p. (intraperitoneal) into a BALB/c mouse and then 3.2 x 106 alive hybrid cells injected i.p.
Fs (FsPgA2) clone is subcloned by limiting dilution once. One subclone, named PlB6, is selected for its specific reactivity in ELISA with PVLCl-ov and no cross-reactivity to PSKLCs-ov. Hybridoma FsPgA2-PIB6 (ATCC HB10471) is abbreviated to Fs.

3. Purification of F5 Immunoglobulin Harvested tissue culture supernatant is concentrated 7 fold and precipitated with 50% (NH4)2S04. Fs IgG is recovered in the precipitate after centrifugation a~ 9000 rpm for 30 min. The precipitate or ascitic fluid is dialyzed against protein A buffer (3 M NaCl, 1.45 M glycine pH
9.2) and applied to Sepharose~) 4B-protein A column. Pure IgG is SUBSTITUTE S~EET

.. ..
. .

WO 90~15329 2 o 5 ~ 4 o 7 Pcr/cA90/00l77 25 ; ! , j eluted with low pH buffer such as 0.1 M sodium citrate, pH 3.0, nutraliæd and dialized against PBS

4. DeteImination of F5 immuno~lobulin cla~
The immunoglobulin class of Fs antibody is determined in Ouchterlony~ double immunodiffusion against commercially available anti-immunoglobulin antisera. It was found to be IgGl K.
:
5. Determination of Isoelectrofocusing point (IEF) of Fs IgG
IEF is performed on Phast System~9 (sold by Pharmacia) using pre-cast gels and ampholyte pH standard of ~9. The isoelectric point of Fs is in the pH range of 6.55 - S.85.

6. lmmunospecificity of F5 IgG in ELISA
Specificity o r5 antibody is tested in ELISA under the antigen ;~
excess conditions. Fs IgG reacts with human, porcine or canine VLC
but not with VLC2 nor with myosin heavy chains.
The degree of Fs cross-reactivity to PSkMLC1+2 is calculated from Fs IgG dilution curves in ELISA. Wells are coated with 10 ~lg/ml (Table II), PVLC1-ovalbumin and PSkMLCI +2-ovalbumin and incubated with differen~ quantities of F~ IgG to construct antibody dilution curves. Immune complexes are detected with HRP anti-mouse IgG (1:5000). Fs shows no cross-reactivity to skeletal muscle myosin light chain.

Ps-PlB6 anti-PVLCl IgG dilution cunre in ELISA tested against PVLC~ valbumin and PSkMLCs-ovalbumin Microtitration wells (immulon 1, Dynatech Inc,.) are coated with 10 ~lg/ml of either PVLCl-ovalbumin to-o) or PSkMLCS-.... . . .
- . ~

, ~:. . . . . . . ..
, `: :

WO 90~15329 PCl'/CA90/00177 20~6~1'7 26 ovalbumin (~o) overnight at 4C. The remaining binding sites are blocked w~th 1% TS skim miL~c and the wells reacted for 1 hour at 37C
with Fs IgG added in varying amounts. The bound antibody is detected with pero~tidase-anti-mouse IgG conjugate. The colour reaction is 5 developed upon addition of H22 as a substrate in OPD. After 30 min the blocking agent (2 N H2S04) is added and absorbance is read at 492 nm.

TABLE II
Fs ~ OD ~92 nm ng/ml PVLCl~v PSkMLCs-ov .

0.01 0 0.01 0 0 03 0.01 -0.05 0 0.12 0.03 0.18 0 -- 80 0.24 0.01 200 0.86 0 400 1.92 0.02 Abbreviations: PVLCI - porcine ventricular myosin light chain 1 ov - ovalbumin PSkMLCs - porcine skeletal myosin light chain 1~2 ~ Fs - antibody secreted by the hybridoma F1PgA2-PlB6 Cross-reactivity to SkMLCs is also tested in a competition ELISA, where free light chains compete with solid phase bound light chains for binding to Fs in solution. First, the conditions for PVLCI
binding are established. Immulon 10 plates are coated with 1, 0.45 and 30 0.1 llg/ml concentration concommitantly with free PVLC1 at increasing concentrations (0-10 ~g/ml). The percent inhibition of binding of Fs to a solid phase bound PVLC1 by free psrLcl is calculated. The best results are obtained when PVLCI concentration for coating is 0.45 llg/ml and - ... .
: .. . . ~ . . -.,. ~ :

wo 90/ls329 PCr/CA90/OOt77 27 2~)!ss~407 ':5 0.1 ~lg/ml. The curve is linear in PVLC range of 50 to 600 ng/ml and quantitites as small as 50 ng/ml of P~C~ could be detected in -unknown samples of patients' sera.

To calculate the degree of cr~)ss-reactivity of Fs with SkMLCs, 5 ELISA plates are coated with 0.45 ~lg/ml and 0.1 llg/~ of PVLCl-ovalbumin and incubated with Fs IgG at 0.1 ,ug/ml togethe; with either PVLCI or PSkMLCs at different concentrations (Table m). PSkMLCs do not displaoe Fs IgG from binding to P~Cl. Thus, Fs IgC; does not cross-react wi~ SkMLCs ` -Inhibition of binding of F5 IgG to PVLCl-ovalbumin by free PVLCl or PSkMLC~ in ELlSA
Microtitration plate (Immulon 1, l:~,vnatech Inc.) is coated with 0.45 lig/ml of PVLC1-ovalbumin and incubated simultaneously with F5 IgG at 0.1 ~g/ml and either free PV~ -1 (o-o) or PSkMLCs (o-o) at 15 increasi~.g concentrations. Bound Fs IgG is detected with peroxidase anti-mouse IgG conjugate.
TA8LE I~
Antigen % max~num bindin~
ng/ml PVLCl PSk~ILCS
100 96.8 138.9 200 75.6 138.9 400 61.3 39 9 600 34.9 146.3 800 25.4 142.5 1000 21.5 150.2 2000 lC. 136.7 4000 8.4 124'~
Abbreviations: PVLCl - porcine ventricular myosin light chain 1 PSkMLCS- porcine skeletal myosin light chains 1~2 Exam~YI
PRODUCl'I~OEMQUSEMO~OCI.ONALA~ D~._C~ IDIOTYPIC

... ., .. , , ~ , ................ . .

' ` .~ ' -.. . . .

WO 90/15329 PCI'/CA90/00177 2~640'~ 28 1. Immuni~ation of mice with mouse anti-DVLC1 F5 immunoglobulins Six-to-eight week old female BALB/C mice are injected subcutaneously ts.c.) into hind footpads at 14 day intervals wi~ 50ug of mouse anti-DVLCl F5IgG-KLH conjugate. The first inoculation is 5 applied in complete Freund's adjuvant, while the subsequent ones in incomplete Freund's adjuvant. The immune sera titer is tested in ELISA against F5 IgG (idiotype) and normal mouse IgG. Animals with high serum titers are boosted i.v. with 100 ug of idiotype in saline three days before fusion.

10 2. Preparation of F(ab')2 fragments from Fs IgG

50 mg of Fs IgG in 50 ml of 0.1 M sodiu n atrate buffer pH 3.9 is mixed with 1 ml of I mg/ml pepsin (Worthington) and incubated for 4 hrs a~ 37C. The digestion is terrninated by adjusting pH to 8.0 with 1 M
Tris and dialysis against protein A binding buffer. Undigested IgG and 15 Fc fragment are removed by chromatography on HPLC-protein A.

3. Pusion and hybAdoma production Both fusion and cloning are performed as described above for anti-VLC antibodies. However popliteal and inguinal lymph node lyrnphocytes instead of spleen cells were used for fusion.
20 4. Selection of clones seaeting intemal image anti-idiotypic antibodies Anti-idiotypic antibody (Ab2 ) that structurally and/or functionally mimic the nominal antigen (DV~C) iS called an internal ~nage anti-idiotypic antibody.

` ' ,' ' : ' wo 90/15329 PCI'/CA90/00177 29 2 0~ 6~0~

Supernatants from growing clones are tested in ELISA
simultaneously against Fs anti-~VLC IgG F(ab')2 and normal mouse IgG
F(ab')2. Supematants, positive for anti-DVLC idiotype and negative for normal IgG, detected using peroxidase labelled anti-Fc antiserum, are 5 subsequently assayed for an internal image of D~LC in a competiti~re E~SA. Microtitration wells, coated with F(ab')2 fragments of Fs anti-DVLC IgG accordingly, are incubated with sample tissue culture supernatants concurrently with varying amount of DVLC standard for 1 hr at 37C. The bound anti-idiotype was detected with peroxidase an~-10 mouse IgG Fc conjugate. The hybridoma supernatants containinginternal image anti-idiotypes competed with the antigen (DVLC) for the binding sites on solid phase bound idiotype and the maximum binding (expressed as OD4g2nm) were lowered and eventually inhibited completely.

~xample VI~
Desa~iption of Assay 1 In a direct approach (Figure lA), antigen (DvLCl or DVLC2) is immobilized on a solid phase and may react with the enzyme-labelled anti-DVLC antibody (but not in excess). Free antigen (HVLCs), if present, 20 in the sample will compete for this antigen and lower the amount of enzyme bound. Free labelled antigen-antibody immune complexes will be eliminated by washing.

In an indirect approach (Figure lB), DvLCs are immobilized as above and may react with unlabelled anti-DVLC antibody. If the free 25 HVLCs are present in the sample, they will compete for the immobilized antigen and lower the amount of an~body bound. Free HVLC-anti-DVLC

:

:

wo gn/l5329 PCr/CA90/~Ot77 20~)64Q~ 30 immune complexes will be removed by washing and bound antibody detected with enzyme bound anti-immunoglobulin antiserum. In both cases, the colour reaction will develop upon the addition of substrate in a chromogen solution.

In both approaches, the amount of free HVLCs in the sample can be calculated from the calibration curve where known amounts of free DVLCs compe~e for the immobilized DVLCs. A typical calibration curve is presented in Figure 3.
Percentage maximum binding ( B/Bo) is calculated according to the formula:
% B = 100 X OD492nm in the presence of free antigen - BG
Bo OD492nm in the absence of free antigen - BG
where, BG = background binding (buffer instead of antibody) B = absorbance in the present of free, competing Bo = maximum binding (absorbance in the absence of free DVLC

In detail, polystyrene microtitration plates (Immulon 1 flat-bottom 9~well plates, Dynatech Lab.) are coated with 10 ug/ml of DVLCl in 0.05 M sodium carbonate/bicarbonate buffer pH 9.6 overnight at 4C.
The unbound antigen is washed off with TS Tween and the remaining 20 binding sites saturated with 1% low fat millc in Tris buffered saline (TS) for 1 hr at 37C. After washing, the plates are incubated simultaneously with 50 ul of either rising concentrations of DVL~ (calibration curve) or undiluted patients' sera and 50 ul of anti-DYLCl antibody at 50%
maximum binding (rabbit IgG at 4.5 ug/rnl). Antibody dilution curve is 25 prepared before hand. An example of such a curve is presented in Figure 2 where the bound antibody is detected with peroxidase labelled anti-immunoglobulin anti-serum and H22 as a substrate.

. . . ~. , : ~ :

- . . . . .

wo 90/15329 PCI/CA90/00177 31 2056~7 After the unbound antibody :igen immune complexes are washe~ .f, horse radish peroxidase (~P)-conjugated goat anti-rabbit IgG is added and incubated for an additional 1 hr at 37C. The colorime~ic reaction is developed upon the addition of 30% H22 (1 ul/ml) in 0.1 M sodium citrate buffer pH 5.0, containing 0.1% ~
phenylened~amine dihydrochloride (OPD). The enzyme-substrate reaction is stopped by 3 N H22 and the absorbance read at 490 nm.

After the percentage of maximum binding has been calculated for each patient, the amount of free HVLCl in patients sera is extrapolated from the calibration curve. The calibration curve from one of such experiments is presented in igure 3. The kinetics of ventricular light chain 1 (H~ ~C~ lease into circulation after myocardial infarction is foll .~ ed in two patients with oven myocardial mfarction. The releas~ of HVLCI (Figure 4) is biphasic. After an initial rise on day 1, the level of HVL - drops to normal -ange and then rises a~ain and persists for a few days. The initial peak probably represents t~e endogeneoL p~ol of unassembled HVLCI and the subsequent one, the gradual release of HVLCl due to proteolytic degradation of myosin.

The hike in VLC- is e~en more pronounced in a rat isoprotrenolol induced ~A mode, (Figure 4), demonstrating that the described assay can accurately measure serum VLCl.

Example VIII

In detail, polysterenè microtitration plates (Immulrn~ 1 flat bottom 96 well plates, Dynatech Labs) are coated ~lth 0.45 ~g/ml of .
.

. ' ' ' ~

wo 90/15329 PCr/CA90~00l77 PVLCl~valbumin (porcine ventricular light chain 1) in 0.05 M sodium carbonate/bicarbonate buffer pH 9.6 overnight at 4C. The nonbound antigen is washed off with l~Tween~ and the remaining binding sites saturated with 1% low fat miL~c in TS for 1 hr at 37C. After washing, the plates are incubated simultaneously with rising concentrations of PVLCl (calibration curve) and anti-PVLCI Fs antibody at 0.15 or 0.3 g/ml.

The nonbound antigen antibody immune complexes are washed off and horse radish peroxidase (HRP)-conjugated goat anti-mouse lgG added and incubated for an additional 1 hr at 37C. The colormetric reaction is developed upon the addition of 30% H22 (1 Ill/ml~ in 0.1 M sodium citrate buffer pH 5.0, containing 0.1~ o- - -phenylenediamine dihydrochloride (OPD). The enzyme-substrate reaction is stopped by 3 N H2S04 and the absorbande read at 492 nm.
Percentage maximum binding ( B/Bo) is calculated accord;ng to the formula:
% B = loo X OD492nm in the presenceof freeantigen- BG
Bo OD492nm in the absence of free antigen- BG
where, BG = bac}cground binding (buffer instead of antibody~
1~ = absorbanoe in ~e present of free, competing PVLC
Bo = maximum binding (absorbance in the absence of free PVL

A calibration curve is constructed by measuring out PVLC
concentration on x-axis and % B/Bo on y-axis and is presented in Table IV.
:~ .

- . . - : .
, . . .. . .. . . . .. .. . . .. . ..
~ ., : . .. . .

.: . , .
, . . , : .-.. , -- ; .. , . ~ ~ .
- i - .

WO 90/15329 2 ~ ~ 6 4 0 7 Pcr/cA90/00177 33 j Takle lY
Inhibition of binding of F5 IgG to PVLC~ by free P~LCl in ELISA
.
PVLCI concentration % maximum binding (ngSml) 0.3 ~g/ml F5 IgG û.15 ~g/ml F5 IgG
.
510 000 2.40 1.47 8 000 3.75 2.67 ~, ooo 3.94 2.27 4 000 9.13 7.35 2 ~00 16.71 11.50 10 1 000 17.10 18.32 ~W 21.42 18.85 600 28.05 25.00 400 38.62 æ.46 200 47.84 24.47 100 67.15 47.19 82.80 61.63 81.94 6g.39 :-- 60 89.91 67.91 95.00 94.79 107.59 98.66 104.~1 87.83 99.81 96.~2 102.59 92.25 109.03 95.52 115.95 94.25 .
102.02 98.66 Microtitration plate is coated with 0.45 ~g/ml of PVLC1-ov and incubated with F5 IgG at 0.1~ llg/ml and 0.3 ,ug/ml in parallel with free PVLCl at increasing concentrations.

~cam~le IX
Description of Assay 2 Double sandwich ELISA is depicted in Pigure 5. Pirst anti-DVLC
antibody is bound to a solid phase and then reacted with standard DVLC
preparation or an antigen contained in a serum sarnple. The bound 35 antigen is detected with enzyme labelled second anti-DVLC antibody .
:: . : , -; - .

.
~. . . - .

9 PCl /CA90/0017~
2~6407 that recognizes non-overlapping epitope on DVLC molecule. The amount of antigen in the sample is determined directly from the standard curve.

In detail, microtitratlon wells (Immulon~)1, Dynatech Labs) are coated with 0.5 llg/ml of F5 IgG F(ab')2 in 0.1 M sodium carbonate/bicarbonate buffer pH 9.6 overnight at 4C. The excess of F(ab')2 is washed off with T~Tween~ and ~e remaining binding sites saturated with T~1% low fat milk for 1 hour at 37C. Non-bound antigen is washed off and the bound one sandwiched with rabbit anti-DVLCI polyclonal IgG at 1 ~lg/ml for 1 hour at 37C The excess of second antibody is removed by washing and bound rabbit antibody detected with peroxidase conjugated to anti-rabbit IgG antiserum diluted 1:4000. Substrate for peroxidase (H202) in OPD solution is added for 30 min and the enzym~substrat~ reaction is stopped by 3 N H2SO4 -and ~e absorbande read at 492 nm. Results are presented in Table V.

Table V
PVLC1 standard cur~e results in 2 double sandwich Fs-rabbit anti-DVLC1 EL1SA
PVLC1 concentration O.D. 492 nm (ng/ml) ~.
1.6 1.56 2.18 0.8 0.44 0.07 . .
Microtitration wells are coated with 0.5 ~Lg/ml of F5 F(ab')2 overnight at 4C. Excess of antibody is washed off and wells are incubated with PVLCI in varying amounts. Bound antigen is detected -- - , .. . ~.

WO 90/153t9 PCI/CA90/00177 35 2~6407 with 1 ~Lg/ml rabbit anti-DVLCI IgG and then with HRP anti-rabbit IgG
diluted 1:4000.

Description ~f Assay 3 The competitive, solid phase ELISA is also possible when antibody is immobillzed o n the solid phase. The principle of this direct assay is presented in Figure 6.

In this case, anti-~rLC1 antibody is immobilized on the solid phase which binds enzym~labelled antigen (VLCl) or if present, antigen contained in the serum sample. Unlabelled antigen in the sample will compete with the labelled ar ~,en for limited binding sites on antibody and will lower the amount of enzyme bound. Unbound labelled antigen is washed off and enzyme substrate added. HVLCs in sera samples will be calculated from the replacement curves.

VLC1 can be labelled either directly with an enzyme ~such as peroxidase), or indirectly with biotin and avidin-biotin-peroxidase complex. Biotin-avidin-biotin bridge method has several important advantages over direct peroxidase labelling; (i) av :in has an exceptionally high affinity for (+)-biotin (1015 M~ ii) (+) - biotin is very easily coupled to an~. bodies and to many enzymes and proteins often without any loss of activity; and (iii) avidin is very stable and has several binding sites so that it may be l~sed as a bridging molecule between t~ biotinylated molecules. The use of the avidin/biotin system produces superior dotection Ibilities and low background levels.

, .

wo 90/15329 PCr/CA90/00177 20~6~07 In detail, microti$ration wells (Immulon~9 1, Dynatech Labs) are coated with 10 llg/ml of F5 IgG and the excess of antibody is washed off with T~Tween~. The remair~ing binding sites are saturated with T~
1% low fat miL~c and the wells are incubated for 1 hour at 37C wi$h 100 5 ng/ml of peroxidase labelled P'VLCI. Unbound antigen is washed off and peroxidase substra~e added as for the preceding Example IX.
Results are expressed as % ma~amum binding and presented in Table VI.

Takle Inhibition of PVLCl -HRP binding to F5 lgG by ~ee PVLC
PVLCI concentration % maxiimum binding (ng/ml) ~90 ~9 1.
Microtitration wells are coated with 10 llg/ml of Fs IgG and incubated simultaneously with equal volumes of 200 ng/ml of P~ILCI-HRP and free PVLC at ~arying concentrations for 1 hour at 37C. H22 20 is added for 30 min as peroxidase substrate.

Examvle XI :
Description of Assay 4 The schematic representation of assay No. 4 is depicted in Figure 7.
Since an internal image anti-idiotypic antibody can mimic structurally the antigen, it can replace this anffgen from binding to its corresponding idiotype. The solid phase bound idiotype-anti-idiotype . . .
. ; , . . ., .

.

WO 90/15329 PCl`/CA90/00177 37 20~6~07 complex is detected with peroxidase labelled antiserum to Pc fragment of mouse IgG. The free antigen in patient's serum will lower the amount of anti-idiotype bound to idiotype.

Technically, microtitration plates are coated with anti-VLC
5 immunoglobulin fragments (F(ab')2) and then reacted concurrently with anti-~LC anti-idiotypic antibody and H~LCs containing patients' sera for 1 hour at 37C. After the unbound HVLC5 and anti-idiotypic antibody have been washed off, the plates are incubated with Fc fragment specific peroxidase labelled anti-mouse IgG for further 1 hour 10 and then with H22 for 30 min. HVLC content in a sample is calculated from a standard VLC replacement curve.

The use of an internal image anti-idiotypic antibody has an important advantages over the use of VLCs as a standard. It sigruficantly reduces the utilization of fresh autopsy hearts from which 15 myosin light chains are purified. There is no biological variability between different lots of anti-idiotypic antibody while a consaiderable ~7ariation may exist between lots of myosin light chains isolated from different animal hearts. The cost of production and purification of monoclonal anti-idiotypic antibody is much lower than the labour-20 intensive cost of myosin light chain purification. The final cost of thekit could thus be lowered by the employement of anti-idiotypic antibody.

, . . ,. ~ ~ - :
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. , . . , ~ .
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36. Gere JB, Krauth GH, Trahern CA, Bigham DA. Am J Clin Pathol, 1978; 71: 309-318.
37. Nagai R, Ueda S, Yazaki Y., In: Advances in Myocardiology, vol. _ edited by M. Tajuddin, B. Bhatia, HH Siddigui, G. Rona.
Baltimore, University Park Press, 1980: 415 420.
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43. Katus HA, Yasuda T, Gold ~C, Leinbach RC, Strauss HW, WaksmonskiC,HaberE,Khaw BA., AmJCardiol, 1984;
54: 964-970.

.: . ;: .:.. . ;, . . : - . . , wo so/1s32s Pcr/cAso/ool77 41 ~ 2~6.~37 44. Horvath B, Gaetjens E.) Biochem. Biophys. Acta, 1972;
263:77g 7g3-45. Morkin E, Yazaki Y, Katagiri T, LaRaia PJ., Biochem Biophys Acta, 1973; 324: 42~429.
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47. Zak R, Martin AF, Prior G, Rabinowitz M., J Biol Chem, 1977; 252: 343~3435.

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Claims (8)

WE CLAIM:

1. A competitive method of measuring human ventricular myosin light chain one (HVLC1) in a patient with myocardial cell damage, which comprises incubating a fixed amount of a polyclonal or monoclonal anti-HVLC1 antibody, a fixed amount of HVLC1 standard and an unknown amount of HVLC1 analyte present in the serum of a patient;
and detecting one member selected from the group consisting of anti-HVLC1 antibody and HVLC1 standard either directly by means of enzyme labeling of said anti-HVLC1 antibody or enzyme labelling of said HVLC1 standard or indirectly by means of enzyme-anti-mammalian Ig conjugate, whereby the amount of HVLC1 analyte in the serum of a patient, is determined by comparing the extent to which said HVLC1 analyte displaces said HVLC1 standard from binding to said anti-HVLC1 antibody, with a calibration curve obtained from known amounts of unlabelled HVLC1 calibration standards.

2. The competitive method according to claim 1, which is a solid phase assay and wherein one member selected from the group consisting of anti-HVLC1 antibody and HVLC1 standard, is solid phase bound.

3. A sandwich method of measuring human ventricular myosin light chain one in cardiac patients, HVLC1 analyte, which comprises the steps of:
a) providing a first anti-HVLC1 monoclonal antibody attached to a solid surface;

b) contacting said antibody-coated solid surface with a sample to be tested for a sufficient time to allow an immunologic reaction to occur;
c) contacting the coated-surface according to step b) with a second polyclonal or monoclonal anti-HVLC1 antibody; and d) detecting said second anti-HVLC1 antibody either directly by means of enzyme labelling of said second anti-HVLC1 antibody or indirectly by means of an enzyme-anti-mammalian Ig conjugate, whereby the amount of HVLC1 analyte bound to the said first anti-HVLC1 antibody is determined from a calibration curve obtained from known amounts of unlabelled HVLC1 calibration standards.

4. A diagnostic kit for measuring human ventricular myosin light chain one (HVLC1) in a sample adapted to be used according to the method of claim 3 comprising:
I) a solid surface having bound thereto an anti-HVLC1 antibody which is produced by the hybridoma cell line having the ATCC accession number HB10471;
II) known amounts of HVLC1 calibration standards; and III) an other anti-HVLC1 polyclonal or monoclonal antibody which recognizes at least a different epitope on said HVLC1 and which is detected by means of an enzyme-anti-mammalian Ig conjugate.

5. The kit according to claim 4, wherein said antibody of I) is a F(ab')2 fragment of anti-HVLC1 monoclonal antibody and said antibody of m) is produced by the hybridoma cell line having the ATCC
accession number HB10471 which is detected by means of an enzyme-anti-mouse IgG Fc fragment specific conjugate.

6. A diagnostic kit for measuring human ventricular myosin light chain one in a sample adapted to be used according to the method of claim 1 comprising:
I) a solid surface having bound thereto an anti-HVLC1 antibody which is produced by the hybridoma cell line having the ATCC accession number HB10471;
II) a known amount of enzyme labelled HVLC1 standard;
and III) known amounts of unlabelled HVLC1 calibration standards.

7. A diagnostic kit for measuring human ventricular myosin light chain one in a sample adapted to be used according to the method of claim 1 comprising:
I) a solid surface having bound thereto an HVLC1 standard;
II) known amounts of unlabelled HVLC1 calibration standards; and III) an anti-HVLC1 antibody which is produced by the hybridoma cell line having the ATCC accession number HB10471 and which is detected by means of an enzyme-anti-mouse IgG conjugate.

8. A monoclonal antibody which specifically binds human ventricular myosin light chain one and does not substantially bind skeletal muscle myosin light chains in the method of any of Claims 1 to 3, and which is produced by the hybridoma cell line having the ATCC
accession number HB10471.