WO1997008551A1

WO1997008551A1 - Methods and antibodies for detecting creatine kinase

Info

Publication number: WO1997008551A1
Application number: PCT/US1996/013755
Authority: WO
Inventors: Robert F. Zuk; Maje B. Perryman
Original assignee: First Medical, Inc.
Priority date: 1995-08-31
Filing date: 1996-08-26
Publication date: 1997-03-06
Also published as: AU6764196A

Abstract

Assays and antibodies are provided for detecting creatine kinase (CK) enyzmes. Total CK isoenzyme may be detected with an antibody specific for an epitope within a conserved region of the M subunit and B subunit of CK isoenzymes as well as to mitochondrial CK. The ratio of CK-MB isoenzyme to total CK isoenzyme may be determined simultaneously using a solid phase assay device having a first reaction zone and a second reaction zone. Antibody specific for total CK isoenzymes is present in the first reaction zone while antibody specific for CK-MB isoenzyme is present in the second reaction zone. A single patient plasma or serum sample may then be reacted with the solid phase to effect the simultaneous determination of the relative amounts of CK isoenzyme.

Description

METHODS AND ANTIBODIES FOR DETECTING CREATINE KINASE

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to methods and compositions for detecting creatine kinase isoenzymes in patient samples. More particularly, the present invention relates to immunoassays for detecting the presence of total CK isoenzyme and CK-MB isoenzyme in a single patient sample and relating the ratio of such amounts to the likelihood that the patient has suffered an acute myocardial infarction.

Acute myocardial infarction is a leading cause of mortality and morbidity in the United States and other Western societies. The proper treatment of acute myocardial infarction requires early detection and confirmation that an infarction has occurred. Frequently, chest pains and other symptoms associated with myocardial infarction are caused by other conditions, and the electrocardiograms administered to a patient may be inconclusive. With such patients, it has been found that the relative amounts of certain creatine kinase (CK) isoenzymes and isoforms in their blood can be diagnostic of an infarction. While CK isoenzymes are normally present in blood and serum as a result of muscle cell turnover, the amount of CK isoenzyme MB (CK-MB) is substantially higher in the myocardium than in other muscle tissues. Thus, the release of excessive amounts of CK-MB isoenzyme into the blood is diagnostic of myocardial infarction.

Creatine kinase is a dimer consisting of two molecular subunits, designated as the M subunit and B subunit, which combine to form three isoenzymes. The first isoenzyme includes a pair of the B subunits and is designated as the CK- BB isoenzyme. The second isoenzyme combines a pair of the M subunits and is designated as CK-MM isoenzyme. The third isoenzyme combines one of each of the M subunit and B subunit and is designated as CK-MB isoenzyme. A mitochondrial form of CK exists and will comprise up to about 15% of the total CK released as a result of acute myocardial infarction At present, if a patient is suspected of having suffered a myocardial infarction, a diagnostic assay comprising a number of cardiac markers may be performed. Exemplary assays might include detection of total CK isoenzyme, CK-MB isoenzyme, troponin I, troponin T, myoglobin, myosin light chain, and the like. Of particular interest to the present invention, the total CK isoenzyme level is usually determined using an enzymatic assay, while the CK-MB isoenzyme level is determined using an immunoassay. The relative levels of CK-MB and isoenzyme and total CK isoenzyme are then compared to determine the likelihood that the patient has suffered an infarction.

Such assays are problematic since they compare a level based on mass, i.e., the immunological detection of CK- MB isoenzyme, with a level based on enzyme activity, i.e., the enzyme assay of total CK isoenzyme. Moreover, the need to determine cardiac markers using such disparate techniques complicates the assay protocols and increases the chance of error. As the results of these assays will determine the prescribed treatment for the patient, there is a need to obtain accurate results in a rapid manner.

For these reasons, it would be desirable to provide improved methods and substances for the detection of cardiac markers in patient samples, including at least total CK isoenzyme and CK-MB isoenzyme. It would be particularly desirable if such assays provided for the immunologic detection of the amounts of both total CK isoenzyme and CK-MB isoenzyme. Such assays will preferably be run using a single patient sample, more preferably employing a single solid phase assay device which permits the rapid detection of total CK isoenzyme, CK-MB isoenzyme, and optionally other cardiac markers. 2. Description of the Background Art

U. S. Patent No. 4,912,033 describes antibodies and assays for the specific detection of CK-MB isoenzyme in patient serum. U. S. Patent No. 4,353,982 describes a two- site immunoradiometric assay for detecting CK-MB isoenzyme utilizing a triple antibody technique. TJ. S. Patent Nos. 5,009,996 and 5,009,997 describe two-site immunoradiometric assays for detecting CK-MB, where the assays employ cross- reactive antibodies raised against CK-MM and CK-BB isoenzymes. U. S. Patent 4,624,961 describes two-site immunoradiometric assays for detecting CK-MB isoenzyme where cross-reactivity is reduced by performing the assay in the presence of pooled human sera. The detection of specific isoforms of the MM, BB and MB CK isoenzymes is described in the following U. S. Patent Nos. 4,810,639; 4,900,662; 5,202,234; 5,382,515;

5,382,522; and in EP 288 179 and EP 467 782. Apple et al. (1987) describes the measurement of total CK and certain CK isoenzymes (including CK-MB, designated CK-2) in patient samples as an indicator of myocardial infarction. Monoclonal antibodies which are specific for CK-MB are available from International Immunoassay Laboratories, Inc., Santa Clara, California.

SUMMARY OF THE INVENTION The present invention provides novel and improved assays and compositions for the detection of creatine kinase (CK) enzymes in patient samples, such as blood, plasma, serum, urine, CSF, and the like. In the first aspect of the present invention, total CK isoenzyme is measured in the patient sample by exposing the sample to a binding substance which binds all CK isoenzyme, including mitochondrial CK, and thereafter detecting binding between said binding substance and the CK isoenzymes. Preferably, the binding substance binds to a conserved region common to the M and B subunits of the CK isoenzymes. Usually, the binding substance will be an antibody raised against a conserved region of the subunits . More preferably, the antibody will have a substantially equal binding affinity for each of the CK-MM, CK-BB, and CK-MB isoforms, and mitochondrial CK. The use of such assays permits detection of total CK isoenzyme using only a single antibody which is cross-reactive with each of the three CK isoenzymes. Alternatively, although less preferably, mixtures of antibodies specific for the M subunit and B subunit, respectively, can be used for binding total CK isoenzyme in the methods of the present invention.

In a second aspect, the present invention provides an assay for assessing cardiac tissue damage in patients suspected of suffering acute myocardial infarction. Such methods comprise applying an undivided volume of blood plasma, or serum to a solid phase, where the solid phase concludes at least a first reaction zone and a second reaction zone. The first reaction zone includes immobilized binding substance which binds total CK isoenzymes, preferably being a cross- reactive antibody described above. The second reaction zone comprises an immobilized binding substance which specifically binds CK-MB isoenzyme. The amounts of both the total CK isoenzyme and the CK-MB isoenzyme are then detected, and may be compared to assess the likelihood that the patient suffered an infarction. The binding substance in the second reaction zone will typically be an antibody specific for the CK-MB isoenzyme and not cross-reactive with other CK isoenzymes. In a preferred protocol, the amounts of total CK isoenzyme and CK-MB isoenzyme will be determined simultaneously by exposing the solid phase in a single step to a labeling reagent which is capable of non-specifically binding to all CK isoenzymes. After the simultaneous labeling step, each of the reaction zones may be separately detected to determine the amount of label found therein. The labeling reagent may comprise a mixture of antibodies directed against the M and B subunits of the CK enzyme.

In a third aspect, the present invention provides a single antibody which binds to all CK isoenzymes with substantially equal affinities. Preferably, the antibody will bind each of the isoenzymes with an affinity in the range from 10⁷ M^"1 to IO¹² M usually from IO⁸ M^"1 to 10¹⁰ MA Usually, the antibody will bind to a conserved region of the CK enzyme, which is common to both the M and B subunits as well as to mitochondrial CK.

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a schematic illustration of a solid phase useful in performance of the assays of the present invention, shown as a rotor having a first reaction zone and a second reaction zone within a common reaction region.

Figs. 2A-2D illustrate an assay performed in accordance with the principles of the present invention utilizing the rotor of Fig. 1.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

Methods and compositions are provided for detecting cardiac markers in patient samples, typically blood samples taken from the patient shortly after experiencing the symptoms associated with acute myocardial infarction. Usually the samples will be taken within 8 hours from the onset of the symptoms, preferably within 4 hours of onset, and more preferably within 1 hour of onset. The patient blood sample will usually be treated prior to assay according to the present invention, typically having cellular components removed to form plasma and optionally having cellular components and clotting factors removed to form serum. Blood samples may be obtained initially using conventional techniques, such as phlebotomy, with plasma and serum formed according to well known techniques.

In a first aspect of the method of the present invention, total CK isoenzyme may be measured in such patient samples using a binding substance which binds all three CK isoenzymes as well as mitochondrial CK. In a second aspect of the method present invention, both the amount of total CK isoenzyme in the sample and the amount of CK-MB isoenzyme in the sample are determined, where the ratio or other relationship of these amounts can be used to determine the likelihood of the patient having suffered an infarction. In a third aspect of the present invention, antibodies and other equivalent binding substances are provided which bind to an epitope within a conserved region of the M and B subunits of the CK enzymes as well as mitochondrial CK. Optionally, the methods of the present invention may be used to simultaneously detect other known cardiac markers, including troponin I, troponin T, myoglobin, myosin light chain, isoforms of the CK isoenzymes, and the like.

In the assays of the present invention, the total CK isoenzyme (including all three isoenzymes) and the CK-MB isoenzyme will be measured by reacting the sample with at least one binding substance which binds to the target enzyme, usually an antibody, an antibody fragment, or equivalent molecule, as described in more detail below. It will be appreciated that numerous techniques have been developed for producing binding molecules having equivalent specificities to antibodies and antibody fragments. Thus, the terms "binding substance" and "specific binding substance" will refer to intact antibody molecules (immunoglobulins) as well as to portions of the antibody molecule which retain the ability to bind to epitopes on the target enzyme. Such antibody portions include the Fab, Fab', and F(ab')₂ fragments of immunoglobulins. In addition to such antibody fragments, numerous techniques have been prepared for recombinantly producing molecules having the binding specificities of antibodies. Usually, an antibody molecule is prepared in a conventional manner, as described in more detail below, and the gene encoding the antibody molecule isolated. After sequencing the antibody gene, recombinant nucleic acids can be isolated or synthesized and used to prepare binding proteins by a variety of now conventional methods. Total CK isoenzyme will preferably be detected with a single antibody or other binding substance which binds to an epitope defined by a conserved region common to the M subunit and B subunit of the CK enzymes, as well as to a region on mitochondrial CK. A presently preferred region comprises amino acid numbers 271-299 based on the consensus number of the CK isoenzymes (each of which is colinear) . The amino acid sequence of this region is:

271 281 291

FMWNQHLGYV LTCPSNLGTG LRGGVHVKL (SEQ ID No.:l) Antibodies which recognize the epitopes on such conserved regions may be prepared by conventional techniques using antigenic and haptenic peptides which incorporate the epitope (s) of the conserved regions. The peptides may readily be prepared using known solid phase synthesis techniques and automated production equipment available from commercial vendors, such as Applied Biosystems, Foster City, California. The peptides so prepared may then be used as immunogens for producing polyclonal and/or monoclonal antibodies by well- known techniques. The production of monoclonal antibodies is well described in literature. See, e . g. , Harlow and Lane, eds., AntiJbodies: A Laboratory Manual , Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. The production of monoclonal antibodies specific for the CK-MB isoenzyme is described in detail in U.S. Patent No. 4,912,033, the full disclosure of which is incorporated herein by reference. As described in that patent, the antibodies specific for CK-MB isoenzyme will be substantially free from cross-reactivity with the CK-MM and CK-BB isoenzymes. In particular, the 29 amino acid sequence set forth above (SEQ ID No.:l) may be synthesized and used as an immunogen to produce antibodies for use in the methods of the present invention.

The antibodies and other binding substances specific for total CK isoenzyme will preferably have substantially equal binding affinities for each of the three CK isoenzymes, usually having a range of binding affinities with a ratio of less than 100, more preferably less than 10. The binding affinities will typically be from lO'M^"1 to 10¹²MA preferably from lO^M^"1 to ¹²MA In the assays for simultaneously determining the amounts of total CK isoenzyme and CK-MB isoenzyme in a single patient sample, a solid phase will be prepared having a reaction region comprising at least a first reaction zone and a second reaction zone. The first reaction zone will have antibody or other binding substance capable of specifically binding the total CK isoenzyme (i.e., being cross-reactive with all three isoenzymes) . The second reaction zone will have an antibody or other binding substance capable of specifically binding with only the CK-MB isoenzyme (i.e., being non-cross-reactive with the other two isoenzymes) . The two reaction zones will be located within a common reaction volume or receptacle on the solid phase which receives the sample and which may subsequently receive labeling reagent, wash solutions, and other assay components intended for carrying out the assay protocol.

In the exemplary embodiment, the solid phase is a rotor of the type described in copending application Serial No. 08/522,434. The rotor may be used in assays performed with an analyzer system as described in copending application Serial No. 08/521,615. The full disclosure of each of these copending applications has previously been incorporated herein by reference. The antibodies or other binding substances will be bound to the solid phase using conventional binding techniques. Antibodies may be bound covalently or non- covalently. Methods for the immobilization of antibodies and other protein binding substances are well known to those skilled in the art. For the protocols below, the antibodies will typically be bound in an amount greater than that expected to be required for binding the CK isoenzymes. In that way, after incubation with the sample, substantially all of the CK isoenzymes in the sample will be bound. It will be appreciated, of course, since the CK-MB isoenzyme will be bound within both the first and second reaction zones, the total amount of CK isoenzyme will be the sum of binding in both the reaction zones. Additionally, the amount of CK-MB isoenzyme bound within the second reaction zone will be somewhat less than that actually present in the sample since a portion of the CK-MB will be bound within the first reaction zone.

After the total CK isoenzyme and CK-MB isoenzyme are bound within the first and second reaction zones, respectively, the solid phase will be exposed to labeling reagent (s) in order to detect the amount of isoenzyme bound within each zone. Preferably, a single labeling reagent will be introduced to both reaction zones within the reaction region simultaneously in order to reduce the number of steps required in the assay protocol. The label will typically comprise one or more binding substances covalently or non- covalently linked to a detectable label. The binding substance (s) may be one or more antibodies specific for the three CK isoenzymes. For example, it may be possible to employ antibody raised against an epitope in one of the conserved regions of the M and B subunits, as described above. When that is done, the epitope should be sufficiently sterically removed from the epitope which is bound to the solid phase to permit detection with the labeling reagent. Alternatively, a mixture of antibodies specific for the M and B subunits may be employed, where the antibodies are attached to the same label. Suitable labels will be of a type which are separately detectable within each of the reaction zones. After the initial steps of binding the CK isoenzyme and labeling the bound CK isoenzyme, it is necessary to separately detect the amount of isoenzyme bound within each reaction zone. Thus, radio and enzyme labels that do not produce a localized signal will generally not be suitable for use in the present invention. Localized labels, such as fluorescent labels, luminescent labels, and the like will find particular use. An exemplary fluorescent label is described in copending application Serial No. 08/521,860, the full disclosure of which has previously been incorporated herein by reference. Such fluorescent labels may be detected by directing excitation energy to a single one of the reaction zones to initiate a fluorescent emission, and detection of such emission. Since the excitation energy can be localized to each reaction zone in turn, only the isoenzyme bound within that reaction zone (together with fluorescent label) will be detected, and there will be little or no interference from label in the other reaction zone(s) . Referring to Fig. 1, an exemplary solid phase rotor

10 suitable for use in the assays of the present invention will be described. Rotor 10 is a simplified version of the rotors which are described in co-pending application Serial No. 08/522,434, the full disclosure of which has previously been incorporated herein by reference. Rotor 10 includes a rotor body 12 having a reaction chamber 14 including a first reaction zone 16 and a second reaction zone 18. The first reaction zone will have antibody or other binding substance specific for total CK isoenzymes immobilized therein. The second reaction zone 18 will have antibody or other binding substance specific for CK-MB isoenzyme immobilized therein. Reaction chamber 14 may include additional reaction zones for other cardiac markers, but such zones are not shown in order to simplify explanation of the assay protocols. Rotor 10 will further include chambers and flow paths (not shown) for introducing sample, wash reagents, diluents, labeling reagents, and the like. Rotor 10 will further comprise at least one waste chamber for receiving each of the reaction solutions as they pass through the reaction chamber 14 in turn. Each of these aspects of the rotor design is described in detail in copending application Serial No. 08/522,434.

According to the method of the present invention, a sample is first introduced to the solid phase rotor 10 so that a measured volume of the sample, typically patient plasma or serum, enters into the reaction chamber 14, as illustrated in Fig. 2A. The sample will remain within the chamber 14 for a sufficient time to permit binding between the CK isoenzymes therein with each of the reaction zones 16 and 18, typically for a time in the range from one minute to 10 minutes.

After the reaction is complete, the sample will be removed from the reaction chamber, typically by rotating the rotor to cause outward flow of the sample to a waste chamber (not shown) which is located radially outward from the reaction chamber. A labeling reagent is next introduced into the reaction chamber 14, as illustrated in Fig. 2B. The labeling reagent is allowed to incubate with the reaction zones 16 and 18 for a time sufficient to permit binding of the label to the CK isoenzymes previously bound therein.

Typically, the incubation of the labeling reagent will last for one minute to 10 minutes. After the labeling reaction is completed, the labeling reagent will be removed from the reaction chamber by rotating the rotor 10 to cause outward flow to the waste chamber. After washing to remove non-specifically bound label, the amount of label within each of the reaction zones 16 and 18 may be determined. As illustrated in Figs. 2C, the amount of fluorescent label bound within reaction zone 16 may first be determined by exposing the zone 16 to a beam 26 of excitation energy emanating from a suitable lightsource 28.

Emitted fluorescent radiation 30 may be detected by a suitable detector 32 which is oriented to receive such fluorescence.

After the amount of label bound within reaction zone 16 is determined, the rotor 10 may be repositioned, as illustrated in Fig. 2D, to allow detection of label within reaction zone 18. The label bound within the second reaction zone 18 is determined in the same manner as that bound within reaction zone 16. After determining the relative amounts of total CK isoenzyme and CK-MB isoenzyme, the ratio of CK-MB isoenzyme to total CK isoenzyme may be determined.

Although the foregoing invention has been described in some detail by way of illustration and example, for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: First Medical, Inc.

(ii) TITLE OF INVENTION : Methods and Antibodies for Detecting

Creatine Kinase

(iii) NUMBER OF SEQUENCES: 1

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Townsend and Townsend and Crew LLP

(B) STREET: Two Embarcardero Center, 8th Floor

(C) CITY: San Francisco

(D) STATE: California

(E) COUNTRY: USA

(F) ZIP: 94111

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentin Release #1.0, Version #1.25

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER: PCT/US

(B) FILING DATE:

(C) CLASSIFICATION:

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Heslin, James M.

(B) REGISTRATION NUMBER: 29,541

(C) REFERENCE/DOCKET NUMBER: 016415-001300PC

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: 415-326-2400

(B) TELEFAX: 415-576-0300

(2) INFORMATION FOR SEQ ID NO:1 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 29 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1 :

Phe Met Trp Asn Gin His Leu Gly Tyr Val Leu Thr Cys Pro Ser Asn 1 5 10 15

Leu Gly Thr Gly Leu Arg Gly Gly Val His Val Lys Leu 20 25

Claims

WHAT IS CLAIMED IS;

1. An improved assay for detecting total CK isoenzyme in a patent sample, wherein the improvement comprises exposing the sample to a binding substance which binds all CK isoenzymes and detecting binding between said binding substance and said CK isoenzymes.

2. An improved assay as in claim 1 wherein the binding substance binds to a conserved region common to the M and B subunits.

3. An improved assay as in claim 2, wherein the binding substance is an antibody raised against a conserved region of CK-MM, CK-MB, CK-BB, and mitochondrial CK.

4. An improved assay as in claim 3, wherein the antibody has a substantially equal binding affinity for each of the CK-MM, CK-BB, and CK-MB isoforms.

5. An improved assay as in claim 2, wherein only a single binding substance is used.

6. An improved assay as in claim 1, wherein the binding substance is a mixture including binding substance specific for the M subunit and binding substance specific for the B subunit.

7. An assay for assessing cardiac tissue damage in a patient suspected of suffering an acute myocardial infarction, said method comprising: applying an undivided volume of blood from the patient to a solid phase, wherein the solid phase includes a first reaction zone having immobilized binding substance which binds all CK isoenzymes and a second reaction zone having immobilized binding substance which specifically binds CK-MB isoenzyme; detecting the amount of total CK isoenzyme bound in the first reaction zone; and detecting the amount of CK-MB isoenzyme bound in the second reaction zone.

8. An assay as in claim 7, wherein there is a single binding substance in the first reaction zone, which single binding substance binds to a conserved region common to the M and B subunits.

9. An assay as in claim 8, wherein the binding substance is an antibody raised against a conserved region of CK-MM, CK-MB, CK-BB, and mitochondrial CK.

10. An assay as in claim 9, wherein the antibody has a substantially equal binding affinity for each of the CK- MM, CK-BB, and CK-MB isoforms.

11. An assay as in claim 7, wherein the binding substance in the second reaction zone is specific for CK-MB isoenzyme and not cross-reactive with other CK isoenzymes.

12. As assay as in claim 7, further comprising determining the ratio of CK-MB to total CK isoenzyme to assess tissue damage.

13. An assay as in claim 7, wherein the amounts of total CK isoenzyme and CK-MB isoenzyme are determined by exposing the solid phase in a single step to a labelling reagent which is capable of binding to all CK isoenzymes and thereafter separately detecting the amount of label bound in each of the first and second reaction zones as a measure of total CK isoenzyme and CK-MB isoenzyme respectively.

14. An assay as in claim 13, wherein the labelling reagent comprises a mixture of antibodies directed against the M and B subunits of CK enzyme.

15. A single antibody which binds all CK isoenzymes with substantially equal affinities.

16. A single antibody as in claim 15, where said antibody binds each of CK-MM, CK-MB, and CK-BB with an affinity in the range from IO⁷ M^"1 to IO¹² MA

17. A single antibody as in claim 16, wherein the antibody binds to a conserved region of the CK enzyme common to the M and B subunits as well as to mitochondrial CK.

18. A single antibody as in claim 16, wherein the antibody is raised against a polypeptide having the sequence of SEQ ID No. :1.