EP0382725A4

EP0382725A4 - Method and test kit for neutralization/immunoinhibition assay

Info

Publication number: EP0382725A4
Application number: EP19880904874
Authority: EP
Inventors: Frank J. Lucas; Show-Jiuan Ho
Original assignee: Coulter Electronics Inc
Current assignee: Coulter Electronics Inc
Priority date: 1987-05-12
Filing date: 1988-05-11
Publication date: 1990-12-27
Also published as: WO1988008984A1; EP0382725A1; JPH01503565A; CA1320114C; AU1800088A

Abstract

Method and test kit suitable for determination of the level of a diagnostically relevant isoenzyme in a biological fluid sample. This method and test kit are based upon the determination of residual enzyme activity of a biological fluid sample subsequent to inhibition of the diagnostically relevant isoenzyme within the sample. Such neutralization/immunoinhibition of the enzyme is accomplished with a relatively impure, low avidity anti-serum which is specific for the isoenzyme. This assay is unique in that the anti-serum which is employed in the neutralization/immunoinhibition of the isoenzyme is only effective in the neutralization/immunoinhibition of from at least about 50 % to up to about 85 % of the original enzymatic activity of the isoenzymes within the sample. This method provides the unique ability to determine the level of diagnostically relevant isoenzyme from rate data which is inclusive of high residual enzymatic activity of the diagnostically relevant isoenzymes and other interferents. This method is suitable for determination of CK-MB levels and, thus, provides a diagnostic tool for the exclusion of acute myocardial infarction as the cause of patient distress.

Description

Title: Method and Test Kit For Neutralization/Immunoinhibition Assay

BACKGROUND OF THE INVENTTON

1. Field of the Invention - This invention is directed to an improved neutralization assay, based upon the relatively incomplete immunoinhibition of the activity of a diagnostically relevant isoenzyme whose relative concentration can be indicative of an abnormal, physiological state; and, a test kit for performance of this improved assay. This assay has particular application in the determination of CK-MB levels in biological fluids to aid in the accurate identification of patients suffering acute myocardial infarction.

2. Description of the Invention - The study of enzymes and their roles in various physiological processes, has led to a mor complete understanding of the interrelationships between these complex proteins and abnormal bodily functions associated with trauma and/or pathology. For example, the principle creatine kinases (CK) found in biological fluid samples are known to be associated with specific tissues found in the skeletal muscle (CK- MM), smooth muscle (CK-MB), and in the brain (CK-BB). When injury occurs to one or more of these tissues, the creatine kinase (CK) level of the blood will be elevated for the isoenzyme which is most prevalent in the traumatized tissue. If an individual suffers a heart attack (i.e., acute myocardial infarction), the blood level will reflect an increase in CK-MB isoenzyme subsequent to the onset of the attack; the maximum increase in CK-MB level appearing generally 10-12 hours after the initial experience of distress. The patent and technical literature are replete with publications directed to various methods for the determination of the CK isoenzyme activity. Preliminary to a review of these references, a brief glossary of terms would be both appropriate and helpful to a more complete understanding and discussion of the literature pertinent to this area.

The term "neutralization" is used hereinafter, in the context of a clinical assay, to characterize an immunoinhibition assay having two distinct phases: a primary phase in which the anti-serum and the target enzyme rapidly interact, thereby forming an immunocomplex which is essentially devoid of enzymatic activity and, a secondary phase in which there is a gradual decrease in the residual activity of the enzyme after floculation. (Cinader, Ann. Rev. Microbiol. 11, 371-390 (1957).)

The term "agglutination" is used hereinafter, in the context of a clinical assay, to characterize an immunoinhibition assay in which the antigen particles are large enough to be sedimented by centrifugation at 1,000 to 2,000 rpm (500-1,000 g) and, thus, when brought into contact with an anti-serum specific for this antigen, result is the production of an immunocomplex which is in the form of an agglomerate. (Kabat &. Mayers, Experimental

Immunochemistry, Chas. Thomas PubL, Springfield, Illinois, 905 pgs. (1967).)

The term "immunotitration" is used hereinafter, in the context of a clinical assay, to characterize an immunoinhibition assay in which the anti-serum interaction with the target enzyme forms an enzymatically inactive precipitate. (Schinke, Methods in Enzymology, Vol. 40, pgs. 241-251 (1975).) The term "immunoprecipitation" is used hereinafter, in the context of a clinical assay, to characterize an assay in which the anti-serum interaction with the target enzyme forms a precipitate which is then separated by centrifugation and subjected to analysis for its protein content. (Schinke, Methods In Enzymology, Vol. 40, pg. 241-251 (1975).)

The technical literature relating to the interaction of an anti- *ΓO serum and an enzyme, dates back almost 40 years; one of the earlier references being a review by Sevag on immunocatalysis and anti-enzymatic antibodies, Immunocatalysis, 2nd Ed., C. Thomas Co., Springfield, IL, 547 pgs. The interest in immunoinhibition of enzymes, appeared to accelerate in the early

1 5 and mid- 1950' s with several articles and reviews appearing. A review by Cinader in 1955 described the nature of various enzyme/anti-enzyme antibodies, Cinader, Bull. Soc. Chim. Biol., 37(7-8), 761-781 (1955). Cinader thoroughly reviewed and detailed prior work in this area, and proposed models for

2 0 antibody/enzyme neutralization interaction.

A second review by Cinader in 1957 described and discussed the more recent advances in antibody /enzyme interaction, Ann. Rev. 5 Microbiol. 11, 371-390 (1957). This Cinader article, presented a detail review of the studies involving antibody/enzyme interactions as a tool in biological and immunological investigations, and the correlation of loss or change in such enzyme levels and/or activity with specific pathologies. 0

In the early 1960's, two papers appeared which related to immunoinhibition of creatine kinase, Samuels, Biophys. J. 1, 437 (1961), and Arch. Bioch. and Biophys. 92, 497 (1961). Samuels 5 reported the observation that the addition of substrate to a sample containing creatine kinase enzyme does not, unlike other enzyme/antibody systems, preclude immunoinhibition of the enzyme by the antibody. Samuels also reported that Immunoinhibition causes conformational changes in the kinase enzymes and that such conformational changes differed from those resulting where a substrate and the enzyme interact. (Ann. NY Acad. Sci., Vol. 103, 889-963 (1963).)

Watts, et al reported in 1962 what appears to be the first recognition that creatine kinase exists as a complement or a series of different, but related, enzymes (i.e. isoenzymes), Watts, et al, Biochem. J. 82, 412 (1962). The revelations by Watts, et al, paralleled developments relating to the study of LDH, which included the immunochemical differentiation of three distinct LDH isoenzymes (as originating in tissues of the heart, liver and muscle), Cahn, Science 136, 962-969 (1962). The work by Cahn involved the immunoinhibition of various LDH isoenzymes after centrifugation.

A third review was published Cinader in 1963 which was devoted exclusively to anti-enzyme antibodies, Cinader, Introduction for Ann. NY Acad. Sci., Vol. 103, 495-548 (1963). The Cinader review article discussed more than 100 references relating to this topic. The Cinader review article specifically treated the topic of multiple molecular forms of a single enzyme and how immunoinhibition techniques presently available could potentially play a significant role in both differentiation and quantification of these enzymes. The Cinader review article noted that creatine kinase was an exception to the general scene relating to immunoinhibition and compared its immunochemical behavior to a proposed four (4) component model system. Cinader also noted that neutralization measurements were more accurate for determination of isoenzymes in a mixture, than the traditional precipitation base methodologies. In 1964, three distinct forms of creatine kinase enzyme complement were identified, CK-MM, CK-MB and CK-BB, Duel and Van Breeman, Abst. Fed. of Europ. Bioch. Soc, pg. 52 (1964); Clin. Chim. Acta 19, 276 (1964); and, Burger, A. et al., Biochem. Z.

339,305 (1964). In 1965, Dawson explained the relationship and differences between these three isoenzymes as originating with the monomeric units of each and their respective combinations. Dawson continued his elucidation of this subject by publishing two additional articles; one appearing in 1967 (Dawson & Fine, Arch. Neurol. 16:175-180 (1967)); and, a second in 1968, (Dawson, et al., Ann. NY Acad. Sci., 155, 616-626 (1968)). That same year, Perkoff implicated CK-MB with the occurrence of acute myocardial infarction, Perkoff, Ann. Intern. Med. 122, 326 (1968).

The clinical usefulness of the measurement of increased CK-MB activity became fully recognized in 1970 as indicative of acute myocardial infarction, Wilkinson, J. Clin. Chem. 16(9), 773-739 (1970). The predictive value of a CK-MB assay, as indicative of acute myocardial infarction, was confirmed in 1973 by two independent researchers, . Wagner, et al., Circulation 47, 263 (1973); and, Konttinou, Br. Med. J. 1, 386 (1973).

Patent applications for diagnostic assays for determination of CK- MB, began to be applied for in the early and mid-1970's, ultimately maturing as U.S. Patents 3,932,221 (foreign priority, June 9, 1971); and, 4,067,775 (foreign priority, November 3, 1975).

U.S. Patent 3.932.221 (to Pfleiderer) described a diagnostic method for determination of residual activity of diagnostically relevant- isoenzymes. According to Pfleiderer, total enzymatic activity of the diagnostically relevant isoenzyme, vis-a-vis a specific substrate, is initially determined in the sample containing the diagnostically relevant isoenzyme, the sample thereafter contacted with an anti-serum specific for the isoenzyme. The immunochemical interaction of the anti-serum and the diagnostically relevant isoenzyme, forms a precipitate which is then separated from the sample and discarded. The residual enzymatic activity of the sample is then determined against the same substrate and this residual activity compared to the total enzymatic activity for the sample as initially measured. The difference between the total initial enzymatic activity and the residual activity, is presumed to be attributable to the diagnostically relevant isoenzyme. The separation of the precipitating immunocomplex from the sample is necessary in the Pfleiderer method, because of his concerns that the immunocomplex formed between the anti-serum and the diagnostically relevant isoenzyme, retain at least some of its initial enzymatic activity. It is also a requirement of the Pfleiderer method that the anti-serum used, be highly efficient, that is precipitating at least 90%, and preferably 95-100% of the diagnostically relevant enzyme. Presumably, if the resultant immunocomplex formed between his anti-serum and the diagnostically relevant isoenzyme were enzymatically inactive, then no separation would be necessary.

U.S. Patent 4.067.775 (to Wurzburg, et al.) describes an improved technique for determination of CK-MB which involves the use of unique anti-serum for immunoinhibition of the M sub-unit of both CK-MM and CK-MB isoenzymes. The anti-serum, like that of Pfleiderer, has a relatively high avidity and, thus, is capable of substantially complete inhibition of the diagnostically relevant isoenzyme (less than 5 U/L residual activity remaining after such immunoinhibition); however, unlike Pfleiderer, the interaction of the anti-serum with the isoenzyme is effective in this neutralization of the enzyme and without formation of a precipitating immunocomplex. Thus, the residual activity of the diagnostically relevant isoenzyme (in this case the B sub-unit of CK-MB) can be determined without separation of the immunocomplex and with a single measurement.

The value and accuracy of a diagnostic protocol for CK-MB, based upon a single measurement performed on only one patient sample as an aid in the diagnosis or exclusion of acute myocardial infarction (AMI), has recently been challenged, Gerhardt, W. et al., Clin. Chim. 28/2, 277-283 (1982); and, Wu, A.H.B., et al., Clin. Chim. 28/10, 2017-2021 (1982). In each of these articles, the authors question the reliability of a single CK-MB assay as dispositive of the occurrence or non-occurrence of AMI. Each article recommended that multiple assays be conducted after the onset of the patient distress which is suspected as being caused by acute myocardial infarction. Typically, an assay for determination of the level of CK-MB would be conducted upon admission of the patient to the hospital and then a second (and possibly a third) assay conducted at 10 to 12 hour intervals thereafter. The performance of a series of assays over a recommended time course is believed to provide a more reliable method for the early exclusion of AMI.

In each of the diagnostic test kits which are presently marketed for CK-MB determinations, including those utilizing the proprietary method and antibodies of the assignee of the Wurzburg '775 patent, the recommended diagnostic protocol contemplates measurement of multiple enzyme levels of each sample; one measurement of total enzymatic (kinase) activity, and a second, after immunoinhibition, for residual enzymatic activity.

As is evident from the above discussion of the prior art, the complexity involved in the selective immunoinhibition of CK isoenzymes, and the accurate determination of residual activity of the sample containing such isoenzymes, is critical to the clinical exclusion of AMI as the cause of the patients distress. This task is further complicated by the individual patient sample which can contain antibodies to non-primate proteins (anti-goat, anti-rabbit, anti-mouse antibodies), of the type which are commonly used to produce the anti-serum of the test kits used for detection of CK- MB. In addition, the cross-reactivity (immunoinhibition of the B sub-unit of the isoenzyme) of the anti-serum used in such reagents, can further skew a clinically significant result.

Because of the above and associated complexities, the reliability of such test procedures for determination of CK-MB levels has become compromised and the preparation of reagents and methodologies, unduly cumbersome. The previous direction taken by those concerned with these problems, has been to enhance the reliability of such assays by what is perceived to^' have been the best and, presumably, only available avenue (i.e. increasing the specificity, avidity and/or sensitivity of the anti- serum used in such test kits). While improvements in the quality of anti-serum has provided some positive enhancements, it has not been without significant cost. These anti-sera are generally highly purified and the operating window of the assay has become increasingly narrow. The result is a relatively expensive test which must be performed under rigorously controlled conditions with highly skilled operators and/or very expensive equipment.

SUMMARY OF THE TNVENTTON

This invention has, as its principle objective, the reduction in the complexity and expense of performance of an immunoinhibition assay for diagnostically relevant isoenzyme by making use of relatively impure, low quality anti-sera in combination with rate data processing technique. The rate data processing techniques of this invention, thus, provide a means for compensation of the relatively high background levels of enzymatic activity, which can otherwise interfere with an accurate determination of the level of the diagnostically relevant isoenzymes.

More specifically, this invention provides a method for the determination of a diagnostically relevant isoenzyme of an enzyme occurring in multiple molecular configurations in a complex biological fluid. This method utilizes, in combination, relatively impure, low avidity anti-serum specific for the diagnostically relevant isoenzyme, multiple measurement and multiple sampling techniques, and computational correction of rate data, which are derived from measurement of the residual activity of the diagnostically relevant isoenzyme and other enzymatically active sample constituents, with respect to a common substrate. In the preferred embodiments of this invention, the anti-serum utilized in this method is capable of immunoinhibition of at least 50% and up to about 85% of the initial enzymatic activity of the diagnostically relevant isoenzyme in the patient sample. It is a clinical imperative that for a test of this sort to be effective, both multiple measurements of an individual sample is required, as is repetition of the assay over a time course, on different samples, at one or more periodic intervals (i.e. 10 to 12 hour intervals) from the onset of patient distress . DESCRIPTION OF THE INVENTION INCLUDING PREFERRED EMBODIMENTS

Preliminary to further discussion of the method and test kit of this invention, it would be helpful to briefly define the following terms and phrases to aid in an understanding of this invention.

The phrase "diagnostically relevant isoenzyme" is intended as descriptive of an enzymatically active analyte, typically found in a clinical sample or specimen; and, which may be indicative of an abnormal physiological condition or pathology if present at an abnormal level.

The phrase "multiple molecular configurations" is intended as descriptive of an enzymatically active compounds which may differ in one or more of their physical, conformational and/or chemical features, but yet behave in an essentially identical manner when contacted with a common substrate.

The phrase "residual enzymatic activity" is that degree of enzymatic activity in the sample which remains subsequent to contact of the sample with an anti-serum specific for the neutralization/immunoinhibition of the diagnostically relevant isoenzyme.

In one of the preferred embodiments of this invention, the method and test kit of this invention can be used to provide the clinician with a reliable diagnostic tool to exclude the occurrence of an acute myocardial infarction (AMI) as the cause of distress in a patient. Utilization of this method, contemplates the performance of

(a) multiple measurements of an individual sample for initial determination of total enzymatic activity of the multiple molecular configurations of the diagnostically relevant isoenzyme, prior to neutralization/immunoinhibition;

(b) the determination of residual enzymatic activity of the multiple molecular configurations of the isoenzyme, subsequent to neutralization/immunoinhibition; and,

(c) determination of the level of diagnostically relevant isoenzyme of multiple samples from a patient, over a time course (i.e. at 12 and at 24 hour intervals) following the onset of distress.

More specifically, upon admission of the patient to a hospital, his level of diagnostically relevant isoenzyme would , be determined and, thereafter a second and possibly a third biological fluid specimen obtained from the patient at periodic intervals, and the test method repeated thereon. The values for the diagnostically relevant isoenzyme thus obtained would be compared for each specimen, and clinically significant differences noted.

In practice, the determination of diagnostically relevant isoenzyme involves initially determining the total activity of the multiple molecular configurations of the diagnostically relevant isoenzyme. The patient specimen is thereafter incubated with an anti-serum to the isoenzyme for a period sufficient to effect neutralization/ immunoinhibition of at least 50% of the original level thereof. The immunoinhibition reaction which occurs during this incubation period, results in the formation of an immune complex which can be separated from the specimen, if desired, by centrifugation. Since the immune complex is itself enzymatically inactive, its presence in the specimen does not interfere with the determination of residual activity of the multiple molecular configurations of a diagnostically relevant isoenzyme.

Accordingly, the analysis of the sample for residual activity of such isoenzymes can proceed in the presence of this immune complex. The neutralization of the diagnostically relevant isoenzyme, generally requires anywhere from about 30 seconds to about 5 minutes, depending upon its relative concentration in the sample and the relative avidity of the anti-serum for the isoenzyme.

Following neutralization, a substrate for the multiple molecular configurations of the diagnostically relevant isoenzyme, is added to the patient specimen and the rate of consumption of substrate by the enzymes present in the sample monitored kinetically. The rate data are collected for a period sufficient to provide an accurate reflection of the residual enzymatic activity. It is noteworthy that neutralization of the diagnostically relevant isoenzyme generally continues during this period of contact of sample with substrate; and, that the residual enzymatic activity of the sample continues to decline, however, at a much more gradual rate. During this period of monitoring of the sample for residual enzymatic activity, at least about 15% of the total initial enzymatic activity of the sample is retained.

The anti-serum used in the method of this invention is prepared by conventional methods, as described in Methods In Enzymology (Davis, et al, Vol. X, p. 696-699, 1967; Richmond, Vol. 43, pgs. 86- 100, 1973), or obtained through commercial sources, such as Cambridge Medical Diagnostics, Billerica, Massachusetts; DSL, Houston, Texas; or, PEL FREEZ, Rogers, Arkansas. The method and test kits of the invention are suitable for use in the determination of the following diagnostically relevant isoenzymes:

Lactic dehydrogenase beta-Glyceraldehyde-3 phosphate dehydrogenase Xanthine oxidase 1-Glutamic dehydrogenase Catalase

Tryptophanase Tyrosinase alpha-Glucan phosphorylase Hexokinase Creatine-ATP phosphotransferase

RNA polymerase RNA polymerase, form II

Reverse transcriptase (RNA-dependent, DNA polymerase) Lipase

Lecithinase (phospholipase A)

Alkaline phosphatase alpha-Glycerolphosphatase (glycerol-1 -phosphatase)

Lecithinase (phospholipase C) Deoxyribonuclease

Nuclease beta-Ribonuclease C 1-esterase Amylase Lysozyme

Neuraminidase

Acid alpha-glucosidase (a-b-glucoside glucohydrolase) beta-Galactosidase Hyaluronidase Carbohydrase

Bromelain Carboxypeptidase

Try sin

Elastase

Papain Tropomyosinase

Gelatinase

Streptococcus peptidase A

Collagenase c-Asparaginase Crease

Penicillinase

5'-Adenylic acid deaminase

Adenosinetriphosphatase

Glutamic acid decarboxylase Tryptophan synthetase

Phosphoglucose isomerase

Pyruvate carboxylase

The substrates which can be used in the method of this invention are generally available from a variety of commercial sources or can be prepared by conventional synthesis techniques from readily available materials. Substrates for each of the above enzymes are identified in standard reference text, see for example, Methods in Immunology and Immunochemistry, Vol. IV, Academic Press (1977), pp. 316-320.

The method for monitoring the residual enzymatic activity of the sample is preferentially performed on an automated clinical chemistry analyzer, and the rate data collected and processed automatically within the analyzer. The sample is initially obtained in the conventional manner and prepared for analysis. Such preparation can typically involve the separation of the cellular components of a whole blood sample from the serum fraction, and thereafter analyzing the serum fraction. Under some circumstances, it may be appropriate to dilute the sample prior to such analysis. In a typical analysis of the sample for a diagnostically relevant isoenzyme in accordance with the method of this invention, the total enzymatic activity of the sample would be determined for the multiple molecular configurations of the diagnostically relevant isoenzyme. A relative impure, low avidity anti-serum for the diagnostically relevant isoenzyme would then be added to the sample, the anti-serum and isoenzyme allowed to interact (incubate) until at least about fifty percent (50%) of the activity diagnostically relevant isoenzyme was neutralized. During this neutralization process, an enzymatically inactive precipitating immunocomplex may form. The presence of this precipitate can be tolerated in the sample during the performance of the method of this invention. Following this initial neutralization phase of the process, a substrate for diagnostically . relevant isoenzyme is added to the sample and the residual enzymatic activity of the sample recorded.

The measurement . of the enzymatic activity is based upon conversion of the substrate to an indicator, and the relative concentration of the indicator monitored kinetically. It is of course understood that the substrate employed in the method of this invention is also subject to attack by the multiple molecular configurations of the diagnostically relevant isoenzyme. Thus, the rate data for the enzymatic conversion of substrate to an indicator is not a useful measurement and, without further refinement, cannot be used as a basis for diagnosis.

In order to simplify the explanation of the method of this invention, the balance of this discussion will deal with the creatine kinases as the model system upon which this method has been most fully developed. The model system selected for development of this method is based upon the determination of the level of the CK-MB isoenzyme. The analytical protocol used in this determination, is as previously described. The measurement of residual creatine kinase activity in the sample produced rate data which was essentially meaningless without further refinement. In the process of arriving at some rational, reproducible and consistent method of refinement of this data, it was discovered that in a neutralization/immunoinhibition environment, a single correction factor was not adequate. More specifically, it was necessary to empirically determine an individualized correction factor for the specific anti- serum employed in the method; and, that the variations in the anti- serum from animal to animal and from different bleeds of the same animal, differed sufficiently to required empirical determination of individualized correction factors. In the model system (CK-MB), the processing of the rate data involves correction of the data to mathematically delete residual enzymatic activity from CK-MB, CK-MM, cross-reactivity of the anti-serum with the B sub-unit and other interferents.

The standard formula for calculation of CK value in international units is as follows:

Δ A/min x 10^ x Total Volume in mis U/L = _:

6.22 x lθ3 x 1 x Sample Volume in mis

Since the CK-MB isoenzyme of interest is a dimer, the standard formula is modified to reflect this • fact. The value of the numerator is simply multiplied by the appropriate factor (IC=2) to reflect the unique character of the CK isoenzyme. The standard formula is, thus, restated as follows: Δ A/min x 10^ x Total Volume in mis x IC

U/L =

6.22 x 10- x 1 x Sample Volume in mis

Because of the unpredictable variation in result for each batch of anti-sera, an additional correction was required to further compensate for these variations and allow for the development of a reliable and consistent presentation of the analytical data. The following formula provides the capability to interpret rate data from a heterogenous system in which the anti-serum can vary in avidity from batch to batch and, in addition, tolerate a significant level of residual enzymatic activity from not only the diagnostically relevant isoenzyme, but also a number of other interferents.

Δ A/min x 10⁶ x Total Volume in mis x IC x FVCF

U L =

6.22 x 10- x 1 x Sample Volume in mis

EXAMPLES

The Examples which follow further define, describe and illustrate the method of this invention. Apparatus and techniques used in the performance of this method are standard or as hereinbefore described. Parts and percentages appearing in such Examples are by weight, unless otherwise indicated. EXAMPLE 1

A whole blood sample is initially obtained from a patient suspected of suffering acute myocardial infarction. The sample is prepared for analysis in an automated clinical chemistry analyzer, preferably the DACOS® chemistry analyzer available from Coulter Electronics Corporation of Hialeah, Florida. Such sample preparation typically involves separation of the serum from the cellular fraction.

Specimen Handling

If serum is not assayed immediately, it should be kept in a stoppered container and refrigerated. Avoid exposure to bright light. Hemolyzed samples should not be used, although slight hemolysis can be tolerated.

Test Principle

In the DACOS test methodology for CK-MB isoenzyme, a goat antibody is used to inhibit the activity in the patient's serum sample contributed by the M sub-unit of the MB isoenzyme and by the MM isoenzyme. The residual activity is measured using the DART CK reagent (modified Oliver-Rosalki method) (available from Coulter Electronics Corporation, Hialeah, Florida). Since the monitored activity is contributed by the B sub-unit of CK-MB and it is doubled to obtain the CK-MB activity. When present in the serum sample, CK-BB, macromolecular forms of CK and mitochondrial CK will contribute to this residual activity, but the frequency and magnitude for the incidence of any of these interferents is less than 1%. Interference from adenylate kinase is suppressed by AMP and adenosine pentaphosphate in the DART CK (CPK) reagent.

D AR T ® Reagent Package Contents

Anti CK-MM goat antibody 1 x 0.24 ml Tris-HCL buffer 1 x 12 ml

Dart CK reagent 2 x 10 ml

Reagent Preparation

Prepare a 1 :50 dilution of anti-CKMM by taking 50 ul of the antibody and adding 2.45 ml of Tris-HCL buffer. Mix gently. This dilution is stable at 5°C for 10 days.

Gently tap one vial of DART CK (CPK) reagent several times to loosen contents from sides of container. To one vial, add 10.0 ml of water which meets or exceeds the specification for NCCLS Type II water. Mix immediately by gently swirling and inverting, to avoid foaming, until contents are completely dissolved. Reagent is stable for 72 hours.

Expected Values

Normal CK-MB activity: 0 - 16 U/L (See Normal Range Section) Analytical Range

Serum samples with total CK activity exceeding 1200 U/L should be diluted and reassayed.

The rate data processing capability of the data management terminal associated with the DACOS analyzer is able to effectively compare the sample data with a standard curve in its data base. Compensation for high residual enzymatic activity of the sample is automatically factored into the final print out of the results of the assay in the patient sample. In the DACOS analyzer, this is achieved by applying a correction factor (FVCF) to the rate data. The correction factor for the DART CK-MB isoenzyme test kit

(Catalog #7546862) is 1.33, based upon anti-serum lot #72000K.

The above assay is repeated on additional patient samples taken at 12 and 24 hour intervals, and the results compared to the initial determination of CK-MB activity.

Claims

1. A method for determination of the relative concentration of a relevant isoenzyme in a biological fluid sample, wherein said relevant isoenzyme has multiple molecular configurations and can vary in concentration, said method including the steps of: (i) incubating a biological fluid sample, suspected of containing an abnormal level of a relevant isoenzyme, with anti-serum specific for neutralization/immunoinhibition of said relevant isoenzyme in said biological fluid sample; (ii) allowing interaction of said serum and said relevant isoenzyme until said isoenzyme has formed an enzymatically inactive immunocomplex; (iii) adding an isoenzyme specific substrate to said sample under conditions conducive to enzymatic conversion of said substrate to an indicator indicative of the residual enzymatic activity of the sample; and, (iv) monitoring the sample for the presence of the indicator, the method characterized wherein:

(a) said step of incubating under neutralization/immunoinhibition conditions is with a relatively impure, low avidity anti-serum specific for the relevant isoenzyme;

(b) said interaction of the anti-serum and isoenzyme is allowed to proceed until the rate of formation of the resultant immunocomplex approaches equilibrium prior to addition of the isoenzyme specific substrate;

(c) said monitoring comprises making multiple measurements of the sample for the presence of the indicator over a period of time sufficient to derive rate data for the production of the indicator;

(d) processing the rate data to correct for residual enzymatic activity of the sample which is attributable to the isoenzyme and other enzymatically active constituents of the sample which are also specific for the substrate, said rate data processing involving applying a predetermined correction factor to said rate data, said correction factor having been developed specifically for the anti-serum used in step (a); and,

(e) repeating steps (a) to (d) on a subsequent biological fluid sample obtained from the same sample source, at a prescribed interval, and comparing the relative enzymatic activity of the initial sample with that of the subsequent sample.

2. The method of claim 1, characterized wherein the neutralization/immunoinhibition of the isoenzyme reduces the endogenous enzymatic activity of the sample at least 50% up to about 85%.

3. The method of claim 1, characterized wherein the isoenzyme specific substrate is added to the sample after the rate of formation of the resultant immunocomplex approaches equilibrium.

4. The method of claim 1, characterized wherein the substrate is cleaved by the enzyme, releasing a chromophore or fluorophore which is capable of detection by conventional monitoring techniques.

5. The method of claim 1, characterized wherein the anti-serum used in the assay of the initial biological fluid sample and a subsequent biological fluid sample, is from a different animal, or different bleed from the same animal. 6. The method of claim 1, characterized wherein the relevant isoenzyme is CK-MB.

7. The method of claim 6, characterized wherein the prescribed interval between determination of the level of activity of the CK- MB of the initial sample, and of the CK-MM activity of a subsequent sample is about 12 hours.

8. The method of claim 6, characterized wherein the neutralization/immunoinhibition of the enzyme by the anti- , serum forms a precipitating immunocomplex.

9. The method of claim 6, characterized wherein the anti-serum is obtained from a non-primate source and the biological fluid sample contains antibodies to the anti-serum.

10. The method of claim 6, characterized wherein the anti-serum cross-reacts with at least some of the B sub-unit of CK-MB.

11. The method of claim 6, characterized wherein the monitoring of the level of indicator is performed on an automated clinical analyzer and the predetermined correction factor applied to the rate data by the rate data processing logic of the analyzer.

12. A test kit for the determination of a relevant isoenzyme having multiple molecular configuration, said test kit including a high avidity anti-serum specific for neutralization/immunoinhibition of essentially all of said relevant isoenzyme and an isoenzyme specific substrate, the test kit characterized wherein:

(a) said anti-serum comprises a relatively impure, low avidity anti-serum capable of neutralization/immunoinhibition of at least 50% up to about 85% of the relevant isoenzyme; and,

(b) a rate data correction factor specific for the anti-serum.