CA1320114C - Method and test kit for neutralization/immunoinhibition assay - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE

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

~ 3 ~

Title: Method and Test Kit For Neutralization/Immunoinhibition Assay BA~KGRnlJ~D ~)~E 7~1V NTIO~l 1. FieldQf the Invention - This invention is directed to an improved neutralization assay, based upon the relatively incomplete immunoinhibition of the activity of a diagnostically 10 relevant isoenzyme whose relative concentration can be indica~ive of an abnormal, physiological state; and, a test ki~ ~or 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 15 acute myocardial infarction.

2. Pescription of the Invention - The study of enzymes and their roles in various physiological processes~ has led to a more 2 0 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-25 MM), smooth muscle ~CK-MB), and in the brain ~CK-BB). When inJury occurs to one or more of these tissues, ~he creatine lcinase (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 30 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.

~, -~' 2 1 ~ 2 ~
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, brief glossary of terms would be both appropriate and helpful to 5 a more complete understanding and discussion of the literature pertinent to this area.

The term "neutralization" is used hereinafter, in the context of a 10 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 ~orming an immunocomplex which is essentially deYoid of enzymatic activity and, a secondary phase in which there is a gradual decrease in 15 the residual activity of the enzyme after floculation. (Cinader, Ann. Rev. Microbiol. 11, 371-390 (1957).) The term "agglutination" is used hereinafter, in the contex~ of a 20 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 25 the form of an agglomerate. (Kabat & Mayers, Fxperimental Immunochemistry, Chas. Thomas Publ., ~pringfield, Illinois, 905 pgs. (1967~.) 3 0 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. 24I-251 (1975).) 3 132~
The term "immunoprecipitation" is used hereinafter, in the context of a clinical assay, to charac~erize an assay in which the anti-serum interaction with the target enzyme forms a precipitate which is then separated by centrifugation and subjected to 5 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-10 serum and an enzyme, dates back almost 40 years; one of theearlier 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 S 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 ~his area, and proposed models for 20 antibody/enzyme neutralization interaction.

A second review by Cinader in 1957 described and discussed the more recent advances in antibody/enzyme interaction, Ann. Rev.
Microbiol. 11, 371-390 (lg57). This ~inader 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.

In the early 196~'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 3 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 antibocly. Samuels also reported that immunoinhibition causes conformational changes in the kinase enzymes and that such conformational changes differed from 5 those resulting where a substrate and the enzyme interact. (Ann.
NY Acad Sci., Vol. 103, ~89-963 (1963).) Watts, et al reported in 1962 what appears to be the first 1 0 recognition that creatine kinase exists ~s a complement or a series of different, but related, enzymes (i.e. isoenzymes), Watts, et al, Biochem. J. 82, 412 (19623. The revelations by Watts, et al, paralleled developments relating to the study of LDH, which included the immunochemical differentiation of three distinct 15 LDH isoenzymes (as originating in tissues of the heart, liver and muscle), Cahn, ~cience 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-en~yme antibodies, Cinader, Introduction for Ann. NY Acad. Sci., ~ol. 103, ~95-548 (1963). The Cinader review article discussed mvre than 100 references relating to this topic.
25 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 3 0 that creatine kinase was an exception to the general scene relating to immunoinhibitivn 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 3 S traditional precipitation base methodologies.

In 1964, three distinct forms of creatine kinase enzyme complement were identified, CK-MM, CK-MB and CK-13B, Duel and Van Breeman, Abst. Fed. of Europ. Bioch. Soc., pg. 52 (1964~; Clin.
Chim. Acta 19, 276 (1964); and, Burger, ~. et al., ~iochem. Z.
339,305 (1964). In 1965, Dawson explained the relationship and differences between these three isoenzymes as originating with the nnonomeric units of each and their respective combinations.
Dawson continued his elucidation of this subject by publishing two additional articles; one appearing in 1967 (Dawson & ~7ine, 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), 773-73~
2~) (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. Paten~s 3,932,221 (foreign priority, June 9, 1971); and, 4,067,775 (foreign priority, November 3, 1975)-U.~. Patç~2.221 (to Pfleiderer) described a diagnostic method for determination of residual activity of diagnostically 3 5 relevant isoenzymes. According to Pfleiderer, total çnzymatic activity of the diagnostically relevant isoenzyme, vis-a-vis a 6 1 '~ 2 ~
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 5 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 ~otal enzymatic activity for the sample as initially measured. The 10 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 15 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 20 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 30 Pfleiderer, has a relatively high avidity and, thus, is capable of substantially complete inhibition of the diagnostically relevant isoenzyme (less t~an 5 U/L residual activity remaining after such immunoinhibi~ion); however, unlike Pfleiderer, the interaction of the anti-serum with the isoenzyme is effective in this 3 5 neutralization of the enzyme and without formation of a precipitating immunocomplex. Thus, the residual activity of the 7 1~011~

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 C~-MB assay as dispositive of the occurrence or non-occurrence of AMI. Each article recommended that multiple assays be conducted after the l S 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 20 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 AM~.

2S In each of the diagnostic test kits which are presently marketed for CK-M13 determinations, including those utilizing the proprietary method and antibodies of ~he assi~,nee of the Wurzburg '775 patent, the recommended diagnostic protocol contemplates measurement of multiple enzyme levels of each 3 Q sample; one measurement of total enzymatic ~kinase) activity, and a second, after immunoinhibition, for residual enzymatic activity.

35 As is evident from the above discussion of the prior art, the complexity involved in the selective immunoinhibition of CK

8 ~ 3 ~

isoenzymes, and the accurate determination of residual ~cti~/ity 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 S contain antibodies to non-primate proteins (anti-goat, anti-rabbit, anti-mouse antibodies), of the type which are commonly use(l 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 1 () reagents, can further skew a clinieally significant result.

Because of the above and associated complexities, the reliability of such test procedures for determination of CK-MB levels has 1~ 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.
2 0 increasing the specificity, avidity and/or sensitivity of the anti-serum used in such test kits). While improvements in the quality of an~i-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 25 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 .

This invention has, as its principle objective, the reduction in the 3 5 complexity and expense of peTfo~man~e of an immunoinhibition assay for diagnostically relevant isoenzyme by making use of g 1~

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 5 otherwise interfere with an accurate determination of the JeYel of the diagnostically relevant isoenzymes.

More specifically, this invention provides a method for the 10 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 affinity anti-serum specifi¢ for the diagnostically relevant isoenzyme, multiple measurement and 15 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 20 invention, the anti-serum utilized in this method is capable of immunoinhibition of at least 50% and up to about 85% of the initial en~ymatic activity of the diagnostically relevant isoen~yme in the patient sample. It is a clinical imperative that for a test of this sort to be effective, both multiple measurements of an 25 individual sample is reguired, 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 .

s~
,. .

10 l32all~

DESCRIPTION OF THE INVENTION
INC~L~pING PREFERRED EM~B~M~N'rS

Prel;millary 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 specirnen; and, which may be indicative of an 15 abnormal physiological condition or pathology if present at an abnormal level The phrase "multiple molecular configurations" is intended as 2 0 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 esselltially identical manner when contacted with a common substrate.

The phrase "residual enzymatic activity" is that degree of enzymatic activity in the sample which remains subsequen~ to contact of the sample with an anti-serum specific for the neutraliæation/immunoinhibition of the diagnostically relevant 3 0 isoenzyme.

In one of the preferred embodiments of this invention, the method and test kit of this invention can be used to provide the 35 clinician with a reliable diagnostic tool to exclude the occurrence of an acute myocardial infarction (Al~II) as the cause of distress in ~32~

a patien$. 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 ~o 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, ovel 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 ~he 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 3 0 isoenzyme involYes 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 3 5 level thereof The immunoinhibition reaction which occurs during this incubation period, results in the formation of an immune 12 13~011~
complex which can be separated from the specimen, if desired, by centrii~ugation. 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 5 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 isoellzyme, generally requires anywhere from about 30 seconds 10 to about 5 minutes, depending upon its relative concentration in the sample and the relative avidity of the anti-serum for the isoenzyme.

15 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 20 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 25 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.

3 0 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), ~r obtained ~hrough commercial sources, such as Cambridge Medical Diagnostics, ~3illerica, Massachusetts; DSL, 35 Houston, Texas; or, PEL FREEZ, Rogers, Arkansas.

13 1~0~1~

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 l-Glutamic dehydrogenase 1 0 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 l-esterase Amylase 3 0 Lysozyme Neuraminidase Acid alpha-glucosidase (a-b-glucoside glucohydrolase) beta-Galactosidase Hyaluronidase 3 5 Carbohyd:rase Bromelain 1~ 132~
Carboxypeptidase Trypsin ~lastase Pap ain Tropomyosinase Gelatinase Streptococcus peptidase A
(~ollagenase c-A~sparaginase 1 0 Crease Penicillinase 5'-Adenylic acid deaminase ~denosinetriphosphatase Glu~amic 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 fron~.
readily available materials. Substrates for each of the above enzymes are identified in standard reference ~ext, see for 2 5 example, Methods in Immunology and Immunochemistry, Vol. 1~, Academic Press (1977), pp. 316-32U.

The method for monitoring the residual enzymatic activity of the 3 0 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 tlle 3 5 cellular components of a whole blood sample *om the serum fraction, and thereafter analyzing the serum fraction. Under 13~

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 5 be determined for the multiple molecular configurations of the diagnostically relevant isoenzyme. A relative impure, low affinity anti-serum for the lliagnostically 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 10 activity diagnostically relevant isoenzyme was neutralized.
During this neutralization process, an enzymatically inactive precipitating immunocomplex may form. Tlle presence of this precipitate can be tolerated in the sample during the performance of the method of this invention. Following this initial 15 neutralization phase of the process, a substrate for diagnostically relevant isoenzyme is added to the sample and the residual enzymatic activity of ihe sample recorded.

20 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 25 configurations of the diagnostically relevant isoenzyme. Thus, th 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 wi~h the creatine kinases as the model system upon wllich this method has been most fully developed. The model system selected for 3 5 development of this method is based upon the determination of the level of the CK-MB isoen7.yme. The analytical protocol used in 16 ~.32~
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, 5 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 10 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. ~n the model system (CK-MB), the processing of the rate data involves 15 correction of the data to mathematically delete residual enzymatic activity from CK-MB, CK-MM, cross-reactivity of the anti-serum with the le sub-unit and other interferents.

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

~ A/min x 106 x Total Volume in mls U/L= _ _ _ 6.22 x 103 x 1 x Sample Volume in mls Since ~he CK-MB isoenzyme of interest is a dimer, the standard 30 formula is modified to re~lect ~his fac~. The value of the numerator is si~ply multiplied by the appropriate fac~or (IC=2) to reflec~ the unique character of the CK isoenzyme. The standard formula is, thus, restated as follows:

17 1~2011~

~ A/min x 106 x Total Volume in mls x IC
U/L = ., , ,~
6.22 x 103 x 1 x Sample Volume in mls Because of the unpredictable variation in result for each batch of anti-sera, an additional correction was required to further 10 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 sys$em in which the anti-serum can vary in avidity from batch to batch and, in addition, ~olerate a significant 15 level of residual enzymatic activity *om not only the diagnostically relevant isoenzyme, but also a number of other interferents .

~ A~min x 106 x Total Volume in mls x IC x ~VCF
~J/L = , 6.22 x 103 x 1 x Sample Volume in mls E2~kl~

The Examples which follow filrther define, describe and illustrate the method of this invention. Apparatus and techniques used in 3 0 the performance of this method are standard or as hereinbefore described. Parts and percentages appearing in such Examples are by weight, unless o~herwise indicated.

~32011~

E~AMPLE 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 10 Coulter Electronics Corporation of Hialeah, Florida. Such sample preparation typisally involves separation of the serum from the cellular fraction.

15 ~

If serum is not assayed immediately, it should be kept in a stoppered container and refrigerated. Avoid exposure to bright 2 0 light. Hemolyzed samples should not be used, although slight hemolysis can be tolerated.

~~

In the I)ACOS 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 isoen~yme and 30 by the MM isoenzyme. The residual activity is measured using the DART CK reagent (modified Oliver-Rosalki method) (available ~rom 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 35 serum sample, CK-BB, macromolecular forms of CK and mitochondrial CK will contribute to this residual activity, but the , . . .

19 132~
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.

lû Anti CK-MM goat antibody 1 x 0.24 ml Tris-HCL buffer 1 x 12 ml Dart CK reagent 2 x 10 ml 1 5 4c~

Prepa~e 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 20 dilution is stable at 5C 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 25 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.

Normal CK-MB activity: 0 - 16 U/L (See Normal Range Sec~ion) 132~114 Analvtical Ran~e Serum samples with total CK activity exceeding 1200 U/L should 5 be diluted and reassayed.

The rate data processing capability of the data management terminal associated with the DACOS analyzer is able to effectively 10 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 I)ACO~ analyzer, this is achieved by applying a correction factor (FVCF) to the rate data.
15 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 20 at 12 and 24 hour intervals, and the results compared to the initial determination of CK-MB activity.

Claims (18)

1. A method for determination of a diagnostically relevant isoenzyme having multiple molecular configurations, said isoenzyme being present in biological fluid samples and varying in relative concentration, based upon the physiological condition of the patient, or the presence of pathology, said method comprising:

(a) incubating an initial biological fluid sample, suspected of containing an abnormal level of a diagnostically relevant isoenzyme, with a relatively impure, low affinity anti-serum specific for neutralization/immunoinhibition of the diagnostically relevant enzyme;

(b) allowing the anti-serum and isoenzyme to interact until the rate of formation of the resultant immunocomplex approaches equilibrium;

(c) adding an isoenzyme specific substrate to the sample under conditions conducive to enzymatic conversion of the substrate to an indicator which is of residual enzymatic activity in the sample;

(d) monitoring the sample for the presence of indicator, said monitoring comprising 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;

(e) 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, (f) repeating steps (a) to (e) on a subsequent biological fluid sample obtained from the same patient 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, 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, wherein the isoenzyme specific substrate is added to the sample upon completion of the primary phase of the neutralization/immunoinhibition reaction.

4. The method of claim 1, 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, 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, wherein the diagnostically relevant isoenzyme is (CK-MB.

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

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

11. The method of claim 6, 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 the level of a diagnostically relevant isoenzyme in a biological fluid sample, said kit comprising:

(a) an anti-serum for neutralization/immunoinhibition of at least 50% up to about 85% of the diagnostically relevant isoenzyme;

(b) a substrate for the diagnostically relevant isoenzyme;
and, (c) a rate data correction factor for the anti-serum.

13. A method for determination of the diagnostically relevant isoenzyme, creatine kinase MB, said isoenzyme being present in biological fluid samples, along with other creatine kinase isoenzymes of somewhat different molecular configuration and varying in relative concentration, based upon the physiological condition of the patient, or the presence of pathology, said method comprising:

(a) incubating an initial biological fluid sample, suspected of containing an abnormal level of CK-MB isoenzyme, with a relatively impure, low affinity anti-serum specific for neutralization/immunoinhibition up to about 85% of the diagnostically relevant enzyme;

(b) allowing the anti-serum and isoenzyme to interact until the rate of formation of the resultant immunocomplex approaches equilibrium;

(c) adding an isoenzyme specific substrate to the sample under conditions conducive to enzymatic conversion of the substrate to an indicator which is indicative of the residual enzymatic activity in the sample;

(d) monitoring the sample for the presence of indicator, said monitoring comprising 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;

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

14. The method of claim 13, wherein the neutralization/immunoinhibition of the enzyme by the anti-serum forms a precipitating immunocomplex.

15. The method of claim 13, wherein the anti-serum is obtained from a non-primate source and the biological fluid sample contains antibodies to the anti-serum.

16. The method of claim 13, wherein the anti-serum cross-reacts with at least some of the B sub-unit of CK-MB.

17. The method of claim 13, 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.

18. In a method for the determination of a diagnostically relevant isoenzyme having multiple molecular configuration involving the neutralization/immunoinhibition of the isoenzyme with an anti-serum and determination of the residual activity of the isoenzyme, the improvement comprising:

(a) incubating the biological fluid sample with a relatively impure, low affinity anti-serum which is specific for the neutralization/immunoinhibition of up to about 85% of the diagnostically relevant isoenzyme; and, (b) determining the level of diagnostically relevant isoenzyme by monitoring the enzymatic activity of the sample as a function of the rate of production of an indicator which is produced as a result of the action of the isoenzyme upon an enzyme specific substrate, and correcting the rate data for the anti-serum utilized in step (a).