EP0382725A4 - Method and test kit for neutralization/immunoinhibition assay - Google Patents
Method and test kit for neutralization/immunoinhibition assayInfo
- Publication number
- EP0382725A4 EP0382725A4 EP19880904874 EP88904874A EP0382725A4 EP 0382725 A4 EP0382725 A4 EP 0382725A4 EP 19880904874 EP19880904874 EP 19880904874 EP 88904874 A EP88904874 A EP 88904874A EP 0382725 A4 EP0382725 A4 EP 0382725A4
- Authority
- EP
- European Patent Office
- Prior art keywords
- isoenzyme
- sample
- serum
- immunoinhibition
- neutralization
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
- G01N33/542—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with steric inhibition or signal modification, e.g. fluorescent quenching
Definitions
- 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.
- 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.
- 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.
- immunoinhibition 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.
- 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.
- Cinader 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
- 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.
- 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.
- 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.
- 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 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.
- the residual activity of the diagnostically relevant isoenzyme in this case the B sub-unit of CK-MB
- 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.
- 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.
- 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.
- 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.
- 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.
- diagnosis 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.
- 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.
- 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.
- 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.
- AMI acute myocardial infarction
- 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.
- 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.
- 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:
- Alkaline phosphatase alpha-Glycerolphosphatase (glycerol-1 -phosphatase)
- 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.
- 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.
- 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.
- 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.
- 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 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.
- 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.
- serum 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.
- 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.
- 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.
- DART CK DART CK
- 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.
- FVCF correction factor
- 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.
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
Description
Claims
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US4871287A | 1987-05-12 | 1987-05-12 | |
US48712 | 1987-05-12 |
Publications (2)
Publication Number | Publication Date |
---|---|
EP0382725A1 EP0382725A1 (en) | 1990-08-22 |
EP0382725A4 true EP0382725A4 (en) | 1990-12-27 |
Family
ID=21956040
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP19880904874 Withdrawn EP0382725A4 (en) | 1987-05-12 | 1988-05-11 | Method and test kit for neutralization/immunoinhibition assay |
Country Status (5)
Country | Link |
---|---|
EP (1) | EP0382725A4 (en) |
JP (1) | JPH01503565A (en) |
AU (1) | AU1800088A (en) |
CA (1) | CA1320114C (en) |
WO (1) | WO1988008984A1 (en) |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
ATE120551T1 (en) * | 1990-06-21 | 1995-04-15 | Axis Biochemicals As | ANALYSIS OF VARIANTS OF ANALYTES. |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
DE2548963C3 (en) * | 1975-11-03 | 1982-03-11 | Merck Patent Gmbh, 6100 Darmstadt | Method and means for determining the activity of creatine kinase MB |
-
1988
- 1988-05-11 EP EP19880904874 patent/EP0382725A4/en not_active Withdrawn
- 1988-05-11 AU AU18000/88A patent/AU1800088A/en not_active Abandoned
- 1988-05-11 WO PCT/US1988/001553 patent/WO1988008984A1/en not_active Application Discontinuation
- 1988-05-11 CA CA000566474A patent/CA1320114C/en not_active Expired - Fee Related
- 1988-05-11 JP JP50458888A patent/JPH01503565A/en active Pending
Non-Patent Citations (2)
Title |
---|
CLINICAL CHEMISTRY, vol. 32, no. 6, 1988, page 1137, abstract no. 432, Winston-Salem, N.C., US; C.E. SEWELL et al.: "Automated determination of CK-MB isoenzyme" * |
See also references of WO8808984A1 * |
Also Published As
Publication number | Publication date |
---|---|
CA1320114C (en) | 1993-07-13 |
WO1988008984A1 (en) | 1988-11-17 |
EP0382725A1 (en) | 1990-08-22 |
AU1800088A (en) | 1988-12-06 |
JPH01503565A (en) | 1989-11-30 |
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