JPH01503565A - Neutralization/Immune Inhibition Assay Methods and Test Kits - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] ■■ 1, □ Comparison of the activities of diagnostically relevant isoenzymes whose relative concentrations may indicate abnormal physiological conditions An excellent neutralization assay based on incomplete immune inhibition; This relates to a test kit for carrying out the test. This assay is useful for sexual myocardial infarction. Measurement of CM-MB in biological fluids to aid in accurate identification of patients suffering from disease. It has certain special uses.

2. Matasara 2nd Hall The study of enzymes and their roles in various physiological processes has revealed that these complex Interaction between promiscuous proteins and aberrant body functions associated with trauma and/or medical conditions For example, the relationships found in biofluid samples have become more fully understood. The main taleatin kinase is skeletal muscle (CK-?LM).

smooth muscle (CM-?IB), and certain tissues found in the brain (CM-BB) known to be related. If failure occurs in one or more of these tissues , blood talleatin kinase (CK) levels are most prevalent in traumatized tissue. 0 people with elevated isoenzyme levels had heart attacks (it) and sexual myocardial infarction. ), blood levels of the CK-MB isoenzyme increase with the onset of the attack. results in an increase in CK-11B levels; the greatest increase in CK-11B amounts is generally caused by the first suffering It seems to be dark from 10pm to 12pm.

Patent and technical literature is included, along with literature directed to various methods of measuring CK isoenzyme activity. There are enough. Before reviewing these documents, it is appropriate to provide a brief explanation of terminology, and at the same time It will aid in a more complete understanding and discussion of the literature relevant to this field.

The term "neutralization" hereinafter refers to the formation of two separate parts in the context of an oral n-bed assay. : The antiserum and the target enzyme interact rapidly, resulting in an immune system almost devoid of enzymatic activity. The first part forms a complex, and the second part gradually reduces the remaining activity of the enzyme after aggregation. Used to characterize immunoassays with two parts. [Cinnadar (Ci) nader) + Ann, Shiv. Micropaiol, (Ann, Rev, M icrobiol, ) 11+ 371-390 (1957). ]Here, The term "concentration reaction" hereinafter refers to clinical assays in which the antigen particles are 1.000 to 2. ,000 rpm to be precipitated by centrifugation (500 ~1,000 g), therefore, when contacted with a specific antiserum against this antigen , this features an immunoinhibitory assay in which immune complexes, which are in the form of aggregates, are generated. used to attach. [Kabat and May ers) + Experimental Immunochemist, Lee, Chas, Thomas Bubble, (Chas.

Thomas Publ, ) + Springfield, 4924.905 pages (1 967), ) The term "immunotitration" hereinafter refers to an immunoinhibitory assay in relation to an n-bed assay. It is used to characterize the antiserum's target enzyme, and the interaction of the antiserum with the target enzyme is forms an inert precipitate. [Sehinke + Methods In -Enthymology, Vol. 40, pp. 241-251 (1975). ]Here, The term "precipitation" characterizes immunoinhibitory assays in the context of clinical assays. The interaction of the antiserum with the target enzyme forms a precipitate, which then The precipitate is separated by centrifugation and its protein content is analyzed.

[Sink, Mesosoz Inchimorosii, Vol. 40, pp. 241-251 (197 5). ] The technical literature on the interaction of antisera with enzymes dates back almost 40 years; One of the relatively early references is the immunocatalytic and antifermentic activity of Sevag. Commentary on elementary antibodies, Immunocatalysis, 2nd edition, C., Thomas Kompany. -, Springfield, I., p. 547. Enzymes in immune inhibition Interest was fostered in the early to mid-1950s, with some literature and reviews appearing. It was. A review by Sinagou in 1955 describes the properties of various enzymes/antienzyme antibodies. Listed are pull, tsuk, him, payor, (Bull, Soc.

China, Biol, ), 37 (7-8), 761-781 (19 55). Synagoo thoroughly reviews and details the pioneering research in this field, A model for antibody/enzyme neutralization interaction was proposed.

A second review by Sinagou discusses more recent advances in antibody/enzyme interactions. Written and discussed by Ann and Shiv. Micropaiol, (Ann, Re v8Microbio1. ) 11+371-390 (1957). This China Gu's literature explores antibody/enzyme interaction as a tool in biological and immunological investigations. studies involving the use of such enzymes, and any loss or change in levels and/or activity of such enzymes. It provides a detailed review of the interrelationship with specific medical conditions.

In the early 1960s, two papers appeared, these were creatine kinase. Bioph ys,) J., 1, 437 (1961).

and Arch.

Biocb, and Biophys,) 92.497 (1961), Samuels added the substrate to a sample containing the creatine kinase enzyme. , the observation that unlike other enzyme/antibody systems, does not interfere with immune inhibition of the enzyme by antibodies. reported. Samuels also says that immunity is harmful because of structural changes in kinase enzymes. changes in structure and that result from interactions between substrate and enzyme. reported something different. [Ann, NY, Akado Sai Acad, Sci., ) Vol. 103, pp. 889-963 (1963). ] In 1962, Watts et al. What seems to have been the first thing to be recognized or known to exist as a substance or complement (i.e. Watts et al., Bioche++1., Jie et al. I, 82.412 (1962). The discovery by Watts et al. Immunochemical division of isoenzymes (as those occurring in heart, liver, and muscle tissue) Cahn + service parallel to developments in LDH research, including Jens 136.962-969 (1962).

Studies by Cano involved immunoinhibition of various LD) isoenzymes after centrifugation.

The third review was published by Synagoo in 1963, and was directed exclusively at anti-enzyme antibodies. Kereta, Sinader, Introduction Fore Ann, NY, Acad , Sci., Vol. 103, pp. 495-548 (1963). This is the synagogue More than 100 types of literature related to the topic are discussed. Synagogue's review literature is particularly The topic of multimolecular forms of enzymes and currently available immune inhibition technologies are How can enzymes potentially play a significant role in both differentiation and quantification? I treated Ruka. Sinader's review literature shows that creatine kinase has general implications for immune inhibition. The immunochemical behavior is described as an exception to the typical scene, and the proposed fourth order equation describes its immunochemical behavior. A comparison was made to the model system. Synagoo also uses neutralization evaluation to measure isoenzymes in mixtures. described that it is more accurate than conventional precipitation-based methods.

In 1964, three different forms of creatine kinase enzyme components, CM-Question, C K-MB and CM-BB were identified in Duell and Pumblyman, Abu Sto, Fet, of Yolob.

Bioc, Soc, (Abst, Fed, of Europe, Bioc h, Soc, ) + page 52 (1964); Clin, Chem, Acta (CIi n, Chi+n, Acta) 19, 276 (1964); and bar Gar, stingray, etc., biochem.

Z (Bioche+m, Z,) 339.305 (1964), 1 In 965, Taurine explained the relationships and differences between these three isoenzymes. It was first explained using the monomer units of each conjugate.

Taurine is found in two other publications; the first published in 1967] and taurine. Fine, Arc, 221 Roll, (Arch.

Neurol, ) 16: 175-180 (1967)); the second one is Published in 1968 [Taurine et al. Ann, NY Acad, Sci, +' 155 + 616-626 (1968)) describes his explanation of this subject in the literature. continued. In the same year, Birkoff established that CK-MByF: associated with acute myocardial infarction. Ann, Intern.

Med, ) 122.326 (1968).

The clinical validity of assessing increased CM-MB activity as an indication of sexual myocardial infarction Now fully recognized, Wilkinson, J'-, Taryn, Kem, U, C11n, Chew, ) 16(9), 773-739 (1970 ), e, as an indication of sexual myocardial infarction, the predicted value of the CJ-MB assay is Circulation 4, confirmed in 1973 by researchers of Wagner et al. 7.263 (1973); and Contineau, Puru, Met, J. (B r.

Med, J.) 1.386 (1973).

(A patent application for a diagnostic assay to determine J-MB was filed in the early 1970s and Applications began to be filed in the mid-term, and were eventually awarded U.S. Patent No. 3,932.221 (foreign priority, June 9, 1971); and No. 4.067, 775 (Foreign Priority + 1 It was patented on November 3, 1975).

73,932,221 [Pfleiderer] :1 describes a diagnostic method for determining the residual activity of isoenzymes that is sufficient for diagnosis. . According to Preidler, total fermentation of diagnostically relevant isoenzymes for specific substrates The elementary activity is first determined in a sample containing the isoenzyme appropriate for diagnosis, and then the sample is contact with a specific antiserum against the enzyme. Immunization of antiserum and diagnostically appropriate isoenzymes The chemical interactions form a precipitate which is then separated from the sample and discarded. Measure the residual enzyme activity of the sample against the same substrate and measure this residual activity first. Compare the total enzyme activity for such samples.

The difference between initial total enzyme activity and residual activity is considered to contribute to the appropriate isoenzyme for diagnosis. available. Separation of the precipitated immune complexes from the sample is necessary for the Plypooler method. , this is the immune complex formed between the antiserum and the relevant isoenzyme for diagnosis. Due to his interest in retaining at least some of the original enzymatic activity. also used The antiserum is highly effective, i.e. contains at least 90% of the diagnostically relevant enzyme, preferably Precipitation of 95-100% is a prerequisite for Preidler's method. fear The immune complexes formed between the antiserum and the appropriate isoenzymes are easily detected. If enzymatically inactive, no separation is necessary.

゛　奔-4,067775'' (Woluppag et al.) includes IJ-mon and CK-MB CM- including the use of unique antisera for immunoinhibition of the M subunit of the enzyme An elegant method for measuring MB has been described. similar to that of Pridaler In addition, the antiserum has a relatively high avidity and, therefore, most of the diagnostically relevant isoenzymes. Complete inhibition is possible (residual activity of 5 U/L or less is maintained after such immune inhibition) ;Unlike in the case of Shikaji and Prider, the interaction between antiserum and isoenzyme is due to fermentation. It is effective in neutralizing this element and is not accompanied by the formation of precipitating immune complexes. follow The residual isoenzyme (in this case, the B subunit of CM-?lB) that is relevant for diagnosis is The activity of immunocomplexes can be determined in a single measurement without separation of immune complexes.

in just one patient sample to help diagnose or rule out acute myocardial infarction (AMT) Based on a single measurement carried out (accuracy and value of J-?'lB's has recently been a problem, Gerhard, Duplicate 219 etc., Cl1n, Chim, 28/2+ 277-283 (1982) i and Wu, Ei, H. Bee, (Wu,’A, H.

B) etc., Cl1n, Chin, 28/10.2017-2021 (19 '82), in each of these documents, these patients were diagnosed with or without AMI. We explore the reliability of single-CM-MB surveys as a raw trend.

Each document describes the period after the patient's suffering, thought to have been caused by sexual myocardial infarction, began. It was recommended that several assays be performed. Typically, CM-? The level of lB A definitive assay is performed when the patient is admitted to the hospital, followed by a second (and Assay 3) was then performed at 10-12 hour intervals.

Performing a series of assays over a recommended period of time may be beneficial for early elimination of AMI. It is believed that this provides a more reliable method for

A diagnostic test kit utilizing the methods and antibodies of U.S. Pat. No. 4,067,775 For each diagnostic test kit currently on the market, including Intended for measuring multienzyme levels in a sample; first for measuring total enzyme (kinase) activity; and second, the measurement of residual enzyme activity after immune inhibition.

As is clear from the above description of the prior art, selective immune inhibition of CK isoenzymes Accurate determination of residual activity in samples containing such isoenzymes is difficult. It is absolutely necessary for the critical elimination of iAMI where suffering has occurred. This job is CK- Commonly used to generate antisera for test kits used to detect MB Contains antibodies (anti-goat, anti-rabbit, anti-mouse antibodies) against non-primate proteins in the form of It is further complicated by the individual patient samples obtained. Furthermore, the anti-inflammatory agents used in such reagents Serum cross-reactivity (immune inhibition of isoenzyme B subunit) is important in the BN world. distort the results.

Because of the above and associated complexities, such tests for measuring CM-MB levels Operational reliability is compromised and reagent preparation and methods become unduly burdensome. These The traditional direction taken by those interested in the problem is the best, and perhaps the only, increasing the reliability of such assays (immediately) Specificity, avidity and/or sensitivity of the antiserum used in the relevant test kit (increase). Although improvements in antiserum quality provided some positive enhancement, This was accompanied by significant costs. Generally, these antisera are highly purified and assayed. The operating window for A has become narrower and narrower. This results in relatively expensive testing for highly skilled performed under highly controlled conditions using operators and/or very expensive equipment. It is something that must be done.

! about one A first object of the present invention is to provide rate data processing technology. isoenzyme suitable for diagnosis by using relatively impure low-grade antiserum in combination with The aim is to reduce the complexity and cost of performing immunoinhibitory assays. Therefore, The rate data processing technology of the present invention enables accurate determination of isoenzyme levels suitable for diagnosis. Relatively high background levels of enzyme activity that can interfere with accurate measurements Provide a means of compensation.

More particularly, the present invention is useful for diagnostics of enzymes that occur in multiple molecular configurations in complex biological fluids. A suitable method for measuring isoenzymes is provided. This method is suitable for isoenzymes suitable for diagnosis. Certain relatively impure, low avidity antisera, multiple measurement techniques and multiple sample collection techniques Although it is used in combination to correct calculations of rate data and rate data, these data A sample preparation that is active for diagnostically relevant isoenzymes and other enzymes on common substrates. Derived from the measurement of residual activity in minutes. In preferred embodiments of the invention, this method The antiserum used in the test is based on the initial enzymatic activity of the diagnostically relevant isoenzyme in the patient's sample. can also inhibit immunity by 50% and up to about 85%. This type of test is effective Therefore, multiple evaluations of individual samples are required, and evaluations of different samples at the same time are required. one or more regular time intervals (i.e., 10-12 hour intervals) from the start of the patient's suffering. Therefore, it is necessary to repeat the assay over a certain period of time.

The name of the book that contains 1°゛　 Before further discussing the methods and test kits of the present invention, the following terms and phrases are briefly explained. Definitions will be helpful in understanding the invention.

"Isoenzymes suitable for diagnosis" are those typically found in clinical samples or samples. , and exhibiting an abnormal physiological condition or, if present, an abnormal level of pathology. The purpose of this paper is to describe the enzyme activity specimens that can be used.

"Multiple molecular configuration" refers to the physical, structural and/or may differ in one or more chemical characteristics, but when in contact with a common substrate We intend to describe enzymatically active compounds that behave in a similar manner.

Here, “residual enzyme activity” refers to the neutralization/immune inhibition of isoenzymes that are sufficient for diagnosis. It represents the degree of enzyme activity remaining in a sample after contacting the sample with a given antiserum.

In one preferred embodiment of the invention, the methods and test kits of the invention can be used to When a person suffers from a medical condition, a clinician should be consulted to prevent the occurrence of a sexual myocardial infarction (A?II). We can provide reliable diagnostic tools.

Use of the method of the invention includes: (a) Total enzyme activity of multiple molecular configurations of isoenzymes appropriate for diagnosis before neutralization/immune inhibition. Evaluation of multiple individual samples for initial determination of sex; (b) Following neutralization/immunization, determination of residual enzyme activity of multiple molecular configurations of isoenzymes. fixed; and (c) for a period of time following the onset of suffering (i.e. 12 and 24 hours); Determination of the levels of diagnostically relevant isoenzymes in a number of samples taken from patients (at intervals) Do this.

More specifically, when a patient is admitted to a hospital, the levels of isoenzymes appropriate for the patient's diagnosis are determined. and then obtain a second and possibly a third biofluid sample from the patient at regular intervals. , repeat the test method for this. The appropriate isoenzyme for the diagnosis obtained in this way Values are compared for each sample and clinically significant differences are noted.

In reality, the measurement of diagnostically appropriate isoenzymes is based on the multiple molecular arrangement of diagnostically appropriate isoenzymes. including first measuring the total activity of. Afterwards, the patient's sample was anti-blood-stained against the isoenzyme. at least 50% of the initial level of neutralization/immunoinhibition. Warm up. The immunoinhibitory response that occurs during this warm rjL period allows for long-term Cardiac processing forms immune complexes that can be separated from the sample. immune complex Although the body is inert to the enzyme itself, its presence in the sample is diagnostically relevant. does not interfere with determination of residual activity of multiple molecular configurations of isoenzymes. Therefore, such isoferment Analysis of samples for residual activity of the immune complex can proceed in the presence of this immune complex. Ru. Neutralization of an isoenzyme that is relevant for diagnosis generally depends on its relative concentration in the sample and the isoenzyme. It depends on the relative binding activity of the antiserum to the element, but in any case it is about 30 It takes seconds to 5 minutes.

Following neutralization, substrates are applied to multiple molecular configurations of diagnostically relevant isoenzymes in patient samples. kinetically monitor the rate of substrate depletion by the enzyme present in the sample. . Rate data is collected for a sufficient time to give an accurate reflection of residual enzyme activity. one Neutralization of diagnostic isoenzymes generally does not continue during this period of contact between sample and substrate. It should be noted that the residual enzyme activity of the sample continues to decline at a significantly slow rate. This is something worth noting. During this period of monitoring the sample for residual enzyme activity, At least about 15% of the original total enzyme activity is retained.

The antiserum used in the method of the present invention is manufactured by Methods in Enzymology (David's etc. Vol. 10, pp. 696-699; Richmond, Vol. 43, pp. 86-100; 1973), or Cambridge Medical Diagnostics, Billerica.

? ) USETZ; DINISEL, HOUSTON, TEXAS; OR B-ELF Obtained through commercial sources such as Leeds, Loggers, and Arkansas.

The method and test kit of the present invention are used to measure isoenzymes suitable for the diagnosis of: Suitable for: lactate dehydrogenase β-glyceraldehyde 3-phosphate dehydrogenase xanthine oxidase 1-glutamate dehydrogenase α-glucan phosphorylase hexokinase Creatine-ATP phosphotransferase RNA polymerase RNA polymerase type■ Reverse transcriptase (RNA-dependent, DNA polymerase) Lipase Lecithinase (phospholipase A) alkaline phosphatase ゛α-glycerol phosphatase (glycerol-1-phosphatase) Lecithinase (phospholipase C) deoxyribonuclease nuclease Acid α-glucosidase (ab-glucoside glucocarboxy peptidase) Trobomyosinase gelatinase Streptococcus peptidase A 5'-adenylate deamiguse adenosine triphosphatase glutamate deoxylase tryptophan synthetase phosphoglucose isomerase pyruvate carboxylase Substrates that can be used in the methods of the invention are generally available from a variety of commercial sources. Manufactured from materials that can be manufactured or are commercially available by conventional synthetic techniques can do. Substrates for each of the above enzymes can be found in standard references, e.g. In Immunology and Immunochemistry, Volume ■, Academy Confirmed in Tsuku Press (1977), pp. 316-320.

Methods for monitoring residual enzyme activity in samples are preferentially performed on automated clinical chemistry analyzers and are Port data is automatically collected and processed within the analyzer. Initially, the sample was obtained using the conventional method. and prepared for analysis. Such preparation typically consists of subdividing whole blood samples from serum fractions. and separating the cell components and subsequently analyzing the serum fraction. The amount it takes under some conditions It may be appropriate to dilute the sample before carrying out the analysis. According to the method of the invention A typical method of analyzing a sample for diagnostically relevant isoenzymes involves Enzyme activity is measured on multiple molecular configurations of diagnostically relevant isoenzymes. suitable for diagnosis A relatively impure, low-avidity antiserum directed against a specific isoenzyme is then added to the sample to The activity of at least about 50% of the isoenzymes suitable for diagnosis is neutralized. (warm)

During this neutralization process, enzyme-inactive precipitated immune complexes are formed. The presence of this precipitate is allowed in the sample while carrying out the method of the invention. Following this initial neutralization phase of the method, Add a substrate for the isoenzyme appropriate for diagnosis to the sample to determine the remaining enzyme activity of the sample. Record.

Evaluation of enzyme activity is based on the conversion of substrate to indicator, and the relative concentration of this indicator is varied. Monitor mechanically. Substrates used in the method of the invention may also include isoenzymes suitable for diagnosis. It is, of course, understood that the attack can occur due to the multiple molecular arrangement of . Therefore, to the indicator of the substrate The rate data for enzymatic conversion of Therefore, it cannot be used as a basis for diagnosis.

To simplify the detailed description of the invention, the remainder of this discussion We treat creatine kinase as an exemplary system that has been developed in minutes. Explanation of this method The exemplary system selected for development is to measure the levels of CK-MB isoenzyme. based on. The analysis document used for this measurement is as described above. in the sample Rate data can be obtained by measuring residual taleatin kinase activity; The data was of little value without further improvement. improve this data Neutralization/immune inhibition in a process arrived at in a somewhat reasonably reproducible and harmonized manner. We found that a single modifier is not appropriate under harmful circumstances. More specifically, Individualized modifications to the specific antisera used can be determined experimentally. necessary; variations in antisera from animal to animal and antisera from different blood of the same animal. The difference in gave a change. In the exemplary system (OK-?lB), processing of rate data is done by CM -MB, CLM? l. Cross-reactivity of antisera with B subunits and other interfering bodies arithmetically removing residual enzyme activity from the

The standard formula for calculating CK values in international units is the C-MB isoenzyme of interest. Since it is a dimer, the standard formula is modified to reflect this fact. to the value of the numerator Simply multiply by an appropriate factor (IC = 2) to reflect the unique properties of the CK isoenzyme. Ru. Therefore, the standard formula can be rewritten as follows: Due to changes that cannot be determined, it is necessary to further compensate for these changes and to confirm the reliability of the analytical data. The zero-order equation, which requires further modification to develop a consistent representation, is heterogeneous. Antiserum is bound batch by batch, giving the ability to interpret rate data from the system. The activity changes and, in addition, not only isoenzymes relevant for diagnosis, but also some other interfering substances. Significant levels of residual enzymatic activity from

Sue' L-Plate The following examples further clarify and explain the method of the invention. Implement this method The equipment and techniques used are standard and have been described above. In these examples All parts and percentages are by weight unless otherwise specified.

fiJLl A whole blood sample was initially obtained from a patient believed to be suffering from a myocardial infarction. this test An automated clinical chemistry analyzer, preferably one manufactured in Hialeah, Florida, USA. DACOS available from Luther Electronics Corporation @ Prepared for analysis on a chemical analyzer. Such sample preparation typically involves the use of cellular parts. This involves separating the serum from the blood.

Fudan raw 25 If serum is not to be analyzed immediately, it should be frozen in a covered container. Akira Avoid exposure to bright light.

Although hemolyzed samples should not be used, slight hemolysis is acceptable.

Riyu in armor CM-? Using goat antibodies in the DACO5 test method for the IB isoenzyme hand? of the patient, supported by the M subunit of the IB isoenzyme and the isoenzyme. Inhibits activity in serum samples. Remove remaining activity with DART CK reagent (modified (Coulter Electronics Corporation) (available from ). The monitored activity is the B subunit of CM-MB. The ability to obtain CM-MB activity is doubled since the C in serum sample When M-BB is present, macromolecular CK and mitochondrial CK The frequency and magnitude of the effects of any of these interferers that contribute to residual activity is less than 1%. Interference from adenylate kinase is DART CM (CPK ) adenosine pentaphosphate and A? in the reagent. Controlled by IP.

DART@j-bar-ji6 Anti-CK-Zaigo antibody I Xo, 24 van Tris HC4 buffer I X12 tan Dart CM reagent 2 XIOpi 3μ bath 1 tube Take 50 μE of antibody and 2.45 thl of Tris HC! Adding buffer A 1:50 dilution of anti-CKMM was made. Mix gently. This dilution is It was stable for 10 days at 5°C.

Gently tap the DART (H (CPM) reagent glass bottle several times on the side of the container. The contents were loosened. Glass bottle with NCCLS type ■Water type stick water satisfied or No more than this 10. Ovnll water was added. until the contents are completely dissolved , mix immediately by swirling gently in one direction and in the other direction to avoid foaming. Ta. The reagent was stable for 72 hours.

ヱ…Mamegoko■ Normal CM-MB activity: 0-16 tl/L (see normal range section) country Serum samples with total CK activity greater than 1200 Ll need to be diluted and reanalyzed. be.

The rate data processing capability of the data management terminal associated with the DACO5 analyzer has been tested. price data can be effectively compared to standard curves based on that data. trial Compensation for high residual enzyme activity in the sample is based on the results of the analysis on the patient sample. The factor is automatically added to the component. D.A. For COS analyzers, this is a repair This is accomplished by applying a positive factor (FVCF) to the rate data. D.A.R. TCM-1'lB isoenzyme test kit (catalog; #7546862) The correcting factor for this is T1.33t' for antiserum R) 972,000M.

Repeat the above analysis on other patient samples at 12 and 24 hour intervals. , and compare the results with the initial measurements of CM-MB activity.

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Claims (12)

[Claims] 1. (i) A biological fluid sample believed to contain abnormal levels of the appropriate isoenzymes as described above. Antiserum specific for neutralization/immune inhibition of the above appropriate isoenzymes in biofluid samples. (ii) incubating the serum with the appropriate isoenzyme; (iii) allowing the enzyme to form an inactive immune complex; (iii) Enzymatic transfer of the above substrate into an indicator indicating the remaining enzymatic activity of the sample is performed. (iv) monitoring the sample for the presence of the indicator; in a biological fluid sample that has multiple molecular configurations and can vary in concentration. A method for determining the relative concentration of an appropriate isoenzyme comprising: (a) neutralizing/immunoinhibiting The above step of incubation under conditions produces a relatively impure, low binding activity characteristic of the appropriate isoenzyme. It uses sex antiserum; (b) The rate of formation of the immune complex that generated the above interaction between antiserum and isoenzyme is Proceed to near equilibrium before adding enzyme-specific substrate; (c) for a period sufficient for said monitoring to induce rate data to produce an indicator; Including making multiple measurements of the sample in response to the presence of the indicator: (d) Samples identified for isoenzymes and also substrates by processing rate data. Correct any residual enzyme activity in the sample that contributes to other enzymatically active components of the above rate data. The above rate data was specifically expressed against the antiserum used in step (a). applying a predetermined modification factor; (e) performing steps (a) to (d) on the next biofluid sample obtained from the same sample source; Repeatedly, at defined intervals, compare the relative enzyme activity of the first sample with that of the next sample. A method for determining the relative concentration of a suitable isoenzyme in a biological fluid sample, characterized in that . 2. Neutralization/immunoinhibition of isoenzymes reduces the endogenous enzyme activity of the sample by at least 50% 2. The method of claim 1, wherein the reduction is about 85%. 3. After the rate of formation of the immune complex that produced the isoenzyme-specific substrate approaches equilibrium, The method according to claim 1, characterized in that the method is added to a food. 4. Substrates are split enzymatically and develop a color that can be detected by conventional monitoring techniques 2. A method according to claim 1, characterized in that the fluorophore or fluorophore is liberated. 5. The antiserum used to assay the first biofluid sample and the second biofluid sample is blood from different animals or from different blood from the same animal. The method according to claim 1, characterized in that: 6. 2. Method according to claim 1, characterized in that the suitable isoenzyme is CK-MB. 7. Determination of the level of CX-MB activity in the first sample and CK-MM activity in the next sample 7. The method of claim 6, wherein the defined interval between is about 12 hours. 8. Neutralization of the enzyme/immune inhibition by antiserum leads to the formation of immune complexes that precipitate. 7. The method of claim 6, characterized in that: 9. The antiserum is obtained from a non-primate source and the biofluid sample contains antibodies to the antiserum. 7. The method according to claim 6, further comprising: 10. The antiserum cross-reacts with at least some of the B subunit of CK-MB. 7. The method according to claim 6, characterized in that: 11. Indicator levels are monitored using an automated clinical analyzer and predetermined correction factors are applied to the analyzer. Claim 6, characterized in that the method is applied to rate data by rate data processing logic. Method described. 12. The suitable isoenzyme described above for measuring suitable isoenzymes with multiple molecular configurations. High avidity specific to neutralization/immune inhibition of almost all substrates specific to enzymes and isoenzymes In a test kit containing an antiserum with (a) The above antiserum neutralizes/immunizes up to about 85% with at least 50% of the appropriate isoenzyme. consists of a relatively impure, low-avidity antiserum that is capable of inhibiting infectious diseases; (b) A test kit featuring rate data correction factors specific to the antiserum.