CA1182726A - Incorporation of pyridoxal phosphate in dry analytical elements for the determination of enzymes - Google Patents
Incorporation of pyridoxal phosphate in dry analytical elements for the determination of enzymesInfo
- Publication number
- CA1182726A CA1182726A CA000420478A CA420478A CA1182726A CA 1182726 A CA1182726 A CA 1182726A CA 000420478 A CA000420478 A CA 000420478A CA 420478 A CA420478 A CA 420478A CA 1182726 A CA1182726 A CA 1182726A
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- Prior art keywords
- pyridoxal phosphate
- enzyme
- coverage
- layer
- phosphate
- 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.)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S435/00—Chemistry: molecular biology and microbiology
- Y10S435/805—Test papers
Abstract
Abstract of the Disclosure Enzymes which are activated by pyridoxal phosphate are assayed in a dry analytical element which includes a high coverage of pyridoxal phos-phate. The coverage of pyridoxal phosphate is such that the multiplication product of the coverage and the spreading coefficient of the element results in a reagent mixture concentration of at least about 0.9 mmole per liter when a liquid sample is contacted with the element thereby eliminating the need for a preincubation of the sample with a solution contain-ing pyridoxal phosphate. The reactions for the determination of the enzymes are initiated by contact of the dry elements with a liquid sample.
Description
~82~
INCORPORATION OF PYRIDOXAL PHOSPHAT~ IN DRY
ANALYTICAL ELEMENTS FOR THE DETERMINATION OF ENZYMES
Field of the Invention The present invention rela~es to dry analyt ical elements which are useful in determining the amount of pyridoxal phosphate act;vated enzyme in a liquid sample. The elements of the present invention are particularly useful in quantitating aspartate aminotransferase and alanine aminotransferase enzymes.
Description Relative to the Prior Art Transaminase enzymes are enzymes which catalyze the transfer of an a-amino group from an ~ amino acid to an ~-keto acid. These enzymes are also sometimes referred to as aminotransferases.
Two of the most clinically significant aminotrans-ferase enzymes are L-alanine:-ketoglutarate amino-transferase, EC 2.6.1.2 (commonly referred to as alanine aminotransferase or ALT) and L-aspartate:
a-ketoglutarate aminotransferase, EC 2.6.1.1 (commonly referred to as aspartate aminotransferase or AST) ~including mitochondrial and cy~oplasmic iso-enzymes).
Both of these enzymes are cllnically signi-ficant in that they are found in high concentration in elther the heart muscle or the liver. Thus, their elevated presence in serum ls an indication of a recent myocardial infarction or liver disease.
ALT and AST are similar in that they both contain a prosthetic group. The prosthetic group is pyridoxal phosphate which must be present for the enzyme to be active. The pyridoxal phosphate forms a Schiff base with a lysine group in the active si~e of the enzyme. In the transaminase reaction, the ~-amino group of the substrate displaces the amine portion of the lysine group of the active SitP of tha enzyme forming a transient Schiff base with pyridoxal phosphate. The pyridoxal phosphate then transfPrs
INCORPORATION OF PYRIDOXAL PHOSPHAT~ IN DRY
ANALYTICAL ELEMENTS FOR THE DETERMINATION OF ENZYMES
Field of the Invention The present invention rela~es to dry analyt ical elements which are useful in determining the amount of pyridoxal phosphate act;vated enzyme in a liquid sample. The elements of the present invention are particularly useful in quantitating aspartate aminotransferase and alanine aminotransferase enzymes.
Description Relative to the Prior Art Transaminase enzymes are enzymes which catalyze the transfer of an a-amino group from an ~ amino acid to an ~-keto acid. These enzymes are also sometimes referred to as aminotransferases.
Two of the most clinically significant aminotrans-ferase enzymes are L-alanine:-ketoglutarate amino-transferase, EC 2.6.1.2 (commonly referred to as alanine aminotransferase or ALT) and L-aspartate:
a-ketoglutarate aminotransferase, EC 2.6.1.1 (commonly referred to as aspartate aminotransferase or AST) ~including mitochondrial and cy~oplasmic iso-enzymes).
Both of these enzymes are cllnically signi-ficant in that they are found in high concentration in elther the heart muscle or the liver. Thus, their elevated presence in serum ls an indication of a recent myocardial infarction or liver disease.
ALT and AST are similar in that they both contain a prosthetic group. The prosthetic group is pyridoxal phosphate which must be present for the enzyme to be active. The pyridoxal phosphate forms a Schiff base with a lysine group in the active si~e of the enzyme. In the transaminase reaction, the ~-amino group of the substrate displaces the amine portion of the lysine group of the active SitP of tha enzyme forming a transient Schiff base with pyridoxal phosphate. The pyridoxal phosphate then transfPrs
2~
the ~-amino group to the ~-keto acid and recom-bines with the lysine of the enzyme. Pyridoxfll phos-phate is also the prosthetic group or activator for a number of other enzymes.
Pyridoxal phosphate is a derivative of pyri-doxine (Vitamin B6)- It is also sometimes referred to as pyridoxal-5'-phosphate3 phosphopyri-doxal, P-5-P, PLP or PDP. Pyridoxal phosphat~ has R
molecular weight of about 265.
While serum generally contains a certain amount of endogenous pyridoxal phosphate, in analyses for ALT and AST, as well as other enzymes having this prothetic group, it is desirable to supplement the serum amounts of pyridoxal phosphate by preincubating a sample to be analyzed for AST or ALT with a solu-tion containing additional pyridoxal phosphate.
Thus, any enzyme which mlght be in the apo form, or inactive form, ls activa-ted by this preincubation step. While the need for pyridoxal phosphate pre-incubation ln the ALT and AST assay was debated inthe literature for several years, preincubation is now accepted as being necessary in order to bring out all of the potential enzyme activity which is in a sample~ Thus 9 the International Federation of Clinl~
cal Chemistry recommends a 10 minute incubation of a ~ample in a solution which con~ains 0.1 mmole per liter of pyridoxal phosphate (see "International Federation of Clinical Chemistry, Provisional Recom-mendations on IFCC Methods for the Measurement of Catalytic Concentrations of Enzymes", "Part 2 IFCC
Method for Aspartate Aminotransferase''g Clin Chem 23,8~7 (1977)).
The need for a preincubation step in thi s standard IFCC method has been an impedimant ~o the adoption of this method in automatic analyzers. Many automatic analyzers are adapted to accommodate this preincubation stap only wlth ~reat difficulty. Thusg 7~
there has been a great incen~lve to eliminat , lf possible, preincubation of the sample with pyridoxal phosphate solution. The ul~imate goal is to develop an assay which is sample initiated. That is, an assay wherein all the reagents, including pyridoxal phosphate, are together and the assay is performed by adding only the sample to the reaction mixture with~
out the need for preincubation.
One example of an attempt to dispense with the preincubation step is illustrated in ~he ar~icle of Bruns et al, "Evaluation of the IFCC-Recommended Procedure for Serum Aspartate Aminotransferase as Modified for Use With the Centrifical Analyzer", Clin. Chem., Vol. 27, page 156 (1981). While these authors were able to adap~ a conventional solution automatic analyzer to the IFCC method by substantial-ly reducing the preincubation period, their data does show that there is a signi~icant advantage with even a short random preincubation time. In other words, they found that eliminating the preincubation time entirely produced an inferior assay.
Because of the importance of pyridoxal phos-phate on the activation of the apo-enzyme form of AST
and ALT, this activation has been widely studied.
~5 For example, it is known that in dilute solution, activation o the apo-enzyme occurs a~ low concentra-tions of pyridoxal phosphate. Thus, the IFCC recom-mends that the incubation be carried out in a 0.1 mmole per liter pyridoxal phosphate solution. Other studies have indicated that somewhat higher levels of pyridoxal phosphate might be d~sirable, for example, up to 0.3 mmole per liter. However, in solution, additional pyridoxal phosphate gave little, i~ any3 further activation. In addition, because of the spectral absorption of pyridoxsl phosphate and the possible inhibition of the coupling enzyme us~d in the indicator reaction of ~he assay, higher concen-tra~ions of pyridoxal phosphate are not used in solu-tion assays (see Hafkenscheid et al, "Influence of Pyridoxal-5'-phospha~e on the Determination of the Alanine Aminotransferase and Aspartate Aminotr~ns-ferase of Commercial Test Sera", J. ClinO ~hem. Clin.Biochem.~ Vol. 17, pp. 219-223 9 (1979); Soo-se Chen et al, "Modification of Pig M~, Lactate Dehydro-genase by Pyridoxal 5'-Phosphate" 9 Biochem J., Vol.
149, pp. 107-113, (1975) and "Reversable Modification of Pig Heart Mitochondrial Malate Dehydrogenase by Pyridoxal 5'-Phosphate", Biochem. J.~ Vol. 151, pp.
~97-303 (1975)).
Dry analytical elements which are arl improvement over conventional solution methods have been recently developed. In a typical dry analytical element, the reagents which are needed for the assay of a particular analyte are coa~ed in a layer wh~ch is then dried. The sample, such as serum, is then contacted with the dried element, the reaction takes place and the results are detected.
It is possible to adapt dry analytical ele-ments for the determination of enzyme concentratlons in samples. For example, U.S. Pa~ent 3,992,158 to Przybylowicz and Millikan discloses a useful element for the determination of AST. Such an element would be use~ul; however, in order to comply with the requirements of the IFCC method, the serum would have to be first preincubated with a solution containlng pyridoxal phosphate for 10 minutes. Even though a shorter time migh~ be used, Bruns et al clearly show that an assay having no preincubation is inferior to one having even a short random incubation. The methods which are designed to use the dry analytical element of Przybylowicz and Mlllikan most preerably use undiluted samples. In order to prov~de pre-incubation time, it would be necessary to treat samples for the determination of the transaminases 7~i with a solution containing pyridoxal phosphate before placing them in the analyzer. It is readily apparent that this is undesirable.
A detailed description of dry analytical elements for the determination of transaminase enzymes is found in Research Disclosure 9 Vol. 146, June 19769 Item 1463~. No mention is made of pyri-doxal phosphate in this publication. esearch Dis-closure is a publication of Indus~rial Opportunities Ltd.; Homewell, Havant; Hampshire, P09 lEF, United Kingdom.
A dry analytical element for the determina-tion of transaminase enzymes without a preincubation step would be desirable. Such an element should pro-vide a sensitive and accurate assay which provldes a de&ree of activation which is comparable to the IFCC
solution method (which includes a 10 minute pre-incubation step).
Summary of the Invention We have found that it is highly desirable to include an extremely large quantity of pyridoxal phosphate in a dry analytical element for the deter-mination of enzymes which are activated by pyridoxal phosphate. me coverage and spreading character-istics are such that the reaction mixture which results when the liquid sample is con~acted with the element has a pyridoxal phosphate concentration of greater than about 0.9 mmole per liter. The amount of pyridoxal phosphate which is useful in the ele-ments of the present invention is far in excess ofwh~t would be expected to be useful from dilute solu-tion studies. While most solution methods use 0~1 mmole per liter in the preincubation mixture9 and while it is known that high levels of pyridoxsl phos-phate cause absorption and po~sible coupling enzymedeac~iva~ion, the amount of pyridoxal phosphate in the elements of the present invention is mcre than 2~
three times that which is known to be the upper lim~t in solution methods. Unexpectedly, it has been found that the incorporation of this large quantlty of pyridoxal phosphate produces a dry element which is useful in fln assay which does not require preincuba-tion of the sample prior to contact with the remain-der of the reagents. Thus, ~he elements of the pre-sent invention are useful with samples ~hich have had a minimum of preanalysis preparation, such as undi-luted serum samples.
In use, the amount of a given reagen~ in thereaction mixture in a dry analytical element is related to both the coverage of that reagent on the dry analytical element and the spreading character-istics of the sample on the element. For a givencoverage of reagent, the amount of reagent ln a given volume of reaction mixture depends on the area which that given volume of solution occupies on the ele-ment. For the purposes of the present invention it has been found desirable to describe the spreading characteristics of a dry analytical element in terms of a spreading coefficient. While a dry analytical element does not have a reaction mixture concentra~
tion until contacted with liquid sample, the con-2S centration which would result is determined by multi-plying the coverage (e.g., in mmoles per m2) by the spreading coefficlent (e.g., in m2 per liter). The spreading, coefficient is the area occupied per unlt volume of sample applied to the element. The spread-ing coefficient for any particular element is aninherent characteristic and is easily determined by contacting a sample of the element with a known volume of liquid, measuring the area over which the liquid spreads and then calculating the spreading coefficient by dividîng the area by the volume of liquld. More specific examples and condi~ions for this measurement are given later in the spec;ficationO
ThUs, in one aspect of the present invention there is provided a dry analy~ical element for the determination of an analyte in a sample wherein said analyte is an enzyme which is activated by pyridoxal phosphate, said element comprising a support having thereon a layer comprising reagents capable of producing a detectable change in response to the presence of the activated form of ~aid enzyme wherein said element contains a layer com-prising pyridoxal phosphate at a coverage such thatthe multipllcation product of ~aid coverage and the spreading coefficient of ~he element results in a reaction mixture concentration of pyridoxal phosphate of greater than abou~ 0.9 mmole per 11ter.
~riPf Description of the Drawin~
Fignre 1 is a plot of the response of a dry analytical element for the determination o-E AST as function of the coverage of pyridoxal phosphate on the element for a series oE calibrator solutions.
ThP calibrator solutions contain only the apo-form of the enzyme and thus the plot illustrates the rela-tionship between ~he degree o activatlon of the enzyme and the coverage of the pyridoxal phosphate.
Detailed Description of the Invention The present invention is described in rela-tion to dry analytical elements for the determlnation of AST and ~LT. It will be understood, however, that other enzymes which are activated by pyridoxal phos-phate are quantitated by elements containing a high
the ~-amino group to the ~-keto acid and recom-bines with the lysine of the enzyme. Pyridoxfll phos-phate is also the prosthetic group or activator for a number of other enzymes.
Pyridoxal phosphate is a derivative of pyri-doxine (Vitamin B6)- It is also sometimes referred to as pyridoxal-5'-phosphate3 phosphopyri-doxal, P-5-P, PLP or PDP. Pyridoxal phosphat~ has R
molecular weight of about 265.
While serum generally contains a certain amount of endogenous pyridoxal phosphate, in analyses for ALT and AST, as well as other enzymes having this prothetic group, it is desirable to supplement the serum amounts of pyridoxal phosphate by preincubating a sample to be analyzed for AST or ALT with a solu-tion containing additional pyridoxal phosphate.
Thus, any enzyme which mlght be in the apo form, or inactive form, ls activa-ted by this preincubation step. While the need for pyridoxal phosphate pre-incubation ln the ALT and AST assay was debated inthe literature for several years, preincubation is now accepted as being necessary in order to bring out all of the potential enzyme activity which is in a sample~ Thus 9 the International Federation of Clinl~
cal Chemistry recommends a 10 minute incubation of a ~ample in a solution which con~ains 0.1 mmole per liter of pyridoxal phosphate (see "International Federation of Clinical Chemistry, Provisional Recom-mendations on IFCC Methods for the Measurement of Catalytic Concentrations of Enzymes", "Part 2 IFCC
Method for Aspartate Aminotransferase''g Clin Chem 23,8~7 (1977)).
The need for a preincubation step in thi s standard IFCC method has been an impedimant ~o the adoption of this method in automatic analyzers. Many automatic analyzers are adapted to accommodate this preincubation stap only wlth ~reat difficulty. Thusg 7~
there has been a great incen~lve to eliminat , lf possible, preincubation of the sample with pyridoxal phosphate solution. The ul~imate goal is to develop an assay which is sample initiated. That is, an assay wherein all the reagents, including pyridoxal phosphate, are together and the assay is performed by adding only the sample to the reaction mixture with~
out the need for preincubation.
One example of an attempt to dispense with the preincubation step is illustrated in ~he ar~icle of Bruns et al, "Evaluation of the IFCC-Recommended Procedure for Serum Aspartate Aminotransferase as Modified for Use With the Centrifical Analyzer", Clin. Chem., Vol. 27, page 156 (1981). While these authors were able to adap~ a conventional solution automatic analyzer to the IFCC method by substantial-ly reducing the preincubation period, their data does show that there is a signi~icant advantage with even a short random preincubation time. In other words, they found that eliminating the preincubation time entirely produced an inferior assay.
Because of the importance of pyridoxal phos-phate on the activation of the apo-enzyme form of AST
and ALT, this activation has been widely studied.
~5 For example, it is known that in dilute solution, activation o the apo-enzyme occurs a~ low concentra-tions of pyridoxal phosphate. Thus, the IFCC recom-mends that the incubation be carried out in a 0.1 mmole per liter pyridoxal phosphate solution. Other studies have indicated that somewhat higher levels of pyridoxal phosphate might be d~sirable, for example, up to 0.3 mmole per liter. However, in solution, additional pyridoxal phosphate gave little, i~ any3 further activation. In addition, because of the spectral absorption of pyridoxsl phosphate and the possible inhibition of the coupling enzyme us~d in the indicator reaction of ~he assay, higher concen-tra~ions of pyridoxal phosphate are not used in solu-tion assays (see Hafkenscheid et al, "Influence of Pyridoxal-5'-phospha~e on the Determination of the Alanine Aminotransferase and Aspartate Aminotr~ns-ferase of Commercial Test Sera", J. ClinO ~hem. Clin.Biochem.~ Vol. 17, pp. 219-223 9 (1979); Soo-se Chen et al, "Modification of Pig M~, Lactate Dehydro-genase by Pyridoxal 5'-Phosphate" 9 Biochem J., Vol.
149, pp. 107-113, (1975) and "Reversable Modification of Pig Heart Mitochondrial Malate Dehydrogenase by Pyridoxal 5'-Phosphate", Biochem. J.~ Vol. 151, pp.
~97-303 (1975)).
Dry analytical elements which are arl improvement over conventional solution methods have been recently developed. In a typical dry analytical element, the reagents which are needed for the assay of a particular analyte are coa~ed in a layer wh~ch is then dried. The sample, such as serum, is then contacted with the dried element, the reaction takes place and the results are detected.
It is possible to adapt dry analytical ele-ments for the determination of enzyme concentratlons in samples. For example, U.S. Pa~ent 3,992,158 to Przybylowicz and Millikan discloses a useful element for the determination of AST. Such an element would be use~ul; however, in order to comply with the requirements of the IFCC method, the serum would have to be first preincubated with a solution containlng pyridoxal phosphate for 10 minutes. Even though a shorter time migh~ be used, Bruns et al clearly show that an assay having no preincubation is inferior to one having even a short random incubation. The methods which are designed to use the dry analytical element of Przybylowicz and Mlllikan most preerably use undiluted samples. In order to prov~de pre-incubation time, it would be necessary to treat samples for the determination of the transaminases 7~i with a solution containing pyridoxal phosphate before placing them in the analyzer. It is readily apparent that this is undesirable.
A detailed description of dry analytical elements for the determination of transaminase enzymes is found in Research Disclosure 9 Vol. 146, June 19769 Item 1463~. No mention is made of pyri-doxal phosphate in this publication. esearch Dis-closure is a publication of Indus~rial Opportunities Ltd.; Homewell, Havant; Hampshire, P09 lEF, United Kingdom.
A dry analytical element for the determina-tion of transaminase enzymes without a preincubation step would be desirable. Such an element should pro-vide a sensitive and accurate assay which provldes a de&ree of activation which is comparable to the IFCC
solution method (which includes a 10 minute pre-incubation step).
Summary of the Invention We have found that it is highly desirable to include an extremely large quantity of pyridoxal phosphate in a dry analytical element for the deter-mination of enzymes which are activated by pyridoxal phosphate. me coverage and spreading character-istics are such that the reaction mixture which results when the liquid sample is con~acted with the element has a pyridoxal phosphate concentration of greater than about 0.9 mmole per liter. The amount of pyridoxal phosphate which is useful in the ele-ments of the present invention is far in excess ofwh~t would be expected to be useful from dilute solu-tion studies. While most solution methods use 0~1 mmole per liter in the preincubation mixture9 and while it is known that high levels of pyridoxsl phos-phate cause absorption and po~sible coupling enzymedeac~iva~ion, the amount of pyridoxal phosphate in the elements of the present invention is mcre than 2~
three times that which is known to be the upper lim~t in solution methods. Unexpectedly, it has been found that the incorporation of this large quantlty of pyridoxal phosphate produces a dry element which is useful in fln assay which does not require preincuba-tion of the sample prior to contact with the remain-der of the reagents. Thus, ~he elements of the pre-sent invention are useful with samples ~hich have had a minimum of preanalysis preparation, such as undi-luted serum samples.
In use, the amount of a given reagen~ in thereaction mixture in a dry analytical element is related to both the coverage of that reagent on the dry analytical element and the spreading character-istics of the sample on the element. For a givencoverage of reagent, the amount of reagent ln a given volume of reaction mixture depends on the area which that given volume of solution occupies on the ele-ment. For the purposes of the present invention it has been found desirable to describe the spreading characteristics of a dry analytical element in terms of a spreading coefficient. While a dry analytical element does not have a reaction mixture concentra~
tion until contacted with liquid sample, the con-2S centration which would result is determined by multi-plying the coverage (e.g., in mmoles per m2) by the spreading coefficlent (e.g., in m2 per liter). The spreading, coefficient is the area occupied per unlt volume of sample applied to the element. The spread-ing coefficient for any particular element is aninherent characteristic and is easily determined by contacting a sample of the element with a known volume of liquid, measuring the area over which the liquid spreads and then calculating the spreading coefficient by dividîng the area by the volume of liquld. More specific examples and condi~ions for this measurement are given later in the spec;ficationO
ThUs, in one aspect of the present invention there is provided a dry analy~ical element for the determination of an analyte in a sample wherein said analyte is an enzyme which is activated by pyridoxal phosphate, said element comprising a support having thereon a layer comprising reagents capable of producing a detectable change in response to the presence of the activated form of ~aid enzyme wherein said element contains a layer com-prising pyridoxal phosphate at a coverage such thatthe multipllcation product of ~aid coverage and the spreading coefficient of ~he element results in a reaction mixture concentration of pyridoxal phosphate of greater than abou~ 0.9 mmole per 11ter.
~riPf Description of the Drawin~
Fignre 1 is a plot of the response of a dry analytical element for the determination o-E AST as function of the coverage of pyridoxal phosphate on the element for a series oE calibrator solutions.
ThP calibrator solutions contain only the apo-form of the enzyme and thus the plot illustrates the rela-tionship between ~he degree o activatlon of the enzyme and the coverage of the pyridoxal phosphate.
Detailed Description of the Invention The present invention is described in rela-tion to dry analytical elements for the determlnation of AST and ~LT. It will be understood, however, that other enzymes which are activated by pyridoxal phos-phate are quantitated by elements containing a high
3~ level of this activator. By choice of suitable de~ecting reagents, enzymes such as other tr~ns-aminases, or example glutamate cysteine trans-aminase, and other transaminases described in table 16-4 on page 684 of Mahler et al 9 Biological Chem-istry, Harper and Row9 1966; isomerases3 for exampleisomers for an~llne 9 glutamate 9 proline, lysine and serine and dicar~oxylases, for xample those des-7~
cribed in Mahler et al~ cited above, at page 685, are determined.
The elements of the present invention include a high coverage of pyridoxal phosphate, iOe., in excess of that coverage which would produce a 0.9 mmole per liter concentration. We have found that the relationship between activation and pyridoxal phosphate concentration in a dry analytical element using undiluted serum îs dramatically differen~ from solution activ~tion. While the general shapes of the activation curves are similar, in solution a plateau region is reached at very low levels of pyridoxal phosphate. For example~ Hafkenschield et al (article cited above) found that the activation curve was substantially flat after about 0.2-0.3 mmoles per liter ln a solution study. We have found that for a dry analytical element using undiluted serum the curve does not begin to flatten out until about a concentration of 0.9 mmole per liter--about three times the maximum solution value ~see Example 2 and Figure 1) is reached.
In particularly preferred embodiments, the elements contain sufficient pyridoxal phosphate to produce a reaction mixture concentration of at least about 2.0 mmoles per liter. Between about 0.9 and 2.0 mmoles per liter, there is some continuing improvement in the degree of ac~ivation~ A~ levels higher th~n about 2.0 mmoles per liter, little fur-ther improvement is seen. Levels higher than 2.0 mmoles per liter are no~ detrimental to the per-formance of the element but the cost of pyridoxal phosphate places a practical limit on the amount incorporated. It is preferred to use a sllght excess above 2.0 mmoles per liter to insure complete activa~
~ion~ The exact amount depends on the particular enzyme being activated. For ALT the optimum i 6 near 7~
g 2 mmoles per liter. For AST the optimum is n~ar 3 mmoles per liter.
The pyridoxal phosphate is included in the element by a varie~y of methods. For example, ~he 5 pyridoxal phosphate is simply dissolved in the coat-ing composi~ion for the desired layer. Alternative-ly, a dispersion of a high boiling liquid having the pyridoxal phosphate dissolved therein is included in the coating composition. In yet ano~her alternative, lQ the pyridoxal phosphate is associated with an amino containing polymer such as albumin which in turn is included in the coating composition.
Pyridoxal phosphate is particularly useful in the assay of AST. The preferred reagent composition 15 which is capable of producing a detectable change in response to the presence of activated AST include NADH, L-aspartate, ~-ketoglutarate and malate dehydrogenase (MDH). AST catalyzes the reaction of L-aspartate and ~-ketoglutarate to oxalacetate and 20 L-glutamate. In the next step, the oxalacetate and NADH react in the presence of the malate dehydrogenase to produce malate and NAD. The amount of AST in the sample is related to the rate of dis-appearance of NADH. Many samples, for example, blood 25 serum, often contain endogenous pyruvate which would interfere with the malate dehydrogenase catalyzed reaction. In order to substantially el~minate the intererence from pyruvate, the reagent composition for the determination of AST frequently con~ains 30 lactate dehydrogenase (LDH). Alternatively oxamate is included to reduce pyruvate interference.
Immediately after the sample is m~xed with the reagent composition, the lactate dehydrogenase catalyses the r~pid consumption of the pyruvate.
35 Subsequently, the rate of NADH disappearance is monitored and is substantially free from the effe~ts of pyruvate.
:.
ALT is another enzyme which uses the ac~i-vator pyridoxal phosphate. A preferred detecting reagent composition for ~he determination of ALT
includes NADH, L-alanine, ~-ketoglutarate and lactate dehydrogenase. In a manner slmilar to the assay for AST, any ALT in a sample catalyzes the reaction of L-alanine and ~-ketoglutarate to L-glutamate and pyruvate. The pyruvate and NADH
react in the presence of ~he lactate dehydrogenase to produce lactate and NAD. Again, the rate of dis-appearance of NADH is monitored and is related to the amount of ALT in the sample.
While the described NADH based methods for ALT and AST are currently preferred, dry analytical lS elements which contain other reagents capable of producing a detectable change in the presence of the activated form of these enzymes are also improved by the incorporation of high levels of pyridoxal phos-phate. For example NAD to NADH indicator reactions are useful. Useful reagents are described, for example, in Henry R. J., Clinical Chemlstry: Princl-ples and Technics, Harper ~ Row, New York, N.Y., Second Ed., 1974, pp 873-893. Useful reagents including pyruvate oxidase are described in U.S.
2S Patent 4,246,342. ~seful reagents including gluta-mate dehydrogenase are described in Ger. Patent 2,431,779. Other useful reagents include citrate synthase/DTNB and are described in Anal. Biochem., 35, pp. 405-410 (1970).
The transaminase dry analytlcal elements of the present invention preferably include~ in addition to the high coverage of pyridoxal phosphate, a high coverage of NAD or NADH. Preferably, the coverage of NAD or NADH is such that the multiplication product of the coverage and the spreading coefficient is sufflcient to provide an NAD or NADH concentration in the reaction mixture of be~ween abou~ 1 mmole per 12~2~
liter and 8 mmoles per liter. The high level of NADH
provides for improvements in dynamic range and linearity of response of a dry analytical element for the determination of a transaminase.
Useful dry analytical elements according to the present invention are made by simply coating the reagent composition which includes the detecting reagents, pyridoxal phosphate and any other reagents, such as a buffer, onto a suitable support. In alter-native embodiments, the support is coated with a num-ber of layers, some of which contain one or more reagents. In particularly preferred embodiments~ a multilayer element is formed which includes a spread-ing layer to improve spreading of the sa~ple on the element. Optionally, the spreading layer contains one or more reagents of the detecting reagent com-position. Useful spreading layers include layers made from fibrous materials such as paper or fabric.
The layer containing the detecting reagents must be in fluid communication with the pyridoxal phosphate reagent. This means that all the reagents are in the same layer with pyridoxal phospha~e or are in separate layers which are adjacent or separated from each other by interlayers; all the layers being permeable to the sample, solubilized reagents and reaction products.
Partlcularly useul dry analytical elements are those which comprise a support having thereon an isotropically porous, nonfibrous spreading layer and, interposed between the support and ~he spreading layer a reagent layer. Isotropically porous, non-fibrous spreading layers are described in U.S. Patent 3,992,158 to Przybylowicz and Millikan and U S.
Patent 4,258,001 to Pierce et al. Compositlons and methods for the formatlon of dry analytical elements are described in these patents.
2~i Where possible, it is particularly desirable to include the substrates for the enzyme under analy-sis in the spreading layer of ~he dry analytical element. Since the enzymes are relatively large 5 molecules, the location of the substrates for the enzymes in ~he porous spreading layer facilitates the contact of the enzyme in the sample with the sub-strates~ As a result 3 the lag time, or the ~ime required for the reaction to begin to take place, is reduced. Further, elements having the substrates ln the spreading layer exhibit improved long term keep-ing.
While it is preferred to have the substrates in the spreading layer, it is preferred to have the pyridoxal phosphate in a separate reagent layer including a binder such as gelatin. By including the pyridoxal phosphate with a binder, the long ~erm keeping oE the element is improved.
The spreading coefficient for any particular element is determined by con~acting the element with a known volume of sample. In calculating the spread~
ing coefficient, the volume of sample should be chosen so that the element does not become saturated with fluid. After the element has been contacted with the known volume of sample, the area over which the sample spreads on the element is measured. To acilitate measurement of the area, the measurement of spreading coefficient is optionally made with a sample containing a dye so that the area is easily visible. The area produced by the sample divided by the volume of the sample is the spre~ding coefficient.
Within broad limits, the spreading coeffi cient for a particular element is substantially independent of the viscosity of the sample which is used to calculate ths spreading coefficient. It ls desirable to use the most ViSCQUS fluid in calculat-ing the spreading coefficient~ The currently pre-2~
-~3-ferred Eluid for measuring the spreading coefficient is blood serum.
Also within broad limits, the spreading coefficient is substantially independent of the 5 method of ~pplying the sample to the element. In the currently preferred method, a stable pendant drop is dispensed from a container and the element is brought up to touch ~he drop onto the element. This method is more completely described in U.S. Patent 4,041,995.
The spreading coefficient for ~ dry analyti-cal element having ~he isotropically porous spreading layer described in ~he Pr~ybylowicz and Millikan patent, cited above, was de~ermined 8S follows: a 10 microliter drop of human serum was spotted on an 15 element similar to the element of Example 1 of the Przybylowicz and Millikan patent except ~hat the spresding layer contained barium sulfate and a cellu-lose acetate binder. The resulting spot had a diameter of about 0.8 cm. Thus, the area that was 20 produced by the 10 microliter drop was 0.50 cm2.
Therefore, thP spreading coefficient for thi~ element is 0.050 cm2 per microllter. In order ~o achieve a reaction mlxture concentration of at least 0.9 mmole per liter, the covera~e of pyridoxal phosphate on an 25 element having these spreading characteristics would have to be at lea6t about 0.05 g per m2.
It is usually desirable ~o include a buffer in the reagent composition for the determination of enzymes. A wide v~riety o buffers are useful (see 3Q for example Good et al, "Hydrogen Ion Buffer~ for Biological Research", Biochemistry, Vol. 5, page 467 (FebO 1966)). Particularly convenlent buffers include N-tris(hydroxymethyl)methylamino ethane sul-fonic acid tTES) and tris(hydroxymethyl)amino e~hane 35 (TRIS). O~her useful buffers include imidazole, di-ethanolamine and trlethanolamine.
.~
..
~2~72~i M~terials and elements which are adapted to use the described reagent composition ineluding the detecting reagents and pyridoxal phospha~e are des-cribed, for example, in U.S. Patents 3,092,465, 3,418,099, 3,418,083~ 2,893,843, 2,893,844, 2,912,309, 3,00~,879, 3,802,842, 3,798,064, 3,298,739, 3,~15,647, 3,~17,453, 3,9g3~594, 3,936,357, 4,270,g20, 4,~48~829, 4,255,384,
cribed in Mahler et al~ cited above, at page 685, are determined.
The elements of the present invention include a high coverage of pyridoxal phosphate, iOe., in excess of that coverage which would produce a 0.9 mmole per liter concentration. We have found that the relationship between activation and pyridoxal phosphate concentration in a dry analytical element using undiluted serum îs dramatically differen~ from solution activ~tion. While the general shapes of the activation curves are similar, in solution a plateau region is reached at very low levels of pyridoxal phosphate. For example~ Hafkenschield et al (article cited above) found that the activation curve was substantially flat after about 0.2-0.3 mmoles per liter ln a solution study. We have found that for a dry analytical element using undiluted serum the curve does not begin to flatten out until about a concentration of 0.9 mmole per liter--about three times the maximum solution value ~see Example 2 and Figure 1) is reached.
In particularly preferred embodiments, the elements contain sufficient pyridoxal phosphate to produce a reaction mixture concentration of at least about 2.0 mmoles per liter. Between about 0.9 and 2.0 mmoles per liter, there is some continuing improvement in the degree of ac~ivation~ A~ levels higher th~n about 2.0 mmoles per liter, little fur-ther improvement is seen. Levels higher than 2.0 mmoles per liter are no~ detrimental to the per-formance of the element but the cost of pyridoxal phosphate places a practical limit on the amount incorporated. It is preferred to use a sllght excess above 2.0 mmoles per liter to insure complete activa~
~ion~ The exact amount depends on the particular enzyme being activated. For ALT the optimum i 6 near 7~
g 2 mmoles per liter. For AST the optimum is n~ar 3 mmoles per liter.
The pyridoxal phosphate is included in the element by a varie~y of methods. For example, ~he 5 pyridoxal phosphate is simply dissolved in the coat-ing composi~ion for the desired layer. Alternative-ly, a dispersion of a high boiling liquid having the pyridoxal phosphate dissolved therein is included in the coating composition. In yet ano~her alternative, lQ the pyridoxal phosphate is associated with an amino containing polymer such as albumin which in turn is included in the coating composition.
Pyridoxal phosphate is particularly useful in the assay of AST. The preferred reagent composition 15 which is capable of producing a detectable change in response to the presence of activated AST include NADH, L-aspartate, ~-ketoglutarate and malate dehydrogenase (MDH). AST catalyzes the reaction of L-aspartate and ~-ketoglutarate to oxalacetate and 20 L-glutamate. In the next step, the oxalacetate and NADH react in the presence of the malate dehydrogenase to produce malate and NAD. The amount of AST in the sample is related to the rate of dis-appearance of NADH. Many samples, for example, blood 25 serum, often contain endogenous pyruvate which would interfere with the malate dehydrogenase catalyzed reaction. In order to substantially el~minate the intererence from pyruvate, the reagent composition for the determination of AST frequently con~ains 30 lactate dehydrogenase (LDH). Alternatively oxamate is included to reduce pyruvate interference.
Immediately after the sample is m~xed with the reagent composition, the lactate dehydrogenase catalyses the r~pid consumption of the pyruvate.
35 Subsequently, the rate of NADH disappearance is monitored and is substantially free from the effe~ts of pyruvate.
:.
ALT is another enzyme which uses the ac~i-vator pyridoxal phosphate. A preferred detecting reagent composition for ~he determination of ALT
includes NADH, L-alanine, ~-ketoglutarate and lactate dehydrogenase. In a manner slmilar to the assay for AST, any ALT in a sample catalyzes the reaction of L-alanine and ~-ketoglutarate to L-glutamate and pyruvate. The pyruvate and NADH
react in the presence of ~he lactate dehydrogenase to produce lactate and NAD. Again, the rate of dis-appearance of NADH is monitored and is related to the amount of ALT in the sample.
While the described NADH based methods for ALT and AST are currently preferred, dry analytical lS elements which contain other reagents capable of producing a detectable change in the presence of the activated form of these enzymes are also improved by the incorporation of high levels of pyridoxal phos-phate. For example NAD to NADH indicator reactions are useful. Useful reagents are described, for example, in Henry R. J., Clinical Chemlstry: Princl-ples and Technics, Harper ~ Row, New York, N.Y., Second Ed., 1974, pp 873-893. Useful reagents including pyruvate oxidase are described in U.S.
2S Patent 4,246,342. ~seful reagents including gluta-mate dehydrogenase are described in Ger. Patent 2,431,779. Other useful reagents include citrate synthase/DTNB and are described in Anal. Biochem., 35, pp. 405-410 (1970).
The transaminase dry analytlcal elements of the present invention preferably include~ in addition to the high coverage of pyridoxal phosphate, a high coverage of NAD or NADH. Preferably, the coverage of NAD or NADH is such that the multiplication product of the coverage and the spreading coefficient is sufflcient to provide an NAD or NADH concentration in the reaction mixture of be~ween abou~ 1 mmole per 12~2~
liter and 8 mmoles per liter. The high level of NADH
provides for improvements in dynamic range and linearity of response of a dry analytical element for the determination of a transaminase.
Useful dry analytical elements according to the present invention are made by simply coating the reagent composition which includes the detecting reagents, pyridoxal phosphate and any other reagents, such as a buffer, onto a suitable support. In alter-native embodiments, the support is coated with a num-ber of layers, some of which contain one or more reagents. In particularly preferred embodiments~ a multilayer element is formed which includes a spread-ing layer to improve spreading of the sa~ple on the element. Optionally, the spreading layer contains one or more reagents of the detecting reagent com-position. Useful spreading layers include layers made from fibrous materials such as paper or fabric.
The layer containing the detecting reagents must be in fluid communication with the pyridoxal phosphate reagent. This means that all the reagents are in the same layer with pyridoxal phospha~e or are in separate layers which are adjacent or separated from each other by interlayers; all the layers being permeable to the sample, solubilized reagents and reaction products.
Partlcularly useul dry analytical elements are those which comprise a support having thereon an isotropically porous, nonfibrous spreading layer and, interposed between the support and ~he spreading layer a reagent layer. Isotropically porous, non-fibrous spreading layers are described in U.S. Patent 3,992,158 to Przybylowicz and Millikan and U S.
Patent 4,258,001 to Pierce et al. Compositlons and methods for the formatlon of dry analytical elements are described in these patents.
2~i Where possible, it is particularly desirable to include the substrates for the enzyme under analy-sis in the spreading layer of ~he dry analytical element. Since the enzymes are relatively large 5 molecules, the location of the substrates for the enzymes in ~he porous spreading layer facilitates the contact of the enzyme in the sample with the sub-strates~ As a result 3 the lag time, or the ~ime required for the reaction to begin to take place, is reduced. Further, elements having the substrates ln the spreading layer exhibit improved long term keep-ing.
While it is preferred to have the substrates in the spreading layer, it is preferred to have the pyridoxal phosphate in a separate reagent layer including a binder such as gelatin. By including the pyridoxal phosphate with a binder, the long ~erm keeping oE the element is improved.
The spreading coefficient for any particular element is determined by con~acting the element with a known volume of sample. In calculating the spread~
ing coefficient, the volume of sample should be chosen so that the element does not become saturated with fluid. After the element has been contacted with the known volume of sample, the area over which the sample spreads on the element is measured. To acilitate measurement of the area, the measurement of spreading coefficient is optionally made with a sample containing a dye so that the area is easily visible. The area produced by the sample divided by the volume of the sample is the spre~ding coefficient.
Within broad limits, the spreading coeffi cient for a particular element is substantially independent of the viscosity of the sample which is used to calculate ths spreading coefficient. It ls desirable to use the most ViSCQUS fluid in calculat-ing the spreading coefficient~ The currently pre-2~
-~3-ferred Eluid for measuring the spreading coefficient is blood serum.
Also within broad limits, the spreading coefficient is substantially independent of the 5 method of ~pplying the sample to the element. In the currently preferred method, a stable pendant drop is dispensed from a container and the element is brought up to touch ~he drop onto the element. This method is more completely described in U.S. Patent 4,041,995.
The spreading coefficient for ~ dry analyti-cal element having ~he isotropically porous spreading layer described in ~he Pr~ybylowicz and Millikan patent, cited above, was de~ermined 8S follows: a 10 microliter drop of human serum was spotted on an 15 element similar to the element of Example 1 of the Przybylowicz and Millikan patent except ~hat the spresding layer contained barium sulfate and a cellu-lose acetate binder. The resulting spot had a diameter of about 0.8 cm. Thus, the area that was 20 produced by the 10 microliter drop was 0.50 cm2.
Therefore, thP spreading coefficient for thi~ element is 0.050 cm2 per microllter. In order ~o achieve a reaction mlxture concentration of at least 0.9 mmole per liter, the covera~e of pyridoxal phosphate on an 25 element having these spreading characteristics would have to be at lea6t about 0.05 g per m2.
It is usually desirable ~o include a buffer in the reagent composition for the determination of enzymes. A wide v~riety o buffers are useful (see 3Q for example Good et al, "Hydrogen Ion Buffer~ for Biological Research", Biochemistry, Vol. 5, page 467 (FebO 1966)). Particularly convenlent buffers include N-tris(hydroxymethyl)methylamino ethane sul-fonic acid tTES) and tris(hydroxymethyl)amino e~hane 35 (TRIS). O~her useful buffers include imidazole, di-ethanolamine and trlethanolamine.
.~
..
~2~72~i M~terials and elements which are adapted to use the described reagent composition ineluding the detecting reagents and pyridoxal phospha~e are des-cribed, for example, in U.S. Patents 3,092,465, 3,418,099, 3,418,083~ 2,893,843, 2,893,844, 2,912,309, 3,00~,879, 3,802,842, 3,798,064, 3,298,739, 3,~15,647, 3,~17,453, 3,9g3~594, 3,936,357, 4,270,g20, 4,~48~829, 4,255,384,
4,256,693, U.K. Pa~ent 2,052,057 and Research Disclosure, Vol. 146, June 1976, I~em 14638.
The following examples are presented.
Example 1 Dry analytical elements for the determlna-tion of AST were prepared. The elements varied in ~he coverage of pyridoxal phosphate (PLP). Repre-sented schematically, the elements had the structure:
-Spreading Layer: barium sulfate, celluloge acetatebinder, polyurethane, Triton X-405~, sodium 2~ ~-ketoglutarate, sodium aspartate _ Subbing Layer: poly(N-isopropyl acrylamide) -Reagent Layer: gelatin, Triton X-405~, buEfer:
N-tris(hydroxymethyl)methyl-2-aminoethane sul~onlc acid (TES), NADH3 MDH, LDH, pyridoxal phosphate various levels and gelatin hardener Support: poly(ethylene ~erephthalate) The polyurethane binder in the spreading layer promotes the coheslon of the spreading layer and is available from the ~. F. Goodrich Co. under the name Estane~. The subbing layer is present to promote the adhesion of the spreading layer to the reagen~ layer.
A 10 microli~er sample of human serum was spotted on one of the elements described above. The sample was completely absorbed into thP coated layers of the element and produced a spot having a diameter of 0.8 cm. The spreading coefficient for these ele-ments was therefore 0.050 cm2 per microliter.
Aliquots from forty eight samples o hospi~
tal patient blood serum were assayed for AST activity by a modified IFCC reference method (37C, 30 minutes pyridoxal phosphate incubation). This establ-lshed an AST reference value for each patient sample.
Four aliquots (10 microliters each) from each patient sample were then spotted on four sepa-rate samples of each dry analytical element. The reflection density (DR) of each elemen~ was monl-tored between 150-300 seconds after spotting.
A patient calibration line was established by regressing the activity for each patient sample (as determined by the reference assay) versus the individual rates measured experimentally on the des-cribed elements. Using this regression line the total error vari~nce aE (also referred to as Sy-x) was calculated and from the four replicates, the method preclsion oO (also known as pooled precision) was calcula~ed. Random bias ae was also calculated from oO and uE (see Lawton et al, "Statistical Comparison of ~lultiple Analytic Procedures: Application to Clinical Chemistry", Technometrics, Yol. 21, No. 4, page 397 ~1979))~
Sensitivlty is the rPciprocal of the slope of the regression line. Table I summar~zes the results.
2~
h u~
~ , r~ C) O O
,~
~ ~rl E3 ~ PC ~
I~ oo o ~::
U~ U~
1~
I O ~o oo 1~1 ~`I o b ~ ~1 I ~D O~
o . . .
b ~t ~ ~) Q~ ~1,, o c~
~ b I ~1 ~1 a~
,~ ,1 U~ oO
a~ o o a~
~ ~0 O
,:~
P~
n ~ ~
O O o , X ~ C`l The data show that each element was c~pable oE producing an excellent AST assay. The fact that the aE ~or all elements is low indicates that substantlally all of the apo-form of the enzyme in the serum has been converted to the active form.
Otherwise, because of the varying ratio of active to inactive enzyme in serum samples one would expect higher variability in comparison with the standard assay which completely activates all enzyme by way of a 10 minute preincubation with pyridoxal phosphate.
Further, increasing coverages of pyridoxal phosphate from 0.9S mmoles per liter to 2.08 mmoles per l~ter improved the ~E and sensitivity. Further increases in pyridoxal phosphate level showed lesser improvements in aE and sensitivity.
Example 2 This example illustrates the ability of the elements of the present invention to activate the apo-form of AST without the need for preincubation.
The ability of elements of the invention to activate the apo-form of the enzyme was tested by preparing samples which contained only the apo-Eorm of AST and then measuring the response of elements with varying amounts of pyridoxal phosphate.
Dry analytical elements for the determina-tion o~ AST were prepared and are represented sche-matically as follows:
Spreadlng Layer: Avicel9, poly(vinylpyrrolidone), binder, ~-ketoglutarate and L aspartate . . _ Reagent Layer: gelatin, buffer: imidazole, oxamate, NADH, pyridoxal phosphate various coverages Support: poly~ethylene tereph~halate) _ 2~
Table II lists the coverages of pyridoxal phosphate in the various elements in this example.
Table II
P~ridoxal Pho 6 phate
The following examples are presented.
Example 1 Dry analytical elements for the determlna-tion of AST were prepared. The elements varied in ~he coverage of pyridoxal phosphate (PLP). Repre-sented schematically, the elements had the structure:
-Spreading Layer: barium sulfate, celluloge acetatebinder, polyurethane, Triton X-405~, sodium 2~ ~-ketoglutarate, sodium aspartate _ Subbing Layer: poly(N-isopropyl acrylamide) -Reagent Layer: gelatin, Triton X-405~, buEfer:
N-tris(hydroxymethyl)methyl-2-aminoethane sul~onlc acid (TES), NADH3 MDH, LDH, pyridoxal phosphate various levels and gelatin hardener Support: poly(ethylene ~erephthalate) The polyurethane binder in the spreading layer promotes the coheslon of the spreading layer and is available from the ~. F. Goodrich Co. under the name Estane~. The subbing layer is present to promote the adhesion of the spreading layer to the reagen~ layer.
A 10 microli~er sample of human serum was spotted on one of the elements described above. The sample was completely absorbed into thP coated layers of the element and produced a spot having a diameter of 0.8 cm. The spreading coefficient for these ele-ments was therefore 0.050 cm2 per microliter.
Aliquots from forty eight samples o hospi~
tal patient blood serum were assayed for AST activity by a modified IFCC reference method (37C, 30 minutes pyridoxal phosphate incubation). This establ-lshed an AST reference value for each patient sample.
Four aliquots (10 microliters each) from each patient sample were then spotted on four sepa-rate samples of each dry analytical element. The reflection density (DR) of each elemen~ was monl-tored between 150-300 seconds after spotting.
A patient calibration line was established by regressing the activity for each patient sample (as determined by the reference assay) versus the individual rates measured experimentally on the des-cribed elements. Using this regression line the total error vari~nce aE (also referred to as Sy-x) was calculated and from the four replicates, the method preclsion oO (also known as pooled precision) was calcula~ed. Random bias ae was also calculated from oO and uE (see Lawton et al, "Statistical Comparison of ~lultiple Analytic Procedures: Application to Clinical Chemistry", Technometrics, Yol. 21, No. 4, page 397 ~1979))~
Sensitivlty is the rPciprocal of the slope of the regression line. Table I summar~zes the results.
2~
h u~
~ , r~ C) O O
,~
~ ~rl E3 ~ PC ~
I~ oo o ~::
U~ U~
1~
I O ~o oo 1~1 ~`I o b ~ ~1 I ~D O~
o . . .
b ~t ~ ~) Q~ ~1,, o c~
~ b I ~1 ~1 a~
,~ ,1 U~ oO
a~ o o a~
~ ~0 O
,:~
P~
n ~ ~
O O o , X ~ C`l The data show that each element was c~pable oE producing an excellent AST assay. The fact that the aE ~or all elements is low indicates that substantlally all of the apo-form of the enzyme in the serum has been converted to the active form.
Otherwise, because of the varying ratio of active to inactive enzyme in serum samples one would expect higher variability in comparison with the standard assay which completely activates all enzyme by way of a 10 minute preincubation with pyridoxal phosphate.
Further, increasing coverages of pyridoxal phosphate from 0.9S mmoles per liter to 2.08 mmoles per l~ter improved the ~E and sensitivity. Further increases in pyridoxal phosphate level showed lesser improvements in aE and sensitivity.
Example 2 This example illustrates the ability of the elements of the present invention to activate the apo-form of AST without the need for preincubation.
The ability of elements of the invention to activate the apo-form of the enzyme was tested by preparing samples which contained only the apo-Eorm of AST and then measuring the response of elements with varying amounts of pyridoxal phosphate.
Dry analytical elements for the determina-tion o~ AST were prepared and are represented sche-matically as follows:
Spreadlng Layer: Avicel9, poly(vinylpyrrolidone), binder, ~-ketoglutarate and L aspartate . . _ Reagent Layer: gelatin, buffer: imidazole, oxamate, NADH, pyridoxal phosphate various coverages Support: poly~ethylene tereph~halate) _ 2~
Table II lists the coverages of pyridoxal phosphate in the various elements in this example.
Table II
P~ridoxal Pho 6 phate
5 Experimen~ ~!m2 mmoles/L
Comparative 0 Comparative 0.02 0.6 4 0004 1.2 0.064 1.9 10 6 0.086 2.5 Mitochondrial AST (mAST) was isolated from human liver using the procedure described ln DiCola et al, Acta. Vitamin Enzymol, Yol. 30, page 28 (1976).
Cytoplasmic AST (cAST) was isolated from 15 outdated red blood cells obtained from the Red Cross using the procedure described in Clin Chem; Vol. 18, page 374 (1972).
Preparation of the apo form of each iso-enzyme was according to the following procedure:
A 15 U/mL solution of thP isoenzyme was made 4 millimolar in aspartate. This solution was held at room temperature for 2 hours. Then, the solu~ion was made 500 millimolar in sodium phosphate pH 6.0 and lncubated overnight at 25C. The solutions were then 25 ultrafiltered and buffered to pH 7.8 with 10 milli-molar TES.
Samples of mAST apoenæyme were spiked into human serum in which all AST activity had been elimi-nated and which was substantially free from endo~
30 genous pyridoxal phosphate. Solutions were prepared which contalned 243, 163 and 121 U/L ~AST.
Aliquots of each solution were spot~ed on samples of each element described above. The rPfl~c-tion density was monitored and for each solution-element pair a ~DR per minu~P was determined.Figure 1 is a plo~ of ~D~ per minu~e against ~i pyridoxal phosphate concentration for each sample solution of mAST~
The data for the apo mitochondrial isoenzyme show the following:
(1) The enzyme in the solutions was sub-stantially inactive. While some small activity was observed with ~he element con~aining no pyridoxal phosphate, this may have been due to some small amount of residual active AST caused by residual pyridoxal phosphate in the serum into which the enzyme was spiked.
(2) The apo enzyme was incompletely acti-vated in this dry ~lement format having no preincuba~
tion by 0.6 mmole per liter pyridoxal phosphate in ~he reaction mixture. This concentration represents double the maximum level which has been suggested for solution-type assays.
(3) The da~a show a plateau region for activation beginning at about 0.9 mmole per liter ~ pyridoxal phosphate in the reaction mixture.
The data for th apo cytoplasmic isoenzyme were plo~ted in a similar manner. While the overall lPvel of activation was lower for this isoenzyme, the same conclusions with respect to the activation by 2S pyridoxal phosphate in the elements were made.
The invention has been described in de~ail with par~icular reference to preferred embodiments thereof, but it will be understood that varia~ions and modifications can be effected withln the spirit and scope of the invention.
Comparative 0 Comparative 0.02 0.6 4 0004 1.2 0.064 1.9 10 6 0.086 2.5 Mitochondrial AST (mAST) was isolated from human liver using the procedure described ln DiCola et al, Acta. Vitamin Enzymol, Yol. 30, page 28 (1976).
Cytoplasmic AST (cAST) was isolated from 15 outdated red blood cells obtained from the Red Cross using the procedure described in Clin Chem; Vol. 18, page 374 (1972).
Preparation of the apo form of each iso-enzyme was according to the following procedure:
A 15 U/mL solution of thP isoenzyme was made 4 millimolar in aspartate. This solution was held at room temperature for 2 hours. Then, the solu~ion was made 500 millimolar in sodium phosphate pH 6.0 and lncubated overnight at 25C. The solutions were then 25 ultrafiltered and buffered to pH 7.8 with 10 milli-molar TES.
Samples of mAST apoenæyme were spiked into human serum in which all AST activity had been elimi-nated and which was substantially free from endo~
30 genous pyridoxal phosphate. Solutions were prepared which contalned 243, 163 and 121 U/L ~AST.
Aliquots of each solution were spot~ed on samples of each element described above. The rPfl~c-tion density was monitored and for each solution-element pair a ~DR per minu~P was determined.Figure 1 is a plo~ of ~D~ per minu~e against ~i pyridoxal phosphate concentration for each sample solution of mAST~
The data for the apo mitochondrial isoenzyme show the following:
(1) The enzyme in the solutions was sub-stantially inactive. While some small activity was observed with ~he element con~aining no pyridoxal phosphate, this may have been due to some small amount of residual active AST caused by residual pyridoxal phosphate in the serum into which the enzyme was spiked.
(2) The apo enzyme was incompletely acti-vated in this dry ~lement format having no preincuba~
tion by 0.6 mmole per liter pyridoxal phosphate in ~he reaction mixture. This concentration represents double the maximum level which has been suggested for solution-type assays.
(3) The da~a show a plateau region for activation beginning at about 0.9 mmole per liter ~ pyridoxal phosphate in the reaction mixture.
The data for th apo cytoplasmic isoenzyme were plo~ted in a similar manner. While the overall lPvel of activation was lower for this isoenzyme, the same conclusions with respect to the activation by 2S pyridoxal phosphate in the elements were made.
The invention has been described in de~ail with par~icular reference to preferred embodiments thereof, but it will be understood that varia~ions and modifications can be effected withln the spirit and scope of the invention.
Claims (9)
1. A dry analytical element for the deter-mination of an analyte in a liquid sample wherein said analyte is an enzyme activated by pyridoxal phosphate, said element comprising a support having thereon a layer comprising a reagent composition capable of producing a detectable change in response to the presence of the activated form of said enzyme wherein said element contains a layer com-prising pyridoxal phosphate at a coverage such that the multiplication product of said coverage and the spreading coefficient of the element results in a reaction mixture concentration of pyridoxal phosphate of greater than about 0.9 mmole per liter.
2. A dry analytical element for the deter-mination of an analyte in a liquid sample wherein said analyte is an enzyme which is activated by pyri-doxal phosphate, said element comprising a support having thereon, in order, a reagent layer and a spreading layer wherein said layers comprise a reagent composition capable of producing a detectable change in response to the presence of the activated form of said enzyme and wherein at least one of said layers comprise pyridoxal phosphate at a coverage such that the multiplication product of said coverage and the spreading coefficient of the element results in a reaction mixture concentration of pyridoxal phosphate of greater than about 0.9 mmole per liter.
3. A dry analytical element for the deter-mination of an analyte in a liquid sample wherein said analyte is an enzyme which is activated by pyri-doxal phosphate, said element comprising a support having thereon, in order, a reagent layer and an iso-tropically porous spreading layer wherein said layers comprise a reagent composition capable of producing a detectable change in response to the presence of the activated form of said enzyme wherein said reagent layer comprises a binder and pyridoxal phosphate wherein the coverage of said pyridoxal phosphate is such that the multi-plication product of said coverage and the spreading coefficient of the element results in a reaction mixture concentration of pyridoxal phosphate of greater than about 0.9 mmole per liter.
4. A dry analytical element according to claims 1, 2 or 3 wherein the spreading coefficient and pyridoxal phosphate coverage are such that the reaction mixture concentration of pyridoxal phosphate is greater than about 2.0 mmoles per liter.
5. A dry analytical element according to claim 1, 2 or 3 wherein said enzyme is aspartate aminotransferase and said detecting reagents comprise NADH, L-asparate, .alpha.-ketoglutarate and malate de-hydrogenase.
6. A dry analytical element according to claim 1, 2 or 3 wherein said enzyme is aspartate aminotransferase and said detecting reagents comprise NADH, L-asparate, .alpha.-ketoglutarate, lactate dehydro-genase and malate dehydrogenase.
7. A dry analytical element according to claim 1, 2 or 3 wherein said enzyme is alanine amino-transferase and said reagents comprise NADH, L-ala-nine, .alpha.-ketoglutarate and lactate dehydrogenase.
8. A method for activating an enzyme in a liquid sample with pyridoxal phosphate, said method comprising the step of contacting said sample with a dry element comprising a support having thereon at least one layer comprising pyridoxal phosphate at a coverage such that the multiplication product of said coverage and the spreading coefficient of the element results in a reaction mixture concentration of pyri-doxal phosphate is greater than about 0.9 mmole per liter.
9. A method for the determination of an analyte in a liquid sample wherein said analyte is an enzyme which is activated by pyridoxal phosphate said method comprising the steps of (a) contacting said sample with a dry ana-lytical element comprising a support having thereon a layer comprising a reagent composition capable of producing a detectable change in response to the presence of the activated form of said enzyme said element containing at least one layer comprising pyridoxal phosphate at a coverage such that the multiplication product of said coverage and the spreading coefficient of the element results in a reaction mixture concentration of pyridoxal phosphate is greater than about 0.9 mmole per liter, and (b) measuring the rate of said detectable change.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US06/345,844 US4450232A (en) | 1982-02-04 | 1982-02-04 | Incorporation of pyridoxal phosphate in dry analytical elements for the determination of enzymes |
| US345,844 | 1982-02-04 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1182726A true CA1182726A (en) | 1985-02-19 |
Family
ID=23356719
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000420478A Expired CA1182726A (en) | 1982-02-04 | 1983-01-28 | Incorporation of pyridoxal phosphate in dry analytical elements for the determination of enzymes |
Country Status (5)
| Country | Link |
|---|---|
| US (1) | US4450232A (en) |
| EP (1) | EP0085954B1 (en) |
| JP (1) | JPS58155100A (en) |
| CA (1) | CA1182726A (en) |
| DE (1) | DE3372787D1 (en) |
Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4575488A (en) * | 1982-06-25 | 1986-03-11 | Technicon Instruments Corp. | Interference free transaminase assay |
| JPS63137699A (en) * | 1986-11-28 | 1988-06-09 | Fuji Photo Film Co Ltd | Analytical element for measuring enzymic activity |
| GB8900924D0 (en) * | 1989-01-17 | 1989-03-08 | Amco Chemie Handel | Compositions for use in enzyme deficiencies |
| US4990445A (en) * | 1988-01-20 | 1991-02-05 | Beckman Instruments, Inc. | Stable reagent and kinetic assay for alpha-amylase |
| US5441739A (en) * | 1990-06-22 | 1995-08-15 | The Regents Of The University Of California | Reduced and controlled surface binding of biologically active molecules |
| US5462858A (en) * | 1993-12-29 | 1995-10-31 | Eastman Kodak Company | Dry multilayer analytical elements for assaying transaminases |
| US6565738B1 (en) | 1999-01-28 | 2003-05-20 | Abbott Laboratories | Diagnostic test for the measurement of analyte in abiological fluid |
Family Cites Families (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB1143835A (en) * | 1965-05-11 | 1900-01-01 | ||
| US3992158A (en) * | 1973-08-16 | 1976-11-16 | Eastman Kodak Company | Integral analytical element |
| CA1054034A (en) * | 1975-06-20 | 1979-05-08 | Barbara J. Bruschi | Multilayer analytical element |
| US4242446A (en) * | 1978-07-26 | 1980-12-30 | Coulter Electronics, Inc. | Method for determining a substance in a biological fluid and reagent combination for use in the method |
| US4241179A (en) * | 1978-08-14 | 1980-12-23 | Coulter Electronics, Inc. | Method for determining a transaminase in a biological fluid and reagent combination for use in the method |
| DE2932973A1 (en) * | 1978-08-14 | 1980-02-28 | Fuji Photo Film Co Ltd | INTEGRAL MULTILAYERED MATERIAL FOR CHEMICAL ANALYSIS OF BLOOD |
| US4235962A (en) * | 1978-08-21 | 1980-11-25 | The Dow Chemical Company | Combination kit for transaminase assay of a body fluid |
| US4318982A (en) * | 1979-06-04 | 1982-03-09 | Miles Laboratories, Inc. | FMN-Labeled specific binding assay |
-
1982
- 1982-02-04 US US06/345,844 patent/US4450232A/en not_active Expired - Lifetime
-
1983
- 1983-01-28 CA CA000420478A patent/CA1182726A/en not_active Expired
- 1983-02-03 DE DE8383101021T patent/DE3372787D1/en not_active Expired
- 1983-02-03 EP EP83101021A patent/EP0085954B1/en not_active Expired
- 1983-02-04 JP JP58016356A patent/JPS58155100A/en active Granted
Also Published As
| Publication number | Publication date |
|---|---|
| US4450232A (en) | 1984-05-22 |
| JPH0249718B2 (en) | 1990-10-31 |
| EP0085954B1 (en) | 1987-07-29 |
| DE3372787D1 (en) | 1987-09-03 |
| EP0085954A2 (en) | 1983-08-17 |
| JPS58155100A (en) | 1983-09-14 |
| EP0085954A3 (en) | 1985-04-17 |
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