Enzymatic method for determining analyte concentrations

Abstract: An improved enzymatic method for determining the concentration of an analyte in a sample body fluid including CO₂ and ammonia, the novel reagent compositions used in this method, and diagnostic test kits useful for determining various analytes according to the method of the invention.

Description: The instant invention provides an improved enzymatic method for determining the concentration of analyte in a sample body fluid which improvement comprises measuring the change in absorbance due to the re-oxidation of reduced coenzyme which is generated in situ. The change in absorbance is proportional to the concentration of analyte in the sample. The invention also provides methods for determining total CO₂ and ammonia using the method of the invention, as well as all of the novel reagents used in these methods, and diagnostic test kits useful for the determination of total CO₂ and ammonia in sample body fluids. The quantitation of analytes in sample body fluids by enzymatic methods is a fairly recent phenomenon. The basic procedure involves determining the sample "blank" by mixing a sample containing the analyte with the enzyme substrates known to be used for the quantitation of that particular analyte. Substrate specific enzymes are then added to the reaction mixture. The enzymatic conversion of the substrates and analyte results in a change in the reaction composition which can be quantitated by various methods which measure the change in absorbance due to the action of substrate specific enzymes on the substrates. This change in absorbance is then correlated with the concentration of analyte in the sample. For example, the enzymatic quantitation of total CO₂ in serum or plasma involves mixing a sample containing CO₂ with the substrate phosphoenolpyruvate (PEP). After a blank reading is taken, the substrate specific enzyme phosphoenolpyruvate carboxylase (PEPC) is added causing the conversion of PEP to oxaloacetate (OAA) and phosphate according to the following reaction: The OAA produced is then correlated with the concentration of CO₂ in the sample by various methods. For example, in Wilson et al., Clinical Chemistry , 19 :640 (1973) and Munson et al., Clinical Chemistry , 20 :872 (1974), the OAA is simultaneously coupled with reduced nicotinamide adenine dinucleotide (NADH) and malate dehydrogenase (MDH) such that the amount of NADH oxidized is directly proportional to the CO₂ in sample. In Norris, et al., Clinical Chemistry , 21 :8, 1093 (1975), the OAA is quantitated by reaction with the diazonium salt of Fast Violet B. One principle disadvantage of enzymatic methods for CO₂ is that they are very sensitive to interference from atmospheric CO₂, especially at the alkaline pH required for optimum reaction and stability of NADH. Plasma ammonia may also be determined enzymatically according to the method of Van Anken et al., Clinical Chemica Acta , 56 :151 (1974). This method is based upon the following reaction: The NADP produced is proportional to the concentration of ammonia in the sample and may be quantitated by measuring the change in absorbance due to the oxidation of the coenzyme NADPH to NADP⁺. The enzymatic reagents in this method are also very unstable, particularly the coenzyme NADPH. French Patent Publication No. 2232760 describes a method for determining ammonia concentration in blood samples based on the same reaction as above but conducted at a slightly basic pH. The instant invention comprises an improvement to those enzymatic methods which determine the concentration of an analyte in a sample body fluid by measuring the change in absorbance due to the action of substrate specific enzymes on a sample-substrate reaction mixture, which improvement comprises measuring the change in absorbance due to the re-oxidation of a reduced coenzyme generated in situ after the addition of substrate specific enzymes to sample-substrate reaction mixture to which the coenzyme has been added, whereby a first absorbance reading is taken after mixing the sample with the coenzyme, the enzyme substrate and the substrate specific enzymes, the second absorbance reading being taken an appropriate time after the first absorbance reading, with the coenzyme and enzyme substrate, the second absorbance reading being taken an appropriate time after the substrate specific enzymes have been added, or with substrate specific enzymes, the second absorbance reading being taken an appropriate time after the coenzyme and enzyme substrate have been added. This invention is applicable to the determination of a variety of analytes such as CO₂, ammonia, Aspartate Transaminase (AST), Alanine Transaminase (ALT), Lactic Dehydrogenase (LDH, pyruvate to lactate). Triglyceride, salicylate, and urea. The instant invention also comprises a method for determining total CO₂ in a sample body fluid according to this improved method, including the novel reagent compositions used as well as a diagnostic test kit useful for the determination of total CO₂ in a sample. The instant invention also comprises a method for determining ammonia concentration in a sample body fluid according to this improved method, including the novel reagent compositions used as well as a diagnostic test kit useful for the determination of ammonia in a sample. The coenzymes preferably used in the method of the invention are nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP), although coenzyme analogs such as nicotinamide hypoxanthine dinucleotide phosphate or thio-NAD would also be suitable. The preferred substrate/enzyme system for generating the reduced coenzyme is the glucose-6-phosphate (G6P)/glucose-6-phosphate dehydrogenase (G6PD) system, although many other substrate/enzyme systems such as glucose/glucose dehydrogenase and formate/formate dehydrogenase may also be used. G6P is added to the sample-substrate reaction mixture along with the coenzyme and enzyme substrates used for quantitation of the particular analyte of interest. A first absorbance reading comprising the sample blank is taken. The substrate specific enzymes are then added. An alternative procedure would involve addition of sample subsequent to the in situ production of reduced coenzyme resulting from mixing substrate specific enzymes and the appropriate substrates and coenzyme, with the initial absorbance reading taken either immediately prior or subsequent to the addition of sample. Mixing of the two reagents can be accomplished either by hand to form a single working reagent, or in situ by automated analyzer. G6PD catalyzes the conversion of G6P to 6-phospho-gluconic acid (6PGA), generating the reduced coenzyme. The change in absorbance due to the re-oxidation of the in situ generated reduced coenzyme is proportional to the concentration of analyte in the sample. The unique feature of the invention is the in situ generation of reduced coenzyme, with either simultaneous or subsequent re-oxidation of coenzyme by analyte, substrate, and specific enzymes. The in situ generation of the reduced coenzyme greatly improves the stability of the reagents. The method of the invention is easily applied to the determination of total CO₂ in comprising the steps of: (a) mixing the sample with the coenzyme and enzyme substrates, and substrate specific enzymes, (b) measuring the absorbance of the solution at 320-380 nm, (c) measuring the absorbance of the solution at about 320-380 nm following an appropriate time subsequent to step (b), wherein the total CO₂ in the sample is proportional to the change in absorbance due to the re-oxidation of the reduced coenzyme which is generated in situ. As mentioned previously the coenzymes and enzyme substrates may be mixed with the substrate specific enzymes prior to addition of sample, or the coenzyme and enzyme substrates may be mixed with sample and a first absorbance reading taken, after which substrate specific enzymes are added. Step (a) above is meant to encompass both alternatives and the first alternative is preferred. For example, determination of total CO₂ according to the method of the invention is performed according to the following reaction sequence: A body fluid sample such as serum or plasma is mixed with the coenzyme NAD and the enzyme substrates known to be used in the quantitation of total CO₂, G6P and PEP. The absorbance of the mixture is measured within a wavelength range of 320-380 nm. The enzymes are then added to this reaction mixture and a second absorbance reading is taken. In this reaction sequence NAD is reduced to NADH due to conversion of G6P to 6PGA by G6PD. The analyte and PEP are converted into OAA and phosphate by the catalytic action of PEPC. The OAA produced is then reduced to ma late by the catalytic action of MDH. The concentration of CO₂ in the sample is proportional to the change in absorbance due to the re-oxidation of NADH which absorbs strongly at around 340 nm or within a range of from 320-380 nm. The appropriate time for the second absorbance measurement is preferably three minutes although a wider range is suitable such as 2-6 minutes. The instant invention also provides for two novel reagent compositions used in the above method. The first reagent composition consists essentially of the coenzyme and enzyme substrates. This reagent [Reagent 1] is comprised of PEP, G6P, and NAD. The second reagent composition consists essentially of the enzymes which catalyze the conversion of the substrates and generate the co-enzyme NADH. This reagent [Reagent 2] is comprised of PEPC in an amount sufficient to catalyze the conversion of PEP to OAA; G6PD in an amount sufficient to catalyze the conversion of G6P to 6PGA; MON in an amount sufficient to catalyze the conversion of OAA to malate. Reagents 1 and 2 may additionally contain buffers, preservatives, chelating agents, surface active agents, protease inhibitors, LDH inhibitors, antibacterials and other constituents which perform stability enhancing functions but do not materially affect the characteristics of the invention. Suitable buffers are potassium phosphate, ammonium phosphate, HEPES , 4-morpholine propanesulfonic acid (MOPS) or 2-[tris(hydroxymethyl)methylamino]-1-ethane-sulfonic acid (TES). The sample to be tested may be diluted with any suitable diluent if desired, such as deionized water or saline. Preservatives such as sodium azide (NaN₃), hydroxybenzoic acid or gentamicin are suitable. Non-ionic surface active agents such as octyl phenoxypolyethoxy ethanol or a polyoxyethylene fatty alcohol ether are suitable. PMSF or Aprotinin are known protease inhibitors and, sodium oxamate, oxalic acid or gossypol will effectively inhibit interference due to Lactic Dehydrogenase. A variety of chelating agents such as EDTA, EGTA, N-(2-hydroxyethyl)ethylenediaminetriacetic acid (HEDTA), etc. are also suitable. Suitable defoaming agents may also be added if desired. Enzyme stabilizers and activators such as dithiothreitol (DTT), bovine gamma globulin (BGG), Mg²⁺, N-acetyl cysteine (NAC) and glycerol may be suitable. The instant invention also comprises a diagnostic test kit for use in determining total CO₂ in a sample body fluid which test kit contains: (a) a container of reagent 1, (b) a container of reagent 2. In the preferred embodiment, 1 to 5 microliters of serum or plasma is mixed with about 100 to 300 microliters of Reagent 1. The absorbance is read at about 340 nm. The reaction mixture is then mixed with about 20 to 94 microliters of Reagent 2 and 1-75 µl of diluent. The second absorbance reading is taken at about 340 nm and the difference in absorbance is proportional to the concentration of total CO₂ in the sample. The best results are achieved when about 2 microliters of sample, about 20 microliters of a diluent such as deionized water or saline, and about 150 microliters of Reagent 1 are mixed and the absorbance of the solution is measured at about 340 nm. Then about 50 microliters of Reagent 2 and about 30 microliters of diluent are added to this reaction mixture and a second absorbance reading is taken. It should also be noted that the order in which Reagent 1 and Reagent 2 are added to the sample may be interchanged, Namely, the sample may be first mixed with Reagent 2 and the absorbance read, after which Reagent 1 is added to the reaction mixture and a second absorbance reading taken. Alternatively, Reagents 1 & 2 may be added together, the absorbance read and a second absorbance reading taken following sample addition. The essential constituents of Reagent 1 are PEP, G6P and NAD. Reagent 1 may additionally contain a buffer such as potassium phosphate, magnesium chloride or sulfate, and a preservative. Magnesium chloride in the form of MgCl₂·6H₂O works well, and a preservative such as sodium azide (NaN₃) is suitable. Reagent 1 may also contain a chelating agent, a nonionic surface active agent, an inhibitor of LDH and a silicon based defoaming agent. The range of concentrations of the various ingredients are about 2 to 50 millimolar PEP, 1.0 to 2.0 millimolar G6P, 2 to 10 millimolar NAD, and 2 to 25 millimolar MgCl₂ and 0.1 to 1% NaN₃, 10-100 mM Potassium Phosphate, 0.1-0.5 mM EGTA, .01-0.1% of nonionic surface active agent and 0.1-10 mM sodium oxamate and 0.01-0.1% of a silicon based defoaming agent. In a preferred embodiment Reagent 1 should contain about 50 millimolar potassium phosphate at pH 6.9, about 6.7 millimolar MgCl₂, 0.1% NaN₃, 1.2 millimolar G6P, 8.0 millimolar NAP, 8.0 millimolar PEP, 3.3 millimolar oxamate, 0.2 millimolar EGTA, and 0.01% of a non-ionic surface active agent such as octyl phenoxy polyethoxy ethanol and 0.05% of a silicone-based defoaming agent. Reagent 2 consists essentially of PEPC in an amount sufficient to catalyze the conversion of PEP to OAA; G6PD in an amount sufficient to catalyze the conversion of G6P to 6PGA; and MDH in an amount sufficient to catalyze the conversion of OAA to malate. Reagent 2 may additionally contain buffers and various other ingredients such as chelating agents, surface active agents, antibacterials, or preservatives, etc., for example, bovine gamma globulin (BGG), NaN₃, EDTA, PMSF, EGTA, and glycerol. Reagent 2 preferably contains 1000 to 2200 U/L PEPC; 5,000 to 62,000 U/L G6PD; and 2400 to 11,000 U/L MDH. Reagent 2 may further contain the following ranges of ingredients: 20 to 200 millimolar ammonium phosphate, 0.1 to 1% BGG, 1 to 30 millimolar DTT, 1 to 40% glycerol, 0.1-1% NaN₃, 0.1- 0.5 mM EDTA and 0.1-0.5 mM PMSF. In a preferred embodiment Reagent 2 contains about 1400 U/L PEPC, 50,000 U/L G6PD, 7000 U/L MDH, 100 millimolar ammonium phosphate at PH 7.2, 20 millimolar DTT, 0.1% NaN₃, 30% glycerol; 0.1% BGG; 0.2 mM EDTA and 0.1 mM PMSF. Ideally Reagents 1 and 2 should be formulated at about pH 7 to minimize the absorption of atmospheric CO₂. These reagents work particularly well on automated clinical chemistry analyzers such as the COBAS BIO®, COBAS FARA® , and COBAS MIRA™ (Hoffmann-La Roche Inc., Nutley, New Jersey). The Reagent 2 components such as BGG, DTT, and glycerol act as enzyme stabilizers and activators. Sodium oxamate is an inhibitor of the enzyme lactate dehydrogenase and is included to prevent endogenous pyruvate, normally found in human body fluids, from interfering in the assay. Magnesium ion assists the conversion of PEP to OAA by PEPC. The improved method of the invention is also easily applied to the determination of ammonia in a sample body fluid, and the instant invention is also directed to a method for the determination of ammonia in a sample body fluid comprising the steps of: (a) mixing the sample with substrates specific enzymes, (b) measuring the absorbance of the solution at 320-380nm, (c) mixing the reaction mixture of step (a) with the coenzyme and enzyme substrates, (d) measuring the lowest absorbance at 320-380 nm after the coenzyme has been completely reoxidized. wherein the concentration of ammonia in the sample is proportional to the change in absorbance due to the re-oxidation of the reduced coenzyme generated in situ. The preferred coenzyme is NADP⁺ in the determination of ammonia according to the instant invention. The ammonia containing sample, preferably serum, plasma or urine, is mixed with α-ketoglutarate, NADP⁺, and G6P and a blank reading is taken. G6PD and Glutamate Dehydrogenase (GIDH) are then added to the reaction mixture causing a rapid increase in absorbance due to the formation of NADPH which absorbs strongly at about 340 nm or within a range of 320-380 nm. The absorbance then slowly decreases due to the subsequent oxidation of NADPH in the ammonia reaction: A second absorbance reading is taken at the point of lowest absorbance after the NADPH has been oxidized. The change in absorbance between the first and second reading is proportional to the concentration of ammonia in the sample. Alternatively, Reagents 3 and 4 may be mixed first allowing the NADPH to be generated. Then sample is added. The instant invention also comprises the novel reagent compositions used in the determination of ammonia according to the method of the invention. The first ammonia reagent, [Reagent 3] consists essentially of NADP⁺ and G6P. Reagent 3 may also contain MES buffer (2-[N-morpholino]ethane sulfonic acid) or other buffers or stabilizers. For example Reagent 3 may contain 50-150 mM MES buffer, pH 6.0, 10-50 mM NADP+ and 10-20 mM G6P. In a preferred embodiment Reagent 3 contains approximately: 80 mM MES buffer pH 6.0 26.4 mM NADP⁺ 7.9 mM G6P The second ammonia reagent [Reagent 4] consists essentially of α-ketoglutarate, G6PD and GlDH. Reagent 4 may also contain Tris (Tris(hydroxymethyl)aminomethane) buffer or other buffers or stabilizers. For example, Reagent 4 may comprise 25-500 mM Tris buffer, pH 8.0, 2-15 mM a-ketoglutarate, 5-15,000 U/L G6PD and 5-40,000 U/L GLDH. In a preferred embodiment Reagent 4 contains approximately: 320 mM Tris, pH 8.0 8,800 U/L G6PD 22,000 U/L GlDH 7.9 mM α-ketoglutarate The determination of ammonia in a sample may be made on the COBAS MIRA in two ways. In the first, single working reagent is prepared by mixing 1 volume of Reagent 3 with 10 volumes of Reagent 4. Then 25-75 µl sample is pipetted simultaneously with 100-200 µl working reagent in the first cycle. An absorbance reading at 340 nm is taken at 4.5 sec (Tl) after reaction has begun, and again at cycle 10 ( 4 min.) when the reaction is complete. The difference in absorbance readings correlates with the quantity of ammonia in the sample. Rather than prepare a working reagent, another alternative is to let the COBAS MIRA pipet Reagents 3 and 4 separately in a 1:10 ratio, respectively. For example 10-20 µl Reagent 3 and 100-200 µl Reagent 4 are pipetted in the first cycle, and complete generation of NADPH is achieved by cycle 3 (as verified by a maximum A₃₄₀). Then 25-75 µl sample is pipetted in cycle 4. A final absorbance reading at 340 nm is taken 10 cycles later, and the difference in A₃₄₀ between cycles 14 and 3 is proportional to the ammonia concentration. An equally acceptable variation in the pipetting sequence is to add sample and Reagent 4 together in cycle 1, take a A₃₄₀ reading, and then add Reagent 3 in cycle 2. As before, the final A₃₄₀ is measured about 10 cycles later. This variation was used below in EXAMPLE 3. The instant invention also comprises a diagnostic test kit for use in determining ammonia in a sample body fluid which test kit contains: (a) a container of Reagent 3, (b) a container of Reagent 4, The present invention will be further described in connection with the following examples which are set forth for the purposes of illustration only. EXAMPLE 1 Reagent Preparation Reagent 1 (1 liter): 8.71 g potassium phosphate (dibasic, anhydrous), 1 g NaN₃, 0.076 g EGTA (acid form), 0.366 g Oxamate (Monosodium salt) are dissolved in about 950 ml deionized water. The final components are fully dissolved in the reagent solution in the following order: first, 2.8 g Phosphoenolpyruvate (Mono-cyclohexylammonium salt); second, 5.86 g NAD·3H₂O (acid form); third, 1.36 g MgCl₂·6H₂O; fourth, 0.34 g Glucose-6-phosphate (Monosodium Salt). Finally 0.1 g Triton ® X-100 (reduced) and 0.5 g Foamaster FLD are added, the pH adjusted to 6.9 with KOH, and the volume brought to 1 liter with deionized water. The concentrations of the components in Reagent 1 are: 50 mM potassium phosphate, pH 6.9, 1.2 mM G6P, 8.0 mM NAD⁺, 8.0 PEP, 6.7 mM MgCl₂, 3.3 mM oxamate, 0.2 mM EGTA, 0.1% NaN₃, 0.01% Triton® X100, and 0.05% Foamaster FLD. Reagent 2 (1 liter): 3.91 g of monobasic ammonium phosphate (anhydrous) and 8.72 g dibasic ammonium phosphate (anhydrous) are dissolved in about 650 mls deionized water along with 1 g NaN₃, and 0.058 g EDTA (acid form). This is mixed with 300 ml. glycerol, and the pH adjusted to 7.2 with NH₄OH, following which 1 g of Bovine gamma-globulin (BGG) and 3.09 g dithiothreitol (DTT) are added and dissolved. Then 50,000 units of G6PD (L. Mesenteroides), 1400 U of PEPC (Maize leaf) and 7000 units of MDH (pig heart cytosol) are added with gentle stirring, followed by addition with stirring of 1 ml of a 0.1 M solution of phenylmethylsulfonyl fluoride (PMSF) in isopropanol. The pH is adjusted to 7.2 with NH₄OH if necessary, and the volume adjusted to 1 liter with deionized water. The final concentrations of the components in Reagent 2 are: 100 mM ammonium phosphate, pH 7.2, 20 mM DTT, 0.2 mM EDTA, 0.1 mM PMSF, 0.1% NaN₃, 30% glycerol, 50,000 U/L G6PD, 1400 U/L PEPC, and 7000 U/L MDH. THe final concentration of the critical reagents in the reaction mixture are 0.72 mM G6P, 4.8 mM PEP, 4.8 mM NAD⁺, 4.0 mM MgCl₂, 10,000 U/L G6PD, 280 U/L PEPC, and 1400 U/L MDH. EXAMPLE 2 CO ₂ Determination on the COBAS - MIRA The assay is conducted at 37°C. 150 µl of Reagent 1, 50 µl of reagent 2, and 30 µl of diluent are mixed in the reaction cuvette and allowed to incubate for three minutes. Then, 2 µl of sample and 18 µl of diluent are added and the absorbance at 340 nm measured immediately after mixing. Three minutes later, the absorbance at 340 nm is measured a second time. The absorbance change is proportional to the concentration of CO₂ in the sample. Standards and controls are run in conjunction with the unknowns, and the CO₂ concentration in the unknown samples is calculated from the standard curve in the usual manner. EXAMPLE 3 Ammonia determination on the COBAS MIRA The assay is conducted at 37°C and 130 µl of Reagent 4 is mixed with 50 µl sample in the first cycle and the absorbance is read at 340 nm. 13 µl Reagent 3 is then added and after 4 minutes (cycle 11) a second absorbance reading is made at 340 nm. From the change in absorbance between the two readings the ammonia concentrations are determined. The table below illustrates the absorbance changes when ammonia is determined according to the method of the invention: SAMPLE A(cycle 1) A(cycle 11) ΔA(cycle 11-cycle 1) 50 uM std. 0.6601 0.6026 -0.0575 88 uM 0.6568 0.5785 -0.0783 132 uM 0.6460 0.5455 -0.1005 175 uM 0.6391 0.5202 -0.1188 A(cycle 1) is the absorbance of Reagent 4 and sample. A(cycle 11) is the absorbance after Reagent 3 was added. ΔA(cycle 11-cycle 1) is the change in absorbance due to the ammonia reaction and is used to calculate the ammonia concentration in the sample.

Claims: Claims for the following Contracting States : AT, BE, CH, DE, FR, GB, IT, LI, NL, SE An enzymatic method which determines the concentration of an analyte in a sample body fluid by measuring the change in absorbance due to the action of substrate specific enzymes on a sample/substrate reaction mixture, said method comprising determining the concentration of an analyte in a sample body fluid by measuring the change in absorbance due to the re-oxidation of a reduced coenzyme generated in situ after the addition of substrate specific enzymes to a sample/substrate reaction mixture to which the coenzyme has been added, whereby a first absorbance reading is taken after mixing the sample with the coenzyme, the enzyme substrate and the substrate specific enzymes, the second absorbance reading being taken an appropriate time after the first absorbance reading, with the coenzyme and enzyme substrate, the second absorbance reading being taken an appropriate time after the substrate specific enzymes have been added, or with substrate specific enzymes, the second absorbance reading being taken an appropriate time after the coenzyme and enzyme substrate have been added. The method of claim 1 wherein the reduced coenzyme is generated by the oxidation of glucose-6-phosphate (G6P) by Glucose-6-Phosphate Dehydrogenase (G6PD). The method of claim 2 wherein the coenzyme is selected from the group consisting of Nicotinamide adenine dinucleotide (NAD) or Nicotinamide adenine dinucleotide phosphate (NADP). The method of claim 3 wherein the sample body fluid is serum, plasma or urine. The method of claim 4 wherein the analyte is selected from the group consisting of CO₂, NH₃, Aspartate Transaminase (AST), Alanine Transaminase (ALT), Urea, Salicylate, Triglyceride or Lactic Dehydrogenase (LDH). The method of claim 5 wherein the analyte is total carbon dioxide (CO₂). The method of claim 5 wherein the analyte is ammonia (NH₃). A reagent composition for use in an enzymatic method involving re-oxidation by analyte, substrate and specific enzymes of a reduced coenzyme generated in situ, for determining total carbon dioxide (CO₂) in a sample body fluid which reagent comprises Phosphoenolpyruvate (PEP), Glucose-6-Phosphate (G6P) and Nicotinamide Adenine Nucleotide (NAD) [Reagent 1]. The composition of Claim 8 additionally containing a buffer, Magnesium chloride, a preservative, a chelating agent, a non-ionic surface agent, an inhibitor of LDH, and a silicone-based defoaming agent. The composition of Claim 9 wherein the buffer is potassium phosphate, the preservative is sodium azide (NaN₃), the chelating agent is EGTA, the LDH inhibitor is sodium oxamate. The composition of Claim 10 containing 2-50 mM PEP, 1-2 mM G6P, and 2-10 mM NAD. The composition of Claim 11 containing 10-100 mM potassium phosphate, 2-25 mM MgCl2, 0.1-1% Sodium Azide (NaN₃), 0.1-0.5 mM EGTA, 0.01-0.1% of a nonionic surface active agent, 0.01-0.1% of a silicon based defoaming agent, and 0.1-10 mM sodium oxamate. The composition of Claim 12 containing about 50 mM potassium phosphate pH 6.9, 6.7 mM MgCl₂, 0.1% NaN₃, 1.2 mM G6P, 8.0 mM NAD, 8.0 mM PEP, 3.3 mM sodium oxamate, 0.2 mM EGTA, 0.01% of a nonionic surface active agent, and 0.05% of a silicon based defoaming agent. A reagent composition for use in an enzymatic method involving re-oxidation by analyte, substrate and specific enzymes of a reduced coenzyme generated in situ, for determining total CO₂ in a sample body fluid which reagent comprises Phosphoenolpyruvate Carboxylase (PEPC) in an amount sufficient to catalyze the conversion of PEP to Oxaloacetic Acid (OAA), Glucose-6-Phosphate Dehydrogenase (G6PD) in an amount sufficient to catalyze the conversion of Glucose-6-Phosphate (G6P) to 6-phosphogluconate (6PGA), and Malate Dehydrogenase (MDH) in an amount sufficient to catalyze the conversion of OAA to malate [Reagent 2]. The composition of claim 14 additionally containing a buffer, a surface active agent, an antibacterial, a preservative, enzyme stabilizers, a chelating agent, enzyme enhancers and a protease inhibitor. The composition of claim 15 wherein the buffer is ammonium phosphate, the enzyme stabilizers are BGG and DTT, the preservative is NaN₃, the chelating agent is EDTA, and the protease inhibitor is PMSF. The composition of Claim 16 containing 1000-2200 U/L PEPC, 5,000-62,000 U/L G6PD, and 2400-11,000 U/L MDH. The composition of Claim 17 containing 20-200 mM ammonium phosphate, 0.1-1% BGG, 1-30 mM DTT, 20-40% glycerol, 0.1-1% NaN₃, 0.1-0.5 mM EDTA, 0.1-0.5 mM PMSF. The composition of Claim 18 containing about 1400 U/L PEPC, 50,000 U/L G6PD, 7000 U/L MDH, 100 mM ammonium phosphate pH 7.2, 20 mM DTT, 0.1% NaN3, 30% Glycerol, 0.1% BGG, 0.2 mM EDTA and 0.1 mM PMSF. A method of any of claims 1 to 4 for the determination of total CO₂ in a sample body fluid comprising the steps of: a) mixing the sample with the coenzyme, enzyme substrates, substrate specific enzymes, b) measuring the absorbance of the solution at 320-380 nanometers (nm) subsequent to step (a) c) measuring the absorbance of the solution at 320-380 nm following an appropriate time subsequent to step (b), wherein the concentration of CO₂ in the sample is proportional to the change in absorbance due to the re-oxidation of a reduced coenzyme generated in situ. The method of Claim 20 wherein the coenzyme and enzyme substrates are mixed and a first absorbance reading taken, after which sample is added. The method of Claim 21 wherein the sample body fluid is serum or plasma. The method of Claim 22 wherein the sample is comprised of 1-5 µl of serum, plasma or urine. The method of Claim 23 wherein 100-300 µl of Reagent 1 is mixed with 20-94 µl of Reagent 2 and 1-75 µl diluent. The method of Claim 24 wherein the reaction product of step (a) is mixed with sample and 1-93 µl diluent and the absorbance is read at about 340 nm. The method of Claim 25 which is conducted on an automated chemistry analyzer. The method of Claim 26 wherein about 150 µl Reagent 1, about 50 µl Reagent 2, and about 30 µl diluent are mixed, 1-6 minutes after which about 2 µl sample and about 18 µl diluent are added to the reaction mixture and an initial absorbance reading at about 340 nm taken immediately, followed by a second absorbance reading at same wavelength in 2-6 minutes. A diagnostic test kit for use in determining total CO₂ in a sample body fluid by an enzymatic method involving re-oxidation by analyte, substrate and specific enzymes of a reduced coenzyme generated in situ, said kit comprising: a) a container of Reagent 1 (as defined in Claim 8), b) a container of Reagent 2 (as defined in Claim 14). The test kit of Claim 28 for use with automated clinical chemistry analyzers. A reagent composition for use in the determination of ammonia (NH₃) in a sample body fluid by an enzymatic method involving re-oxidation by analyte, substrate and specific enzymes of a reduced coenzyme generated in situ, which reagent comprises NADP and G6P [Reagent 3]. The composition of Claim 30 additionally containing a buffer. The composition of Claim 31 wherein the buffer is 2-[N-morpholino]ethane sulfonic acid (MES). The composition of Claim 32 containing about 50-150 mM MES buffer pH 6.0, 10-50 mM NADP and 10-20 mM G6P. The composition of Claim 33 containing about 80 mM MES buffer pH 6.0, 26.4 mM NADP, and 7.9 mM G6P. A reagent composition for use in the determination of ammonia (NH₃) in a sample body fluid by an enzymatic method involving re-oxidation by analyte, substrate and specific enzymes of a reduced coenzyme generated in situ, which reagent comprises α-ketoglutarate, G6PD and glutamate dehydrogenase (GLDH) [Reagent 4]. The composition of Claim 35 additionally containing a buffer. The composition of Claim 36 wherein the buffer is Tris. The composition of Claim 37 containing about 25-500 mM Tris buffer, pH 8.0, 5-15,000 U/L G6PD, 5-40,000 U/L GLDH, and 2-15 mM α-ketoglutarate. The composition of Claim 38 containing about 320 mM Tris buffer, 8,800 U/L G6PD, 22,000 U/L GLDH, and 7.9 mM α-ketoglutarate. A method of any of claims 1 to 4 for the determination of ammonia in a sample body fluid comprising the steps of: a) mixing the sample with substrate specific enzymes, b) measuring the absorbance of the solution at 320-380 nm, c) mixing the reaction mixture of step (a) with the coenzyme and enzyme substrates, d) measuring the lowest absorbance at 320-380 nm after the coenzyme has been reoxidized, wherein the difference is absorbance between (d) and (b) is proportional to the concentration of ammonia in the sample. The method of claim 40 wherein the sample body fluid is serum, plasma or urine. The method of claim 41 wherein the sample is comprised of 25-75 µl serum or plasma, 100-200 µl Reagent 4 is used and 10-20 µl Reagent 3 is used. The method of any of claims 1 to 4 for the determination of ammonia in a sample body fluid, wherein 130 µl Reagent 4 is first mixed with 10-20 µl Reagent 3, after which sample is added and the absorbance is read about 340 nm. The method of Claim 43 wherein the reaction product of step (a) is mixed with 13 µl Reagent 3 and the lowest absorbance is read at about 340 nm after the NADPH has been oxidized. The method of Claim 44 which is conducted on an automated chemistry analyzer. A diagnostic test kit for use in determining ammonia in a sample body fluid by an enzymatic method involving re-oxidation by analyte, substrate and specific enzymes of a reduced coenzyme generated in situ, said kit comprising: a) a container of Reagent 3 (as defined in Claim 30), b) a container of Reagent 4 (as defined in Claim 35). The test kit of Claim 46 for use with automated clinical chemistry analyzers. Claims for the following Contracting State : ES An enzymatic method which determines the concentration of an analyte in a sample body fluid by measuring the change in absorbance due to the action of substrate specific enzymes on a sample/substrate reaction mixture, said method comprising determining the concentration of an analyte in a sample body fluid by measuring the change in absorbance due to the re-oxidation of reduced coenzyme generated in situ after the addition of substrate specific enzymes to a sample/substrate reaction mixture to which the coenzyme has been added, whereby a first absorbance reading is taken after mixing the sample with the coenzyme, the enzyme substrate and the substrate specific enzymes, the second absorbance reading being taken an appropriate time after the first absorbance reading, with the coenzyme and enzyme substrate, the second absorbance reading being taken an appropriate time after the substrate specific enzymes have been added, or with substrate specific enzymes, the second absorbance reading being taken an appropriate time after the coenzyme and enzyme substrate have been added. The method of claim 1 wherein the reduced coenzyme is generated by the oxidation of glucose-6-phosphate (G6P) by Glucose-6-Phosphate Dehydrogenase (G6PD). The method of claim 2 wherein the coenzyme is selected from the group consisting of Nicotinamide adenine dinucleotide (NAD) or Nicotinamide adenine dinucleotide phosphate (NADP). The method of claim 3 wherein the sample body fluid is serum, plasma or urine. The method of claim 4 wherein the analyte is selected from the group consisting of CO₂, NH₃, Aspartate Transaminase (AST), Alanine Transaminase (ALT), Urea, Salicylate, Triglyceride or Lactic Dehydrogenase (LDH). The method of claim 5 wherein the analyte is total carbon dioxide (CO₂). The method of claim 5 wherein the analyte is ammonia (NH₃). A method of any of claims 1 to 4 for the determination of total CO2 in a sample body fluid comprising the steps of: a) mixing the sample with the coenzyme, enzyme substrates, substrate specific enzymes, b) measuring the absorbance of the solution at 320-380 nanometers (nm) subsequent to step (a), c) measuring the absorbance of the solution at 320-380 nm following an appropriate time subsequent to step (b), wherein the concentration of CO2 in the sample is proportional to the change in absorbance due to the re-oxidation of a reduced coenzyme generated in situ. The method of Claim 8 wherein the coenzyme and enzyme substrates are mixed and a first absorbance reading taken, after which sample is added. The method of Claim 9 wherein the sample body fluid is serum or plasma. The method of Claim 10 wherein the sample is comprised of 1-5 µl of serum, plasma or urine. The method of Claim 11 wherein 100-300 µl of Reagent 1 is mixed with 20-94 µl of Reagent 2 and 1-75 µl diluent. The method of Claim 12 wherein the reaction product of step (a) is mixed with sample and 1-93 µl diluent and the absorbance is read at about 340 nm. The method of Claim 13 which is conducted on an automated chemistry analyzer. The method of Claim 14 wherein about 150 µl Reagent 1, about 50 µl Reagent 2, and about 30 µl diluent are mixed, 1-6 minutes after which about 2 µl sample and about 18 µl diluent are added to the reaction mixture and an initial absorbance reading at about 340 nm taken immediately, followed by a second absorbance reading at the same wavelength in 2-6 minutes. A method of any of claims 1 to 4 for the determination of ammonia in a sample body fluid comprising the steps of: a) mixing the sample with substrate specific enzymes, b) measuring the absorbance of the solution at 320-380 nm, c) mixing the reaction mixture of step (a) with the coenzyme and enzyme substrates, d) measuring the lowest absorbance at 320-380 nm after the coenzyme has been reoxidized, wherein the difference in absorbance between (d) and (b) is proportional to the concentration of ammonia in the sample. The method of claim 16 wherein the sample body fluid is serum, plasma or urine. The method of claim 17 wherein the sample is comprised of 25-75 µl serum or plasma, 100-200 µl Reagent 4 is used and 10-20 µl Reagent 3 is used. The method of any of claims 1 to 4 for the determination of ammonia in a sample body fluid, wherein 130 µl Reagent 4 is first mixed with 10-20 µl reagent 3, after which sample is added and the absorbance is read at about 340 nm. The method of Claim 19 wherein the reaction product of step (a) is mixed with 13 µl Reagent 3 and the lowest absorbance is read at about 340 nm after the NADPH has been oxidized. The method of Claim 20 which is conducted on an automated chemistry analyzer.

Inventors: KAUFMAN RICHARD ALLEN, KONOPKA JOHN MICHAEL, ROSENFELD HENRY JOSEPH, SABO JANINE ELIZABETH, KAUFMAN, RICHARD ALLEN, KONOPKA, JOHN MICHAEL, ROSENFELD, HENRY JOSEPH, SABO, JANINE ELIZABETH
Applicants: HOFFMANN LA ROCHE, F. HOFFMANN-LA ROCHE AG
Application year: 1990
Main classification: C12Q1
IPC codes: C12Q1