GB2078369A - Enzymatic assay method and apparatus - Google Patents

Abstract

In assay methods involving converting a first substance in a sample to a second substance, oxidising the thus-formed second substance using an oxidase, and measuring the oxygen consumed or hydrogen peroxide generated during the oxidation, errors which occur due to the presence of amounts of the second substance in the original sample can be eliminated by contacting the sample, prior to the step involving the conversion of the first substance to the second substance, with an immobilised kinase in the presence of ATP to phosphorylate any second substance which may be present in the original sample and which would otherwise by oxidised by the oxidase. The method can be used for the assay of, for example, lactose or saccharose.

Description

SPECIFICATION Assay method and apparatus This invention relates to an assay method and apparatus.

Enzymatic analysis has been commonly used for the quantitative determination and analysis of a component in sample. Enzymatic analysis is, however, uneconomical due to the use of a large amount of expensive enzyme. Also it is disadvantageous because it can require the use of complicated procedures, the assay can take a long time and problems can occur due to the lability of enzyme.

The use of an immobilized enzyme has been introduced to improve enzymatic analysis, especially in combination with an electro-chemical measurement means to form a so-called enzyme electrode. The assay method using an enzyme electrode has number of advantages. For example, it is simple to operate, requires less amount of reagents, shortens the assay time, and less enzyme is consumed. Many enzyme electrodes have been reported, especially in the field of clinical biochemistry. Further this technique has become popular for analysis of starting materials, products and compositions in the field of fermentation, foods and others. The application of the enzyme electrode for fermentation process measurement and quality control process measurement in the fermentation and food industries has been much noticed.

In the fermentation industry, for the purpose of proper cultivation of fungi or yeast and higher productivity, accurate and rapid measurement for saccharose in a medium is required. In the food industry, quality control for dairy products containing saccharose and lactose requires rapid and accurate analysis of these sugars. In clinical biochemistry, for example an assay for amylase activity is performed by hydrolysing a starch substrate to maltose and the maltose to hexose. Hence it is necessary to measure maltose and hexose rapidly and accurately.

Examples of the assay of saccharose, lactose, maltose and amylase activity, and related invertase, a-glucosidase or B-galactosidase activity are as follows:

invertase mutarotase Saccharose aD-fructose + a-glucose ,P-D-glucose O2 > glucose oxidase H gluconic acid -galactosidase Lactose D-galactose + ss-D-glucose 2 > glucose oxidase H202 oxidase gluconic acid amylase a-glucosidase Starch 3 maltose * ss-D-glucose 2 - glucose oxidase H2O2 gluconic acid In these reaction systems, glucose is formed by the action of mutarotase, ss-galactosidase or a-glucosidase. The glucose is oxidised by glucose oxidase and the amount of the decrease in oxygen or of the hydrogen peroxide generated measured, thereby determining the amount of each objective component.

In general, the assay of a component in a sample proceeds as follows.

enzyme second substance Component to be assayed in sampler 2 corresponding oxidase H2O2 oxidized second substance By this reaction scheme, the enzyme activity or component in the sample is assayed by using the oxidase of the second substance (corresponding oxidase for the second substance). The examples of this reaction scheme are not limited to the above analysis of sugar related substances. For example: (1) To be assayed: phospholipid, phosphoryl chlorine, activity of phospholipase D, phosphatase.

the second substance: chlorine.

oxidase: chlorine oxidase.

measure: oxygen consumed or hydrogen peroxide generated.

(2) To be assayed: neutral fat activity of lipase, lipoprotein lipase.

the second substance: glycerol.

oxidase: glycerol oxidase.

measure: oxygen consumed or hydrogen peroxide generated.

(3) To be assayed: activity of leucine-aminopeptidase, synthetic substrate containing amino acid.

the second substance: amino acid.

oxidase: amino acid oxidase.

measure: oxygen consumed or hydrogen peroxide generated.

(4) To be assayed: lactic acid, lactate dehydrogenase (LDH) activity.

the second substance: pyruvic acid.

oxidase: pyruvate oxidase.

measure: oxygen consumed or hydrogen peroxide generated.

(5) Others: [pyruvic acid formed as final (second) substance, refer to Japanese Patent Laid-Open Publication No. 55-13068] (a) ADP, phosphoenolpyruvate and pyruvate kinase.

(b) Alanine, c:-ketoglutarate and glutamic-pyruvic-transaminase (GPT).

(c) aspartate, -ketoglutarate and glutamic-oxaloacetic-transaminase (GOT) and oxaloacetate decarboxylase.

(d) glycerol, ATP, glycerol kinase and pyruvate kinase.

(e) creatinine, creatininase, ATP, creatinphosphokinase and pyruvate kinase.

In orderto obtain good and accurate results, the second substance such as glucose, galactose, fructose, choline, glycerol, amino acid and pyruvic acid should be completely formed, and additional amounts of the second substance should be not present with the component to be assayed in the original assay sample. The presence of these additional amounts of the second substance can cause errors in the analysis. Generally a sample for assay, excepting standard samples for use in producing standard curves, will be contaminated by amounts ofthe second substance such as glucose and chlorine.

To avoid errors, the contamination of the original sample for assay by a second substance such as glucose and galactose should be determined first. Subsequently, after finishing the assay of the sample, the first value obtained for the contamination of the sample must be deleted from the total value to obtain the correct value. This is a disadvantage. Further, the contaminating second substance such as glucose is oxidised as a pretreatment by an oxidase such as glucose oxidase prior to the assay ofthe sample. This pretreatment using an oxidase can have disadvantages also. For example, dissolved oxygen in a sample is consumed by the action of the oxidase and hydrogen peroxide is generated. These phenomena affect the final determination.

We have investigated the determination of saccharose, lactose, maltose and amylase activity and have found that the problems caused by the presence of glucose in the original sample to be assayed can be overcome by treating the sample first with hexokinase in the presence of ATP to phosphorylate the glucose to glucose-6-phosphate. When the assay is subsequently effected there is no decrease in the level of dissolved oxygen and no hydrogen peroxide is generated attributable to the original amounts of glucose in the sample. However, as a result of this pretreatment, hexokinase remains in the reaction mixture, which causes phosphorylation of the glucose formed in the actual assay. To avoid this effect, denaturation or removal of the remaining hexokinase is required.However, heat or pH denaturation will cause the denaturation of the substance to be assayed, and removal of hexokinase is quite difficult.

We have found that the difficulties caused by the hexokinase remaining after the phosphorylation of the glucose in the original assay sample can be overcome using an immobilized hexokinase. The thus treated sample can then be assayed without errors caused by the original glucose. This technique has general application to assays conducted by converting a first substance to a second substance, oxidising this second substance using an oxidase and measuring the oxygen consumed or hydrogen peroxide generated during the oxidation.

Accordingly, the present invention provides an assay method which comprises: (i) converting a first substance in a sample to a second substance, (ii) oxidising the thus formed second substance using an oxidase, and (iii) measuring the oxygen consumed or hydrogen peroxide generated in the oxidation step (ii), wherein, prior to step (i), the sample is contacted with an immobilised kinase in the presence of ATP to phosphorylate any second substance which may be present in the sample and which would otherwise be oxidised in step (ii).

The present invention also provides apparatus for use in assaying a sample which comprises a first substance and which may also contain a second substance, which apparatus comprises: an immobilised kinase for phosphorylating any second substance in the original sample in the presence of ATP, means for converting the first substance to the second substance, an oxidase for oxidising the thus-formed second substance and means for measuring either the oxygen consumed or the hydrogen peroxide generated during the oxidation.

For ease of reference, any second substance present in the sample to be assayed prior to step (i) will be referred to herein as admixed second substance.

In an embodiment of the present invention, a sample to be assayed is treated in step (i) by one or more enzymes to form the second substance in one or more reactions. This second substance is oxidised by the corresponding oxidase in step (ii) and the oxygen consumed or hydrogen peroxide generated is measured.

The enzymatic reaction system are not limited. Similarly, examples of samples to be analysed which contain an admixed second substance which can cause an error on assay are not limited. Clinical diagnostic samples such as serum and urine, fermentation media, culture filtrates, food raw materials and food products can be analysed.

Examples of the component in a sample to be analysed, the first substance, include saccharose, lactose, starch, maltose, phospholipid, phosphorylcholine, neutral fat, glycerol, lactic acid and creatinine. The enzyme used in the conversion step (i) may be amylase, invertase, ss-galactosidase, a-glucosidase, phospholipase-D, phosphatase, lipase, lipoprotein lipase, leucine aminopeptidase, LDH, GPT, GOT, pyruvate kinase, glycerokinase, oxaloacetic acid decarboxylase, creatininase or creatinephosphokinase.

The present invention can be used for the quantitative determination of the first substance, and for assaying the activity of an enzyme employed in the conversion step (i). The reaction system can be selected and multiplied.

The second substance is the substance produced from the first substance in step (i) and should not be identical to the first substance. Examples of the second substance are substances which are produced by enzymatic action on the first substance and can be oxidised by a corresponding oxidase. The second substance can be glucose, galactose, choline, glycerol or pyruvic acid.The second substances, enzymes and the appropriate first substances are illustrated as follows: Second substance: enzyme: first substance: gluxose invertase saccharose galactose, glucose ss-galactosidase lactose glucose amylase, a-glucosidase starch choline phospholipase D phospholipid choline phosphatase phosphorylcholine choline choline esterase benzoylcholine glycerol lipase, lipoproteinlipase neutral fat pyruvic acid LDH lactic acid pyruvic acid pyruvate kinase phosphoenol pyruvate and ADP pyruvic acid GPT alanine and a-ketoglutarate pyruvic acid GOT and oxaloacetate decarboxylase a-ketoglutarate and aspartate pyruvic acid glycerol kinase, glycerol and pyruvate kinase phosphoenolpyruvate and ATP pyruvic acid creatininase, creatinephosphokinase creatinine, ATP and and pyruvate kinase phosphoenolpyruvate The admixed second substance therefore refers to a substance in the original assay sample which is identical with the second substance produced from the first substance in step (i) of the process of this invention.

The kinase for the admixed second substance is an enzyme which phosphorylates the said admixed second substance in the presence of ATP. The kinase must correspond to whatever the admixed second substance is. Examples are glucokinase or hexokinase when glucose is the admixed second substance, galactokinase when galactose is the admixed second substance and similarly hexokinase for the other hexoses, cholinekinase for choline, glycerolkinase for glycerol and pyruvate kinase for pyruvate. The immobilized kinase can be prepared by conventional methods for immobilizing enzymes. Preferable techniques are immobilization by acrylamide, protein cross-linking after mixing the enzyme with a protein such as albumin, entrapping with collagen and fibroin or covalently linking therewith, adsorption on polyporous organic polymer or covalent linking therewith, and entrapping by photopolymerization.The immobilized kinase, or any immobilized enzyme used in the present invention, may be in the form of a membrane, fiber, pellet or tube. After contact with the immobilized kinase the sample to be assayed is fed to step (i) of the process of the invention.

The oxidase employed in step (ii) of the process of the invention is an enzyme which at least catalyzes the oxidation of the second substance in the presence of oxygen, generating hydrogen peroxide. Examples are glucose oxidase for glucose, galactose oxidase or hexose oxidase for galactose, hexose oxidase for the other hexoses, glycerol oxidase for glycerol, choline oxidase for choline and pyruvate oxidase for pyruvic acid. The oxidase is preferably immobilized and by this immobilization automatic analysis by electrochemical means can be used, such as an oxygen electrode, hydrogen peroxide electrode or enzyme electrode.

Electrochemical measuring means for an oxygen decrease or for hydrogen peroxide formation involve electrical detection of variations in the amount of oxygen or hydrogen peroxide. A Clark or Galvanic oxygen electrode or hydrogen peroxide electrode are preferably used. Immobilization of the enzyme can save the amount of expensive enzyme. Also an electrode connecting the immobilized enzyme with an electric detector, for example an enzyme electrode such as an enzyme electrode for an oxygen electrode and an enzyme electrode for a hydrogen peroxide electrode is quite preferable because of the rapid detection that results. Also, there is no requirement for reagents, the electrode can be repeatedly used and there is no inhibitory effects caused by colored substances in a sample.

An embodiment of the apparatus of the invention will now be described, by way of example only, with reference to Figure 1 of the accompanying drawings. In Figure 1,the numberals represent various components as follows: 1: buffer solution vessel, 2: pump, 3: sample injector, 4: kinase column, 5: enzyme column for conversion of the first substance to the second substance, 6: immobilized oxidase column, 7: electrode for electrochemical detection means, 8: flow cell, 9: amplifier for electric current change from electrode, 10: recorder, 11: constant temperature vessel, 12: the second injector and 13: exhaust. The second injector is not needed when an immobilised enzyme column 5 is used.The second injector is used for injection of a non-immobilized enzyme solution employed for conversion of the first substance to the second substance, or for injection of a substrate for assay of enzyme activity.

In an embodiment for lactose assay, column 4 is an immobilized hexokinase or galactokinase, column 5 is an immobilized B-galactosidase, column 6 is an immobilized glucose oxidase or galactose oxidase, and electrode 7 is an oxygen electrode or hydrogen peroxide electrode which is connected to flow cell 8. In an embodiment for saccharose assay, column 4 is an immobilised hexokinase, column 5 is a double linked column of immobilized invertase and mutarotase and column 6 is an immobilized glucose oxidase, or column 5 is an immobilized invertase and column 6 is an immobilized mutarotase and glucose oxidase. In an embodiment for assay or amylase activity, column 4 is an immobilized glucokinase or hexokinase, column 5 is an immobilized a-glucosidase, and colum 6 is an immobilized glucose oxidase.

The amount of immobilized enzyme depends on the flow rate of the reaction medium, the Km value of enzyme, the amount of admixed second substance and the dilution of the sample and is not limited.

Preferably the amount is 20 - 200 mg (wet wt.) of an immobilized enzyme containing hexokinase [above 100 U/g (wet wt.)], glucose oxidase [above 30 U/g (wet wt.)i, invertase [above 2000 U/g (wet wt.)], mutarotase [above 800 U/g (wetwt.)], P-galactosidase [above 30 U/g (wetwt.)] ora-glucosidase [above 140 Ulg (wetwt.)j As explained below in the Examples 13.8 U (total activity) of hexokinase can remove 400 mg/dl of glucose in a sample.

The buffer solution in the buffer solution vessel should be a stable pH buffer for enzymes, such as dimethylglutarate buffer or phosphate bluffer. ATP can be contained in the buffer solution for use in phosphorylation by the kinase. The amount of ATP is preferably slightly larger than content of the admixed second substance in the sample. Salts which keep the enzymes stable may be added. Examples are magnesium chloride, potassium chloride or manganese chloride.

In operation, a constant volume of buffer is supplied at 0.1 - 2 ml/min. and the device is stabilized. After stabilization a sample to be assayed is injected at an aliquot of 5 - 50 yl by using micro-syringe or a micro-pipette. The injected sample is transferred to the immobilized kinase column and the admixed second substance is phosphorylated. The reaction temperature is preferably kept at 25 - 40"C, more preferably at 37"C. Admixed glucose, for example, is removed by conversion to glucose-6-phosphate. The sample is passed through the next column and the first substance in the sample is converted to the second substance.

For example, saccharose or lactose (to be assayed) is enzymatically changed to glucose (as the second substance) by an enzyme, preferably an immobilized enzyme.

In the case where an immobilized enzyme is not used in step (i) of the process of the invention, an enzyme solution is injected into the system via the second injector 12. For assaying enzyme activity, operation differs depending on the number of the enzymatic reaction steps, i.e. one step or two or more steps. In a one step enzymatic reaction, if a substrate is injected into the system via the sample injector 3, this substrate passes through the kinase column and hence is affected by the kinase which can cause unexpected results.

Therefore, the substrate should be injected in via the second injector 12. In a two or more step enzymatic reaction, the substrate is treated as a first step in a column for assaying enzymatic acitivity, and the treated substrate is subjected to further enzymatic action or other reagents in order to convert it to the second substance. In this case at the first step the substrate is not reacted with the kinase, and hence the substrate can be injected into the buffer solution or injected into the second injector. The substrate is converted to the second substance in the second or further step of the column.

The second substance is preferably oxidized by an immobilized oxidase column. The reactant solution is introduced into the flow cell, preferably 0.05 - 0.5 my in volume, and the oxygen consumed or hydrogen peroxide generated is detected by an electrode as a change in electric current change, which is recorded through an amplifier.

In the above reactor system, the column 6 and electrode 7 can be replaced by an enzyme electrode of the same immobilized oxidase and set up at the detector part of electrode. A multiple detecting system set up using a plurality of enzyme electrodes in one system can be employed. In this system, in a part of column 5 a corresponding immobilized enzyme column is connected.

Further, a computer controlled or a computerized automatic device can be set up. For example, sample injection can be preformed by an auto-sampler, each valve set up with an electro-magnetic valve. Other possible attachments include a micro-computer with channels for automatic input and control, etc.

connected to a display panel and an operation key board which can respectively display and input, for example, reaction-times and conditions. The device can be automatically controlled by the computer throughtthis interface.

The following Examples illustrate the present invention: EXAMPLE 1 (1) Removal of the admixed second substance, glucose, by immobilized hexokinase: Referring to Figure 1 of the accompanying drawings, a buffer solution containing 3 mM ATP and 2 mM magnesium chloride in 0.1 M dimethylglutarate buffer (pH 6.5) was prepared in buffer solution vessel 1. The flow rate in pump 2 was set up at 1 ml/min. Other components of the apparatus of Figure 1 were as follows.

Sample injector: 3. Column 4: (2.8 x 30 mm) immobilized hexokinase (138 U/g, wet. wt., 100 mg). Column 5: (2.8 x 30 mm) immobilized B-galactosidase (35 U/g, wet wt., 100 mg). Column 6: (2.8 x 15 mm) immobilized glucose oxidase (70 U/g, wet wt., 50 mg). Electrode 7: oxygen electrode connected to the flow cell 8 (vol. 0.1 ml). A constant temperature of 37"C was maintained. The oxygen electrode was connected to digital recorder 11 through amplifier 9. The apparatus was to be used for lactose assay.

The buffer solution was supplied by the pump at a flow rate of 1 ml/min. After confirming the stabilization of the level of dissolved oxygen by the oxygen electrode set in the flow cell, a 5 ll1 aliquot of each standard glucose solution (concentration 100,200,300,400,500,600 and 700 mg/dl.) was injected into the system via the sample injector. Each standard glucose solution passed into the immobilized hexokinase column where the glucose was converted to glucose-6-phosphate by ATP. If glucose remained, the amount of dissolved oxygen in the standard solution decreased as a result of oxidation of the glucose by the immobilized glucose oxidase column. The oxygen decrease was recorded by the oxygen electrode as a current change through the amplifier.As shown in Figure 2 of the accompanying drawings hexokinase (total activity 13.9 U) can remove at least 400 mg/dl. of admixed glucose. Therefore, at least 800 mg/dl. of admixed glucose can be removed by 30 U of hexokinase. The same result was obtained by replacing the immobilized ss-galactosidase column 5 by a column without P-galactosidase.

(2) Lactose assay in sample containing lactose admixed with glucose: The apparatus and same conditions in (1 ) were used. The flow rate of the buffer was 1 ml/min. After the amount of dissolved oxygen had stabilised, a sample (5 Fi) was injected. The sample was an aliquot of lactose of concentration 100, 200, 300, 400 or 500 mg/dl. containing glucose (300 mg/dl.). Controls having the same lactose concentration but containing no glucose were also tested. The glucose in the sample was converted to glucose-6-phosphate by the action of the immobilized hexokinase column, and the lactose was hydrolysed by the immobilized frgalactosidase column to glucose. The glucose was oxidized by the immobilized glucose oxidase column and the electric current change caused by the decrease in the amount of dissolved oxygen was recorded.

The results are shown in Figure 3 of the accompanying drawings, in which - represents the results obtained for the glucose-containing samples and 0-0 represents the results obtained for the control samples without glucose. As can be seen, lactose was assayed with good accuracy even in the presence of glucose.

(3) Saccharose array in sample containing saccharose admixed with glucose: In the apparatus employed for the lactose assay (2) the immobilized ss-galactosidase column was replaced by an immobilized invertase column (2950 Ulg, wetwt., 100 mg) (2.8 x 30 mm) and the immobilized glucose oxidase column was replaced by a column on which both glucose oxidase and mutarotase had been immobilised (glucoseoxidase 43 Ulg, wetwt., mutarotase 1200 Ulg, wetwt.,50 mg) (2.8 x 15 mm). A buffer solution consisting of 3 mM ATP and 2 mM magnesium chloride containing 0.1 M phosphate buffer (pH 7.0) was used. The samples to be tested were aliquots of saccharose (concentration 100, 200, 300, 400 or 500 mg/dl.) containing 300 mg/dl: glucose.Saccharose solutions without glucose were used as controls. The assay was effected in the same way as in the lactose assay (2) above. The results are shown in Figure 4 of the accompanying drawings. In this Figure, - represents the results obtained for samples with glucose and 0-0 represents the results obtained for the controls without glucose. As can be seen, saccharose can be assayed even in the presence of glucose.

(4) Amylase assay in sample containing amylase admixed with glucose: In the apparatus employed for the lactose assay (2), the immobilized ss-galactosidase column was replaced by an immobilized a-glucosidase column (160 U/g, wetwt., 100 mg) (2.8 x 30 mm). A buffer solution consisting of 3mM ATP and 2 mM magnesium chloride containing 0.1 M phosphate buffer (pH 7.0) was used.

The substrate was soluble starch in 0.1 M phosphate buffer (pH 7.0) (500 mgldl.). Aliquots of amylase (product of Boehringer G.m.b.H., porcine pancrease a-amylase, 100 U/ml: 1,2,3,4,5,6 ml/lit.) containing glucose 200 mg/dl were employed.

The a-amylase solution (10 iil) was added to the substrate solution (100 yl) and incubated at 38"C for 10 minutes. The buffer solution was supplied at a flow rate 1 ml/min. After stabilizing the level of dissolved oxygen, the above incubated sample (5 yl) was injected into the system via the sample injector. Maltose produced from the starch by the a-amylase was decomposed into glucose through the immobilized a-glucosidase column. This glucose was oxidized by the immobilized glucose oxidase column and the electric current change caused by a decrease of the level of dissolved oxygen was recorded. The admixed glucose was converted into glucose-6-phosphate through the immobilized hexokinase column. If completely converted, no effect on the change in electric current will be observed.The results are shown in Figure 5 of the accompanying drawings.

EXAMPLE 2 In the amylase activity assay apparatus of Example 1-(4), the a-amylase activity in an irregular control serum sample (trade name, seraclea NA: Nihon Shoji Co., a-amylase: 1360 lUll, glucose: 230 mg/dl.) was assayed. The incubation time of the substrate and sample was 10 min., 20 min. and 30 min. Each 20 yl sample was injected into the system via the injector. Operation was in the same way as in Example 1-(4).

Buffer solutions without ATP were employed as controls.

The results are shown in Figure 6 of the accompanying drawings in which - represents the results for the buffer solution with ATP and 0-0 represents the results for the controls without ATP. In the case of the controls without ATP, no conversation of glucose to glucose-6-phosphate occurred, resulting in a higher change of electric current. In the case of the buffer solution with ATP, no effect of glucose was observed and the changes in electric current were proportional to the reaction time. amylase activity in the serum can be assayed with good accuracy.

a-Amylase activities in unknown human blood sera were assayed, with the control of the above, as compared with a commercially available kit (trade name: amylase test Daiichi). The results of both assays are identical (assay by the present invention 185 lU/lit., commercially available kit: 181 lU/lit.) EXAMPLE 3 Saccharose in a culture filtrate was assayed by using the saccharose assay apparatus of Example 1-(3).

Culture liquid (at starting time of cultivation: molasses 25%, yeast cultivation medium) was collected at certain times after the beginning of cultivation of the culture. The supernatant of each culture liquid was diluted 10 times by adding water. A 5 ul sample was injected into the system via the sample injector.

Operation was the same as in Example 1-(3). The saccharose concentration, shown by electric current change, was calculated by the standard curve in Figure 4. Also, a commercially available saccharose assay kit (Boehringer G.m.b.H., F-kit, saccharose/glucose, lot. 139041, UV absorption method) was used to assay the same sample. The results areshown in Figure 7 of the accompanying drawings in which represents the result of the assay according to the present invention and 0-0 represents the result of the assay using the commercially available assay kit.

In spite of the presence of glucose- in molasses, the saccharose in the culture liquid can be assayed. In the UV absorption assay method in a commercially available assay kit, previously admixed glucose is oxidized by glucose oxidase to remove glucose. Removal of glucose by the present invention requires only 2 minutes, whereas the commercially available assay kit requires 75 minutes for the removal of glucose and thereafter 15 minutes for denaturation of the glucose oxidase. Further constant dissolved oxygen should be supplied and pH control is required if necessary. In addition to these complicated operations, the effect of colored substances in the sample causes error on assay. The present invention, on the other hand, is simple to operate, effects an assay rapidly, does not cause errors due to the presence of colored substances and accurate assays can be achieved.

Claims (15)

1. An assay method which comprises: (i) converting a first substance in a sample to a second substance.

(ii) oxidising the thus-formed second substance using an oxidase, and (iii) measuring the oxygen consumed or hydrogen peroxide operated in the oxidation step (ii), wherein, prior to step (i), the sample is contacted with an immobilised kinase in the presence of ATP to phosphorylate any second substance which may be present in the sample and which would otherwise be oxidised in step (ii).

2. An assay method according to claim 1 wherein an immobilised oxidase is used in step (ii).

3. An assay method according to claim 1 or 2 wherein measurement is effected in step (iii) by electrochemical means.

4. An assay method according to claim 3 wherein an oxygen electrode, a hydrogen peroxide electrode or an enzyme electrode is employed.

5. An assay method according to any one of the preceding claims wherein the kinase is glycerol kinase, pyruvate kinase, chlorine kinase, glucokinase, galactokinase or hexokinase.

6. An assay method according to any one of the preceding claims wherein the oxidase in step (ii) is glycerol oxidase, pyruvate oxidase, choline oxidase, glucose oxidase, galactose oxidase or hexose oxidase.

7. An assay method according to any one of the preceding claims wherein an enzyme is employed to convert the first substance to the second substance instep (i) and it is the activity of this enzyme which is assayed.

8. An assay method according to any one of claims 1 to 6 which is employed for the quantitative determination of the first substance.

9. An assay method according to any of the preceding claims wherein, prior to step (i), the sample is contacted with the immobilised kinase in the presence of ATP substantially as hereinbefore described in Example 1(1).

10. A method of assaying lactose substantially as herein before described in Example 1(2).

11. A method of assaying saccharose substantially as hereinbefore described in Example 1(3) of Example 3.

12. A method of assaying amylase substantially as hereinbefore described in Example 1(4) or Example 2.

13. An assay method substantially as hereinbefore described with reference to Figure 1 of the accompanying drawings.

14. Apparatus for use in assaying a sample which comprises a first substance and which may also contain a second substance, which apparatus comprises: an immobilised kinase for phosphorylating any second substance in the original sample in the presence of ATP, means for converting the first substance to the second substance, an oxidase for oxidising the thus-formed second substance and means for measuring eitherthe oxygen consumed or the hydrogen peroxide generated during the oxidation.

15. Apparatus for use in assaying sample which comprises a first substance and which may also contain a second substance, said apparatus being substantially as hereinbefore described with reference to and as illustrated by Figure 1 of the accompanying drawings.