CA1084393A

CA1084393A - Process for the determination of substrates or enzyme activities

Info

Publication number: CA1084393A
Application number: CA276,113A
Authority: CA
Inventors: Josef Danninger; Ulfert Deneke; Gunter Lang; Gerhard Michal; Peter Roschlau
Original assignee: Boehringer Mannheim GmbH
Current assignee: Roche Diagnostics GmbH
Priority date: 1976-06-09
Filing date: 1977-04-13
Publication date: 1980-08-26
Also published as: NL169503C; AU500988B2; DE2625834C3; BR7703008A; US4168205A; BE854407A; FR2354561A1; IE45546B1; DE2625834B2; FR2354561B1; SE7705303L; ES458544A1; NL169503B; IT1075527B; GB1519134A; DK202677A; DD130178A5; JPS52150692A; FI60719C; FI771356A

Abstract

ABSTRACT OF THE DISCLOSURE

A reagent for the enzymatic determination of sub-strates or enzyme activities. comprises a system for the determination of a substrate or enzyme with a redox reaction as the measurement reaction, wherein ascorbate oxidase is present the reagent permits investigation of fluids and foodstuffs which contain ascorbic acid which disturbs enzymatic determination by virtue of its strong reducing action.

Description

The present invention is concerned with a process for the determination of substrates or enzyme activities with the use of a redox re~ction as measurement reaction and is also concerned with a reagent for carrying out this process.
In clinical and pharMaceutical chemistry, bio-chemistry and foodstuff chemistry, redox reactions are of great interest for the determination of en~yme or substrate concentrations. These reactions can be evalu-ated by photometric measurements. If, in addition to the redox components of interest, the test system contains other reducing substances, disturbances can occur.
Ascorbic acid is found especially frequently in sample material. Since it is a strong reducing agent, it gives . .~
rise to disturbances when investigating phanmaceuticals or physiological fluids, such as serum or urine, ascorbic acid-containing plant juices or other foodstuffs which contain ascorbic-acid or to which ascorbic acid has been added. Thus, for example, it is known that the following reactions are disturbed by ascorbic acid:
A. reactions in which hydrogen peroxide and a hydrogen donor are reacted wit'n peroxidase (PoD3 are disturbed by the reaction:
ascorbic acid + H202 POD~ dehydroascorbic acid + H20 B. reactions in which tetrazolium salts are reduced with reducing agents to give formazanes are disturbed by the reaction:
ascorbic acid + tetrazolium salt formazane + dehydroascorbic acid C. reactions in which phenols are oxidatively coupled with

-2-, ." ~ ' ~' ' ..

~343~3 nucleoph]lic reagents are disturbed because ascorbic acicl gives rise to side reactions, which have not yet been elucidated, so that non-uniform coupling products are formed, the absorption behaviour of which deviates in normal and ultra-violet light from the coupling products normally formed.
Conventional oxidation methods are, as a rule, unsuitable for the removal of ascorbic acid from the sample material.
Oxidation agents also attack and destroy substrates and enzymes.
Their reduction products, which are mostly di- and trivalent metal ions, frequently inhibit the enzymes used for the indica-tor reaction. The destruction of ascorbic acid in the presence of oxygen necessitates the use of strongly alkaline media, for example a 25% aqueous solution of sodium hydroxide. This also ~; leads to the destruction of substrates and enzymes.
`~~ Therefore, the problem forming the basis of the present ... .
- invention is to provide a process for the determination of ; substrates or enzyme activities, with the use of a redox reac-tion as measurement reaction, which is no longer disturbed by - 20 ascorbic acid.
According to the present invention, this problem is solved by adding ascorbate oxidase to the reaction batch.

~ . , Ascorbate oxidase catalyses the reaction:

~; ascorbic acid + 1/2 2 ascorbate oxidase dehydroascorbic acid + H20 According to the statements in the literature concerning .
ascorbic acid oxidase, it was to have been expected that this enzyme could not be employed for the ~
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purpose according to the present inven-tion. Thus, all previously obtained enzyme preparations produce hydrogen peroxide in a side reaction. At the same time, ascorbate oxidase undergoes a very rapid inactivation which is ascribed to the simultaneously formed hydrogen peroxide (Biochemical Copper Proceedings Symposium Harriman, New York 1965, pp. 305 - 337). This also applies to the most highly purified preparations which, in the ultracentrifuge and by electrophoresis, no longer show the presence of any impurities (Biochem. 4, 1362-1370/1965). ~he reaction inactivation is ascribed to the formation o~ hydrogen peroxide (Biochem. Biophys. Acta 5~, 427 to 439/1962).
According to the calculations of the lat~er authors, lU
ascorbate oxidase in 1 mMol/litre ascorbic acid solution can produce 0.7 x 10 2 mMol/litre of hydrogen peroxide, such ascorbate concentrations being frequently present in sample solutions. The hydrogen peroxidè formed is certainly available for enzymatic reactions, which has been demonstrated in the case of, for example, catalase.
In the system:-hydrogen donor + ~202~PoD~ coloured material ~ 2H20 used for the photometric determination of hydxogen ,~
., peroxide, at a molar extinction coefficient of about 20 cm / ~Mol, it brings about an extinction difference of 0.14. Since the measurement range of normal photo-metric reactions extends from about 0.01 - 1.00, this gi~es rise to an unacceptable error. In the same way, reactions are disturbed in which, instead of hydrogen peroxide, use is made of organic hydroperoxides and instead of POD, use is made of haemoglobin or of other ;

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oxidation catalysts.
However, apart from these errors brought about by hydrogen peroxide, it is known from the above last-mention~d literature reference that the reaction of the ascorbic acid already comes to a stop long before its ~ .
complete removal.
This would be especially serious in the case of all those redox reactions in which hydrogen peroxide is formed since this hydrogen peroxide would certainly, on the one hand, further accelerate the inactivation of the enzyme and, on the other hand, the new formation of hydrogen peroxide by the ascorbate oxidase itself leads to completely unpredictable phenomena. Therefore, it is most surprising that, contrary to expectations, it is possible completely to remove the disturbances brought ;~
about by the presence of ascorbate by the addition of ascorbate oxidase, without other disturbances arising which would again nullify the advantage of:the ascorbate removal.
., The process according to the present invention is preferably used in the case of enzymatic reactions, especially those in which hydrogen peroxide is formed and thus in the case of reactions which are catalysed by oxidases, such as'glucose oxidase, uricase, cholesterol oxidase and the like, as w~ll as in the case of reactions in which tetrazolium salts are reduced and in the case of reactions in which phenols are oxidatively coupled with nucleophi:Lic reagents. The amount of ascorbate oxidase added depends upon the amount of ascorbic acid to be :
e~pected in the sample. As a rule, O.002 to 100 U

; _5_ 39~

ascorbate oxidase/ml. and preferably 0.01 to 30 U/ml., are added to the -test batch. The pH value of the reaction depe~ds, in the first place, upon the pH value which is necessary for the other participating enzyme or enzymes.
pH values of from 4.0 to 8.5 are usually appropriate for the process of the present invention. Accordlng to the - invention, it is especially preferred to use an ascorbate oxidase obtained from small marrows (Curcurbita ~e~
medullosa). However, ascorbate oxidase of other origin can also be used.
The present invention also provides a reagent for the determination of substrates or enzyme activities, com-prising a system for the determination of a substrate or enzyme with a redox reaction as measurement reaction, the reagent additionally containing ascorbate oxidase.
Preferred reagents of this type contain, when glucose is the substrate to be determined, peroxidase, glucose oxidase, o-dianisidine or azino-di-3-ethylbenzyl-thiazoline-6-suIphonate, together with buffer, or hexo-kinase (~), glucose-6-phosphate dehydrogenase (G6P-DH), diaphorase, nicotinamide-adenine-dinucleotide phosphate ~ ;
(NADP), a tetrazolium salt, such as 3-(4-iodophenyl)-2-(4-nitrophenyl)-5-phenyl-2H-tetrazolium chloride (INT) and buffer. When uric acid is the substrate to be determined, the system preferabIy comprises peroxidase, uricase, a phenol1 such as 2,4-dichlorophenol, amino-antipyrine and buffer. In the case of the determination of glutamate, the system preferably comprises glutamate dehydrogenase (GlDH), diaphorase, nicotinamide-adenine-dinucleotide (NAD~, INT and buffer. For the determin-: .

343~3 ation of -tyrosine, the system preferably comprises tyrosinase, 3-methyl-6-po-tassium sulphonyl-benzthiazolone-2-hydrazone and for the detenmination of pyrocatechol, the system preferably comprises diphenol oxidase and

3-methyl-6-potassium sulphonyl-benzthiazolone-2-hydrazone, in each case together with a buf~er. All these systems for the determination of substrates are known. However, there can also be used instead other known systems for the determination of substrates or enzymes within the scope of the reagent according to the present invention.
The present invention is also of especial irnportance for use in the field of rapid diagnostics. As a rule, such rapid diagnostics contain the various reagents needed -for carrying out the process either impregnated in an absorbent carrier or applied" together with an appropriate binding agent, to a carrier filmO A preferred embodi-.
mental form is the addition of the ascorbate oxidase to ~ ~
the mixture of the other reagents, with subsequent impreg- --nation into an absorbent carrier~ In this way, there can be obtained test papers which are practically not dis-turbed, for example, by ascorbic acid when used for the detection of glucose in urine (for example, according to ~erman Patent Specification No. 23 38 932) or of blood in urine (for example, according to German Patent Spec-ifications ~os.2,460,903 or 2,235,152) or of blood in faeces. That the ascorbate oxidase in test papers for the detection of blood in urine remains capable of functioning and is also storage-stable is most su~prising because these test papers contain large amounts of organic hydroperoxides which, similarly to hydrogen peroxide, . - ~ ....... .
. :: , .~'~:;'' ' ', ' : , 3~3 would have been expected to cause an inactivation of the ascorbate oxidase.
The ascorbate oxidase can, however, also be applied to a separate carrier which is then combined with the carrier for the other reagents, for example, laid there-over, stuck thereto or jointly sealed in between approp-riate materials~. In this case, an especially preferred carrier for the ascorbate oxidase is a so-called water-soluble paper (for example, according to German Patent Specification ~o.2,436,598) which allows the colour reaction to be observed especially well on the carrier paper containing the other reagants. This embodiment is ;~-especially advantageous when, in the reagent combination, substances are present which are incompatible with the ascorbate oxidase, for example strongly acidic reagents ~ -such as are used in processes for the determination of urobilinogen, bilirubin and nitrite.
In the case of the above embodiments, there can be used up to 5000 U and preferably up to 2000 U ascorbate , .
oxidase per ml. impregnation solution for the preparation of the reagent. In general, less than 1 U/ml. will not ensure the desired effect.
Furthermore, separate zones of a carrier material can also be impregnated with the ascorbate oxidase or the other-test reagents. In this case, the carrier is prefer-. .
ably brouyht into contact with the solution to beinvestigated in such a manner that the solution first comes into contact with the ascorbate oxidase-containing ; zone and from there is drawn into -the zone which contains ; the other test reagents.

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, L3~3 According to a further ernbodiment, the ascorbate ox.idasiie can be bound to an insoluble carrier material by ~ -methods known for enzyme fixing, methods of this type being described, for exc~ple, in German Patent Specific-~; ations Nos.1,768,512, 2,128,7~3; 2,260,185: 2,260,184;
: 2,426,988, 2,603,158 ancl 1,915,970.
-~ . The process according to the present invention i5 preferably used for the determination of glucose with peroxidase, glucose oxidase and o-dianisidine or 2,2~-. azino-di-3-ethylbenzthiazoline-5-sulphonate (ABTS), for the determination of glucose by the hexokinase/glucose - 6-phosphate dehydrogenase method, for the detection of urlc acld by means of phenol, aminoantipyrine, peroxidase and uricase, for the determination of tyrosine or pyro-catechol by means of SMBT~I (4-methyl-6-potassium sulphonyl- -~
~.., ~`i benzthiazolone-2-hydrazone~ and tyrosinase or diphenol `~l oxidase, respectively.

The following Examples are given for the purpose --: of illustrating the present invention~
i Example 1. ~ :`

.- ~ Determination of glucose with o-dianisidine, :~ peroxidase (POD) and glucose oxidase (GOD), measured in `~ a photometer; wavelength 432 nm, measurement temperature:

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., 3~3 cuvette No. 1 2 3 4 5 pH 7.01 M 2.5 2.5 2.5 2.5 2.5 o-dianisidine 0.1 0.1 0.1 0.1 0.1 5 mg./ml POD, 180 U/ml. 0.05 0.05 0.050.05 0.05 glucose solution 0 03 0 03 0 03 0 03 0 03 1 mg./ml.

ascorbic acid 0.1 0.01 0.1 0.01 solution 10 mM
water 0.12 0.02 0.11 - 0.09 500 U/ml. 0.02 0.02 .
incubate for 1 min.at 25C., read off El, then start with GOD 70 U/ml~ 0.1 0.1 0.1 0.1 0.1 incubate for 30 min. at 25C., read off E2, calculate ~E from E2 E

~E 0~541 0.010 00316 0.540 0.543 Cuvette 1 corresponds to an undisturbed measuremen~

(no ascorbic acid). Cuvettes 2 and 3 show that 1 or 0.1 ~Mol ascorbate practically completely inhibit or 41.5%
inhibit the test and cuvettes 4 and 5 show the complete destruction of these ascorbate concentrations by the addition, in each case, of 10 U ascorbate oxidase.
.

Detection of glucose with 2,2'-azino di-(3-ethyl-benzthiazoline-6-sulphonate) (ABTS), POD and GOD in a photometer, wavelength: 432 nm' measurement temperature:

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. . _ cuvette No. l 2 3 4 5 phosphate buffer 2.75 2~75 2.75 2.7S 2.75 ABTS 50 mM 0.05 0.05 0.05 0.05 0.05 POD 250 U/ml. 0.02 0.02 0.02 0.02 0.02 glucose solution 0.1 mg./ml. 0.1 0.1 0.1 0.1 0.1 solution lmM 0.1 0.01 0.1 0.01 water 0~12 0.02 0.11 - 0.09 ascorbate oxidase 500 U/ml. ~ ~ - 0.02 0.02 incubate for l min. at 25C., read off El, start with GOD 70 U/ml. 0.1 0.1 0.1 0.1 0.1 ~
incubate for 30 min. at 25C., read off E2, calculate ~E from E2-E~
., .
~E 0.50S 0.000 0.40 0.502 0.504 -~
::.
Cuvette l corresponds to an undisturbed measurement (no ascorbate). Cuvettes 2 and 3 show that 0.1 or 0.01 ~ Mol ascorbate completely inhibit or 21% inhibit the test and cuvettes 4 and 5 show the complete destruction of these ascorbate concentrations by the addition, in each , . .
~- case, of 10 U ascorbate oxidase.

Example 3.

~ Detection o~ uric acid by means of phenol, amino-- antipyrine, POD and uricase in an automatic analyser ; (AutoAnalyser).

; ~ nciple uric acid ~ 2 ~ H20 _ ~ e~ allantoin ~ ~22 .~ -11-~ , :~"'`

.
3~3 2H202 + 2,4-dichlorophenol -~ 4-aminoantipyrine PO
quinoid coloured material ~ 2H20 Preparation of the solutio _ l. Ascorbate oxidase rea~ent _ .
In 600 ml. double distilled water, there is dissolved the content of flask 1 and 0.3 ml. Brij-35 is added. The solution can be stored in a dark bottle at about 4C. for four weeks and at about 25C. one week.
2. Uricase reaqent.
In 800 ml. double distilled water, there is dissolved the content of flask 2 and 2.0 mlO Brij-35 are added. ~he : ~
solution can be stored in a dark bottle at about 4C. Eor four weeks and at about 25 C. for one week.
_. ;
1. 50 mM phosphate buffer, pH 5.6 ascorbate oxidase ~ in the amounts which can be seen from Fig.2 of the accompanying drawings.

2. 31 m~ tris-(hydroxymethyl3-aminomethane/citric acid, pH 8.9 uricase ,~ 0.08 U/ml. ~ ;
.- ,. .
POD ~ 0.015 U/ml. `
3.0 mM 2,4-dichlorophenol ;

4.0 mM 4-aminoantipyrine For the carrying out oE the determination, the flow - systems of the automatic device is assembled according to the flow diagram illustrated in Fig.l of the accompanying ;
drawings.
Fig.2 of the accompanying drawings s~nmarises, in graphic :Eorm, the e~perimen~al results obtained.

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.
Detection of glucose with ~/G6P-DH, NADP, IN~ and diaphorase in a photometer; measurement wavelength:
492 nm' incubation temperature: 25G.

:
cuvette No. 1 2 3 phosphate buffer, pH 7.5 O.lM1.7 1.7 1.7 NADP/INT each 1 mg./mlØ1 0.1 0.1 diaphorase 5 U/ml. 0.1 0.1 0.1 glucose solution 0.05 mg.~mlØ1 0.1 0.1 ascorbate solution 10 mM - 0.01 0.01 ` ascorbate oxidase 35 U/ml. - - 0.03 ... :
water 0.04 0.03 incubate for 3 min. at 25C., read off El, start with .'~`' ~ .
~ HK/G6P-DH solution each 56 U/ml. 0.05 0~05 0.05 .. -- ... - _ _ _ ,. _ .
incubate for i5 min., read off E2, calculate QE from ~ E2-El - QE 0.234 0.307 0.230 `

; .
; Cuvette 1 corresponds to an undisturbed measurement, cuvette 2 shows that 0.01 ~Mol ascorbate gives a test result which is 34% too high and cuvette 3 shows that 1 U ascorbate oxidase completely overcomes this disturbance.
Example 5.

Determination of ylutamate by means of GlDH, NAD/
INT/diaphorase in a pho~ometer, measurement wavelength 492 nm; temperature 25C.

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1L3~3 _ cuvette No. 1 2 3 4 5 phosphate buffer, 1.3() 1.20 1.29 1.18 1.27 glutamate solution 0.1 0.1 0.1 0.1 0.1 0.2 mg./ml.
ascorbate solution 0 1 0.01 0.1 0.01 ascorbate oxidase 0.02 0.02 incubate for 3 min. at 25C., then pipette into the individual cuvettes 0. 2 M TRA, 0.05 M ~-, potassium phosphate 1.0 1.0 1.0 1.0 1.5% Triton-X-100 NAD solution 5 mg/ml. 0.2 0.2 0.2 0.2 002 diaphorase solution 0.05 o,05 o,05 0.05 0.05 ~ , GlDH 1000 U/ml.O.05 0.05 0.05 0.05 0.05 . ~ :
read off El, start reaction with - - - - - -- :
I~T solution, 2 mg/mlO 0.05 0.05 0.05 0.05 0.05 incubate for 15 min. at 25C., read off E2, calculate ~E from E2-El ~ ~E 0.184 1.560 0.380 0.186 0.182 - Cuvette 1 corresponds to an undisturbed measurement, cuvettes 2 and 3 show that 0.5 or 0.05 ~ ol ascorbate give test results which are 700 or 100% too high and cuvettes 4 and 5 show the complete removal of this dist--^ urbance by 2 ~ ascorbate oxidase.

Determination of tyrosine with 3-methyl-6-potassium ~ -14-,:

. .

~013~393 sulphonyl-benzthia~olone-2~hydrazone (SMB'm) and tyro-sinase in a photometer, measurement wavelength 492 nm, measurement temperature 25C~C.

cuvette No. 1 2 3 phosphate buffer, pH 5.2 2.77 2.77 2.77 0.25 M
SMBTH 0.lM O.05 0.05 0.05 ascorbic acid solution 0.1 0.1 water 0.12 0.02 ascorbate oxidase 500 U/ml. - - 0.02 tyrosinase 60 U/ml. 0.05 0.05 0.05 incubate for 1 min. at 25C., read off El, start with tyrosine 2 mM 0.05 0.05 0.05 .,;. - . .......... . .
incubate for 1 hr. at 25C., read off E2,,calculate ~E

,~ from E2-El -,,~
~ ~ ~E 1.113 0.728 1.100 _ ~
,~ Cuvette 1 corresponds to an undisturbed measurement, ~-. . ~
in cuvette 2, 1 ~Mol ascorbate lowers -the theoretical value by 35% and in cuvette 3 this disturbance is completely removed by 10 U ascorbate oxidase.
~ Example 7 ; Determination of pyrocatechol wikh SMBTH and - diphenol oxidase.

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3~3 cuvette No. 1 2 3 phosphate bufEer, 0.25 M 2.80 2.70 2.68 SMBTH 0.1 M - 0.05 0.05 0.05 pyrocatechol 0.5 M 0.10 0.10 0.10 ascorbate solution 20 mM - 0.1 0.1 ascorbate oxidase 500 U/ml. - - 0.02 incubate for l min. at 25~C., read off El, start with ,, ,_ , , diphenol oxidase 200 U/ml. 0~05 0.05 0.05 , incubate for 17 min. at 25C., read off E2, calculate QE ~
. ~ .
frcm E2-El ~E 0.890 0.485 0.896 --Cuvette 1 corresponds to an undisturbed measurement, in cuvette 2, 2 ~Mol ascorbate lowers the theore-tical value by 47% and in cuvette 3 this disturbance is completely removed by 10 U ascorbate oxidase.
~ Example 8.
-- Test paper for the detection of ~lucose ~n the urine.
Filter paper is impregnated with a solution of the - following composition and dried at 50C.:
;~ 1.2 M citrate buffer pH 5 50.0 ml.
,~
9-(~-dimethylaminopropyl)-6-chloro- -3-aminocarbazole dihydrochloride 0'75 g' glucose oxidase ~104 U/mg.) 0.25 g.
peroxidase (63 U/mg.) 0.05 g.
ascorbate oxidase ~100 U/mg.) l.00 g.
- water lO0.0 ml.
The test paper reacts with glucose-containing urines . .~

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3g3 with red-orange to black-red colour shades. After a reaction time of 2 minutes, urines with the same glucose contents but also with ascorbic acid contents of up to 150 mg./dl. give practically the same reaction colours.
With test papers of analogous cornposition but with-:
out ascorbate oxidase, depending upon the glucose content, the reaction colours are weakened or completely suppressed in the case of ascorbic acid concentrations above 50 mg./dl.
.` ~ .
~ Test paper for the detection of blood in the urine.

- Filter paper is successively impregnated with the ;. ~ :. , following solutions and dried at 40C.

Solution 1 1.2M citrate buffer pH 5.25 35.0 ml ethylenediamine-tetraacetic acid 0 1 g disodium salt dioctyl sodium sulphosuccinate 0.5 g.

2,5-dimethylhexane-2,5-dihydro-1 6 peroxide ~about 70%) " g' phosphoric acid trimorpholide 12.7 gO
ascorbate oxidase (100 U/mg.) 0.3 g.
methanol 30.0 ml water ad lOOo O ml Solution 2 ~: ~ ... ...
3,3a,5,5J-tetramethylbenzidine 0,3 g.

phenanthridine 0.2 g.
toluene/petroleum ether (30:70 v/v) ad 100.0 ml.
With this test paper, there can be detected the .
presence of 5 erythrocytes/mm3 in the urine, even in the ~;~ presence of 30 to 50 mg. ascorbic acid/100 ml. With a test paper o~ the same composi~ion but without ascorbate -.

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1~34~3 oxidase, this detection is no longer possible even in the presence of only 10 - 20 mg. ascorbic acid/100 ml.
Example 10 Test paper A
Test paper as in Example 9 but without ascorbate oxidase.

.
Test paper B
Water-soluble carboxymethylcellulose paper (weight 60 g.jm2) is impregnated with a solution of 20% glacial acetic acid in methanol for the purpose of neutralisation and then dried. Thereafter, it is sprayed with an aqueous solution of ascorbate oxidase (103 U/ml.) and dried immediately.
Test paper B is laid upon test paper A and both are sealed in together between a polyester film and a nylon mesh. The urine to be,investigated is dropped on to the test strips so produced.
The results obtained are analogous to those of Example 9. , Test paper for the detection of blood in_faeces Filter paper is successively impregnated with the following solutions and dried at 40C.
Solution 1 1.2 M citrate buffer, pH 5.2510 ml. -~
ascorbate oxidase 100 U/mg. 0.3 g. ~ ~' water ad 100.0 ml.
Solution 2 "
_ __ gum guaiac 3 g.

, toluene ad 100.0 ml.

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3~3 The solution is filtered and the filtrate is used for the impregnation.
The test paper obtai:ned is coated with faeces. If, on the rear side, 'chere is dropped on a 3% aqueous solution of hydrogen perox.ide, then a blue coloration is obtained when the faeces contain about 2% blood.
This coloration also occurs in the case of ascorbic acid-containing faeces (about 15 mg./100 g.), whereas it does not occur when the test paper does not contain ascorbate oxidase.

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Claims

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A process for the determination of substrates or enzyme activities with the use of a redox reaction as measurement reaction, wherein the process is carried out in the presence of ascorbate oxidase.

2. A process according to claim 1, wherein 0.002 to 100 U ascorbate oxidase/ml. test batch are used.

3. A process according to claim 2, wherein 0.01 to 30 U ascorbate oxidase/ml. test batch are used.

4. A process according to claim 1, 2 or 3, wherein the ascorbate oxidase is used together with an enzyme forming hydrogen peroxide.

5. A process according to claim 1, 2 or 3, wherein the ascorbate oxidase is used together with a tetrazolium salt.

6. A process according to claim 1, 2 or 3, wherein the ascorbate oxidase is used together with a phenol which can be coupled oxidatively with a nucleophilic reagent.

7. A process according to claim 1, 2 or 3, wherein it is carried out at a pH of from 4.0 to 8.5.

8. A process according to claim 1, 2 or 3, wherein said ascorbate oxidase is one obtained from small marrows (Curcurbita pepo medullosa).

9. Process according to claim 1, 2 or 3, wherein said ascorbate oxidase is one obtained from Curcurbita pepo medullosa and the process is carried out at a pH of 4.0 to 8.5 in the presence of an additive selected from the group consisting of -a) an enzyme which forms hydrogen peroxide, b) a tetrazolium salt, and c) a pehnol which can be coupled oxidatively with a nucleophilic reagent.

10. A reagent for the enzymatic determination of sub-strates or enzyme activities, comprising a system for the determination of a substrate or enzyme with a redox reaction as measurement reaction, wherein ascorbate oxidase is additionally present.

11. A reagent according to claim 10, wherein the system for the determination of a substrate serves for the determination of glucose and comprises peroxidase, glucose oxidase, o-dianisidine or 2-azino-di-3-ethylbenzthiazoline-6-sulphonate and buffer.

12. A reagent according to claim 10, wherein the system for the determination of a substrate serves for the deter-mination of uric acid and comprises peroxidase, uricase, a phenol, aminoantipyrine and buffer.

13. A reagent according to claim 12, wherein the phenol is 2,4-dichlorophenol,

14. A reagent according to claim 10, wherein the system for the determination of a substrate serves for the deter-mination of glucose and comprises hexokinase, glucose-6-phosphate dehydrogenase, nicotinamide-adeninedinucleotide phosphate, diaphorase, a tetrazolium salt and buffer.

15. A reagent according to claim 10, wherein the system for the determination of a substrate serves for the deter-mination of glutamate and comprises glutamate dehydrogenase, diaphorase, nicotinamide-adeninedinucleotide, a tetrazolium salt and buffer.

16. A reagent according to claim 10, wherein the system for the determination of a substrate serves for the deter-mination of tyrosine and comprises tyrosinase, 3-methyl-6-potassium sulphonyl-benzthiazolone-2-hydrazone and buffer.

17. A reagent according to claim 10, wherein the system for the determination of a substrate serves for the deter-mination of pyrocatechol and comprises diphenol oxidase, 3-methyl-6-potassium sulphonyl-benzthiazolone-2-hydrazone and buffer.

18. A reagent according to claim 10, wherein the system for the determination of a substrate and the ascorbate oxidase are impregnated in an absorbent carrier material.

19. A reagent according to claim 18, comprising an absorbent carrier impregnated with glucose oxidase, peroxidase, ascorbate oxidase, a 9-(.gamma.-dimethylaminopropyl)-6-chloro-3-aminocarbazole salt and buffer.

20. A reagent according to claim 18, comprising an absorbent carrier impregnated with an ethylenediaminetetra-acetic acid salt, dioctyl sodium sulphosuccinate, 2,5-dimethylhexane-2,5-dihydroperoxide, phosphoric acid tri-morpholide, ascorbate oxidase, 3,3',5,5'-tetramethylbenzidine, phenanthridine and buffer.

21. A reagent according to claim 18, 19 or 20, wherein the absorbent carrier is a paper.

22. A reagent according to claim 10, wherein the ascorbate oxidase is impregnated into a water-soluble sheet of carrier material and the system for the determination of a substrate or enzyme activity is impregnated into a water-insoluble sheet of absorbent carrier material, the water-soluble and the water-insoluble carrier materials being placed one on top of the other.

23. A reagent according to claim 18, comprising ascorbate oxidase, gum guaiac and buffer.

24. A reagent according to claim 10, 18 or 22, wherein said ascorbate oxidase is one obtained from Curcurbita pepo medullosa.

25. A reagent according to claim 19, 20 or 23, wherein said ascorbate oxidase is one obtained from Curcurbita pepo medullosa.