CA1044582A - Diagnostic agent for cholesterol and cholesterol esters - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE
A diagnostic agent for the detection and determination of cholesterol and cholesterol esters in body fluids comprises an absorbent carrier or a synthetic resin film impregnated with or having embedded therein cholesterol oxidase, a system for the detection of hydrogen peroxide, buffer and at least one surface-active compound with lipophilic and hydrophilic properties; a p??cess for the activation of cholesterol oxidase is also pro-vided.

Description

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The present invention is concerned with a process for the activation of cholesterol oxidase and with a diagnostic agent for the detection and determination of cholesterol and cholesterol esters in body fluids.
German Patent Specification No. 2,224,132 describes a process for the determination of cholesterol in which cholesterol is incubated in an aqueous medium with cholesterol oxidase, followed by the determination either of the oxygen consumption or of the amount of hydrogen peroxide or of cholestenone formed. This German Patent Specification also describes a reagent for the determination of cholesterol which comprises cholesterol oxidase and a system for the determination of hydrogen peroxide or a system for the determination of cholestenone.
We have now found that cholesterol oxidase has an insufficient storage stability and is inactivated relatively quickly. In the course of the investigation of this instability, we have found that the inactivation is brought about by ~mall amounts of detergent which are present as an accompanying sUbstance and originate from the process used for the preparation of the enzyme. This preparation of the c enzyme can be carried out, for example, by the process described in German Patent Specification ~o. 2,224,131 in which a micro-organism which metabolises cholesterol is digested by destruction of the cell walls with a non-ionic, surface-active agent present in a buffer solution and then extracted, whereafter the extract is centrifuged, the precipitate obtained is discarded and the supernatant ~-liquid is applied to an anion exchanger, the enzyme then being eluted with a buffer solution containing the non-ionic surface-active agent and thereafter isolated from the eluate.

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We have also found that, upon removal of the~e residual traces of detergent, a completely ~atisfactory storage stability of the enzyme can be achieved. ~owever, we have also ascertained that the enzyme freed from detergent ha~ a remarkably reduced acitivity. If, for example, the enzyme is used for the determination of cholesterol, then it is necessary either to use a compara-tively freshly prepared preparation or considerably larger amounts of enzyme must be employed for the test or the time - 10 required for-carrying out the determination must be con-siderably increased.
Consequently, it is an object of the present invention to provide a proce~s for the activation of cholesterol oxidase. More particularly, it is an object of the present invention to provide such a process which, on the one hand, permits the use of cholesterol oxidase ~ which is completely free from traces of detergent and, "J therefore, is sufficiently storage-stable and, on the other~ hand, provide9 an activity of the enzyme during use thereof;~ 20 which corresponds to that of the freshly prepared enzyme but which has not been subjected to the removal of small ~- amounts of detergents.
Thus, according to the present invention, there i8 provided a proce~s for the activation of cholesterol oxidase, wherein at least one ~urface-active compound with ~;~ lipophilic and hydrophilic propertieY i~ added to the x cholesterol oxidase before the u~e thereof.
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As surface-active compounds with lipophilic and hydrophilic properties, there are preferably used non-ionic detergents which contain at least one hydroxyl group in the ... .
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molecule. Polyoxyethylene derivativeq of alkyl, aryl and aralkyl alcohols are preferably employed. Example~ of such preferred surface-active compounds include polyoxy-ethylene alkyl ethers, polyoxyethylene alkyl-carboxylic acid ester~, polyoxyethylene-sorbitan alkyl carboxylic acid esters, polyoxyethylene-glycerol alkyl carboxylic acid esters, polyoxyethylene-alkylamines, polyoxyethylene-polyoxypropylene block polymers, polyoxyethylene-alkyl thio-ethers ànd polyoxyethylene alkyl aryl ethers, Specific - 10 examples thereof include hydroxy-polyethoxydodecane, ethyleneoxy adducts of alkyl-phenols, polyethoxy-ethylene derivative~ of sorbitan anhydrides and the like. Polyoxy-ethylene derivatives modified with mercaptans are also equally suitable.
According to the present invention, the above-mentioned surface-active compounds are preferably used in an amount of between about 0.005 to 0.1 wt.%, referred to the aqueous enzyme solution.
Considerable number~ of the above-mentioned surface-; 20 active materials are commercially available and differ from one another by the number of oxyethylene groups .. . .
~' present therein, The most useful ones are those which are water-soluble and which contain, on average, 5 to 20 oxy-ethylene groups per molecule. Lower oxyethylated types of compounds are usually only dispersible and are, therefore, less useful. Higher ethoxylated compounds (greater than 25) r~ .
are admittedly water-soluble but are ~o hydrophilic that -their effectiveness is reduced.
Similarly good results are also obtained with physiological surface-active substances which satisfy . . .
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the above-given de~inition, for example, desoxycholates.
In addition, other ~urface-active substances with lipophilic and hydrophilic propertie~ which contain at least one hydroxyl group, can al~o be used. Examples of such compounds include ethanol, butyl-diglycol and hexylene-glycol. However, lower mono- and dialcohols of this type must be added in relatively large amounts of 10 to 20 vol. %
for the achievement of the desired activation and the suitability thereof must be ascertained experimentally in each case. Thus, for example, no activation is achieved with methanol, ethylene glycol, polyethylene glycol, cyclohexanol, glycerol, lecithin and saponin. Surfacé-active compound~ which have lipophilic and hydrophilic properties but which do not contain a hydr~xyl group in the molecule can a}so be used.
Of the anionic wetting agents, the salts of bile acids, such as cholic acid, taurocholic acid and desoxy-cholic acid, are espejcially preferred, as well as fatty acid salts and fatty acid sarcoside~. Of especial importance are the sulphuric acid derivatives which, a~ is known, have the additional property of stabilising the coloured radical oxidation products of some indicators, such as o-tolidine ~ and heterocyclic azines. These include, for example, the ; following classes of compounds: sulphosuccinic acid esters, alkyl aryl ~ulphonate~, alkyl sulphates and alkyl polyoxy-ethylene sulphate~.
Of the cationic wetting agents, there can be used, `~ for example, alkyl pyridinium and trimethyl ammonium salt~, as well as more complex compounds, for example, benzethonium chloride.

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Of the amphoteric wetting agents, there can be used, for example, the imidazolium betaines.
< Particular example~ of surface-active compounds which can be u~ed according to the present invention include sodium (2-ethyl-hexyl)-sulphosuccinate, sodium dodecyl sulphate, sodium oleate, benzalkonium chloride and cetyl-pyridinium chloride.
The ionic or amphoteric detergents are preferably used in admixture with the above-mentioned non-ionic ~ 10 detergents, the appropriate amounts thereof corresponding - to those of the non-ionic detergent.
The non-ionic, surface-active detergents containing -;
at least one hydroxyl group in the molecule are preferred becauae they provide an approximately 5 to 10 times greater ~ activation and thus a corresponding increase of the reaction ; velocity or shortening o the period of reaction in comparison with the other ~urface-active substances which can be employed.
They are alJo effective in smaller amounts.
By alkyl radicals, there are here to be understood those containing up to about 20 carbon atoms and especially those containing 12 to 18 carbon atoms.
The inactivation of cholesterol oxidase in the pre~ence of trace~ of detergent is e~pecially marked when the enzyme i~ present in ammonium ~ulphate ~olution. The following Table I shows the ~torage stability of the enzyme in LM ammonium sulphate ~olution at 33C. in the i presence of differing amounts of detergent. The experiments `~ were carried out with an octyl-phenol-ethylene oxide adduct.

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TQBLE I
Activitv of cholesterol oxidase in %, referred to freshlv prepared enzyme .
storage detergent period _ ; in day~ without 0 02%0.05% 0.1% 0.6%
89% 60Yo lO~o 7% 3%
. 22 85% 4~h 1% 1% 1%

The effective amount of surface-active compound used in the process according to the present invention depend~
upon it~ molecular weight and upon the degree of its j hydrophobic and hydrophilic properties. ~oo small an amount does not bring about an activation but too large an amount leads to the result generally known for surface-active substances of denaturing ths enzyme. The appropriate concentration range, as well as the optimum concentration, can be experimentally determined for all appropriate ourface-active agento. Such ranges are, for example, 0.15 r to 0~6 wt.% for hydroxypolyethoxydodecane, 0.03 to 0.1 wt.%
for octylphenol-ethylene oxide adducts, 0.15 to 0.6 wt.%
for polyoxyethylene derivatives of sorbitan anhydrides, 0.03 to 0.05 wt.% for sodium desoxycholate and 0.005 to ` 0.02 wt.% for mercaptan-modified alkyl-phenol ethylene oxide adducts (for example Sterox SE*).
Of the non-ionic surface-active polyoxy-ethylene compounds, those are especially useful which have a balance `` ratio of hydrophobic residues to polyoxyethylene chain and ~ are water-soluble. A measurement value for the ratio of `f hydrophobic residue and polyoxyethylene chain is the so-*trademark .
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called HLB value (cf. W. C, Griffin, J. Soc. Co~metic. Chem., 1, 311/1950 and 5, 249/1954).
Those ~urface-active materials with HLB values between about 10 and 17 are especially useful. These value~ are, however, only to be regarded as being optimum standard value~ ~ince the effectivene~s also depends upon the nature of the hydrophobic residue.
~he present invention i8 also concerned with a diagno~stic agent for the detection and determination of cholesterol and of cholecsterol esters in body fluids which can be used for carrying out the above-deocribed process.
; The determination of cholesterol is of conssiderable importance in medical diagnosi~, In clinical chemistry, rapid tests are being used more and more for the detection of substances in body fluids.
Although they frequently do not provide sufficiently accurate results, nevertheless, they do permit a quicker and cheaper indication of routine and large-scale invest-igations. The diagnostic agent~ employed for rapid tests are either absorbent carriers or water-stable films which contain all of the reagents needed for the reaction. When ~ -these diagnostic agents are brought into contact with the body fluids to be te~ted, then colour reactions are obtained which can be evaluated either on the basis of compartative colours or with the u~e of simple reflection photometers, Experiments for the production of agents for , cholesterol rapid tests by impregnation of absorbent paper with cholesterol oxidase, peroxida~e, an Qxidation indicator and a buffer do not give satisfactory results because the .

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teqt papers thu~ obtained do not react with cholesterol-~ontaining ~erum of the usual concentration.
We have now found that useful test papers or test films are obtained, which react in graduated stage-q with cholesterol-containing serum when the carrier additionally contain~ at least one of the above-described ~urface-active agents.
~ hus, according to the present invention, there is also provided a diagnostic agent for the detection and determination of cholesterol and of cholesterol esterq in body fluids which comprise~ a carrier or a synthetic resin , film which i~ impregnated with or has embedded therein doleqterol oxidase, a system for the detection of hydrogen peroxide, a buffer and at lea~t one of the above-described surface-active agents. me surface-active agent is prefer-ably present therein in a concentration of 2 to 3~/0 and more preferably of 10 to 20Yo~ referred to the solid reagents.
'i A preferred Jystem for the detection of hydrogen peroxide compri~es peroxidase and an oxidation indicator 20 optionally together with a swelling agent and/or a stabiliser.
For the detection of cholesterol in serum, test papers are outstandingly useful which have been obtained by the impregnation of absorbent papers with the necessary reagent~. If, however, it is desired to detect cholesterol in whole blood, then the test papers are preferably rendered - hydrophobic, for example, in the manner described in German Patent Specification No, 1,598,048 or are coated with a ~emi-permeable membrane of celluloqe esters. However, for .. .

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the detection of cholesterol in whole blocsd, it iisi ei~pecially preferred to use test films, such ais are obtainable according to German Patent Specification No. 1,598,153 containing the neceissary reagents.
For the preparation of such teist films, the reagents, for example, cholesterol oxidase, peroxidase, buffer, indicator and optionally a swelling agent, are stirred, together wlth the surface-active materials to be used according to the preisent invention, into an aqueous dis-persion of a film,forming polymer. This dispersion is then coated as a thin film and left to dry. When cholesterol-containing blood i3 dropped onto such a reagent-containing film and then wiped off after about one minute, colorations are al~o obtained, the colour depth of which depends upon the amount of cholesterol present in the blood. The3e colorations are particularly suitable for a quantitative evaluation with simple remission photometers.
The diagnostic agent according to the present invention can be used for the detection and determination of free choleisterol. If, however, cholesterol eisters are also present, then the choleisiterol must firist be liberated therefrom. Thiisi can be carried out in known manner, for example, by saponification with aqueous alkali. However, it iis particularly advantageous to split the ester with choleisterol eisterase, which iisi preferably isolated from micro-organisms, since it is then possible to work under very mild conditions.
The choleisterol esterase can be added to the body fluid, ~ollowed by incubation. This process can be . ~

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carried out, for example, by drawing the body fluid into capillarie~, the inner walls of which are coated, in a manner analogous to that described in German Patent Specification ~o. 2,240,672, with chole~terol esterase and optionally with adjuvant materials. After incubation in the capillarie~, the body fluid is then applied to the test strips in the above-described manner. If free and esterified cholesterol were both present in the body fluid, then, of course, the sum of the two is detected. However, the cholesterol esterase can also be incorporated espec-ially advantageou31y into the test strips to give diagnostic agent~ with which, in one step, the sum of the free and e3terified cholesterol can be detected and determined Systems which can be used for the detection of the hydrogen peroxide formed by the oxidation of the choleRterol, for example, peroxidase, buffer, oxidation indicator and optionally a swelling agent and the like, are known, for example,from the descriptions of rapid tests for glucose.
Example~ of component8 for thi8 preferred system are given in the following:
Of the peroxidases, that from horseradish i~
especially preferred but lactoperoxidase and the like can also be employed.
As buffer~, there can be u~ed those which are conventional, for example, phosphate, citrate and borate buffers. The pH value which they give on the diagnostic agent should be between 4 and 9 and preferably between 5 and 8.
As oxidation indicators, there can be used various classes of compounds, namely benzidene derivatives, for ~, . ~ .. . .
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i~4582 example o-tolidine and o-vanillin derivatives according to German Patent Specification No. 1,598,133, or heterocyclic azines according to German Patent Specification No. 1,648,840.
The formulation~ for the diagnostic agent~ and especially tho~e for te~t film_, can alqo contain a con~
ventional _welling agent, such as 30dium alginate, carboxy-methyl-cellulo3e or the like, a~ well aq a stabilising agent for the enzyme, for example, dithioerythritol.
Substrate materials for test films are synthetic re9in di9persion~ of, for example, polyvinyl propionate or acetate. Into these are stirred all the necessary reagents, preferably in dissolved form, whereafter the ~;-mixture obtained is then coated into thin films and dried.
Test papers can be produced by dissolving the re-~` agents in water or in a mixture of water and organic ~olvents with which filter papers are impregnated and then dried.
However, the paper can first be impregnated with the water-~oluble reagent~ and then impregnated with, for example, the indicator~ in organic solution.
The following Examples are given for the purpo~e ~1 of illu~trating the present invention:--~ Exam~le 1.
0,05 ml. of serum are added to 10 ml. 0.5M potassium `~ phosphate buffer of pH 7.5 which contains 0.4% hydroxy-,4 polyethoxydodecane. The extinction (El) is read off at 2~0 nm in a suitable spectrophotometer and the reaction started with 0.02 ml. (0.1 U) of storage-stable cholesterol oxidase, freed from traces of detergent, in lM aqueous ammonium ~ulphate ~olution. After 3 minutes, the extinction ' . . , . , : -1~)44S8'~
(E2) is read off. The concentration of the cholestenone formed and thus of the cholesterol is given by the difference between the first and second readings, having regard to the molar extinction coefficient~ for cholesterol at 240 nm. The mea~urement of a typical qample gave 62 mg.%
free cholesterol and 167 mg.% total cholesterol (after saponification).
A comparative determination but without the addition of the surface-active agent required a reaction time of 15 minutes.
The separation of traces of detergent for the improvement of the storage stability of the enzyme is preferably carried out with the use of hydrophobic adsorption resins, Especially preferred for this purpoQe are, for example, the material which is commercially available as "Bio-Beads"* from Biorad, as well as the products obtainable from Rohm & Haas under the designation XAD*-re~ins, Example 2.
0.05 ml. serum are added to 10 ml. 0.5M potassium phosphate buffer of pH 7.5 which contain~ 0.02% hydroxy-polyethoxy-dodecane and 0.03% qodium desoxycholate. The extinction (El) is read off at 240 nm in a suitable spectro-photometer and the reaction is started with 0.02 ml.
(= 0.1 U) storage-stable cholesterol oxidase, freed from traces of detergent, in LM aqueous ammonium sulphate solution.
J . After 3 minutes, the extinction (E2) is again read off, The concentration of the cholestenone formed and ` 30 thus of the chole3terol i~ given from the difference *trademark - 13 _ .--~, ' - ' :.- - .

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between the first and second readings, having regard to the molar extinction coefficients for cholestenone at '~ ~
240 nm (~ = 15.5 cm2/~mol). ~ -- The meaqurement of a typical sample gave 65 mg.%
free cholesterol and 170 mg.% total cholesterol (after saponification) Example 3.
0.05 ml. serum are added to 10 ml. 0.5M potassium , pho~phate buffer of pH 7.5 which contains 0.02% hydroxy-polyethoxy-dodecane and 0.1% secondary alkyl sulphate.
' The extinction (El) is measured at 240 nm in a suitable ,' spectrophotometer and the reaction is started with 0.02 ml.
;' (= 0.1 U) storage-stable cholesterol oxidaqe, freed from " traces of detergent, in LM aqueous ammonium sulphate j ~olution.
, After 3 minute~, the extinction (E2) is again read " off. The concentration of the cholestenone formed and , thuA of the cholesterol is given by the difference between j the fir~t and second readings, having regard to the molar ¦ 20 extinction coeffici,ent9 of cholestenone at 240 nm.
The measurement of a typical sample gave 62 mg.%
free cholesterol and 167 mg.% total cholesterol (after saponification).
Example 4.
' Filter paper (Schleicher & Schull No. 597 ~F Ind.*)

3~ is impregnated with a solution of the following composition .3, and dried at 40C.:
~ 1 M citrate buffer, pH 5.25 20 ml.
; cholesterol oxidase (60 U/mg.) 0.1 g.
peroxidase (70 U/mg.) 0.05 g.
distilled water ad 100 ml.
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This paper i~ then impregnated with ~olutions of 0.2 g. o-tolidine in 100 ml. methylene chloride which, in addition, each contain 1 g. of the following surface-active materials:
a) polyoxyethylene tributyl-phenol ether b) polyoxyethylene ~orbitan monolaurate c) polyoxyethylene-nonyl-phenol ether d) polyoxyethylene lauryl ether e) polyoxyethylene cetyl ether f) polyoxyethylene stearate g) polyoxyethylene dodecyl thioether.
After drying, te~t papers are obtained which react with chole~terol-containing sera with a green colour. If the sera also contain cholesterol eætero, then stronger green colorations are obtained if the sera have previously been mixed with a drop of cholesterol esterase qolution.
A test paper which does not contain one of the above-mentioned ~urface-active materials does not react with the sera.
Example 5.
Filter paper (Schleicher & Sch~ll No. 597 NF Ind.*) i~ impregnated with a solution of the following composïtion and dried at 40C,:
, 1 M citrate buffer, pH 7 20 ml.
cholesterol oxidase (60 U/mg.) 0.1 g.
chole~terol e~terase (18 U/mg.) 0.25 g.
, peroxidase (70 U/mg.) 0.05 g-distilled water 100 ml.
This paper is impregnated with solutions of 0.2 g.
o-tolidine in 100 ml. methylene chloride which, in addition, *trademæk .' ~ - 15 -each contain 0.5 g. of one of the following surface-active materials:
a) polyoxyethylene cocoate b) polyoxyethylene oleate c) polyoxyethylene polypropylene glycol d) polyoxyethylene stearylamine e) polyoxyethylene glycerol monolaurate.
After drying, test papers are obtained which react with a green colour with tho~e ~era which contain chole9terol and/or chole~terol esters. Test papers without surface-active agent show no reaction.
Practically the same behaviour is shown by test paperQ
which contain the same amount of citrate buffer with a pH
of 5.25 or 6.
le 6.
Paper pre-impregnated in the manner described in Example 4 iJ ~ubsequently impregnated with ~olutions of 0.3 g. o-tolidine in 100 ml. acetone which contain 1.5 g.
of one of the following surface-active material3:
20, a) dioctyl sodium Julphosuccinate b) sodium dodecyl-benzene-sulphonate c) ~odium lauryl polyglycol ether ~ulphate d) sodium lauryl sarco3inate e) ~odium laurate f) cholic acid g) desoxycholic acid h) sodium taurocholate These test papers possess practically the ~ame properties as the test papers produced according to ' 30 Example 4 but the reaction colours are stable for a longer period of time.

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~)4~S82 - Te~t papers with a phosphate buffer of pH 7 posseAs similar propertie~.
Example 7.
Paper pre-impregnated in the manner described in Example 5 is impregnated with ~olutions which contain 0.4 g. o-tolidine and 0.5 g. of one of the following surface-active materials:
a) lauryl pyridinium chloride b) benzethonium chloride c) cetyl trimethyl ammonium chloride d) l-hydroxyethyl-l-carboxymethyl-2-alkyl-imidazolinium betaine.
The properties of these test papers correspond to those of Example 5.
, Example 8.
Filter paper (Schleicher & Sch~ll No. 597 ~F Ind.) i9 impregnated with a ~olution of the following compo~ition ~i and dried at 40C.:
lM pho~phate buffer, pH 6 25 ml.
cholesterol oxida~e (60 U/mg.)0.1 g.
peroxidase (70 U/mg.) 0.05 g.
cholic acid 1.0 g, ' acetone 10 ml.
distilled water ad 100 ml.
!,, This paper i~ subsequently impregnated with solutions of oxidation indicators in 100 ml. acetone.
The amount of indicator used, the chemical name thereof and the colour reaction with cholesterol-containing era are summarised in the following Table II:

T~BLE II
. ., amount indicator reaction :
O.3 g. o-vanillylidene-E~vanilloyl-violet hydrazone 0.2 g. azino-bis-(N-ethyl-benzthia- green zolone-2-sulphonic acid-5) diammonium salt 0,1 g. azino-bis-(N-alkylquinolone- blue-2-~ulphonic acid-6) di- violet ammonium salt 0,1 g. bii-(N-alkyl-quinolone-2)- violet 0,1 g. (~-methyl-benzthiazolone-2)- blue-~-ethyl-quinolone-2)-azine green 0,3 g, (~-methyl-benzthiazolone-2)- blue l-phenyl-3l,4-dimethyl-tri-azolone-5)-azine Exam~le 9, A mixture is prepared from the following components:
polyvinyl propionate dispersions 45 g, sodium alginate, 1,85% in O,SM 35 g.
phosphate buffer, pH 5,5 cholesterol oxidase (60 TJ/mg~ ) 0.5 g-peroxidase (70 U/mg,) 0.25 g, dioctyl sodium sulphosuccinate 1 g, o-tolidine, dissolved in 6 ml. acetone0.6 g.
water 50 ml.
This mixture is either spread out to give a film ~ with a wet film thickness of about 300 nm or is painted o~ 30 on to a solid carrier and dried at 35C. When blood containing chole~terol is dropped on to the film and the blood wiped off after one minute, then, depending upon ~ .

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the cholesterol concentration, green coloration~ of varying intensity are obtained.
If, in addition, 1.0 g. cholesterol esterase is added to the above-de~cribed formulation, then films are obtained with which it is also possible to determine ; increased cholesterol ester content~.

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Claims (12)

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

1. A process for the activation of cholesterol oxidase, wherein at least one surface-active compound with lipophilic and hydrophilic properties is added to the cholesterol oxidase before use in a cholesterol oxidase activating amount.

2, A process according to claim 1, wherein the surface-active compound is non-ionic and contains at least one hydroxyl group.

3. A process according to claim 2, wherein the non-ionic compound is a polyoxyethylene derivative of an alkyl, aryl or aralkyl alcohol.

4. A process according to claim 1, 2 or 3, wherein the surface-active compound is used in an amount of between 0.005 and 0.1 wt.%, referred to an aqueous enzyme solution.

5, A process according to claim 2, wherein said surface-active compound is water-soluble and contains, on average, 5 to 20 oxyethylene groups per molecule, said surface-active compound being used in an amount of about 0.005 to 0.1 wt.%, referred to an aqueous solution of the enzyme,

6. A diagnostic agent for the detection and determination of cholesterol and cholesterol esters in body fluids by oxidation of the cholesterol with oxygen in the presence of cholesterol oxidase to give cholestenone and hydrogen peroxide and detection of the hydrogen peroxide by peroxidase-catalysed oxidation of an oxidation indicator, which comprises an absorbent carrier or a synthetic resin film impregnated with or having embedded therein cholesterol oxidase, a system for the detection of hydrogen peroxide, a buffer and at least one surface-active com-pound with lipophilic and hydrophilic properties, said surface-active compound being present in a cholesterol oxidase activating amount.

7. A diagnostic agent according to claim 6, wherein the surface-active compound is a water-soluble non-ionic polyoxy-ethylene derivative of an alkyl, aryl or aralkyl alcohol con-taining at least one hydroxyl group, and on average, 5 to 20 oxyethylene groups per molecule.

8. A diagnostic agent according to claim 6, wherein the surface-active agent is present therein in a concentration of 2 to 30%, by weight, referred to the solid reagents.

9, A diagnostic agent according to claim 7 or 8, wherein the surface-active agent is present therein in a concentration of 10 to 20%, by weight, referred to the solid reagents,

10. A diagnostic agent according to claim 6, 7 or 8, wherein cholesterol esterase is also present.

11, A diagnostic agent according to claim 6, 7 or 8, wherein at least one of a swelling agent and a stabilising agent is also present.

12. A method for the detection and determination of cholesterol and cholesterol esters in body fluids which com-prises applying the body fluid to a diagnostic agent, as defined in claim 6, 7 or 8, and evaluating any colour change.