CA1254116A - Analytical process and agents for the detection of esterolytic and/or proteolytic enzymes - Google Patents
Analytical process and agents for the detection of esterolytic and/or proteolytic enzymesInfo
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- CA1254116A CA1254116A CA000476408A CA476408A CA1254116A CA 1254116 A CA1254116 A CA 1254116A CA 000476408 A CA000476408 A CA 000476408A CA 476408 A CA476408 A CA 476408A CA 1254116 A CA1254116 A CA 1254116A
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Abstract
ABSTRACT OF THE DISCLOSURE
An agent for the detection of esterolytic or proteolytic enzymes containing (a) an amino acid ester or peptide ester of a phenol, as the chromogenic enzyme substrate, and (b) a substance, which accelerates the enzymatic cleavage of the aminoacid ester or peptide ester bond of component (a), which is a polyaminoacid with molecular weight of between 1,000 and 2,000,000, preferably a polyaminoacid which is built up from aminoacids of the general formula
An agent for the detection of esterolytic or proteolytic enzymes containing (a) an amino acid ester or peptide ester of a phenol, as the chromogenic enzyme substrate, and (b) a substance, which accelerates the enzymatic cleavage of the aminoacid ester or peptide ester bond of component (a), which is a polyaminoacid with molecular weight of between 1,000 and 2,000,000, preferably a polyaminoacid which is built up from aminoacids of the general formula
Description
The present invention relates to agents for analyti-cal detection of esterolytic and/or proteolytic enzymes, for example in body fluids, the esters being incorporated into test agents, ;n particular test strips, in a suitable S manner. Bes;des chromogenic en7yme substrates (aminoacid esters or pept;de esters of suitable pheno~s) and, if ap-propriate, diazonium salts which couple with the phenols to form a co~our, the agents according to the invention also contain polyaminoacids as acce~erators fer the enzymatic cleavage of the aminoacid esters or peptide esters. The agents are preferably used for the detection of leucocytes, ;n particular in urine.
The detection of leucocytes in body fluids, in par-ticular in urine, is of great ;mportance ;n the d;agnost;cs of diseases of the k;dneys and of the urogenital tract.
This detection was originally carried out by counting the leucocytes in the non-centrifuged ur;ne or in the urine sediment. In both methods, only intact leucocytes can be recorded. However, it is known that the rate of leucocyte-lys;s ;s subject to wide variations, depending on the urinemedium; thus, for example, ;n strongly alkaline urines the leucocyte half-life is only 6û m;nutes. This means that the leucocyte counts determ;ned are too low. Apart from th1s lysis error, quantitative microscopic determ;nat;on of the leucocytes in the non-centrifuged, homogenised urine gives very accurate values in the counting chamber. Never-theless, th;s method ;s only rarely used ;n practice, since it ;s laborious and t;me-consuming and requ;res trained personnel.
The preferred process for leucocyte determinations in the urine in medical practice was therefore the so-called field of view method in the urine sediment. For this, the sample (sediment) first had to be obtained by centrifugation. However, other constituents of the urine were also thereby concentrated, and these - such as, for Le A 22 899 ,
The detection of leucocytes in body fluids, in par-ticular in urine, is of great ;mportance ;n the d;agnost;cs of diseases of the k;dneys and of the urogenital tract.
This detection was originally carried out by counting the leucocytes in the non-centrifuged ur;ne or in the urine sediment. In both methods, only intact leucocytes can be recorded. However, it is known that the rate of leucocyte-lys;s ;s subject to wide variations, depending on the urinemedium; thus, for example, ;n strongly alkaline urines the leucocyte half-life is only 6û m;nutes. This means that the leucocyte counts determ;ned are too low. Apart from th1s lysis error, quantitative microscopic determ;nat;on of the leucocytes in the non-centrifuged, homogenised urine gives very accurate values in the counting chamber. Never-theless, th;s method ;s only rarely used ;n practice, since it ;s laborious and t;me-consuming and requ;res trained personnel.
The preferred process for leucocyte determinations in the urine in medical practice was therefore the so-called field of view method in the urine sediment. For this, the sample (sediment) first had to be obtained by centrifugation. However, other constituents of the urine were also thereby concentrated, and these - such as, for Le A 22 899 ,
- 2 -example, salts and epithelial cells - make microscopic counting of the leucocytes considerably more difficult.
A vary;ng sed;ment content, ;nhomogene;t;es of the sed;-ment and a d;fferent opt;cal des;gn of the m;croscopes led to relatively largP errors tup ~o several hundred percent) in stating the leucocyte count.
In order to avoid these difficulties, several at-tempts have already been made to use enzymatic reactions as the detection principle for leucocytes in various body flu;ds, s;nce leucocytes have a w;dely spread enzyme spectrum.
Thus, for example, agents for the detect;on of leucocytes ;n body flu;ds are known from German Offen-Legungsschriften (German Published Specif;cation) 2,826,965 and 2,836,644, in which the esterolytic and/or proteolyt;c activity present in the leucocytes is utilised for analytical purposes. Sulphonphthaleine esters or azo dyestuff esters are used as substrates for the leucocyte esterases and/or proteases. The dyestuffs released in the enzymatic reaction are then determined by known methods. However, the agents descr;bed in these publ;cat;ons are st;ll too ;nsens;tive for practical purposes, since their react;on t;mes are too long with low leucocyte concentrations.
Various methods for the detection of proteases and esterases are also known from histochemical and phyto-chemical enzymology (compare, for example, A.G.E. Pearse, Histochemistry, Theoret;cal and Appl;ed, 3rd edition, Church;ll L;vingstone, Ed;nburgh-London-New York 1968).
In general, colourless or sl;ghtly coloured esters are used for the detect;on, these be;ng spl;t by the enzymes ;nto a colourless ac;d and a s;m;larly colourless alcohol (phe-nol) component. The phenol component ;s then converted ;nto coloured products ;n a subsequer,t react;on, for example by coupling with diazon;um salts or by ox;dation.
F. Schmalzl and H. Braunsteiner, for example, describe ;n Kl;n. Wschr. 46, 642 (1968) a spec;f;c phytochem;cal Le A 22 899 leucocyte esterase detect;on w;th naphthol-AS-D-chloro-acetate as the substrate and a d;azon;um salt wh;ch forms a coloured azo compound with the naphthol liberated.
However, two-component systems of this type have 5 proved to be unsuitable for rapid and simple detection of leucocytes in body fluids, such as, for example, in the urine, since they are much too insensitive: samples con-taining 5,000 leucocytes/~l still do not give a reaction.
3ritish Patent A-1,128,371 and European Patent A-10 12,957 descr;be the use of indoxyl and th;oindoxyl esters as chromogen;c substrates for the detection of hydrolyt;c enzymes in body flu;ds. On enzymatic cleavage of the sub-strate, free indoxyl is formed, which is subsequently o~
dised to the easily detectable blue dyestuff indigo. A
15 commercially available test based on European Patent A-12,957 consists of a strip of filter paper impregnated with N-tosyl-L-alan;ne L-indoxyl ester. When the test strip ;s immersed in a urine sample containing leucocytes, ;t turns blue ;n colour. However, the long wa;ting time Z0 (about 15 minutes) before the end colouration is reached and the test can be evaluated is a considerable disadvan-tage of this product.
European Patent A-14,9Z9 describes various accele~
Fators (pyridine derivatives; imidazole derivatives; alco-25 hols; metal complexes) for the enzymatic cleavage reac-tion. However, the relatively long time before complete oxidation of the ;ndoxyl and the low sensitivity of the test (detectlon lim;t: a few thousand leucocytes/lJl) re-main a d;sadvantage. The same applies to the use of es-30 ters of leuco-indoanilines as substrates for leucocyte enzymes according to European Patent A-34,323.
European Paten A-39,880 provides a combination of the substrates according to European Patent A-12,957 and 14,929 w;th the detect;on principal of coupling with 35 diazon;um salts which has been discussed above. Although it ;s poss;ble considerably to reduce the detection Le A 22 899 1~4ii~;
L;m;ts for leucocytes ;n th;s manner, the detect;on sensit;v;ty of 15-20 leucocytes/lul wh;ch ;s des;red for use in pract;ce ;s st;ll not ach;eved.
The object of the present invention was thus to discover new accelerators for ester-cleaving enzymes which, as a result of accelerat;on of the enzymatic cleav-age of the substrates by the leucocyte enzymes, permit more sens;tive and more rapid detection of the leucocytes in urine. This object is achieved by using polyamino-acids to the reagent system. Surprisingly, the polyamino-ac;ds have a super;or accelerat;ng act;on on the leucocyte enzymes to that of the accelerators descr;bed ;n European Patent A-14,929 (pyridine derivatives, imidazole deriva-tives, metal complexes and alcohols~. In addition, they can also be used as detergents, so that simultaneous addi-tion of detergents to the reagent systems used in practice (for example reaction solutions or formulations for the coat;ng of test strips) is superfluous.
The ;nvent;on relates to agents for the detect;on of esterolyt;c and/or proteolytic enzymes, conta;ning (a) an am;noac;d ester or pept;de ester of a phenol, as the chromogenic enzyme substrate, ~b) a substance which accelerates the enzymatic cleavage of the am;noacid ester bond or peptide ester bond of component (a), if approp-riate ~c) a diazonium salt, if appropriate td) a buffer, and if appropriate (e) a carr;er and/or the usual addi-tives, characterised in that component ~b) is a polyam;no-acid, preferably conta;n;ng bas;c groups, w;th a molecular we;ght ~number-average) of between 1,000 and 2,000,000.
F;nally, the ;nvent;on also relates to a process for the detection of esterolytic and/or proteolytic en-zymes ;n liquid samples, in particular body fluids, wh;ch is character;sed ;n that the sainple ;s brought ;nto con-tact w;th the agent accord;ng to the ;nvent;on and the colour react;on wh;ch occurs is determ;ned.
Both polyam;noac;ds wh;ch are bu;lt up from only Le A 22 899 a single aminoacid (homopolyaminoacids) and polyconden-sates of two or more different aminoacids in the form of copolyaminoacids with a randomised sequence of the con-stituent aminoacids or in the form of sequence polymers are suitable accelerators to be employed according to the invention. Sequence polymers are obtained in the poly-condensat;on of pept;des or their derivatives and have a defined, recurr;ng aminoacid sequence. For the accelerating action of the polyaminoacids, it is particu-larly advantageous for at least one of the aminoacids usedfor the polymerisation or copolymerisation to carry a basic group, for example an amino group or guanido group.
Preferably, the polyaminoacids to be used accord-;ng to the invent;on are bu;lt up from identical or diffe-rent monomers of the general formula H2N-CH-COO~ (I) in which R represents hydrogen or an optionally branched alkyl, cycloalkyl or aralkyl radical which has 1 to 15 C atoms, preferably 1 to 9 C atoms, and is opt;onally subst;tuted by 1 or 2, in particular 1, hydroxyl, mercapto, carboxyl, am;no or guanido groups.
The polyaminoac;ds to be used according to the invent;on as accelerators preferably contain S to 100 25 moleZ, in part;cular 10 to 100 moleX, of monomer units with a basic groùp. Particularly preferred monomer units of this type are aminoacids of the general formula (I) wh;ch contain an am;no or guanido group ;n the radical R.
Examples of such bas;c aminoacids are, in particular, arg;n;ne, lys;ne and orn;th;ne, ~h;ch can be e;ther ;n the racem;c form or ;n the L- or D-form. The polyam;noacids and the sequence polymers can furthermore also conta;n as un;ts bas;c am;noac;ds wh;ch do not occur in natural Le A 22 899 proteins. Examples which may be mentioned here are ~,~-diaminobutyric acid, ~X,~-diaminopropionic acid and the diaminopimelic acids.
The polyaminoacids to be used according to the invention as acceLerators of the enzymatic cleavage are known from the l;terature and are commercially available, or they can be prepared by processes which are known per se (see, for example, E. Katchalski and M. Sela in: Ad-vances of Protein Chemistry 13, 243-492 81958; and C.L.
Anfinsen, M.L. Anson, J.T. Edsall and K. Bailey (Editors) in Academic Press Inc. Publishers, New York).
The average molecular weights ~number-average) of the polyaminoacids should be between 1,000 and 2,ûOO,OOO, and polyaminoac;ds with average molecular weights of 5,000 to 500,000 are preferably used, those with average molecular weights between 10,000 and 300,000 being parti-cularly preferred.
The polyaminoacids are preferably employed ;n the homogeneous liqu;d test in concentrations of 0.0001X by weight to 1% by weight, and the concentrations are parti-cularly preferably in the range from 0.001 to 0.01X by weight. In the preparation, described below, of test de-vices, they are used in concentrations of 0.05X by weight to 10X by weight, preferably 1 to 5X by weight, based on the impregnating solution. The polyaminoacids to be used accord-ing to the invention accelerate the enzymatic cleavage of the substrates described in European Patents A-7,407, 8,428, 12,957, 14,929, 34,323 and 39,880 by the leucocyte enzymes, as well as the cleavage of the sub-strates which have already been descr;bed previous.y (G. Gomori, J. Histochem. Cytochem.
6 469 (1953); H. L8ffler, Klin. Wochenschr. 39, 1120 (1961);
L. Visser and E. 8lout, Fed.-Proc. 28, 407 (1969) and Bioch;m. Biophys. Acta 2~a, 257 (1972), and F. Sweetman and L. Ornste;n, J. H;stochem. Cytochem. 23, 327 (1974)).
The preferred chromogenic substrates in the agents according to the invention also include the compounds Le A 22 899 _ .
~25411~;
descr;bed in a parallel Application, of the general formula (II) 2 11 Ç-A-~c)x2 in which X1 and X2 are identical or different and denote nitrogen or sulphur, with the proviso that X1 and X2 do not simultaneously represent sulphur;
R1 represents hydrogen or an optionally branched alkyl group which has 1 to 6 carbon atoms and can optionally be substituted by halogen or hydroxyl;
R2 and R3 are identical or different and repre-sent hydrogen, C1-C6-alkyl groups, C1-C6-alkoxy groups, C1-C6-acyl groups, halogen, tri-fluoromethyl, nitro, S03H, cyano, C1-C8-acyl-amino groups, C1-C6-dialkylamino groups or C6-c10-arYl groups, uhich can in turn be further substituted by C1-C6-alkyl groups, C1-C6-alkoxy groups, halogen, cyano, nitro, trifluoro-methyl, S03H, C1-C6-acyl groups or C1-C6-dialkylamino groups, or R2 and R3 together form a fused-on aromatic ring, preferably a benzene ring, which can in turn be substituted by 1 or 2 radicals R2;
A denotes an aminoac;d radical or peptide radical and G represents hydrogen or, preferably, a nitrogen-protective group which is usual in peptide chemis-try or derived from such a group.
Ple~erred compounds of the general formu~a (II) are those in which X1 represents sulphur and X2 repre-sents nitrogen. Compounds of the formula (II) in which R1 represent~ hydrogen, and those in which R2 and R3, which are identical or different, represent hydrogen, Le A 22 899 C1-C2-alkyl, C1-Cz-alkoxy, halogen, C1-C4-dialkyl-amino groups or benzene radicals are furthermore preferred.
The ester radical in the compounds of the formu~a (II) is particularly preferably in the 5-position.
Other chromogenic substrates which are preferred according to the invent;on are the compounds likewise des-cribed in a parallel Appl;cation, of the general formula (III) R4 ~ O-A-G
R6 ~ i X ~ x4 ;n ~hich X3 and X4 represent N or CH, with the proviso that ;n each case either X3 or X4 represent N, R4, R5 and R6 are identical or different and represent hydrogen, C1-C6-alkyl groups, C1-C~-alkoxy groups, C1-C6-acyl groups, halogen, trifluoromethyl, nitro, S03H, cyano, C1-C8~
acylamino groups, C1-C6-dialkylamino groups or C6-C10-aryl groups, which can in turn be further substituted by C1-C6-alkyl groups, C1-C6-alkoxy groups, halogen, cyano, nitro, tri-fluoromethyl, S03H, C1-C6-acyl groups or C1-C6-dialkylamino groups, or R5 and Rf together form a fused-on aromatic ring, preferably a benzene ring, which can in turn be substituted by 1 or 2 radicals R4, and A and G have the meaning given above in the case of the formula (II).
In the compounds according to the general formula ~III), X3 preferably represents CH and X4 preferably represents nitrogen. R4, Rs and R6, which can be Le A Z2 899 _ 9 _ identical or different, preferably represent hydrogen, C1-C4-alkyl, C1-C4-alkoxy, acylamino (where the acid radical can be aliphatic or aromatic with 1 to 6 C
atoms), C1-C4-d;alkylam;no, n;~ro, cyano, halogen, or aryl, wh;ch is optionally substituted by c1-c4-alkYl, C1-C4-alkoxy or halogen.
Particularly preferably, R4, Rs and R6 are hydrogen, C1-C4-alkyl, phenyl or halogen, or Rs and R6 together form a fused-on benzene ring.
Suitable chromogenic substrates for the agents according to the invention are moreover also compounds of the general formula ~ (IV) wherein R4, R5, R6, A and G have the meaning given above in the case of formula (III).
In the general formulae (II)~ (III) and (IV), G-A - preferably represents a radical of the general formu-la n R ~ H21 ~ CH ~ C ~
in which R7 represents hydrogen or an optionally branched alkyl, cycloalkyl or aryl radical which has 1-15 C atoms, preferably 1-9 C atoms, and is optionally substituted by a hydroxyl, mercapto or carboxyl group, and R8 represents hydrogen or, preferably, -C0-alkyl, Le A 22 899 - 1 o --CO-aralkyl, -CO-aryl, -SOz-a~kyl or -S02-aryl, the alkyl rad;cals being straight-chain or branched with 1-9 C atoms, preferably 1-6 C
atoms, and the aryl radicals preferably represent-ing benzene rings, which are optionally substitu-ted by C1-C4-alkyl groups, C1-C4-alkoxY
groups or halogen.
G-A- particularly preferably represents a radical, provided w;th a customary nitrogen-protective group, of a naturally occurring aminoacid or of a peptide of 2 to 8 such aminoacids.
The aminoacid radicals can be in their L- or D-form or in their racemic form here. Particularly pre-ferred radica;s are those of glycine, alanine, valine, leucine, isoleucine, phenylalanine.and tyrosine, the L-form being particularly preferred in each case. Any free hydroxyl group present can be acylated, preferably acetylated.
A peptide rad;cal ;n the definition of A is to be understood as meaning, for example, di-, tr;-, tetra- and penta-pept;des, preferably d;- and tri-peptides, pre-ferred poss;ble aminoac;d components being the abovemen-tioned aminoacids.
The substrates of the general formulae (II), (III) and tIY) are obtained by reacting the corresponding phe-nols with aminoac;ds or peptides of the general formula G-A-OH
;n which G and A have the abovement;oned mean;ng, or suitable reactive derivatives thereof, by methods cus-tomary in peptide chemistry.
Examples of suitable reactive derivatives are the acid chlorides and the mixed anhydrides usually employed in peptide synthesis, for example with ethyl chloroformate or active esters, such as, for example, pentachlorophenyl esters or N-hydroxybenzotriazole esters.
Le A 22 899 lZS~
The agents according to the invention preferably contain, as colour-forming agents which react with the phènols (liberated during enzymatic cleavage), diazonium salts of the general formula R~ R'1 R; 3 ~ X~ ) (V) in which ~4 R'5 R'1 denotes a lower alkyl, a lower alkoxy, a lower alkylmercapto, a hydroxy, n;tro, cyano, tri-fluoromethyl, C1-C8-alkylsulphonam;do, arylsul-phonamido, C1-C8-alkylsulphone, arylsulphone, sulphonic ac;d or carboxyl;c acid, an N-morpho-lino, an N-thiomorpholino, an N-pyrrolidino, an optionally N'-alkylated N-piperazino or N-piperi-dino group, halogen or hydrogen, R'3 denotes a lower alkyl, a lower alkoxy, an aryloxy, a lower alkylmercapto, alkylamino or di-alkylamino, a hydroxyl, nitro, cyano, C1-C8-alkylsulphonam;do, arylsulphonam;do, C1-C8-alkyl-sulphone, arylsuLphone, sulphon;c ac;d or carboxy-l;c acid, an N-morphol;no, N-thiomorpholino or N-pyrrolidino, an optionally N'-alkylated N-p;pera-zino or N-piperidino or phenylamino group, a phe-nyl group which is optionally substituted by a lower alkyl or lower alkoxy radical, halogen or hydrogen, R'2, R'4 and R'5, which can be ;dent;cal or d;fferent, each denote a lower alkyl, a lower al-koxy, nitro, C1-C8-alkylsulphonam;do, arylsul-phonam;do, C1-C8-alkylsulphone, arylsulphone, sulphon;c acid or carboxylic acid or a lower alkyl mercapto group, halogen or hydrogen and Le A 22 899 411t~
X denotes a stabilising anion, it being possible for in eacl1 case 2 adjacent radicals R'1 to R'5 to be cyclised to form a benzene ring which ;s optionally subst;tuted by halogen, a C1-C6-alkyl, S a C1-C6-alkoxY or a n;tro, sulphon;c acid or carboxy-lic acid group, so that a diazonium salt of the naphtha-lene ser;es ;s formed.
Preferably, in the general formula (V) R'1 represents C1- to C4-alkyl, c1-c4-alkoxY~
hydroxyl, nitro, halogen or hydrogen, R'3 represents a C1- to C4-alkyl, C1-C4-alkoxy, aryloxy, C1-C4-alkylamino, C1-C4-d;alkylam;no, n;tro, C1-C4-alkylsulphonamido, arylsulphonam;do, C1-C4-alkylsulphone, aryl-sulphone, N-morpholino, N-pyrrolidino, phenylamino or sulphon;c ac;d group or hydrogen; and R'2, R'4 and R'5, which can be identical or different, represent C1- to C4-alkyl, C1- to C4-alkoxy, C1- to C4-alkylamino, C1 to C4-dialkylamino, nitro, C1- to C4-alkylsulphonamido, arylsulphonamido or sulphonic acid groups, halo-gen or hydrogen.
In each case 2 adjacent radicals R'1 to R'5 can here optionally be cyclised to give a benzene ring which ;s opt;onally substituted by halogen or a C1- to C4-alkyl or C1- to C4-alkoxy or a n;tro or sulphon;c acid group~
In the context of the formula (V), aryl in each case represents an aromatic radical wh;ch has 6 to 12 C
atoms, preferably 6 C atoms, and is optionally substituted by halogen or a C1-C4-alkyl or C1-C4-alkoxy group.
The diazon;um salts of the gene al formula tV) are known per se, or they can be synthesised by methods which are known per se (Houben-Weyl, Methods of Organic Chemis-5 try, volume X/3).The agents, according to the invention, for the Le A 22 899 1~25~1i6 detection of proteolytic enzymes and, in particular, leucocyte enzymes preferably contain a suitable buffer system. Possible systems for this purpose are, for example, phosphate, borate, carbonate/b;carbonate, carbo-nate, barb;turate, tr;s-~hydroxymethyl)-am;nomethane (=
tris), 2 am;no-2-methy~-propane-1,3-diol (= amed;ol) or am;noac;d buffer, the pH value and capacity as a rule be;ng chosen such that a pH value of 6-10, preferably of 7-9, ;s estab~;shed in the measurement solution or on the test strip.
In some cases, it may be advantageous also to con-jo;ntly use detergents ;n the agents according to the in-vent;on, ;n addition to the accelerating polyaminoacids.
These detergents on the one hand effect disinte-gration of the leucocytes present in the test solution andthus ~iberate the enzymes, and on the other hand act as solubilising agents for the substrates and the substances formed during cleavage, and, where relevant, intensify the colour. Poss;ble detergents are both cationic and anionic detergents, as ~ell as amphoteric and non-ion;c detergents.
Examples of these which may be mentioned are ben-zyl-d;methyl-tetradecyl-ammon;um chloride, Na dodecyl-sulphate, zephirol, polyv;nylpyrrolidone and hepar;noid, and if appropriate mixtures of two or more of the above-mentioned detergents can also be used. Since the poly-a0;noacids employed according to the ;nvention as activa-tors also have a detergent action, ho~ever, the add;t;on of extra detergents can in principle be dispensed with.
It is particularly advantageous to use the above detergents ;n the determ;nat;on of leucocytes by means of reagents f;xed to a solid phase (for example a-t~st strip), since a more homogeneous colour d;stribu~ion and a 00re ;ntensive colouration can thereby be achieved.
In the agents according to the ;nvent;on, the re-agents described above are preferab~y ;ncorporated ;n an Le A 22 899 1~5'11~t;
;nert carrier of the type which is known per se, part;cu-larly preferred carrier matrices being porous materials, such as, in particular, f;lter paper, and also membranes made of plastic, glass-fibre mats (U.S. Patent Specifica-tion 3,846,247), porous ceramic strios, synthetic non-woven fibres, spongy materials ~U.S. Patent Specification
A vary;ng sed;ment content, ;nhomogene;t;es of the sed;-ment and a d;fferent opt;cal des;gn of the m;croscopes led to relatively largP errors tup ~o several hundred percent) in stating the leucocyte count.
In order to avoid these difficulties, several at-tempts have already been made to use enzymatic reactions as the detection principle for leucocytes in various body flu;ds, s;nce leucocytes have a w;dely spread enzyme spectrum.
Thus, for example, agents for the detect;on of leucocytes ;n body flu;ds are known from German Offen-Legungsschriften (German Published Specif;cation) 2,826,965 and 2,836,644, in which the esterolytic and/or proteolyt;c activity present in the leucocytes is utilised for analytical purposes. Sulphonphthaleine esters or azo dyestuff esters are used as substrates for the leucocyte esterases and/or proteases. The dyestuffs released in the enzymatic reaction are then determined by known methods. However, the agents descr;bed in these publ;cat;ons are st;ll too ;nsens;tive for practical purposes, since their react;on t;mes are too long with low leucocyte concentrations.
Various methods for the detection of proteases and esterases are also known from histochemical and phyto-chemical enzymology (compare, for example, A.G.E. Pearse, Histochemistry, Theoret;cal and Appl;ed, 3rd edition, Church;ll L;vingstone, Ed;nburgh-London-New York 1968).
In general, colourless or sl;ghtly coloured esters are used for the detect;on, these be;ng spl;t by the enzymes ;nto a colourless ac;d and a s;m;larly colourless alcohol (phe-nol) component. The phenol component ;s then converted ;nto coloured products ;n a subsequer,t react;on, for example by coupling with diazon;um salts or by ox;dation.
F. Schmalzl and H. Braunsteiner, for example, describe ;n Kl;n. Wschr. 46, 642 (1968) a spec;f;c phytochem;cal Le A 22 899 leucocyte esterase detect;on w;th naphthol-AS-D-chloro-acetate as the substrate and a d;azon;um salt wh;ch forms a coloured azo compound with the naphthol liberated.
However, two-component systems of this type have 5 proved to be unsuitable for rapid and simple detection of leucocytes in body fluids, such as, for example, in the urine, since they are much too insensitive: samples con-taining 5,000 leucocytes/~l still do not give a reaction.
3ritish Patent A-1,128,371 and European Patent A-10 12,957 descr;be the use of indoxyl and th;oindoxyl esters as chromogen;c substrates for the detection of hydrolyt;c enzymes in body flu;ds. On enzymatic cleavage of the sub-strate, free indoxyl is formed, which is subsequently o~
dised to the easily detectable blue dyestuff indigo. A
15 commercially available test based on European Patent A-12,957 consists of a strip of filter paper impregnated with N-tosyl-L-alan;ne L-indoxyl ester. When the test strip ;s immersed in a urine sample containing leucocytes, ;t turns blue ;n colour. However, the long wa;ting time Z0 (about 15 minutes) before the end colouration is reached and the test can be evaluated is a considerable disadvan-tage of this product.
European Patent A-14,9Z9 describes various accele~
Fators (pyridine derivatives; imidazole derivatives; alco-25 hols; metal complexes) for the enzymatic cleavage reac-tion. However, the relatively long time before complete oxidation of the ;ndoxyl and the low sensitivity of the test (detectlon lim;t: a few thousand leucocytes/lJl) re-main a d;sadvantage. The same applies to the use of es-30 ters of leuco-indoanilines as substrates for leucocyte enzymes according to European Patent A-34,323.
European Paten A-39,880 provides a combination of the substrates according to European Patent A-12,957 and 14,929 w;th the detect;on principal of coupling with 35 diazon;um salts which has been discussed above. Although it ;s poss;ble considerably to reduce the detection Le A 22 899 1~4ii~;
L;m;ts for leucocytes ;n th;s manner, the detect;on sensit;v;ty of 15-20 leucocytes/lul wh;ch ;s des;red for use in pract;ce ;s st;ll not ach;eved.
The object of the present invention was thus to discover new accelerators for ester-cleaving enzymes which, as a result of accelerat;on of the enzymatic cleav-age of the substrates by the leucocyte enzymes, permit more sens;tive and more rapid detection of the leucocytes in urine. This object is achieved by using polyamino-acids to the reagent system. Surprisingly, the polyamino-ac;ds have a super;or accelerat;ng act;on on the leucocyte enzymes to that of the accelerators descr;bed ;n European Patent A-14,929 (pyridine derivatives, imidazole deriva-tives, metal complexes and alcohols~. In addition, they can also be used as detergents, so that simultaneous addi-tion of detergents to the reagent systems used in practice (for example reaction solutions or formulations for the coat;ng of test strips) is superfluous.
The ;nvent;on relates to agents for the detect;on of esterolyt;c and/or proteolytic enzymes, conta;ning (a) an am;noac;d ester or pept;de ester of a phenol, as the chromogenic enzyme substrate, ~b) a substance which accelerates the enzymatic cleavage of the am;noacid ester bond or peptide ester bond of component (a), if approp-riate ~c) a diazonium salt, if appropriate td) a buffer, and if appropriate (e) a carr;er and/or the usual addi-tives, characterised in that component ~b) is a polyam;no-acid, preferably conta;n;ng bas;c groups, w;th a molecular we;ght ~number-average) of between 1,000 and 2,000,000.
F;nally, the ;nvent;on also relates to a process for the detection of esterolytic and/or proteolytic en-zymes ;n liquid samples, in particular body fluids, wh;ch is character;sed ;n that the sainple ;s brought ;nto con-tact w;th the agent accord;ng to the ;nvent;on and the colour react;on wh;ch occurs is determ;ned.
Both polyam;noac;ds wh;ch are bu;lt up from only Le A 22 899 a single aminoacid (homopolyaminoacids) and polyconden-sates of two or more different aminoacids in the form of copolyaminoacids with a randomised sequence of the con-stituent aminoacids or in the form of sequence polymers are suitable accelerators to be employed according to the invention. Sequence polymers are obtained in the poly-condensat;on of pept;des or their derivatives and have a defined, recurr;ng aminoacid sequence. For the accelerating action of the polyaminoacids, it is particu-larly advantageous for at least one of the aminoacids usedfor the polymerisation or copolymerisation to carry a basic group, for example an amino group or guanido group.
Preferably, the polyaminoacids to be used accord-;ng to the invent;on are bu;lt up from identical or diffe-rent monomers of the general formula H2N-CH-COO~ (I) in which R represents hydrogen or an optionally branched alkyl, cycloalkyl or aralkyl radical which has 1 to 15 C atoms, preferably 1 to 9 C atoms, and is opt;onally subst;tuted by 1 or 2, in particular 1, hydroxyl, mercapto, carboxyl, am;no or guanido groups.
The polyaminoac;ds to be used according to the invent;on as accelerators preferably contain S to 100 25 moleZ, in part;cular 10 to 100 moleX, of monomer units with a basic groùp. Particularly preferred monomer units of this type are aminoacids of the general formula (I) wh;ch contain an am;no or guanido group ;n the radical R.
Examples of such bas;c aminoacids are, in particular, arg;n;ne, lys;ne and orn;th;ne, ~h;ch can be e;ther ;n the racem;c form or ;n the L- or D-form. The polyam;noacids and the sequence polymers can furthermore also conta;n as un;ts bas;c am;noac;ds wh;ch do not occur in natural Le A 22 899 proteins. Examples which may be mentioned here are ~,~-diaminobutyric acid, ~X,~-diaminopropionic acid and the diaminopimelic acids.
The polyaminoacids to be used according to the invention as acceLerators of the enzymatic cleavage are known from the l;terature and are commercially available, or they can be prepared by processes which are known per se (see, for example, E. Katchalski and M. Sela in: Ad-vances of Protein Chemistry 13, 243-492 81958; and C.L.
Anfinsen, M.L. Anson, J.T. Edsall and K. Bailey (Editors) in Academic Press Inc. Publishers, New York).
The average molecular weights ~number-average) of the polyaminoacids should be between 1,000 and 2,ûOO,OOO, and polyaminoac;ds with average molecular weights of 5,000 to 500,000 are preferably used, those with average molecular weights between 10,000 and 300,000 being parti-cularly preferred.
The polyaminoacids are preferably employed ;n the homogeneous liqu;d test in concentrations of 0.0001X by weight to 1% by weight, and the concentrations are parti-cularly preferably in the range from 0.001 to 0.01X by weight. In the preparation, described below, of test de-vices, they are used in concentrations of 0.05X by weight to 10X by weight, preferably 1 to 5X by weight, based on the impregnating solution. The polyaminoacids to be used accord-ing to the invention accelerate the enzymatic cleavage of the substrates described in European Patents A-7,407, 8,428, 12,957, 14,929, 34,323 and 39,880 by the leucocyte enzymes, as well as the cleavage of the sub-strates which have already been descr;bed previous.y (G. Gomori, J. Histochem. Cytochem.
6 469 (1953); H. L8ffler, Klin. Wochenschr. 39, 1120 (1961);
L. Visser and E. 8lout, Fed.-Proc. 28, 407 (1969) and Bioch;m. Biophys. Acta 2~a, 257 (1972), and F. Sweetman and L. Ornste;n, J. H;stochem. Cytochem. 23, 327 (1974)).
The preferred chromogenic substrates in the agents according to the invention also include the compounds Le A 22 899 _ .
~25411~;
descr;bed in a parallel Application, of the general formula (II) 2 11 Ç-A-~c)x2 in which X1 and X2 are identical or different and denote nitrogen or sulphur, with the proviso that X1 and X2 do not simultaneously represent sulphur;
R1 represents hydrogen or an optionally branched alkyl group which has 1 to 6 carbon atoms and can optionally be substituted by halogen or hydroxyl;
R2 and R3 are identical or different and repre-sent hydrogen, C1-C6-alkyl groups, C1-C6-alkoxy groups, C1-C6-acyl groups, halogen, tri-fluoromethyl, nitro, S03H, cyano, C1-C8-acyl-amino groups, C1-C6-dialkylamino groups or C6-c10-arYl groups, uhich can in turn be further substituted by C1-C6-alkyl groups, C1-C6-alkoxy groups, halogen, cyano, nitro, trifluoro-methyl, S03H, C1-C6-acyl groups or C1-C6-dialkylamino groups, or R2 and R3 together form a fused-on aromatic ring, preferably a benzene ring, which can in turn be substituted by 1 or 2 radicals R2;
A denotes an aminoac;d radical or peptide radical and G represents hydrogen or, preferably, a nitrogen-protective group which is usual in peptide chemis-try or derived from such a group.
Ple~erred compounds of the general formu~a (II) are those in which X1 represents sulphur and X2 repre-sents nitrogen. Compounds of the formula (II) in which R1 represent~ hydrogen, and those in which R2 and R3, which are identical or different, represent hydrogen, Le A 22 899 C1-C2-alkyl, C1-Cz-alkoxy, halogen, C1-C4-dialkyl-amino groups or benzene radicals are furthermore preferred.
The ester radical in the compounds of the formu~a (II) is particularly preferably in the 5-position.
Other chromogenic substrates which are preferred according to the invent;on are the compounds likewise des-cribed in a parallel Appl;cation, of the general formula (III) R4 ~ O-A-G
R6 ~ i X ~ x4 ;n ~hich X3 and X4 represent N or CH, with the proviso that ;n each case either X3 or X4 represent N, R4, R5 and R6 are identical or different and represent hydrogen, C1-C6-alkyl groups, C1-C~-alkoxy groups, C1-C6-acyl groups, halogen, trifluoromethyl, nitro, S03H, cyano, C1-C8~
acylamino groups, C1-C6-dialkylamino groups or C6-C10-aryl groups, which can in turn be further substituted by C1-C6-alkyl groups, C1-C6-alkoxy groups, halogen, cyano, nitro, tri-fluoromethyl, S03H, C1-C6-acyl groups or C1-C6-dialkylamino groups, or R5 and Rf together form a fused-on aromatic ring, preferably a benzene ring, which can in turn be substituted by 1 or 2 radicals R4, and A and G have the meaning given above in the case of the formula (II).
In the compounds according to the general formula ~III), X3 preferably represents CH and X4 preferably represents nitrogen. R4, Rs and R6, which can be Le A Z2 899 _ 9 _ identical or different, preferably represent hydrogen, C1-C4-alkyl, C1-C4-alkoxy, acylamino (where the acid radical can be aliphatic or aromatic with 1 to 6 C
atoms), C1-C4-d;alkylam;no, n;~ro, cyano, halogen, or aryl, wh;ch is optionally substituted by c1-c4-alkYl, C1-C4-alkoxy or halogen.
Particularly preferably, R4, Rs and R6 are hydrogen, C1-C4-alkyl, phenyl or halogen, or Rs and R6 together form a fused-on benzene ring.
Suitable chromogenic substrates for the agents according to the invention are moreover also compounds of the general formula ~ (IV) wherein R4, R5, R6, A and G have the meaning given above in the case of formula (III).
In the general formulae (II)~ (III) and (IV), G-A - preferably represents a radical of the general formu-la n R ~ H21 ~ CH ~ C ~
in which R7 represents hydrogen or an optionally branched alkyl, cycloalkyl or aryl radical which has 1-15 C atoms, preferably 1-9 C atoms, and is optionally substituted by a hydroxyl, mercapto or carboxyl group, and R8 represents hydrogen or, preferably, -C0-alkyl, Le A 22 899 - 1 o --CO-aralkyl, -CO-aryl, -SOz-a~kyl or -S02-aryl, the alkyl rad;cals being straight-chain or branched with 1-9 C atoms, preferably 1-6 C
atoms, and the aryl radicals preferably represent-ing benzene rings, which are optionally substitu-ted by C1-C4-alkyl groups, C1-C4-alkoxY
groups or halogen.
G-A- particularly preferably represents a radical, provided w;th a customary nitrogen-protective group, of a naturally occurring aminoacid or of a peptide of 2 to 8 such aminoacids.
The aminoacid radicals can be in their L- or D-form or in their racemic form here. Particularly pre-ferred radica;s are those of glycine, alanine, valine, leucine, isoleucine, phenylalanine.and tyrosine, the L-form being particularly preferred in each case. Any free hydroxyl group present can be acylated, preferably acetylated.
A peptide rad;cal ;n the definition of A is to be understood as meaning, for example, di-, tr;-, tetra- and penta-pept;des, preferably d;- and tri-peptides, pre-ferred poss;ble aminoac;d components being the abovemen-tioned aminoacids.
The substrates of the general formulae (II), (III) and tIY) are obtained by reacting the corresponding phe-nols with aminoac;ds or peptides of the general formula G-A-OH
;n which G and A have the abovement;oned mean;ng, or suitable reactive derivatives thereof, by methods cus-tomary in peptide chemistry.
Examples of suitable reactive derivatives are the acid chlorides and the mixed anhydrides usually employed in peptide synthesis, for example with ethyl chloroformate or active esters, such as, for example, pentachlorophenyl esters or N-hydroxybenzotriazole esters.
Le A 22 899 lZS~
The agents according to the invention preferably contain, as colour-forming agents which react with the phènols (liberated during enzymatic cleavage), diazonium salts of the general formula R~ R'1 R; 3 ~ X~ ) (V) in which ~4 R'5 R'1 denotes a lower alkyl, a lower alkoxy, a lower alkylmercapto, a hydroxy, n;tro, cyano, tri-fluoromethyl, C1-C8-alkylsulphonam;do, arylsul-phonamido, C1-C8-alkylsulphone, arylsulphone, sulphonic ac;d or carboxyl;c acid, an N-morpho-lino, an N-thiomorpholino, an N-pyrrolidino, an optionally N'-alkylated N-piperazino or N-piperi-dino group, halogen or hydrogen, R'3 denotes a lower alkyl, a lower alkoxy, an aryloxy, a lower alkylmercapto, alkylamino or di-alkylamino, a hydroxyl, nitro, cyano, C1-C8-alkylsulphonam;do, arylsulphonam;do, C1-C8-alkyl-sulphone, arylsuLphone, sulphon;c ac;d or carboxy-l;c acid, an N-morphol;no, N-thiomorpholino or N-pyrrolidino, an optionally N'-alkylated N-p;pera-zino or N-piperidino or phenylamino group, a phe-nyl group which is optionally substituted by a lower alkyl or lower alkoxy radical, halogen or hydrogen, R'2, R'4 and R'5, which can be ;dent;cal or d;fferent, each denote a lower alkyl, a lower al-koxy, nitro, C1-C8-alkylsulphonam;do, arylsul-phonam;do, C1-C8-alkylsulphone, arylsulphone, sulphon;c acid or carboxylic acid or a lower alkyl mercapto group, halogen or hydrogen and Le A 22 899 411t~
X denotes a stabilising anion, it being possible for in eacl1 case 2 adjacent radicals R'1 to R'5 to be cyclised to form a benzene ring which ;s optionally subst;tuted by halogen, a C1-C6-alkyl, S a C1-C6-alkoxY or a n;tro, sulphon;c acid or carboxy-lic acid group, so that a diazonium salt of the naphtha-lene ser;es ;s formed.
Preferably, in the general formula (V) R'1 represents C1- to C4-alkyl, c1-c4-alkoxY~
hydroxyl, nitro, halogen or hydrogen, R'3 represents a C1- to C4-alkyl, C1-C4-alkoxy, aryloxy, C1-C4-alkylamino, C1-C4-d;alkylam;no, n;tro, C1-C4-alkylsulphonamido, arylsulphonam;do, C1-C4-alkylsulphone, aryl-sulphone, N-morpholino, N-pyrrolidino, phenylamino or sulphon;c ac;d group or hydrogen; and R'2, R'4 and R'5, which can be identical or different, represent C1- to C4-alkyl, C1- to C4-alkoxy, C1- to C4-alkylamino, C1 to C4-dialkylamino, nitro, C1- to C4-alkylsulphonamido, arylsulphonamido or sulphonic acid groups, halo-gen or hydrogen.
In each case 2 adjacent radicals R'1 to R'5 can here optionally be cyclised to give a benzene ring which ;s opt;onally substituted by halogen or a C1- to C4-alkyl or C1- to C4-alkoxy or a n;tro or sulphon;c acid group~
In the context of the formula (V), aryl in each case represents an aromatic radical wh;ch has 6 to 12 C
atoms, preferably 6 C atoms, and is optionally substituted by halogen or a C1-C4-alkyl or C1-C4-alkoxy group.
The diazon;um salts of the gene al formula tV) are known per se, or they can be synthesised by methods which are known per se (Houben-Weyl, Methods of Organic Chemis-5 try, volume X/3).The agents, according to the invention, for the Le A 22 899 1~25~1i6 detection of proteolytic enzymes and, in particular, leucocyte enzymes preferably contain a suitable buffer system. Possible systems for this purpose are, for example, phosphate, borate, carbonate/b;carbonate, carbo-nate, barb;turate, tr;s-~hydroxymethyl)-am;nomethane (=
tris), 2 am;no-2-methy~-propane-1,3-diol (= amed;ol) or am;noac;d buffer, the pH value and capacity as a rule be;ng chosen such that a pH value of 6-10, preferably of 7-9, ;s estab~;shed in the measurement solution or on the test strip.
In some cases, it may be advantageous also to con-jo;ntly use detergents ;n the agents according to the in-vent;on, ;n addition to the accelerating polyaminoacids.
These detergents on the one hand effect disinte-gration of the leucocytes present in the test solution andthus ~iberate the enzymes, and on the other hand act as solubilising agents for the substrates and the substances formed during cleavage, and, where relevant, intensify the colour. Poss;ble detergents are both cationic and anionic detergents, as ~ell as amphoteric and non-ion;c detergents.
Examples of these which may be mentioned are ben-zyl-d;methyl-tetradecyl-ammon;um chloride, Na dodecyl-sulphate, zephirol, polyv;nylpyrrolidone and hepar;noid, and if appropriate mixtures of two or more of the above-mentioned detergents can also be used. Since the poly-a0;noacids employed according to the ;nvention as activa-tors also have a detergent action, ho~ever, the add;t;on of extra detergents can in principle be dispensed with.
It is particularly advantageous to use the above detergents ;n the determ;nat;on of leucocytes by means of reagents f;xed to a solid phase (for example a-t~st strip), since a more homogeneous colour d;stribu~ion and a 00re ;ntensive colouration can thereby be achieved.
In the agents according to the ;nvent;on, the re-agents described above are preferab~y ;ncorporated ;n an Le A 22 899 1~5'11~t;
;nert carrier of the type which is known per se, part;cu-larly preferred carrier matrices being porous materials, such as, in particular, f;lter paper, and also membranes made of plastic, glass-fibre mats (U.S. Patent Specifica-tion 3,846,247), porous ceramic strios, synthetic non-woven fibres, spongy materials ~U.S. Patent Specification
3,552,928), felt, textiles, wood, cellulose or silica gel.
For this purpose, the carriers mentioned are im-pregnated with a solut;on of the reagents descr;bed above in a suitable solvent which can easily be removed, for example water, methanol, ethanol, acetone, dimethylforma-m;de or d;methylsulphox;de. Th;s ;s preferably effected ;n two separate steps: the material is first impregnated ~ith an aqueous solution containing the buffer and other water-soluble additives. It is then impregnated with a solution of the chromogenic enzyme substrates of the gene-ral formula (V) and activators. However, the impregnation can also be carr;ed out ;n another sequence, or w;th a d;fferent compos;t;on of the two ;mpregnating solutions.
Preferably, the ;mpregnat;ng solut;on or the fluid to be investigated contains the chromogenic substrate and the diazonium salt in each case in a concentrat;on of 10 4 to 10~1 mole/litre, in part;cular 10-3 to 10-2 mole/
litre, and the polyaminoacid ;n a concentration of O.O5X
Z5 by weight to 10% by weight, in part;cular 1% by we;ght to 5% by we;ght.
When f;lter paper is used as the matrix, the fin;shed test papers can be used as such or they can be stuck onto handles ;n a manner wh;ch ;s known per se or, 30 preferably, sealed between plast;cs and f;ne-mesh net-works, for examp~e accord;ng to D~-OS (German Published Spec;f;cat;on) 2,110,455.
To produce test strips coated ~ith film, prefer-ably all the reagents are introduced ;nto the solut;on or d;spers;on of a f;lm-form;ng substance, such as, for example, a polyv;nyl ester or polyamide, and are Le A 22 899 1;~5~116 homogeneously m;xed. A th;n layer of the m;xture ;s brushed onto a carrier made of plastic and dried. After drying, the film-coated test strips thus produced are cut and can be used as such or stuck onto handles in a manner which is known per se, or, for example, sealed between plastics and fine-mesh networks according to DE-OS (Ger-man Published Specification) 2,118,455.
A diagnostic agent according to the ;nvention for the detection of esterolytic and/or proteolytic enzymes, in part;cular leucocyte enzymes, can be prepared in the form of powder mixtures or reagent tablets by adding the usual pharmaceutical additives to the abovementioned con-stituents of the test agent and granulating the mixture.
Examples of additives of this type are carbohydrates, such as, for example, mono-, oLigo- or poly-saccharides, or sugar-alcohols, such as, for example, mannitol, sorbi-tol or xylitol, or other soluble inert compounds, such as polyethylene glycols or polyvinylpyrrol;done. The powder m;xtures or reagent tablets have, for example, a f;nal weight of about 50-200 mg, preferably 50-80 mg.
To prepare lyophilisates with a total weight of in each case about 5-20 mg, preferably about 10 mg, a so-lution which, in addit;on to all the reagents required for the test, contains the usual structure-forming agents, such as, for example, polyvinylpyrrolidone, and if approp-riate other fillers, such as, for example, mannitol, sor-bitol or xyl;tol, is freeze-dried.
A diagnost;c agent according to the invention in the form of a solution preferably contains all the re-agents required for the test. Possible solvents are waterand mixtures of water with a water-soluble organic sol-vent, such as, for example, methanol, ethanol, acetone or d;methylformamide. For storage reasons, it may be advan-tageous to divide the reagents required for the test into two or more solutions, which are only brought together during the actual investigation.
Le A 22 899 ` ~5~11tj The diagnostic agents thus prepared permit, after immersion in the body fluid to be investigated or after addition to the body fluid in question, rapid and simple detection of the presence of esterolytic and/or proteoly-tic enzymes, ;n particular leucocyte enzymes, via colourformation, which can be measured v;sually or photometri-cally, for example by reflectance photometry or in a cell.
Since the activity of the leucocyte enzymes per cell can be regarded as an essentially constant parameter, the leucocyte concentration of the body fluid investigated can be determined from the intensity of the colour formation.
Both intact and lysed leucocytes are thereby recorded with the diagnostic agent according to the invention, since the act;v;ty of the leucocyte enzymes ;s fully retained even after lysis of the leucocytes. Consequently, no lysis error occurs.
The following examples serve to illustrate the present invention. Unless indicated otherwise, the amounts given are to be understood as parts by weight or percentages by weight.
General procedure Depending on the substrate, 50-250 jul of N-methyl-pyrrolidone were added, as the solubilising agent, to 2.25 ml of the buffer in question and the volume of the solution was made up to 2.5 ml w;th buffer. 5 /ul of a solution of 5-100 mg of polyaminoacid in 1 ml of water or N-methylpyrrolidone and S ~l of a solution of 2 mg of Na dodecylsulphonate ~SDS) or 4 mg of the other detergents in 1 ml of water or N-methylpyrrolidone were then added.
After good thorough mixing, 5 yl of a 10 1 molar sub-strate solution in N-methylpyrrolidone or the stated sol-vent ~ere added and, after addition of the leucocyte sus-pension, the increase in extinction at the stated wave-length was monitored continuously. In a parallel batch without the addition of leucorytes, the increase in ex-tinction caused by spontaneous hydrolysis is determined.
Le A 22 899 ~ 17 -To determine the rate of reaction, the increase in extinction obtained in the enzyme reaction is reduced by the value found for spontaneous hydrolysis. Absolute values for cleavage of the substrate (moles/m;nute) can be calculated from the ext;nc~;on differences w;th the a;d of the molar extinction coefficients.
Example 1 Influence of the add;t;on of polyaminoacids on the rate of cleavage of tosyl-L-alanine indoxyl ester by leucocytes in 0.1 M tris-~hydroxymethyl)-aminomethane buffer, pH 8.8 (addition of 50 ~l of N-methylpyrrolidone per test batch). The cleavage rates were determined by continuous measurement of the increase in extinction at 360 nm.
Le A 22 899 Table 1 _ _ . . . ._.
Activator yg/~est relative rate of cleavage .
Decanol 12 5 Poly-L-Arg ( 4 0,0 0 0 ) .. 1. 7 5 Po l y--D--Lys ( 1 0 0,0 0 0 ) n 2.05 Poly - DL - Lys (37,000) n 3.10 Poly-L--Lys (14~000) n 1. 82 n (2~000) n 1.94 n (55,000) n 2.37 ~ (100~000) S0 2.10 n (1003000) 125 2.35 n (100~000) 250 2.50 n (260~000) 125 1. 82 n (520,000) 50 1.75 n (520J000) 125 2.20 n (520,000) 250 2.10 Poly- (L-Lys, L-Ala) 2:1 (32)000) 50 1.75 n (32~000) 125 1.90 n (32~000) 250 2.00 Poly-L-Orn (30,000) 125 1.80 : 2.90 The average molecular weights ~number-average) of the polyaminoacids are given in parentheses.
10 jug of SDS per test batch were also used in each case as the detergent.
ExampLe 2 Acceleration of the cleavage of 5-~N-tosyl-L-alanyloxy]-1,2-benzisothiozole by leucocytes on addition Le A 22 899 .L~
of polyaminoacids in 0.1 M tris-thydroxymethyl)-amino-methane buffer, pH 8.4, in the presence of 100 ~l of N-methylpyrrolidone per test batch. The rates of cleavage were determined by continuous measurement of the increase in extinction at 325 nm.
Table 2 Activator ~g/Test r5latLve r Decanol 125 1.0 Poly-L-A g (40~000) 25 ; 2 4 Poly-L-Lys (14~000) 125 7.8 Poly-L-Orn (30~000) 125 7.0 . .
Poly-L-Arg (40~000) 50 2 8 . ,. 125 3-3 n 500 3. 2 Poly-L-Lys (14~000) . 500 5.6 Poly-L-Orn (30~000) 500 4 . 3 Poly-L-Arg (40~000) 500 2 . ~5; 2. 4 Poly-L-Lys (1~000) 500 3. 4 Poly-L-Orn (30,000) 500 3. 0 Le A 22 899 1~25'~116 The average mo~ecular we;ghts (number-average) of the polymers are given in parentheses.
SDS or, in the last three experiments of Table 2, zephirol (as an approximately 50% strength aqueous solu-tion) were added as the detergent, in each case in anamount of 10 lug per test batch.
Example 3 Acceleration of the cleavage of tosyl-L-alanine 3-hydroxy-5-phenylpyrrole ester by leucocytes on addition of polyaminoacids in 0.1 M tris-~hydroxymethyl)-amino-methane buffer, pH 8.8, in the presence of 250 ~l of N-methylpyrrolidone per test batch. The rates of cleevage were determined by continuous measurement of the increase 1n extinction at 330 nm.
Table 3 Activator ~g/Test relative rate of cleavase Decanol 125 1.0 Poly-L-Arq(40,000) 125 4 64 Poly-DL-Lys(37,000) 125 4.3 Poly-D-Lys(100,000) 125 4.5 Poly-L-Lys(14~000) 20 3.7 125 4.8; ~.9 .
~ (260,000) 125 4.55 Poly-(L-Lys2,L-Ala) (32~000) 125 4.65 Poly-L-Orn (30,000) 20 3.7 125 5.75; 6.9 . .
The average molecular we;ghts (number-average) of the polyam;noac;ds are given in parentheses.
Le A 22 899 10 lug of SDS per test batch was added in each case as the detergent.
F.xample 4 Acceleration of the enzymatic cleavage of tosyl-L-alanine 3-hydroxy-5-phenolpyrrole ester by leucocytes on addition of polyaminoacids in 0.1 M tr;s-(hydroxy-methyl)-aminomethane buffer, pH 8.4, ;n the presence of 250 ~l of N-methylpyrrolidone per test batch. The rates of cleavage were determined by continuous measurement of the increase in extinction at 325 nm.
Table 4 Activator YglTest Detersent ~g/Test relative rate f cleavaae __ . .
Decanol 125 SDS 10 Poly-L-Arg (40~000) 125 _ _ 4.8 Poly-DL-Lys(37,000) 125 _ _ 4.8 Poly-L-Lys (14,000) 125 _ 3.7 ~Poly-L-Arg (40,000) 50 SDS ~ 10 6.0 _ 125 SDS 10 6.65 Poly-L-Arg (40,000) 125 BDTA 20 4.05 ~ 125heparinoi ¦20 5.1;
Poly-DL-Lys(37,000) 1 125 7 20 6.5;
Poly-L-Arg (4~000) ! 50zephirol 20 6.35 SDS = sodium dodecyl-~ulphate ~DTA = benzyl-dimethyl-tetradecyl-ammonium chloride Zephirol ~as used as an approximately 50X strength solut;on.
Le A 22 899 .
~ 22 -The average molecular weights (number-average) of the polymers are given in parentheses.
General operating instruct;ons for the pr_paration of the N-tosyl-L-alanyl esters:
The esters were in each case prepared by react;ng N-tosyL-L-alanyl chloride with the phenols in absolute methyl ethyl ketone or absolute toluene in the presence of powdered potassium carbonate. After stirring at about 55C for 6 to 12 hours, between 40 and 70X of the phenol had reacted. The molar ratio of phenol:K2C03 acid chlo-ride was usually 1:1.5:1.5. The pH value was about 7 throug~out the entire reaction time. For working up, the potassium carbonate was filtered off at 50C and the sol-vent was then distilled off in vacuo. The product was purified via column chromatography with silica gel-eluant (petroleum ether:acetone = about 9:1) and subsequent re-crystallisation.
p-Tosyl-L-alanine Literature: E. Fischer and W. Lipschitz, B. 48, 36Z
~1915~.
83.7 9 ~0.93 mole) of L-alanine are dissolved in 465 ml of approximately 2 N sodium hydroxide solution.
186 9 ~0.976 mole) of p-toluenesulphonyl chloride are added to the soCution in portions at 70-72C in the course of 20 minutes. During the addition of the sulphonyl chlo-ride, the reaction mixture is kept at pH 10 with approxi-mately 2 N sodium hydroxide solution by means of an auto-matic titrator; 560 ml of 2 N sodium hydroxide solution are consumed here. When the pH of the reaction mixture no longer changes, the mixture is cooled to 15-5C and brought to pH 3 with 37X strength hydrochloric acid.
The product which has separated out is filtered off with suction and the moist filter cake is recrystallised from 2,350 ml of water.
Yield: 185.5 9 (82X of theory) of L-p-tosylalanine of melting point 132-135C.
Le A 22 899 p-Tosyl-L-alanyl chloride 158.1 9 (0.65 mole) of p-tosyL-L-alanine are stirred in 350 ml of thionyl chloride at 40C, until a clear solution has formed. The excess thionyl chloride is then distilled off under a waterpump vacuum. The resi-due in the flask is taken up in 300 ml of distilled toluene. A clear, slightly yellowish solution is ob-tained, which is poured into 900 ml of stirred ligroin The ac;d chloride precipitates. The following day, it ;s filtered off with suction, washed with light gasolene and dried in a vacuum des;ccator over calcium chloride/potas-sium hydroxide.
Yield: 155 9 ~91X of theory) of almost colourless cry-stals of melting point 81-83C.
Le A 22 899
For this purpose, the carriers mentioned are im-pregnated with a solut;on of the reagents descr;bed above in a suitable solvent which can easily be removed, for example water, methanol, ethanol, acetone, dimethylforma-m;de or d;methylsulphox;de. Th;s ;s preferably effected ;n two separate steps: the material is first impregnated ~ith an aqueous solution containing the buffer and other water-soluble additives. It is then impregnated with a solution of the chromogenic enzyme substrates of the gene-ral formula (V) and activators. However, the impregnation can also be carr;ed out ;n another sequence, or w;th a d;fferent compos;t;on of the two ;mpregnating solutions.
Preferably, the ;mpregnat;ng solut;on or the fluid to be investigated contains the chromogenic substrate and the diazonium salt in each case in a concentrat;on of 10 4 to 10~1 mole/litre, in part;cular 10-3 to 10-2 mole/
litre, and the polyaminoacid ;n a concentration of O.O5X
Z5 by weight to 10% by weight, in part;cular 1% by we;ght to 5% by we;ght.
When f;lter paper is used as the matrix, the fin;shed test papers can be used as such or they can be stuck onto handles ;n a manner wh;ch ;s known per se or, 30 preferably, sealed between plast;cs and f;ne-mesh net-works, for examp~e accord;ng to D~-OS (German Published Spec;f;cat;on) 2,110,455.
To produce test strips coated ~ith film, prefer-ably all the reagents are introduced ;nto the solut;on or d;spers;on of a f;lm-form;ng substance, such as, for example, a polyv;nyl ester or polyamide, and are Le A 22 899 1;~5~116 homogeneously m;xed. A th;n layer of the m;xture ;s brushed onto a carrier made of plastic and dried. After drying, the film-coated test strips thus produced are cut and can be used as such or stuck onto handles in a manner which is known per se, or, for example, sealed between plastics and fine-mesh networks according to DE-OS (Ger-man Published Specification) 2,118,455.
A diagnostic agent according to the ;nvention for the detection of esterolytic and/or proteolytic enzymes, in part;cular leucocyte enzymes, can be prepared in the form of powder mixtures or reagent tablets by adding the usual pharmaceutical additives to the abovementioned con-stituents of the test agent and granulating the mixture.
Examples of additives of this type are carbohydrates, such as, for example, mono-, oLigo- or poly-saccharides, or sugar-alcohols, such as, for example, mannitol, sorbi-tol or xylitol, or other soluble inert compounds, such as polyethylene glycols or polyvinylpyrrol;done. The powder m;xtures or reagent tablets have, for example, a f;nal weight of about 50-200 mg, preferably 50-80 mg.
To prepare lyophilisates with a total weight of in each case about 5-20 mg, preferably about 10 mg, a so-lution which, in addit;on to all the reagents required for the test, contains the usual structure-forming agents, such as, for example, polyvinylpyrrolidone, and if approp-riate other fillers, such as, for example, mannitol, sor-bitol or xyl;tol, is freeze-dried.
A diagnost;c agent according to the invention in the form of a solution preferably contains all the re-agents required for the test. Possible solvents are waterand mixtures of water with a water-soluble organic sol-vent, such as, for example, methanol, ethanol, acetone or d;methylformamide. For storage reasons, it may be advan-tageous to divide the reagents required for the test into two or more solutions, which are only brought together during the actual investigation.
Le A 22 899 ` ~5~11tj The diagnostic agents thus prepared permit, after immersion in the body fluid to be investigated or after addition to the body fluid in question, rapid and simple detection of the presence of esterolytic and/or proteoly-tic enzymes, ;n particular leucocyte enzymes, via colourformation, which can be measured v;sually or photometri-cally, for example by reflectance photometry or in a cell.
Since the activity of the leucocyte enzymes per cell can be regarded as an essentially constant parameter, the leucocyte concentration of the body fluid investigated can be determined from the intensity of the colour formation.
Both intact and lysed leucocytes are thereby recorded with the diagnostic agent according to the invention, since the act;v;ty of the leucocyte enzymes ;s fully retained even after lysis of the leucocytes. Consequently, no lysis error occurs.
The following examples serve to illustrate the present invention. Unless indicated otherwise, the amounts given are to be understood as parts by weight or percentages by weight.
General procedure Depending on the substrate, 50-250 jul of N-methyl-pyrrolidone were added, as the solubilising agent, to 2.25 ml of the buffer in question and the volume of the solution was made up to 2.5 ml w;th buffer. 5 /ul of a solution of 5-100 mg of polyaminoacid in 1 ml of water or N-methylpyrrolidone and S ~l of a solution of 2 mg of Na dodecylsulphonate ~SDS) or 4 mg of the other detergents in 1 ml of water or N-methylpyrrolidone were then added.
After good thorough mixing, 5 yl of a 10 1 molar sub-strate solution in N-methylpyrrolidone or the stated sol-vent ~ere added and, after addition of the leucocyte sus-pension, the increase in extinction at the stated wave-length was monitored continuously. In a parallel batch without the addition of leucorytes, the increase in ex-tinction caused by spontaneous hydrolysis is determined.
Le A 22 899 ~ 17 -To determine the rate of reaction, the increase in extinction obtained in the enzyme reaction is reduced by the value found for spontaneous hydrolysis. Absolute values for cleavage of the substrate (moles/m;nute) can be calculated from the ext;nc~;on differences w;th the a;d of the molar extinction coefficients.
Example 1 Influence of the add;t;on of polyaminoacids on the rate of cleavage of tosyl-L-alanine indoxyl ester by leucocytes in 0.1 M tris-~hydroxymethyl)-aminomethane buffer, pH 8.8 (addition of 50 ~l of N-methylpyrrolidone per test batch). The cleavage rates were determined by continuous measurement of the increase in extinction at 360 nm.
Le A 22 899 Table 1 _ _ . . . ._.
Activator yg/~est relative rate of cleavage .
Decanol 12 5 Poly-L-Arg ( 4 0,0 0 0 ) .. 1. 7 5 Po l y--D--Lys ( 1 0 0,0 0 0 ) n 2.05 Poly - DL - Lys (37,000) n 3.10 Poly-L--Lys (14~000) n 1. 82 n (2~000) n 1.94 n (55,000) n 2.37 ~ (100~000) S0 2.10 n (1003000) 125 2.35 n (100~000) 250 2.50 n (260~000) 125 1. 82 n (520,000) 50 1.75 n (520J000) 125 2.20 n (520,000) 250 2.10 Poly- (L-Lys, L-Ala) 2:1 (32)000) 50 1.75 n (32~000) 125 1.90 n (32~000) 250 2.00 Poly-L-Orn (30,000) 125 1.80 : 2.90 The average molecular weights ~number-average) of the polyaminoacids are given in parentheses.
10 jug of SDS per test batch were also used in each case as the detergent.
ExampLe 2 Acceleration of the cleavage of 5-~N-tosyl-L-alanyloxy]-1,2-benzisothiozole by leucocytes on addition Le A 22 899 .L~
of polyaminoacids in 0.1 M tris-thydroxymethyl)-amino-methane buffer, pH 8.4, in the presence of 100 ~l of N-methylpyrrolidone per test batch. The rates of cleavage were determined by continuous measurement of the increase in extinction at 325 nm.
Table 2 Activator ~g/Test r5latLve r Decanol 125 1.0 Poly-L-A g (40~000) 25 ; 2 4 Poly-L-Lys (14~000) 125 7.8 Poly-L-Orn (30~000) 125 7.0 . .
Poly-L-Arg (40~000) 50 2 8 . ,. 125 3-3 n 500 3. 2 Poly-L-Lys (14~000) . 500 5.6 Poly-L-Orn (30~000) 500 4 . 3 Poly-L-Arg (40~000) 500 2 . ~5; 2. 4 Poly-L-Lys (1~000) 500 3. 4 Poly-L-Orn (30,000) 500 3. 0 Le A 22 899 1~25'~116 The average mo~ecular we;ghts (number-average) of the polymers are given in parentheses.
SDS or, in the last three experiments of Table 2, zephirol (as an approximately 50% strength aqueous solu-tion) were added as the detergent, in each case in anamount of 10 lug per test batch.
Example 3 Acceleration of the cleavage of tosyl-L-alanine 3-hydroxy-5-phenylpyrrole ester by leucocytes on addition of polyaminoacids in 0.1 M tris-~hydroxymethyl)-amino-methane buffer, pH 8.8, in the presence of 250 ~l of N-methylpyrrolidone per test batch. The rates of cleevage were determined by continuous measurement of the increase 1n extinction at 330 nm.
Table 3 Activator ~g/Test relative rate of cleavase Decanol 125 1.0 Poly-L-Arq(40,000) 125 4 64 Poly-DL-Lys(37,000) 125 4.3 Poly-D-Lys(100,000) 125 4.5 Poly-L-Lys(14~000) 20 3.7 125 4.8; ~.9 .
~ (260,000) 125 4.55 Poly-(L-Lys2,L-Ala) (32~000) 125 4.65 Poly-L-Orn (30,000) 20 3.7 125 5.75; 6.9 . .
The average molecular we;ghts (number-average) of the polyam;noac;ds are given in parentheses.
Le A 22 899 10 lug of SDS per test batch was added in each case as the detergent.
F.xample 4 Acceleration of the enzymatic cleavage of tosyl-L-alanine 3-hydroxy-5-phenolpyrrole ester by leucocytes on addition of polyaminoacids in 0.1 M tr;s-(hydroxy-methyl)-aminomethane buffer, pH 8.4, ;n the presence of 250 ~l of N-methylpyrrolidone per test batch. The rates of cleavage were determined by continuous measurement of the increase in extinction at 325 nm.
Table 4 Activator YglTest Detersent ~g/Test relative rate f cleavaae __ . .
Decanol 125 SDS 10 Poly-L-Arg (40~000) 125 _ _ 4.8 Poly-DL-Lys(37,000) 125 _ _ 4.8 Poly-L-Lys (14,000) 125 _ 3.7 ~Poly-L-Arg (40,000) 50 SDS ~ 10 6.0 _ 125 SDS 10 6.65 Poly-L-Arg (40,000) 125 BDTA 20 4.05 ~ 125heparinoi ¦20 5.1;
Poly-DL-Lys(37,000) 1 125 7 20 6.5;
Poly-L-Arg (4~000) ! 50zephirol 20 6.35 SDS = sodium dodecyl-~ulphate ~DTA = benzyl-dimethyl-tetradecyl-ammonium chloride Zephirol ~as used as an approximately 50X strength solut;on.
Le A 22 899 .
~ 22 -The average molecular weights (number-average) of the polymers are given in parentheses.
General operating instruct;ons for the pr_paration of the N-tosyl-L-alanyl esters:
The esters were in each case prepared by react;ng N-tosyL-L-alanyl chloride with the phenols in absolute methyl ethyl ketone or absolute toluene in the presence of powdered potassium carbonate. After stirring at about 55C for 6 to 12 hours, between 40 and 70X of the phenol had reacted. The molar ratio of phenol:K2C03 acid chlo-ride was usually 1:1.5:1.5. The pH value was about 7 throug~out the entire reaction time. For working up, the potassium carbonate was filtered off at 50C and the sol-vent was then distilled off in vacuo. The product was purified via column chromatography with silica gel-eluant (petroleum ether:acetone = about 9:1) and subsequent re-crystallisation.
p-Tosyl-L-alanine Literature: E. Fischer and W. Lipschitz, B. 48, 36Z
~1915~.
83.7 9 ~0.93 mole) of L-alanine are dissolved in 465 ml of approximately 2 N sodium hydroxide solution.
186 9 ~0.976 mole) of p-toluenesulphonyl chloride are added to the soCution in portions at 70-72C in the course of 20 minutes. During the addition of the sulphonyl chlo-ride, the reaction mixture is kept at pH 10 with approxi-mately 2 N sodium hydroxide solution by means of an auto-matic titrator; 560 ml of 2 N sodium hydroxide solution are consumed here. When the pH of the reaction mixture no longer changes, the mixture is cooled to 15-5C and brought to pH 3 with 37X strength hydrochloric acid.
The product which has separated out is filtered off with suction and the moist filter cake is recrystallised from 2,350 ml of water.
Yield: 185.5 9 (82X of theory) of L-p-tosylalanine of melting point 132-135C.
Le A 22 899 p-Tosyl-L-alanyl chloride 158.1 9 (0.65 mole) of p-tosyL-L-alanine are stirred in 350 ml of thionyl chloride at 40C, until a clear solution has formed. The excess thionyl chloride is then distilled off under a waterpump vacuum. The resi-due in the flask is taken up in 300 ml of distilled toluene. A clear, slightly yellowish solution is ob-tained, which is poured into 900 ml of stirred ligroin The ac;d chloride precipitates. The following day, it ;s filtered off with suction, washed with light gasolene and dried in a vacuum des;ccator over calcium chloride/potas-sium hydroxide.
Yield: 155 9 ~91X of theory) of almost colourless cry-stals of melting point 81-83C.
Le A 22 899
Claims (88)
1. Agent for the detection of esterolytic and/or pro-teolytic enzymes, containing a) an amino acid ester or peptide ester of a phenol, as the chromogenic enzyme substrate, and b) a substance which accelerates the enzymatic clea-vage of the amino acid ester or peptide ester bond of component (a), characterized in that it con-tains a polyamino acid with a molecular weight of between 1,000 and 2,000,000 as the accelerating substance.
2. Agent according to claim 1, characterized in that the molecular weight of the polyamino acid is between 5,000 and 500,000.
3. Agent according to claim 1, characterized in that the polyamino acid is built up from only one amino acid.
4. Agent according to claim 2, characterized in that the polyamino acid is built up from only one amino acid.
5. Agent according to claim 1, characterized in that the polyamino acid is built up from two or more different am-ino acids in random sequence.
6. Agent according to claim 2, characterized in that the polyamino acid is built up from two or more different am-ino acids in random sequence.
7. Agent according to claim 1, characterized in that the polyamino acid is built up from two or more different am-ino acids with a recurring amino acid sequence.
8. Agent according to claim 2, characterized in that the polyamino acid is built up from two or more different amino acids with a recurring amino acid sequence.
9. Agent according to claim 1, characterized in that the polyamino acid is built up from amino acids of the general formula in which R represents hydrogen or an optionally branched alkyl, cycloalkyl or aralkyl radical which has 1 to 15 C atoms and is optionally substituted by 1 or 2 hydroxyl, mercapto, carboxyl, amino or guanido groups.
10. Agent accordingly to claim 9, in which R represents an optionally branched alkyl, cycloalkyl or aralkyl radical which has 1 to 9 C atoms.
11. Agent according to claim 2, characterized in that the polyamino acid is built up from amino acids of the general formula in which R represents hydrogen or an optionally branched alkyl, cycloalkyl or aralkyl radical which has 1 to 15 C atoms and is optionally substituted by 1 or 2 hydroxyl, mercapto, carboxyl, amino or guanido groups.
12. Agent according to claim 11, in which R represents an optionally branched alkyl, cycloalkyl or aralkyl radical which has 1 to 9 C atoms.
13. Agent according to claim 1, characterized in that 5 to 100 mole % of the amino acid units of the polyamino acid carry a basic group.
14. Agent according to claim 13, in which 10 to 100 mole %
of the amino acid units of the polyamino acid carry a basic group.
of the amino acid units of the polyamino acid carry a basic group.
15. Agent according to claim 2, characterized in that 5 to 100 mole % of the amino acid units of the polyamino acid carry a basic group.
16. Agent according to claim 15, in which 10 to 100 % of the amino acid units of the polyamino acid carry a basic group.
17. Agent according to claim 3, characterized in that S to 100 mole % of the amino acid units of the polyamino acid carry a basic group.
18. Agent according to claim 17, in which 10 to 100 mole %
of the amino acid units of the polyamino acid carry on a basic group.
of the amino acid units of the polyamino acid carry on a basic group.
19. Agent according to claim 4, characterized in that 5 to 100 mole % of the amino acid units of the polyamino acid carry a basic group.
20. Agent according to claim 19, in which 10 to 100 mole %
of the amino acid units of the polyamino acid carry a basic group.
of the amino acid units of the polyamino acid carry a basic group.
21. Agent according to claim 5, characterized in that 5 to 100 mole % of the amino acid units of the polyamino acid carry a basic group.
22. Agent according to claim 21, in which 10 to 100 mole %
of the amino acid units of the polyamino acid carry a basic group.
of the amino acid units of the polyamino acid carry a basic group.
23. Agent according to claim 6, characterized in that 5 to 100 mole % of the amino acid units of the polyamino acid carry a basic group.
24. Agent according to claim 23, in which 10 to 100 mole %, of the amino acid units of the polyamino acid carry a basic group.
25. Agent according to claim 7, characterized in that 5 to 100 mole % of the amino acid units of the polyamino acid carry a basic group.
26. Agent according to claim 25, in which 10 to 100 mole %
of the amino acid units of the polyamino acid carry a basic group.
of the amino acid units of the polyamino acid carry a basic group.
27. Agent according to claim 8, characterized in that 5 to 100 mole % of the amino acid units of the polyamino acid carry a basic group.
28. Agent according to claim 27, in which 10 to 100 mole %
of the amino acid units of the polyamino acid carry a basic group.
of the amino acid units of the polyamino acid carry a basic group.
29. Agent according to claim 9, characterized in that 5 to 100 mole % of the amino acid units of the polyamino acid carry a basic group.
30. Agent according to claim 29, in which 10 to 100 mole %
of the amino acid units of the polyamino acid carry a basic group.
of the amino acid units of the polyamino acid carry a basic group.
31. Agent according to claim 10, characterized in that 5 to 100 mole % of the amino acid units of the polyamino acid carry a basic group.
32. Agent according to claim 31, in which 10 to 100 mole %
of the amino acid units of the polyamino acid carry a basic group.
of the amino acid units of the polyamino acid carry a basic group.
33. Agent according to claim 13, in which the amino acid units of the polyamino acid which carry a basic group consist of amino acid units carrying amino groups or amino acid units carrying guanido groups or mixtures thereof.
34. Agent according to claim 14, in which the amino acid units of the polyamino acid which carry a basic group consist of amino acid units carrying amino groups or amino acid units carrying guanido groups or mixtures thereof.
35. Agent according to claim 15, in which the amino acid units of the polyamino acid which carry a basic group consist of amino acid units carrying amino groups or amino acid units carrying guanido groups or mixtures thereof.
36. Agent according to claim 16, in which the amino acid units of the polyamino acid which carry a basic group consist of amino acid units carrying amino groups or amino acid units carrying guanido groups or mixtures thereof.
37. Agent according to claim 17, in which the amino acid units of the polyamino acid which carry a basic group consist of amino acid units carrying amino groups or amino acid units carrying guanido groups or mixtures thereof.
38. Agent according to claim 18, in which the amino acid units of the polyamino acid which carry a basic group consist of amino acid units carrying amino groups or amino acid units carrying guanido groups or mixtures thereof.
39. Agent according to claim 19, in which the amino acid units of the polyamino acid which carry a basic group consist of amino acid units carrying amino groups or amino acid units carrying guanido groups or mixtures thereof.
40. Agent according to claim 20, in which the amino acid units of the polyamino acid which carry a basic group consist of amino acid units carrying amino groups or amino acid units carrying guanido groups or mixtures thereof.
41. Agent according to claim 21, in which the amino acid units of the polyamino acid which carry a basic group consist of amino acid units carrying amino groups or amino acid units carrying guanido groups or mixtures thereof.
42. Agent according to claim 22, in which the amino acid units of the polyamino acid which carry a basic group consist of amino acid units carrying amino groups or amino acid units carrying guanido groups or mixtures thereof.
43. Agent according to claim 23, in which the amino acid units of the polyamino acid which carry a basic group consist of amino acid units carrying amino groups or amino acid units carrying guanido groups or mixtures thereof.
44. Agent according to claim 24, in which the amino acid units of the polyamino acid which carry a basic group consist of amino acid units carrying amino groups or amino acid units carrying guanido groups or mixtures thereof.
45. Agent according to claim 25, in which the amino acid units of the polyamino acid which carry a basic group consist of amino acid units carrying amino groups or amino acid units carrying guanido groups or mixtures thereof.
46. Agent according to claim 26, in which the amino acid units of the polyamino acid which carry a basic group consist of amino acid units carrying amino groups or amino acid units carrying guanido groups or mixtures thereof.
47. Agent according to claim 27, in which the amino acid units of the polyamino acid which carry a basic group consist of amino acid units carrying amino groups or amino acid units carrying guanido groups or mixtures thereof.
48. Agent according to claim 28, in which the amino acid units of the polyamino acid which carry a basic group consist of amino acid units carrying amino groups or amino acid units carrying guanido groups or mixtures thereof.
49. Agent according to claim 29, in which the amino acid units of the polyamino acid which carry a basic group consist of amino acid units carrying amino groups or amino acid units carrying guanido groups or mixtures thereof.
50. Agent according to claim 30, in which the amino acid units of the polyamino acid which carry a basic group consist of amino acid units carrying amino groups or amino acid units carrying guanido groups or mixtures thereof.
51. Agent according to claim 31, in which the amino acid units of the polyamino acid which carry a basic group consist of amino acid units carrying amino groups or amino acid units carrying guanido groups or mixtures thereof.
52. Agent according to claim 32, in which the amino acid units of the polyamino acid which carry a basic group consist of amino acid units carrying amino groups or amino acid units carrying guanido groups or mixtures thereof.
53. Agent according to claim 1, characterized in that the reagents are incorporated in an inert carrier.
54. Agent according to claim 2, 3 or 4, characterized in that the reagents are incorporated in an inert carrier.
55. Agent according to claim 5, 6 or 7, characterized in that the reagents are incorporated in an inert carrier.
56. Agent according to claim 8, 9 or 10, characterized in that the reagents are incorporated in an inert carrier.
57. Agent according to claim 11, 12 or 13, characterized in that the reagents are incorporated in an inert carrier.
58. Agent according to claim 14, 15 or 16, characterized in that the reagents are incorporated in an inert carrier.
59. Agent according to claim 17, 18 or 19, characterized in that the reagents are incorporated in an inert carrier.
60. Agent according to claim 20, 21 or 22, characterized in that the reagents are incorporated in an inert carrier.
61. Agent according to claim 23, 24 or 25, characterized in that the reagents are incorporated in an inert carrier.
62. Agent according to claim 26, 27 or 28, characterized in that the reagents are incorporated in an inert carrier.
63. Agent according to claim 29, 30 or 31, characterized in that the reagents are incorporated in an inert carrier.
64. Agent according to claim 32, 33 or 34, characterized in that the reagents are incorporated in an inert carrier.
65. Agent according to claim 35, 36 or 37, characterized in that the reagents are incorporated in an inert carrier.
66. Agent according to claim 38, 39 or 40, characterized in that the reagents are incorporated in an inert carrier.
67. Agent according to claim 41, 42 or 43, characterized in that the reagents are incorporated in an inert carrier.
68. Agent according to claim 44, 45 or 46, characterized in that the reagents are incorporated in an inert carrier.
69. Agent according to claim 47, 48 or 49, characterized in that the reagents are incorporated in an inert carrier.
70. Agent according to claim 50, 51 or 52, characterized in that the reagents are incorporated in an inert carrier.
71 . Agent according to claim 1, characterized in that the reagents are incorporated in an inert carrier in the form of a test strip.
72. Agent according to claim 2, 3 or 4, characterized in that the reagents are incorporated in an inert carrier in the form of a test strip.
73. Agent according to claim 5, 6 or 7 , characterized in that the reagents are incorporated in an inert carrier in the form of a test strip.
74. Agent according to claim 8, 9 or 10, characterized in that the reagents are incorporated in an inert carrier in the form of a test strip.
75 . Agent according to claim 11, 12 or 13, characterized in that the reagents are incorporated in an inert carrier in the form of a test strip.
76. Agent according to claim 14, 15 or 16, characterized in that the reagents are incorporated in an inert carrier in the form of a test strip.
77. Agent according to claim 17, 18 or 19, characterized in that the reagents are incorporated in an inert carrier in the form of a test strip.
78. Agent according to claim 20, 21 or 22, characterized in that the reagents are incorporated in an inert carrier in the form of a test strip.
79. Agent according to claim 23, 24 or 25, characterized in that the reagents are incorporated in an inert carrier in the form of a test strip.
80. Agent according to claim 26, 27 or 28, characterized in that the reagents are incorporated in an inert carrier in the form of a test strip.
81. Agent according to claim 29, 30 or 31, characterized in that the reagents are incorporated in an inert carrier in the form of a test strip.
82. Agent according to claim 32, 33 or 34, characterized in that the reagents are incorporated in an inert carrier in the form of a test strip.
83. Agent according to claim 35, 36 or 37, characterized in that the reagents are incorporated in an inert carrier in the form of a test strip.
84. Agent according to claim 38, 39 or 40, characterized in that the reagents are incorporated in an inert carrier in the form of a test strip.
85. Agent according to claim 41, 42 or 43, characterized in that the reagents are incorporated in an inert carrier in the form of a test strip.
86. Agent according to claim 44, 45 or 46, characterized in that the reagents are incorporated in an inert carrier in the form of a test strip.
87. Agent according to claim 47, 48 or 49, characterized in that the reagents are incorporated in an inert carrier in the form of a test strip.
88. Agent according to claim 50, 51, or 52, characterized in that the reagents are incorporated in an inert carrier in the form of a test strip.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DEL0022899 | 1984-04-06 | ||
| DELEA22899 | 1984-04-06 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1254116A true CA1254116A (en) | 1989-05-16 |
Family
ID=7262557
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000476408A Expired CA1254116A (en) | 1984-04-06 | 1985-03-13 | Analytical process and agents for the detection of esterolytic and/or proteolytic enzymes |
Country Status (1)
| Country | Link |
|---|---|
| CA (1) | CA1254116A (en) |
-
1985
- 1985-03-13 CA CA000476408A patent/CA1254116A/en not_active Expired
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