Diagnostic agents for the detection of leukocytes in body fluids

Abstract

This invention relates to diagnositc agents and methods for the detection of leukocytes in body fluids. In additional aspect, the invention provides a process for the production of such diagnostic agents.

Description

The detection of leukocytes in body fluids, especially in urine, occupies an important place in the diagnosis of diseases of the kidneys and of the urogenital tract.

Hitherto, this detection has been carried out by the microscopic counting of the leukocytes in non-centrifuged urine or in urinary sediment.

It is, of course, common to both methods that only intact leukocytes can be counted. On the other hand, it is known that the rate of leukocytes lysis is subject to enormous variations, depending upon the urinary medium: thus, for example, in strongly alkaline urines, the leukocytes half life can be as low 60 minutes. This results in too low leukocyte counts or, in the case of urine samples which have been standing for quite a long time, even in falsely negative findings.

Apart from lysis errors, the quantitative microscopic determination of leukocytes in non-centrifuged, homogenized urine in a counting chamber gives quite dependable values. However, in practice, this method is seldom used since it is laborious, tiring and time-consuming and requires the use of skilled personnel.

In medical practice, the overwhelming majority of leukocyte determinations in urine are carried out according to the so-called viewing field method in the urine sediment. For this purpose, the material to be investigated (sediment) must first be obtained by centrifuging. However, other components of the urine are thereby also enriched, for example, salts and epithelial cells, which can make the microscopic counting of the leukocytes considerably more difficult. Varying sediment content and inhomogeneities of the sediment, as well as, in some cases, differing microscopic enlargement or differing optical equipment of the microscope have the result that the here usual statement of the number of leukocytes per microscopic viewing field can involve errors of several hundred percent.

Therefore, recently attempts have been made to provide a diagnostic agent with which leukocytes can be detected in body fluids in a simple and complete manner, as quickly and completely as possible. For such a leukocyte test, enzymatic reactions could possibly be used since leukocytes possess a broad spectrum of enzymatic activity.

Thus, for example, in Federal Republic of Germany Patent Applications Nos. P 28 26 965.0, P 28 36 644.1 and P 28 54 987.3, diagnostic agents are described which comprise an absorbent carrier which is impregnated with an appropriate buffer substance, conventional adjuvants and a chromogen. As chromogens, there are used, according to Federal Republic of Germany Patent Application No. P 28 26 965.0, sulphonphthalein esters of the general formula: ##STR1## wherein R.sub.1 " is a carboxylic acid residue optionally substituted by halogen or a lower alkoxy radical or is an amino acid or peptide residue provided with a nitrogen protective group conventional in peptide chemistry, R.sub.2 " is a halogen atom or a lower alkyl radical and R.sub.3 " and R.sub.4 ", which can be the same or different, are hydrogen or halogen atoms; according to Federal Republic of Germany Patent Application No. P 28 36 644.1, azo dyestuff esters of the general formula:

A'--N.dbd.N--B'(OR).sub.n (B), wherein A' is a five- or six-membered, optionally benzo-annellated residue with one or two hetero atoms selected from nitrogen, sulphur and oxygen, which can be substituted one or more times by halogen, lower alkyl and/or lower alkoxy radicals or is a phenyl radical substituted one, two or three times by lower alkyl, lower alkoxy, nitro, sulphonate and/or acylamino radicals, B' is a benzene, naphthalene or quinoline radical optionally substituted once or twice by sulphonato, lower alkoxy and/or lower alkoxy-polyalkyleneoxy radicals, R is a carboxylic acid residue or an amino acid or peptide residue provided with a nitrogen protective group conventional in peptide chemistry and n is 1 or 2; and according to Federal Republic of Germany Patent Application No. P 28 54 987.3, indoxyl esters of the general formula: ##STR2## wherein R.sub.1 "', R.sub.2 "', R.sub.3 "' and R.sub.4 "', which can be the same or different, are hydrogen or halogen atoms, lower alkyl, lower alkoxy, aryl, aralkyl, aralkoxy, hydroxyl, carboxyl, carboxy lower alkoxy, aralkoxycarbonyl, aralkoxycarbonyl lower alkoxy, nitro or lower acylamino radicals or wherein two adjacent substituents represent a benzo-annellated residue optionally substituted by halogen, X is a sulphur atom or an imino group optionally substituted by a lower alkyl, aryl, aralkyl or acyl radical, A" is an amino acid or peptide residue and B" is a nitrogen protective group conventional in peptide chemistry or derived therefrom.

With the help of these diagnostic agents, the detection of esterolytic or proteolytic enzymes and especially of the esterases or proteases present in leukocytes can be carried out simply and quickly in body fluids especially in urine, via a color change.

It is an object of the present invention to find, if possible, a way of accelerating the detection reaction forming the basis of this enzymatic test.

Surprisingly, we have now found that the reaction times of these enzymatically carried out leukocyte tests can be considerably shortened when, in addition to the previously conventional adjuvants and chromogens, at least one activator is added.

Therefore, according to the present invention, there is provided a diagnostic agent for the detection of esterolytic and/or proteolytic enzymes comprising conventional adjuvants and a known esterase and/or protease substrate, characterized in that it additionally contains at least one activator.

The present invention is also concerned with the production of these diagnostic agents, as well as with the use thereof for the detection of leukocytes in body fluids and especially in urine.

The diagnostic agent according to the present invention preferably comprises an absorbent carrier, a film layer, a powder mixture, a lyophilizate, a solution or a reagent tablet which contains a known esterase and/or protease substrate, conventional additives and at least one activator.

Examples of activators which can be used according to the present invention include the following:

(a) pyridine derivatives of the general formula: ##STR3## wherein R.sub.1, R.sub.2, R.sub.3, R.sub.4 and R.sub.5, which can be the same or different, are hydrogen or halogen atoms, lower alkyl or lower alkoxy radicals, vinyl radicals which are substituted by an aryl radical optionally substituted one or more times by lower alkoxy, amino, alkylamino or dialkylamino, or by a heterocyclic radical and wherein two adjacent substituents can represent an indeno- or benzo-annellated residue, optionally substituted one or more times by halogen, hydroxyl, lower alkyl or lower alkoxy, which annellated residue can, in turn, carry a benzo- or pyrido-annellated residue optionally substituted by a lower alkyl radical and wherein R.sub.3 can additionally represent a vinyl-quinuclidyl-carbinol radical;

(b) imidazole derivatives of the general formula: ##STR4## wherein R.sub.1 ' is a hydrogen atom, a lower alkyl radical or an aryl radical optionally substituted by a hydroxyl or an acyl radical and R.sub.2 ' is a hydrogen atom, an aminoalkyl, N-acylaminoalkyl or a lower aliphatic, optionally unsaturated carboxylic acid residue or a lower aliphatic .alpha.-amino acid residue, optionally acylated on the nitrogen atom;

(c) alcohols of the general formula:

X--A--OH (III), wherein X is a hydrogen atom or a hydroxyl group and A is a hydrocarbon radical;

(d) metal complexes of the general formula:

D.sub.m [B(CN).sub.n (NO).sub.p ] (IV), wherein D is an alkali metal ion, B is a heavy metal ion, m is 2, 3, 4 or 5, n is 4, 5, 6, 7 or 8 and p is 0 or 1, the number m being given by the valency of the heavy metal ion and the number n.

Halogen in the definition of the substituents R.sub.1, R.sub.2, R.sub.3, R.sub.4 and R.sub.5 is to be understood to be fluorine, chlorine, bromine or iodine and preferably chlorine or bromine.

The lower alkyl and alkoxy radicals in the definition of the substituents R.sub.1, R.sub.2, R.sub.3, R.sub.4 and R.sub.5, as well as the lower alkyl radicals in the definition of the substituents R.sub.1 ' and R.sub.2 ' can be straight-chained or branched and contain up to 5 and preferably up to 3 carbon atoms, the methyl, ethyl, n-propyl, isopropyl, methoxy and ethoxy radicals being especially preferred.

The hydrocarbon radical A can be straight-chained or branched, saturated or unsaturated, cyclic or acyclic and contains up to 30 and preferably 5 to 22 carbon atoms in the case of acyclic compounds and 3 to 20 and preferably 6 to 17 carbon atoms in the case of cyclic compounds.

The acyl radicals in the definition of the substituents R.sub.1 ' and R.sub.2 ' are to be understood to be aliphatic carboxylic acid residues containing up to 5 and preferably up to 3 carbon atoms, the acetyl radical being especially preferred.

The aryl radicals in the definition of the substituents R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5 and R.sub.1 ' are preferably to be understood to be phenyl or naphthyl radicals, the phenyl radical being especially preferred as the substituent R.sub.1 '.

The heterocyclic radical in the definition of the substituents R.sub.1, R.sub.2, R.sub.3, R.sub.4 and R.sub.5 is to be understood to be a five- or six-membered radical containing up to 3 hereto atoms, the hetero atoms being nitrogen, sulphur and oxygen, the pyridyl, furyl and thienyl radicals being especially preferred.

The "lower aliphatic, optionally unsaturated carboxylic acid radical" and the "lower aliphatic .alpha.-amino acid residue optionally acylated on the nitrogen" R.sub.2 ' is to be understood to be a carboxylic acid residue containing up to 5 and preferably up to 3 carbon atoms or an .alpha.-amino derivative thereof, acetic acid, propionic acid, acrylic acid, L-alanine and N-acetyl-L-alanine being especially preferred.

In the activators of general formula (IV), the alkali metal ions D are preferably sodium or potassium ions and the heavy metal ion B is preferably an iron, nickel, chromium, manganese, cobalt, molybdenum or vanadium ion.

Activators which can be used according to the present invention include, for example, the following:

1. pyridine 2. 2-methylpyridine 3. 3-ethylpyridine 4. 2-bromopyridine 5. 3,5-dichloropyridine 6. 4-methoxypyridine 7. 2,6-dimethyl-4-ethoxypyridine 8. quinoline. 9. 2-methylquinoline 10. 8-methylquinoline 11. 7-isopropylquinoline 12. 2-chloroquinoline 13. 4-bromoquinoline 14. 3-methoxyquinoline 15. 6-ethoxyquinoline 16. 2-methyl-6-bromoquinoline 17. 2-methyl-4-methoxyquinoline 18. 5,7-dibromo-8-methoxyquinoline 19. isoquinoline 20. 1-methylisoquinoline 21. 3-propylisoquinoline 22. 7-methylisoquinoline 23. 1-chloroisoquinoline 24. 4-bromoisoquinoline 25. 7-methoxyisoquinoline 26. 1-methoxy-3-chloroisoquinoline 27. 1-chloro-4-methyl-5-methoxyisoquinoline 28. benzo-[b]-quinoline(=acridine) 29. benzo-[c]-quinoline(=phenanthridine) 30. 2-methylphenanthridine 31. 2-ethylphenanthridine 32. 2-propylphenanthridine 33. 2-methoxyphenanthridine 34. benzo-[f]-quinoline 35. 2-isopropyl-benzo-[f]-quinoline 36. 3-methyl-benzo-[f]-quinoline 37. 2,4-dimethylbenzo-[f]-quinoline 38. benzo-[g]-quinoline 39. 4-methylbenzo-[g]-quinoline 40. 2,4-dimethylbenzo-[g]-quinoline 41. benzo-[h]-quinoline 42. 1,7-phenanthroline 43. 2-methyl-1,7-phenanthroline 44. 2,8-dimethyl-1,7-phenanthroline 45. 4,7-phenanthroline 46. 3-methyl-4,7-phenanthroline 47. 3,8-dimethyl-4,7-phenanthroline 48. 1,10-phenanthroline 49. 2,9-dimethyl-1,10-phenanthroline 50. 4-azafluorene 51. quinine 52. quinidine 53. cinchonine 54. cinchonidine 55. cuprein 56. 2-[phenyl]-vinyl-pyridine-(2') 57. 2-[4"-methoxyphenyl]-vinyl-pyridine-(2') 58. 2-[4"-(N,N-dimethylamino)-phenyl]-vinyl-pyridine-(2') 59. bis-[2-(phenyl)-vinyl]-pyridine-(2',4') 60. 2-[naphthyl-(1")]-vinyl-pyridine-(2') 61. 2-[pyridyl-(2")]-vinyl-pyridine-(2') 62. 2-[pyridyl-(3")]-vinyl-pyridine-(2') 63. 2-[pyridyl-(4")]-vinyl-pyridine-(2') 64. 2-[furyl-(2")]-vinyl-pyridine-(2') 65. 2-[pyridyl-(3")]-vinyl-pyridine-(3') 66. 2-[pyridyl-(3")]-vinyl-pyridine-(4') 67. 2-[pyridyl-(4")]-vinyl-pyridine-(4') 68. 2-[thienyl-(2")]-vinyl-pyridine-(4') 69. imidazole 70. 1-ethylimidazole 71. 1-phenylimidazole 72. 1-(4'-hydroxyphenyl)-imidazole 73. 1-(4'-acetylphenyl)-imidazole 74. histamine 75. N-.alpha.-acetylhistamine 76. (imidazolyl-4)-acetic acid 77. .beta.-(imidazolyl-4)-propionic acid 78. .beta.-(imidazolyl-4)-acrylic acid 79. L-histidine 80. N-.beta.-acetyl-L-histidine 81. hexan-1-ol 82. heptan-1-ol 83. octan-1ol 84. nonan-1-ol 85. decan-1-ol 86. dodecan-1-ol 87. tetradecan-1-ol 88. pentadecan-1-ol 89. hexadecan-1-ol 90. heptadecan-1-ol 91. octadecan-1-ol 92. nonadecan-1-ol 93. eicosan-1-ol 94. docosan-1-ol 95. cyclohexanol 96. cyclohex-1-en-1-ol 97. cycloheptanol 98. cyclooctanol 99. cyclononanol 100. cyclodecanol 101. cyclododecanol 102. cycloheptadecanol 103. cycloheptadec-9-en-1ol 104. citronellol 105. geraniol 106. nerol 107. linalool 108. farnesol 109. nerolidol 110. cis-octadec-9-en-1-ol 111. phytol 112. phentane-1,5-diol 113. hexane-1,6-diol 114. heptane-1,7-diol 115. octane-1,8-diol 116. nonane-1,9-diol 117. decane-1,10-diol 118. dodecane-1,12-diol 119. tripotassiumhexacyanoferrate III 120. tetrapotassiumhexacyanoferrate II 121. dipotassiumtetracyanonickelate II 122. trisodiumoctacyanomolybdate V 123. disodiumpentacyanonitrosylferrate II 124. tripotassiumpentacyanonitrosylmanganate I 125. tripotassiumpentacyanonitrosylchromate I 126. tripotassiumpentacyanonitrosylcobaltate I 127. pentapotassiumpentacyanonitrosylvanadate I.

All the activators are either known compounds or can be prepared analogously to known compounds.

The compounds of general formulae (I), (II) and (IV) employed as activators according to the present invention can be used in concentrations of 10.sup.-4 to 1 mol/liter and preferably of 10.sup.-3 to 10.sup.-1 mol/liter of impregnation solution and the activators of general formula (III) can be used in the impregnation solution in a concentration of 0.5 to 10% (w/v) and preferably of 1 to 5% (w/v).

The diagnostic agents according to the present invention contain, in addition to the activators, the otherwise conventionally employed components, for example, known esterase or protease substrates, buffers and wetting agents and optionally complex-forming agents and oxidation agents. These are employed in the manner and in the concentrations described in Federal Republic of Germany Patent Applications Nos. P 28 26 965.0; P 28 36 644.1 and P 28 54 987.3.

Thus, the chromogens used as esterase or protease substrates are usually employed in concentrations of 10.sup.-4 to 1 mol/liter and preferably of 10.sup.-3 to 10.sup.-1 mol/liter of impregnation solution, coating mass or fluid to be investigated.

A further component of the diagnostic agent for the detection of esterolytic and/or proteolytic enzymes, especially of leukocyte proteases, is an appropriate buffer system. For this purpose, there can be used, for example, phosphate, borate, barbiturate, tris-(hydroxymethyl)-aminomethane(tris), 2-amino-2-methylpropane-1,3-diol(amediol) or amino acid buffers, the pH value and the capacity being so choosen that a pH value of 6 to 10 and preferably of 7 to 9 is obtained in the measurement solution or on the test strip.

A further component of a diagnostic agent for the detection of esterolytic and/or proteolytic enzymes and especially of leukocyte esterases or proteases can be a wetting agent. Non-ionic wetting agents are preferably used but amphoteric, cationic and anionic active wetting agents can also be employed, the concentration of the wetting agent being from 0.05 to 2% and preferably from 0.1 to 1%.

A further component of the diagnostic agent according to the present invention can be an appropriate complex former. It is preferable to use metal salts, for example salts of the elements iron, copper, chromium, cobalt, nickel, manganese and zinc. They can be employed in concentrations of 10.sup.-4 to 10.sup.-1 mol/liter and preferably of 10.sup.-3 to 10.sup.-2 mol/liter of impregnation solution.

Furthermore, in the production of the diagnostic agent according to the present invention, oxidation agents can additionally be used, for example potassium hexacyanoferrate III, potassium bromate, potassium chromate, phenazine-methosulphate or tetrazolium salts. These can be used in concentrations of 10.sup.-6 to 1 mol/liter and preferably of 10.sup.-3 to 10.sup.-1 mol/liter of impregnation solution, coating mass or fluid to be investigated.

For the production of the diagnostic agent according to the present invention, for example an absorbent carrier, preferably filter paper, cellulose or synthetic resin fiber fleece, is impregnated with solutions of the necessary reagents conventionally employed for the production of test strips (substrate, buffer, activators, optionally wetting agents, complex formers, oxidation agents, etc.) in readily volatile solvents, for example, water, methanol, ethanol or acetone. This impregnation is preferably carried out in two steps:

Impregnation is first carried out with an aqueous solution which contains the buffer and possibly water-soluble activators. Thereafter, impregnation is carried out with a solution which contains the esterase or protease substrate and possibly water-insoluble activators, as well as other water-insoluble additives. In special cases, the impregnation sequence can be reversed.

The finished test papers can be used as such or can, in known manner, be struck on to handles or preferably sealed between synthetic resins and fine-mesh fabrics in the manner described in Federal Republic of Germany Patent Specification No. 21 18 455.

For the production of film-coated test strips, all the reagents are introduced into a solution or dispersion of a film-forming substance, for example a polyvinyl ester or polyamide, and homogeneously mixed. The mixture is coated in a thin layer on to a synthetic resin carrier and dried. The film-coated test strips according to the present invention are, after drying, cut up and can be used as such or can be stuck, in known manner, on to handles or, for example, sealed between synthetic resins and fine-mesh fabrics in the manner described in Federal Republic of Germany Patent Specification No. 21 18 455.

The diagnostic agent according to the present invention for the detection of esterolytic and/or proteolytic enzymes in the form of powder mixtures or of reagent tablets can be produced by mixing the above-described components of the test with conventional galenical additives and granulated. Additives of this type include, for example, carbohydrates, such as mono-, oligo- and polysaccharides, and sugar alcohols, such as mannitol, sorbitol or xylitol, and other soluble, inert compounds, such as polyethylene glycol and polyvinylpyrrolidone. In general, the powder mixtures or reagent tablets have an end weight of about 50 to 200 mg. and preferably of 50 to 80 mg.

For the production of lyophilizates with a total weight of about 5 to 20 mg. and preferably of about 10 mg., a solution is freeze-dried which, in addition to the reagents needed for the test, contains conventional structure formers, for example polyvinylpyrrolidone, and possibly further filling materials, for example, mannitol, sorbitol or xylitol.

The diagnostic agent according to the present invention in the form of a solution preferably contains all the reagents needed for the test. As solvents, there can be used water or mixtures of water with a water-soluble organic solvent, for example, methanol, ethanol, acetone or dimethylformamide. For reasons of storage stability, it can be advantageous to divide the reagents needed for the test into two or more solutions which are only mixed at the time of carrying out the actual investigation.

The diagnostic agent produced in this manner enables leukocytes to be detected quickly and simply in body fluids to be investigated via a color formation or color change. In comparison with the diagnostic agents according to Federal Republic of Germany Patent Applications Nos. P 28 26 965.0, P 28 36 644.1 and P 28 54 987.3, when using the activators according to the present invention, considerably shortened reaction times are observed.

The following Examples are given for the purpose of illustrating the present invention:

EXAMPLE 1

Filter paper (for example Schleicher & Schull 23 SL) is successively impregnated with the following solutions and then dried at 60.degree. C. or at ambient temperature.

Solution 1

tris-(hydroxymethyl)-aminomethane hydrochloride buffer, 0.2 mol/liter, pH 9.0, in water

Solution 2

substrate solution 10.sup.-3 mol/liter in acetone

The activators according to the present invention are, depending upon their solubility, added to Solution 1 or Solution 2 so that, in the case of activators of general formulae (I), (II) and (IV), there result end concentrations of 10.sup.-2 mol/liter of impregnation solution and in the case of activators of general formula (III), end concentrations of 2% (w/v) of the impregnation solution.

In the following Table 1, there are summarised the experimental results obtained with the following esterase or protease substrates;

A: diacetyl-3',3"-dibromo-5',5"-dichlorophenolsulphonphthalein, B: diacetyl-4,5,6,7,3,',5',3",5"-octabromophenolsulphonphthalein, C: di-(N-benzylcarbonyl-L-alanyl)-3',5',3",5"-tetrabromophenolsulphonphthalein, D: di-(N-benzyloxycarbonyl-L-phenylalanyl)-3',5',3",5"-tetrabromophenolsulphonphthalein.

In Table 1, the reaction times are given which extend from the dipping of the test strips into a standard solution containing 5000 leukocytes/.mu.l. of isotonic sodium chloride solution up to the first distinct color reaction. As reference values, there are used the reaction times of the formulations in question but without the addition of activator. The color change in the case of the four compounds here investigated is from colorless to deep blue.

TABLE 1______________________________________ reaction times for substrate A B C Dactivators sec. sec. sec. sec.______________________________________comparative formulation 210 90 600 550without activator1. pyridine 110 70 410 4502. 3-ethylpyridine 95 65 440 4703. 4-methoxypyridine 110 65 470 4504. quinoline 75 50 320 3405. 2-methylquinoline 45 55 300 3506. 4-bromoquinoline 60 60 370 3307. 3-methoxyquinoline 50 50 310 3808. isoquinoline 85 55 350 3809. benzo-[b]-quinoline 90 65 280 330 (= acridine)10. benzo-[c]-quinoline 75 60 260 360 (= phenanthridine)11. 2-methylphenanthridine 100 65 250 37012. 2-ethylphenanthridine 80 70 300 36013. 2-propylphenanthridine 80 60 280 33014. 2-methoxyphenanthridine 85 60 260 35015. benzo-[f]-quinoline 90 40 310 31016. benzo-[h]-quinoline 90 55 290 34017. 1,7-phenanthroline 75 65 340 41018. 4,7-phenanthroline 80 70 270 38019. 4-azafluorene 120 50 240 29020. quinine 110 70 350 33021. quinidine 70 65 375 28022. cinchonine 60 50 340 30023. cinchonidine 65 55 390 32024. cuprein 70 60 400 30525. 2-[phenyl]-vinyl- 115 70 370 270 pyridine-(2')26. 2-[pyridyl-(3")]-vinyl- 145 65 430 250 pyridine-(2')27. 2-[pyridyl-(4")]-vinyl- 140 55 350 280 pyridine-(4')28. 2-[furyl-(2")]-vinyl- 150 70 410 250 pyridine-(2')29. imidazole 90 55 380 21030. histamine 110 60 440 29031. (imidazolyl-4)-acetic acid 150 75 450 35032. .beta.-(imidazolyl-4)- 170 70 490 380 propionic acid33. L-histidine 130 80 465 32034. octan-1-ol 150 75 500 46035. decan-1-ol 130 70 480 42036. tetradecan-1-ol 105 60 420 40037. cyclooctanol 125 80 530 48038. cyclododecanol 90 55 490 47039. citronellol 100 40 430 44040. farnesol 130 50 440 41041. phytol 135 55 430 45042. tripotassiumhexacyano- 160 70 550 480 ferrate II43. dipotassiumtetracyano- 160 80 530 450 nickelate II44. disodium pentacyano- 150 65 470 460 nitrosyl-ferrate II______________________________________

Similar experimental results are obtained when, instead of substrates A, B, C and D, there are used other sulphonphthalein esters of Federal Republic of Germany Patent Application No. P 28 26 965.0 and/or when, instead of a standard solution of 5000 leukocytes/.mu.l. of isotonic sodium chloride solution, leukocyte-containing urines are used.

EXAMPLE 2

Filter paper (for example Schleicher & Schull 23 SL) is successively impregnated with the following solutions and then dried at 60.degree. C. or at ambient temperature.

Solution 1

tris-(hydroxymethyl)-aminomethane hydrochloride buffer, 0.2 mol/liter, pH 7.0, in water (for substrates E, F and G), or tris-(hydroxymethyl)-aminomethane hydrochloride buffer, 0.2 mol/liter, pH 8.0, in water (for substrate H).

Solution 2

substrate solutions 10.sup.-3 mol/liter in acetone.

Activator additions and the carrying out of the experiments take place as in Example 1.

The following Table 2 summarizes the experimental results obtained with the following protease substrates:

E: thiazole-2-azo-4'-[1'-(N-benzyloxycarbonyl-L-alanyloxy)-5'-methoxynaphthalene] (color change of the test paper from pink to red) F: 6-methoxybenzothiazole-2-azo-2'-[1'-(N-benzyloxycarbonyl-L-alanyloxy)-naphthalene] (color change of the test paper from pink to red) G: 2,4-dinitrobenzeneazo-4'-[1'-(N-benzyloxycarbonyl-L-alanyloxy)-benzene] (color change of the test paper from yellow to red-violet) H: 2,5-dimethoxybenzeneazo-4'-[1'-(N-benzyloxycarbonyl-L-alanyloxy)-naphthalene] (color change of the test paper from bright orange to red)

The reaction times are given which extend from the dipping of the test paper into a standard solution of 5000 leukocytes/.mu.l. of isotonic sodium chloride solution up to the first distinct color reaction. As reference values, there serve the reaction times of the corresponding formulations without the addition of activators.

TABLE 2______________________________________ reaction times for substrate E F G Hactivators sec. sec. sec. sec.______________________________________comparative formulation without 60 180 160 70activator1. pyridine 50 150 130 552. 2-methylpyridine 45 140 135 503. 2-bromopyridine 50 150 125 454. 3,5-dichloropyridine 45 160 130 505. quinoline 50 110 95 456. 8-methylquinoline 45 130 110 407. 2-chloroquinoline 40 130 110 358. 6-ethoxyquinoline 35 150 120 409. 2-methyl-4-methoxy- 40 120 105 40 quinoline10. isoquinoline 40 130 110 4011. 1-methylisoquinoline 45 100 120 3512. 4-bromoisoquinoline 45 140 115 4013. 7-methoxyisoquinoline 50 130 130 4514. benzo-[b]-quinoline 30 150 60 45 (acridine)15. benzo-[c]-quinoline 35 120 80 40 (phenanthridine)16. 2-methylphenanthridine 40 140 90 4017. benzo-[f]-quinoline 50 130 70 3018. 2-isopropyl-benzo-[f]- 45 130 95 35 quinoline19. 3-methyl-benzo-[f]- 40 125 80 40 quinoline20. 2,4-dimethylbenzo-[f]- 40 110 90 40 quinoline21. benzo-[g]-quinoline 40 90 70 3522. 4-methylbenzo-[ g]-quinoline 40 100 80 4023. benzo-[h]-quinoline 35 70 50 3024. 1,7-phenanthroline 45 120 120 4025. 4,7-phenanthroline 40 105 105 4526. 1,10-phenanthroline 45 130 110 4527. quinine 40 120 60 3528. cinchonine 45 100 90 3029. cuprein 40 140 105 3030. 2-[phenyl]-vinyl-pyridine- 35 125 120 50 (2')31. bis-[2-(phenyl)-vinyl]- 30 150 140 60 pyridine-(2',4')32. 2-[furyl-(2")]-vinyl- 30 120 90 55 pyridine-(2')33. imidazole 45 140 120 4034. 1-ethylimidazole 40 130 130 4535. 1-phenylimidazole 35 120 120 4036. 1-(4'-hydroxyphenyl)- 45 140 135 50 imidazole37. histamine 50 130 140 5538. .beta.-(imidazolyl-4)-propionic 50 150 140 50 acid39. .beta.-(imidazolyl-4)-acrylic 50 160 135 55 acid40. L-histidine 45 150 125 5041. N-.alpha.-acetyl-L-histidine 45 140 130 4042. heptan-1-ol 30 130 100 4043. octan-1-ol 30 120 95 3544. decan-1-ol 20 90 80 3045. dodecan-1-ol 20 80 70 3046. hexadecan-1-ol 25 105 80 4047. eicosan-1-ol 30 120 110 4548. cyclohexanol 35 110 105 4049. cyclodecanol 25 95 85 3050. cyclododecanol 25 120 90 2551. cycloheptadecanol 30 130 120 3052. cycloheptadec-9-en-1-ol 30 110 100 3553. citronellol 25 100 70 2554. linalool 20 105 60 3055. farnesol 30 110 75 2556. cis-octadec-9-en-1-ol 25 90 85 4057. phytol 30 80 65 3058. pentane-1,5-diol 35 130 120 4559. heptane-1,7-diol 40 150 125 4060. decane-1,10-diol 30 135 140 5061. dodecane-1,12-diol 40 160 130 5062. tripotassium hexacyano- 45 160 135 55 ferrate III63. tetrapotassium hexacyano- 40 160 140 50 ferrate II64. disodium pentacyano- 40 140 130 40 nitrosyl-ferrate II65. tripotassium pentacyano- 50 145 130 50 nitrosyl-chromate I66. tripotassium pentacyano- 50 160 145 45 nitrosyl-cobaltate I______________________________________

Similar experimental results are achieved when, instead of substrates E, F, G and H, there are used other azo dyestuff esters of Federal Republic of Germany Patent Application No. P 28 36 644.1 and/or when instead of the standard solution of 5000 leukocytes/.mu.l. isotonic sodium chloride solution, leukocyte-containing urines are used.

EXAMPLE 3

Filter paper (for example Schleicher & Schull 23 SL) is successively impregnated with the following solutions and then dried at 60.degree. C. or ambient temperature.

Solution 1

sodium tetraborate hydrochloric acid buffer, 0.2 mol/liter, pH 8.0, in water

Solution 2

substrate solution 10.sup.-3 mol/liter in acetone

Activator additions and the carrying out of the experiments take place as in Example 1.

In the following Table 3, there are summarized the experimental results obtained with the following protease substrates:

I: 3-[N-(diphenylcarbamoyl)-L-alanyloxy]-indole J: 3-[N-(5',5'-dimethyl-3'-oxo-cyclohex-1'-enyl)-L-alanyloxy]-indole K: 3-[N-(benzyloxycarbonyl)-L-alanyloxy]-indole

The reaction times are given which extend from the dipping of the test strips into a standard solution of 5000 leukocytes/.mu.l. of isotonic sodium chloride solution up to the first distinct color reaction. The reaction times of the formulations in question without the additions of activator serve as reference values.

The test papers produced with the three substrates give, after dipping into leukocyte-containing solutions, colour changes from colorless to deep blue.

TABLE 3______________________________________ reaction times for substrate I J Kactivators sec. sec. sec.______________________________________comparative formulation without 300 180 60activator1. pyridine 220 140 452. 2-bromopyridine 205 130 403. 2,6-dimethyl-4-ethoxy- 160 110 40 pyridine4. quinoline 185 90 355. 2-methylquinoline 190 105 406. 7-isopropylquinoline 165 80 357. 3-methoxyquinoline 150 75 308. 2-methyl-6-bromoquinoline 170 80 409. 5,7-dibromo-8-methoxy- 195 90 40 quinoline10. isoquinoline 150 80 4011. 3-propylisoquinoline 170 75 3512. 7-methylisoquinoline 145 105 3013. 1-chloroisoquinoline 180 90 3514. 1-methoxy-3-chloro- 155 95 40 isoquinoline15. 1-chloro-4-methyl-5-methoxy- 190 80 35 isoquinoline16. benzo-[b]-quinoline 170 90 40 (acridine)17. benzo-[c]-quinoline 130 110 35 (phenanthridine)18. 2-ethylphenanthridine 160 100 3019. 2-methoxyphenanthridine 145 120 3520. benzo-[f]-quinoline 130 105 3521. benzo-[g]-quinoline 140 130 4522. 2,4-dimethyl-benzo-[g]- 135 110 40 quinoline23. benzo-[h]-quinoline 110 70 4024. 1,7-phenanthroline 165 130 4525. 2-methyl-1,7-phenanthroline 130 110 4526. 2,8-dimethyl-1,7-phen- 145 105 40 anthroline27. 4,7-phenanthroline 120 120 3528. 3-methyl-4,7-phenanthroline 145 95 4029. 3,8-dimethyl-4,7-phen- 160 80 35 anthroline30. 1,10-phenanthroline 170 90 3531. 2,9-dimethyl-1,10-phen- 145 120 45 anthroline32. 4-azafluorene 90 60 3533. quinine 150 90 3034. cinchonidine 125 85 3035. cuprein 160 95 3536. 2-[4"-methoxyphenyl]-vinyl- 95 90 45 pyridine-(2')37. 2-[4"-(N,N-dimethylamino)- 110 110 45 phenyl]-vinyl-pyridine-(2')38. bis-[2-(phenyl)-vinyl]- 120 95 40 pyridine-(2',4')39. 2-[naphthyl-(1")]-vinyl- 105 90 40 pyridine-(2')40. 2-[pyridyl-(2")]-vinyl- 80 80 30 pyridine-(2')41. 2-[pyridyl-(4")]-vinyl- 60 50 25 pyridine-(2')42. 2-[pyridyl-(3")]-vinyl- 75 70 30 pyridine-(3')43. 2-[pyridyl-(3")]-vinyl- 95 90 25 pyridine-(4')44. 2-[thienyl-(2")]-vinyl- 80 75 35 pyridine-(4')45. imidazole 175 120 3546. 1-phenylimidazole 180 140 4047. histamine 230 150 4548. N-.alpha.-acetyl-histamine 205 130 4049. (imidazolyl-4)-acetic acid 220 150 5050. L-histidine 195 120 4551. N-.alpha.-acetyl-L-histidine 165 110 4052. hexan-1-ol 95 95 3053. octan-1-ol 90 100 3054. nonan-1-ol 90 80 3055. decan-1-ol 80 60 2556. dodecan-1-ol 75 90 2557. pentadecan-1-ol 100 80 3058. heptadecan-1-ol 80 105 3559. octadecan-1-ol 90 95 4060. nonadecan-1-ol 85 120 4061. docosan-1-ol 85 110 4062. cyclohex-1-en-1-ol 95 130 3563. cyclononanol 80 120 3564. cyclodecanol 80 115 4065. cycloheptadecanol 90 130 4066. geraniol 75 85 3067. nerol 90 70 3568. linalool 80 80 3069. nerolidol 105 90 2570. cis-octadec-9-en-1-ol 110 115 2571. hexane-1,6-diol 130 130 4572. octane-1,8-diol 115 125 4073. nonane-1,9-diol 140 130 4574. decane-1,10-diol 130 115 5075. tetrapotassium hexacyano- 230 160 50 ferrate II76. trisodium octacyano- 205 150 50 molybdate V77. tripotassium pentacyano- 180 150 45 nitrosyl-ferrate II78. tripotassium pentacyano- 210 145 40 nitrosyl-manganate I79. pentapotassium pentacyano- 220 155 45 nitrosyl-vanadate I______________________________________

Similar results are obtained with other indoxyl esters of Federal Republic of Germany Patent Application No. P 28 54 987.3 and/or with leukocyte-containing urines instead of the standard solution of 5000 leukocytes/.mu.l. isotonic sodium chloride solution.

EXAMPLE 4

Filter paper (for example Schleicher & Schull 23 SL) is successively impregnated with the following solutions and then dried at 60.degree. C. or at ambient temperature:

Solution 1

tris-(hydroxymethyl)-aminomethane hydrochloride buffer, 0.2 mol/liter, pH 9.0, in water

Solution 2

diacetyl-3',5',3",5"-tetrabromophenylsulphonphthalein, 10.sup.-3 mol/liter in acetone.

The activators according to the present invention are added individually or as mixtures, depending upon the solubility, to Solution 1 and/or Solution 2 so that, in the case of the individual activators of general formulae (I), (II) and (IV), there result end concentrations of 10.sup.-2 mol/liter of impregnation solution and in the case of activators of general formula (III) end concentrations of 2% (w/v) of the impregnation solution.

In the following Table 4, there are summarized the reaction times which extend from the dipping in of the test strips into a standard solution of 5000 leukocytes/.mu.l. isotonic sodium chloride solution up to the first distinct color reaction. The reaction time of the formulation without the addition of activator serves as reference value.

Upon dipping into leukocyte-containing solutions, the test papers change from colourless to deep blue.

TABLE 4______________________________________activators reaction times______________________________________comparative formulation without 160 sec.activators1. 2-methylquinoline 110 sec.2. tetradecan-1-ol 90 sec.activators 1 and 2 60 sec.3. 1,7-phenanthroline 80 sec.4. tetrapotassium hexacyano- 140 sec. ferrate IIactivators 3 and 45. cinchonine 90 sec.6. 2-[phenyl]-vinyl-pyridine-(2') 105 sec.activators 5 and 6 65 sec.______________________________________

Similar experimental results are obtained with leukocyte-containing urines instead of the standard solution of 5000 leukocytes/.mu.l. isotonic sodium chloride solution.

EXAMPLE 5

Test papers are produced in the manner described in Example 4, using the following solutions:

Solution 1

tris-(hydroxymethyl)-aminomethane hydrochloride buffer, 0.2 mol/liter, pH 8.0, in water

Solution 2

2-methoxy-4-nitrobenzeneazo-4'-[1'-(N-benzyloxycarbonyl-L-alanyloxy)-naphthalene], 10.sup.-3 mol/liter in acetone.

Upon dipping into leukocyte-containing solutions, the test papers change color from bright orange to red.

In the following Table 5, there are given the experimental results. The reaction times are given which extend from the dipping of the test strips into a standard solution of 5000 leukocytes/.mu.l. of isotonic sodium chloride solution up to the first distinct color reaction. The reaction time of the formulation without the addition of activator serves as reference value.

TABLE 5______________________________________activators reaction times______________________________________comparative formulation without 80 sec.activator1. benzo-[b]-quinoline (acridine) 60 sec.2. cis-octadec-9-en-1-ol 40 sec.activators 1 and 2 30 sec.3. benzo-[h]-quinoline 50 sec.4. quinine 55 sec.activators 3 and 4 35 sec.5. 1,7-phenanthroline 45 sec.6. farnesol 30 sec.activators 5 and 6 25 sec.______________________________________

Similar experimental results are achieved with the use of leukocyte-containing urines instead of the standard solution of 5000 leukocytes/.mu.l. of isotonic sodium chloride solution.

EXAMPLE 6

Test papers are produced in the manner described in Example 4, using the following solutions:

Solution 1

sodium tetraborate hydrochloric acid buffer, 0.2 mol/liter, pH 8.0, in water

Solution 2

3-[N-(2'-nitrobenzenesulphenyl)-L-alanyloxy]-indole, 10.sup.-3 mol/liter, in acetone.

Upon dipping into leukocyte-containing solutions, the test papers change color from yellow to green.

The experimental results obtained are summarized in the following Table 6. The reaction times are given which extend from the dipping of the test strips into a standard solution of 5000 leukocytes/.mu.l. isotonic sodium chloride solution up to the first distinct color reaction. The reaction time of the formulation without the addition of activator serves as reference value.

TABLE 6______________________________________activators reaction times______________________________________comparative formulation without 100 sec.activators1. quinoline 80 sec.2. cyclododecanol 70 sec.activators 1 and 2 55 sec.3. 2-[furyl-(2")]-vinyl-pyridine-(2') 75 sec.4. phytol 50 sec.activators 3 and 4 40 sec.5. 4,7-phenanthroline 85 sec.6. tetrapotassium hexacyanoferrate II 90 sec.activators 5 and 6 70 sec.______________________________________

Similar experimental results are obtained with leukocyte-containing urines instead of the standard solution of 5000 leukocytes/.mu.l. of isotonic sodium chloride solution.

EXAMPLE 7

Test papers are produced in the manner described in Example 4 with the following solutions:

Solution 1

sodium tetraborate hydrochloric acid buffer, 0.2 mol/liter, pH 8.0, in water

Solution 2

3-[N-(benzoyl)-D,L-alanyloxy]-indole, 10.sup.-3 mol/liter, in acetone.

Upon dipping into leukocyte-containing solutions, the test papers change color from colorless to blue.

The experimental results obtained are summarized in the following Table 7. The reaction times are given which extend from the dipping of the test strips into a standard solution of 5000 leukocytes/.mu.l. of isotonic sodium chloride solution up to the first distinct color reaction. The reaction time of the formulation without the addition of activator serves as reference value.

TABLE 7______________________________________activators reaction times______________________________________comparative formulation without 90 sec.activators1. 2-[phenyl]-vinyl-pyridine-(2') 70 sec.2. hexadecan-1-ol 55 sec.activators 1 and 2 40 sec.3. 2-[pyridyl-4(")]-vinyl-pyridine- 40 sec. (4')4. linalool 50 sec.activators 3 and 4 30 sec.5. cinchonine 75 sec.6. disodium pentacyanonitrosyl- 60 sec. ferrate IIactivators 5 and 6 40 sec.______________________________________

Similar experimental results are obtained with leukocyte-containing urines instead of the standard solution of 5000 leukocytes/.mu.l. of isotonic sodium chloride solution.

EXAMPLE 8

Test papers are produced in the manner described in Example 4 with the following solutions:

Solution 1

sodium tetraborate hydrochloric acid buffer, 0.2 mol/liter, pH 8.0, in water

Solution 2

3-[N-(toluene-4'-sulphonyl)-L-alanyloxy]-indole, 10.sup.-3 mol/liter, in acetone.

Upon dipping into leukocyte-containing solutions, the test papers change color from colorless to blue.

The experimental results obtained are summarized in the following Table 8. The reaction times are given which extend from the dipping of the test strips into a standard solution of 5000 leukocytes/.mu.l. of isotonic sodium chloride solution up to the first distinct color reaction. The reaction time of the formulation without the addition of activator serves as a reference value.

TABLE 8______________________________________activators reaction times______________________________________comparative formulation without 24 sec.activators1. quinine 22 sec.2. disodium pentacyanonitrosyl- 15 sec. ferrate II3. decan-1-ol 18 sec.activators 1, 2 and 3 6 sec.______________________________________

Similar results are obtained with leukocyte-containing urines instead of the standard solution of 5000 leukocytes/.mu.l. of isotonic sodium chloride solution.

EXAMPLE 9

Filter paper (for example Schleicher & Schull 23 SL) is successively impregnated with the following solutions and then dried at 60.degree. C. or at ambient temperature:

Solution 1

laurylpyridinium chloride, 0.2%, in tris-(hydroxymethyl)aminomethane hydrochloride buffer, 0.2 mol/liter, pH 7.0, in water

Solution 2

thiazole-2-azo-4'-[1'-(N-benzyloxycarbonyl-L-alanyloxy)naphthalene], 10.sup.-3 mol/liter, in acetone.

The activator addition and the carrying out of the experiment take place as in Example 1.

Upon dipping into leukocyte-containing solutions, the test papers change color from pink to violet.

The experimental results obtained are summarized in the following Table 9. The reaction times are given which extend from the dipping of the test strips into a standard solution of 5000 leukocytes/.mu.l. of isotonic sodium chloride solution up to the first distinct color reaction. The reaction time of the formulation without the addition of activator serves as reference value.

TABLE 9______________________________________activators reaction times______________________________________comparative formulation without 70 sec.activator1. pyridine 45 sec.2. quinoline 40 sec.3. benzo-[b]-quinoline (acridine) 35 sec.4. 1,10-phenanthroline 40 sec.5. 4-azafluorene 30 sec.6. quinine 35 sec.7. 2-[pyridyl-(4")]-vinyl-pyridine- 30 sec. (4')8. imidazole 50 sec.9. dodecan-1-ol 30 sec.10. phytol 25 sec.11. disodium pentacyanonitrosyl- 60 sec. ferrate II______________________________________

Similar experimental results are obtained with the other substrates and activators mentioned in Examples 1 to 7 and/or with other conventional wetting agents.

EXAMPLE 10

Filter paper (for example Schleicher & Schull 23 SL) is successively impregnated with the following solutions and then dried at 60.degree. C. or at ambient temperature.

Solution 1

tris-(hydroxymethyl)-aminomethane hydrochloride buffer, 0.2 mol/liter, pH 7.0, in water

Solution 2

thiazole-2-azo-1'-[2'-(N-benzyloxycarbonyl-L-alanyloxy)-naphthalene], 10.sup.-3 mol/liter, and zinc acetate dihydrate, 10.sup.-3 mol/liter, in acetone.

The activator addition and the carrying out of the experiment take place as in Example 1.

Upon dipping into leukocyte-containing solutions, the test papers change color from pink to blue-violet.

The experimental results obtained are summarized in the following Table 10. The reaction times are given which extend from the dipping of the test strips into a standard solution of 5000 leukocytes/.mu.l. of isotonic sodium chloride solution up to the first distinct color reaction. The reaction time of the formulation without the addition of activator serves as reference value.

TABLE 10______________________________________activators reaction times______________________________________comparative formulation without 65 sec.activator1. pyridine 50 sec.2. quinoline 45 sec.3. benzo-[b]-quinoline (acridine) 40 sec.4. 1,10-phenanthroline 30 sec.5. 4-azafluorene 35 sec.6. quinine 30 sec.7. 2-[pyridyl-(4")]-vinyl-pyridine- 35 sec. (4')8. imidazole 45 sec.9. dodecan-1-ol 25 sec.10. phytol 30 sec.11. disodium pentacyano-nitrosyl- 50 sec. ferrate II______________________________________

Similar experimental results are obtained with other azo dyestuff esters of Federal Republic of Germany Patent Application No. P 28 36 644.1, with other activators mentioned in Examples 1 to 7 and/or with other conventional complex formers.

EXAMPLE 11

Filter paper (for example Schleicher & Schull 23 SL) is successively impregnated with the following solutions and then dried at 60.degree. C. or at ambient temperature.

Solution 1

potassium bromate, 10.sup.-2 mol/liter, in sodium tetraborate hydrochloric acid buffer, 0.2 mol/liter, pH 8.0, in water.

Solution 2

3-[N-formyl-L-alanyloxy]-indole, 10.sup.-3 mol/liter, in acetone.

The addition of activator and the carrying out of the experiment take place as in Example 1.

Upon dipping into leukocyte-containing solutions, the test papers change color from colorless to blue.

The experimental results obtained are summarized in the following Table 11. The reaction times are given which extend from the dipping of the test papers into a standard solution of 5000 leukocytes/.mu.l. of isotonic sodium chloride solution up to the first distinct color reaction. The reaction time of the formulation without the addition of activator serves as reference value.

TABLE 11______________________________________activators reaction times______________________________________comparative formulation without 120 sec.activators1. pyridine 85 sec.2. quinoline 70 sec.3. benzo-[b]-quinoline (acridine) 100 sec.4. 1,10-phenanthroline 65 sec.5. 4-azafluorene 50 sec.6. quinine 75 sec.7. 2-[pyridyl-(4")]-vinyl-pyridine- 70 sec. (4')8. imidazole 85 sec.9. dodecan-1-ol 35 sec.10. phytol 40 sec.11. disodium pentacyano-nitrosyl- 90 sec. ferrate II______________________________________

Similar experimental results are also obtained with other indoxyl esters of Federal Republic of Germany Patent Application No. P 28 54 987.3, with the other activators described in Examples 1 to 7 and with other conventional oxidation agents.

It will be understood that the specification and examples are illustrative but not limitative of the present invention and that other embodiments within the spirit and scope of the invention will suggest themselves to those skilled in the art.

Claims

1. Diagnostic agent for the detection of esterolytic and proteolytic enzymes in body fluids, which agent comprises at least one substrate, and adjuvants suitable for said substrate, and at least one activator, and wherein said substrate is a sulphonphthalein ester of the formula ##STR5## wherein R.sub.1 " is a carboxylic acid residue optionally substituted by halogen or a lower alkoxy radical or is an amino acid or peptide residue provided with a nitrogen protective group conventional in peptide chemistry;

R.sub.2 " is a halogen atom or a lower alkyl radical; and

R.sub.3 " and R.sub.4 ", which can be the same or different are hydrogen or halogen atoms; or an azo dyestuff ester of the formula

A'--N.dbd.N--B'(OR).sub.n

wherein

A' is a five- or six-membered, optionally benzo-annellated residue with one or two heteroatoms selected from nitrogen, sulfur and oxygen, which is optionally substituted one or more times by halogen, lower alkyl or lower alkoxy radicals or is a phenyl radical substituted one, two or three times by lower alkyl, lower alkoxy, nitro, sulphonato or acylamino radicals;

B' is a benzene, naphthalene or quinoline radical optionally substituted once or twice by sulphonato, lower alkoxy or lower alkoxypolyalkyleneoxy radicals;

R is a carboxylic acid residue or an amino acid or peptide residue provided with a nitrogen protective group conventional in peptide chemistry; and

n is 1 or 2; or an indoxyl ester of the formula ##STR6## wherein R.sub.1 '", R.sub.2 '", R.sub.3 '" and R.sub.4 '", which can be the same or different, are hydrogen or halogen atoms, lower alkyl, lower alkoxy, aryl, aralkyl, aralkoxy, hydroxyl, carboxy, carboxy lower alkoxy, aralkoxycarbonyl, aralkoxycarbonyl lower alkoxy, nitro or lower acylamino radicals or in which two adjacent substituents represent a benzo-annellated residue optionally substituted by halogen;

X is a sulfur atom or an imino group optionally substituted by a lower alkyl, aryl, aralkyl or acyl radical;

A" is an amino acid or peptide residue; and

B" is a nitrogen protective group conventional in peptide chemistry or derived therefrom.

and said activator is selected from the following:

(a) pyridine derivatives of the general formula: ##STR7## in which R.sub.1, R.sub.2, R.sub.3, R.sub.4 and R.sub.5, which can be the same or different, are hydrogen or halogen atoms, lower alkyl or lower alkoxy radicals, vinyl radicals which are substituted by an aryl radical optionally substituted one or more times by lower alkoxy, amino, alkylamino or dialkylamino, or by a heterocyclic radical, whereby two adjacent substituents can represent an indeno- or benzo-annellated residue optionally substituted one or more times by halogen, hydroxyl, lower alkyl or lower alkoxy, which annellated residue can, in turn, carry a benzo- or pyrido-annellated residue optionally substituted by a lower alkyl radical and R.sub.3 can also stand for a vinylquinuclidyl-carbinol radical;

(b) imidazole derivatives of the general formula: ##STR8## in which R.sub.1 ' is a hydrogen atom, a lower alkyl radical or an aryl radical optionally substituted by a hydroxyl group or an acyl radical and R.sub.2 ' is a hydrogen atom, an aminoalkyl, N-acylaminoalkyl or a lower aliphatic, optionally unsaturated carboxylic acid residue or a lower aliphatic .alpha.-amino acid residue optionally acylated on the nitrogen;

(c) alcohols of the general formula:

X--A--OH

in which X is a hydrogen atom or a hydroxyl group and A is a hydrocarbon radical;

(d) metal complexes of the general formula:

D.sub.m [B(CN).sub.n (NO).sub.p ]

in which D is an alkali metal ion, B is a heavy metal ion, m is 2, 3, 4 or 5, n is 4, 5, 6, 7 or 8 and p is 0 or 1, the number m being given by the valency of the heavy metal ion and the number n.

2. Diagnostic agent as claimed in claim 1 wherein said conventional adjuvants are selected from buffers, complex formers, wetting agents, oxidation agents, film formers, galenical additional materials and structure formers.

3. Diagnostic agent as claimed in claim 1 wherein said activator is a pyridine derivative (a).

4. Diagnostic agent as claimed in claim 1 wherein said activator is an imidazole derivative (b).

5. Diagnostic agent as claimed in claim 1 wherein said activator is an alcohol (c).

6. Diagnostic agent as claimed in claim 1 wherein said activator is a metal complex (d).

7. Diagnostic agent as claimed in claim 1 wherein said activator is benzo-[h]-quinoline.

8. Diagnostic agent as claimed in claim 1 wherein said activator is 4-azafluorene.

9. Diagnostic agent as claimed in claim 1 wherein said activator is quinine.

10. Diagnostic agent as claimed in claim 1 wherein said activator is 2-[phenyl]-vinyl-pyridine-(2').

11. Diagnostic agent as claimed in claim 1 wherein said activator is imidazole.

12. Diagnostic agent as claimed in claim 1 wherein said activator is histamine.

13. Diagnostic agent as claimed in claim 1 wherein said activator is decan-1-ol.

14. Diagnostic agent as claimed in claim 1 wherein said activator is tetradecan-1-ol.

15. Diagnostic agent as claimed in claim 1 wherein said activator is tripotassium-hexacyanoferrate III.

16. Diagnostic agent as claimed in claim 1 wherein said activator is disodium-pentacyano-nitrosylferrate II.

17. Method of detecting leukocytes in body fluid which method comprises contacting a sample with a diagnostic agent as claimed in claim 1.

18. Method as claimed in claim 17 wherein said body fluid is urine.

19. Method as claimed in claim 17 wherein said activator is selected from

benzo-[h]-quinoline;

4-azafluorene;

quinine;

2-[phenyl]-vinyl-pyridine-(2');

imidazole;

histamine;

decan-1-ol;

tetradecan-1-ol;

tripotassium-hexacyanoferrate III; and

disodium-pentacyano-nitrosylferrate II.

20. Method as claimed in claim 17 wherein said activator is quinine.

21. Method as claimed in claim 17 wherein said activator is decan-1-ol.

22. Method as claimed in claim 17 wherein said activator is disodium-pentacyano-nitrosylferrate II.

23. Process for preparing a diagnostic agent as claimed in claim 1 which process comprises impregnating an absorbent carrier with at least one said substrate selected from said esterase and protease substrates, with conventional adjuvants, and additionally with at least one said activator.

24. Process as claimed in claim 23 wherein said absorbent carrier is impregnated in two stages.