CA1064024A - Substrate for the determination of desoxy-ribonuclease - Google Patents
Substrate for the determination of desoxy-ribonucleaseInfo
- Publication number
- CA1064024A CA1064024A CA208,328A CA208328A CA1064024A CA 1064024 A CA1064024 A CA 1064024A CA 208328 A CA208328 A CA 208328A CA 1064024 A CA1064024 A CA 1064024A
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- dna
- desoxy
- blue
- solution
- substrate
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Abstract
Abstract of the Disclosure:
A substrate containing a complex compound of highmolecular-weight desoxy-ribonucleinic acid and 2-amino-7-dimethylamino-3-methyldiphenazthionium chloride, its manufacture and its use for diagnostic determination of the enzymatic activity of des-oxy-ribonuclease and its antibodies.
A substrate containing a complex compound of highmolecular-weight desoxy-ribonucleinic acid and 2-amino-7-dimethylamino-3-methyldiphenazthionium chloride, its manufacture and its use for diagnostic determination of the enzymatic activity of des-oxy-ribonuclease and its antibodies.
Description
The present invention relates to a new substrate suitable for a simple process for determining desoxy-ribonuclease and the antibodies thereof, in particular antibodies against des-oxy-ribonuclease of streptococci (antistreptodornase).
The time interval between a bacterial infection and its manifestation, the incubation period, is clinically mute, and even investigations on a laboratory scale rarely lead to an early diagnosis. An early diagnosis would be possible by direct evidence of bacterial metabolic products, and a subsequent increase in the titer of an antibody acting against these metabolic products would additionally confirm such a diagnosis.
Streptococci of Group A generate a great variety of such extra-cellular metabolic products having an enzymatic natur~, some having an immunogenic activity, for example streptolysine O, NAD-glyco-hydrolase, streptodornase, hyaluronidase and strepto-kinase. The determination of the antistreptolysine titer in -~
the case of A streptococci infections is one of the classical methods; if other diseases, such as hepatitis, Lupus erythema- ;
todes, nephrosis and others, are present in the body at the same time, unspecific disturbances caused thereby may account for an increased titer of the antistreptolysine.
When A-streptococci are cultivated in an adequate nutrient ; medium, desoxy-ribonucleases of different electrophoretic mobility can be established in the bacteria-free supernatant ~25 of the culture. These desoxy-ribonucleases are designated as streptodornase A, B, C and D (DNase A, B, C and D). The major portion as to amount and activity is that of streptodornase B, so that this iso-enzyme is the most obvious to appear during a streptococci infection, owing to the formation of antibodies.
. :. , :: ~, .,: : ::,:: : :, .: , . , ,: ............... : .: ,.
.-., .. -, - , .: . - . . - . , , , . , :
-~ HOE 73/B 012 Several clinico-chemical investigations have demonstrated that the antistreptolysine titer in the case of skin infections caused by A streptococci is found to be little or not changed at all, whilst the serum level of the antistreptodornase B is found to be increased substantially.
Increas~ing importance in the differential diagnosis of the antistreptodornase B titer asks for easily manageable methods for the deter~ination of the antistreptodornase level in the blood serum. Accordingly, a method has been proposed to establish antistreptodornage by means of Methyl-Green, according to which a certain amount of desoxy-ribonuclease is incubated with the serum to be examined for antibodies. As a function of the amount of streptodornase antibodies present, the activity of desoxy-ribonuclease is reduced. When a methyl-green desoxy-ribonucleinic acid (DNA) complex is added to this mixture, thismethyl-green DNA complex is split and thereby decolored as a ~result of the still present streptodornase activity. The readabiIity of the reaction to be carried out by means of a dllution series of the serum is not quite easy and fre~uently 20~ leads to an erroneous determination of end points. The pre-paration of this reagent requires a relatively high~amount of high-molecular-weight DNA.
It lS kn~wn that toluidine Blue O is capable oE coloring an~agar medium containing high-molecular-weight DNA in a blue 25~ shade, whereas areas of the agar medium, which contain DNA
:
that has been degradated, for example, by the action of bacteria, are~colored in a~pink shade by toluidine Blue.
I have now~found a substrate for the determination of desoxy-ribonuclease and antibodies thereof, which contain5 a complex compound consisting of high-molecular-weight desoxy-ribonucleinic acid (DNA) and 2-amino-7-dimethylamino-3-methyldi-phenazthionium chloride (toluidine Blue 0~. Using a D~A ocmplex with the meta-chrcmatic dyestuff 2-amino-7-dimethylamano-3-methyl-diphenaz-thionium chloride (toluidine Blue 0) for the quantitativedetermination especially of streptodornase antibodies offers some advantagas over ~he known methods. The action of DNase which is not bound to antibodies causes the dyestuff to be split off from the complex compound to form a blue-colored precipitate: The supernatant solution loses its color~ Hence, when determining the anti-streptodornase titer in the sera of test persons by means of a dilution series, a simple reading ~-~
allows to find that serum dilution which is just sufficient for an antibody present to bind a determined amount of DNase, and thus a blue precipitate does not yet form. It is con-venient to standardi2e the determination of the end point by making availahle a serum which has a determined content of antistreptodornase B~
When determining desoxy-ribonuclease, the DNA-toluidine blue complex compound used allows the end point of the reaction to be fixed comparatively simply. To this effect, the unknown DNase activity may be measured, for example by comparing it with the activity of a determined enzyme standard which ~ives a blue-colored precipitate at the limit value of its activity, or a certain amount of DNase antibodies is used to establish, by means of the DNA-toluidine blue complex compound, that concentration of DNase which is no longer bound by the defined amount of anti-bodies and splits the substrate to form a blue--L~J~
colored precipitate.
This invention moreover relates to the manufacture of a new substrate containing a complex compound of desoxy ribo-nucleinic acid and 2-amino-7-dimethylamino-3 methyldiphenaz-thionium chloride and to its use in deter;mination methods fordesoxy-ribonuclease and antibodies thereof.
The process for the manufacture of the substrate is relatively simple: DNA and toluidine blue 0 are introduced into water or into an aqueous buffer solution, and the slight turbidity formed is dissolved by boiling and simultaneously homogenizing it. It is advantageous to prepare the substrate in a buffer solution with a pH~value approximating the reaction optimum of the DNase to be determined. Adequate pH-values range from about 4 to 9. The buffer substances to be used for this purpose are those employed for biological and physio-logical work, preferably those proposed by N.E. Good et al Biochemistry 5, 467 ~1966). For the neutral pH-range, tris-: , .
hydroxy-methylamino-methane is, for example, appropriate.
For the preparation of the substrate, it is advantageous to use alkaline earth metal ions, for example in the form of .
water-soluble calcium or magnesium salts, in order to ensure -the full activity of the enzyme acting upon the substrate, ~ ., .
that is to say of the DNase.
If isotonicity of the substrate, for example with plasma is to be achieved, this can be managed by adding corresponding saltst commonly sodium chloride. As DNA, a commercial pre-paration having a high molecular weight (more than 1,0Q0,000) s~especially useful, advantageously a DNA obtained from sperms of fish. DNA of lower molecular weight and different origin is, ~ HOE 73/F 012 however, also useful. The concentration of the VN~ suitably ranges from 0.01 to 0.03 ~, pxe~erably it is 0.02 %; the con-centration o toluidine blue 0 ranges from 0.005 to 0.015 ~, preferably it is 0.01 ~. The concentratior- of the calcium or S magnesium salts used preferably as substances, which yield alkaline earth metal ions, advantageously ranges from 0.1 to 0.5%.
These salts are to activate the DNase. The substrate prepared according to ~his method is a clear homogenous blue solution.
The ratio of the two components in this substrate may vary greatly within reasonable limits; advantageously it is about
The time interval between a bacterial infection and its manifestation, the incubation period, is clinically mute, and even investigations on a laboratory scale rarely lead to an early diagnosis. An early diagnosis would be possible by direct evidence of bacterial metabolic products, and a subsequent increase in the titer of an antibody acting against these metabolic products would additionally confirm such a diagnosis.
Streptococci of Group A generate a great variety of such extra-cellular metabolic products having an enzymatic natur~, some having an immunogenic activity, for example streptolysine O, NAD-glyco-hydrolase, streptodornase, hyaluronidase and strepto-kinase. The determination of the antistreptolysine titer in -~
the case of A streptococci infections is one of the classical methods; if other diseases, such as hepatitis, Lupus erythema- ;
todes, nephrosis and others, are present in the body at the same time, unspecific disturbances caused thereby may account for an increased titer of the antistreptolysine.
When A-streptococci are cultivated in an adequate nutrient ; medium, desoxy-ribonucleases of different electrophoretic mobility can be established in the bacteria-free supernatant ~25 of the culture. These desoxy-ribonucleases are designated as streptodornase A, B, C and D (DNase A, B, C and D). The major portion as to amount and activity is that of streptodornase B, so that this iso-enzyme is the most obvious to appear during a streptococci infection, owing to the formation of antibodies.
. :. , :: ~, .,: : ::,:: : :, .: , . , ,: ............... : .: ,.
.-., .. -, - , .: . - . . - . , , , . , :
-~ HOE 73/B 012 Several clinico-chemical investigations have demonstrated that the antistreptolysine titer in the case of skin infections caused by A streptococci is found to be little or not changed at all, whilst the serum level of the antistreptodornase B is found to be increased substantially.
Increas~ing importance in the differential diagnosis of the antistreptodornase B titer asks for easily manageable methods for the deter~ination of the antistreptodornase level in the blood serum. Accordingly, a method has been proposed to establish antistreptodornage by means of Methyl-Green, according to which a certain amount of desoxy-ribonuclease is incubated with the serum to be examined for antibodies. As a function of the amount of streptodornase antibodies present, the activity of desoxy-ribonuclease is reduced. When a methyl-green desoxy-ribonucleinic acid (DNA) complex is added to this mixture, thismethyl-green DNA complex is split and thereby decolored as a ~result of the still present streptodornase activity. The readabiIity of the reaction to be carried out by means of a dllution series of the serum is not quite easy and fre~uently 20~ leads to an erroneous determination of end points. The pre-paration of this reagent requires a relatively high~amount of high-molecular-weight DNA.
It lS kn~wn that toluidine Blue O is capable oE coloring an~agar medium containing high-molecular-weight DNA in a blue 25~ shade, whereas areas of the agar medium, which contain DNA
:
that has been degradated, for example, by the action of bacteria, are~colored in a~pink shade by toluidine Blue.
I have now~found a substrate for the determination of desoxy-ribonuclease and antibodies thereof, which contain5 a complex compound consisting of high-molecular-weight desoxy-ribonucleinic acid (DNA) and 2-amino-7-dimethylamino-3-methyldi-phenazthionium chloride (toluidine Blue 0~. Using a D~A ocmplex with the meta-chrcmatic dyestuff 2-amino-7-dimethylamano-3-methyl-diphenaz-thionium chloride (toluidine Blue 0) for the quantitativedetermination especially of streptodornase antibodies offers some advantagas over ~he known methods. The action of DNase which is not bound to antibodies causes the dyestuff to be split off from the complex compound to form a blue-colored precipitate: The supernatant solution loses its color~ Hence, when determining the anti-streptodornase titer in the sera of test persons by means of a dilution series, a simple reading ~-~
allows to find that serum dilution which is just sufficient for an antibody present to bind a determined amount of DNase, and thus a blue precipitate does not yet form. It is con-venient to standardi2e the determination of the end point by making availahle a serum which has a determined content of antistreptodornase B~
When determining desoxy-ribonuclease, the DNA-toluidine blue complex compound used allows the end point of the reaction to be fixed comparatively simply. To this effect, the unknown DNase activity may be measured, for example by comparing it with the activity of a determined enzyme standard which ~ives a blue-colored precipitate at the limit value of its activity, or a certain amount of DNase antibodies is used to establish, by means of the DNA-toluidine blue complex compound, that concentration of DNase which is no longer bound by the defined amount of anti-bodies and splits the substrate to form a blue--L~J~
colored precipitate.
This invention moreover relates to the manufacture of a new substrate containing a complex compound of desoxy ribo-nucleinic acid and 2-amino-7-dimethylamino-3 methyldiphenaz-thionium chloride and to its use in deter;mination methods fordesoxy-ribonuclease and antibodies thereof.
The process for the manufacture of the substrate is relatively simple: DNA and toluidine blue 0 are introduced into water or into an aqueous buffer solution, and the slight turbidity formed is dissolved by boiling and simultaneously homogenizing it. It is advantageous to prepare the substrate in a buffer solution with a pH~value approximating the reaction optimum of the DNase to be determined. Adequate pH-values range from about 4 to 9. The buffer substances to be used for this purpose are those employed for biological and physio-logical work, preferably those proposed by N.E. Good et al Biochemistry 5, 467 ~1966). For the neutral pH-range, tris-: , .
hydroxy-methylamino-methane is, for example, appropriate.
For the preparation of the substrate, it is advantageous to use alkaline earth metal ions, for example in the form of .
water-soluble calcium or magnesium salts, in order to ensure -the full activity of the enzyme acting upon the substrate, ~ ., .
that is to say of the DNase.
If isotonicity of the substrate, for example with plasma is to be achieved, this can be managed by adding corresponding saltst commonly sodium chloride. As DNA, a commercial pre-paration having a high molecular weight (more than 1,0Q0,000) s~especially useful, advantageously a DNA obtained from sperms of fish. DNA of lower molecular weight and different origin is, ~ HOE 73/F 012 however, also useful. The concentration of the VN~ suitably ranges from 0.01 to 0.03 ~, pxe~erably it is 0.02 %; the con-centration o toluidine blue 0 ranges from 0.005 to 0.015 ~, preferably it is 0.01 ~. The concentratior- of the calcium or S magnesium salts used preferably as substances, which yield alkaline earth metal ions, advantageously ranges from 0.1 to 0.5%.
These salts are to activate the DNase. The substrate prepared according to ~his method is a clear homogenous blue solution.
The ratio of the two components in this substrate may vary greatly within reasonable limits; advantageously it is about
2 parts by weight of DNA to 1 part by weight of toluidine blue 0, a 10% deviation from this ratio being of no consequence.
In many cases, it may be convenient in carrying out the test to dilute the solution wi~h a dilution liquid containing a buffer system, which is to be selected in accordance wi~h the pH optimum of the DNase to be determined. In addition to the buffer substances proposed by Good et al. (1966) and other buffer systems useful for biochemical work, imidazole-hydro chloric acid has proved to be suitable for the determination of DNases at a pH of from 6.0 to 8Ø The dilution liquid advantageously contains one of the stabilizers as frequently used for operations in dilute enzyme solutions. Proteins, for example albumins, gelatins and derivatives thereof, are as well ; useful for this purpose as are polyhydroxy compounds, for example dextrans and su~ar~
~ he substrate~of the invention is especially sultable in micro msthods~for~the determination of streptodornase anti-bodies in the clinical diagnosis, in particular for the micro ;~ -titer plates used in serology.
.
There is, however, no reason why the substrate should not be used in a macro test using increased volumes in the test tubes. The substrate may also be used as disclosed above to determine the DNase.
In a comparative test for the antistreptodornase B titers in healthy people and patients who have gone through an in-fection caused by streptococci, the test system cited in the Examples is used to find a marked increase of antistrepto-dornase B in the patients.
Being normally below a ratio of 1 : 200 in healthy per-sons, serum titers of the streptodornase antibodies exceeding this ratio may, inter alia, allow the diagnosis of rheumatic fever, infection of the upper air passages or Glomerulo-nephritis.
The following Examples illustrate the invention~
`~ E X A M P L E 1:
Preparation of the DNA substrate O.3 Gram of trishydroxymethylamino-me-thane, 0.5 y of NaCl, 15 mg of purified DNA and 0.05 ml of a 0.01M calcium chloride solution were added to 100 ml of distilled water.
The pH-value of the solution was adjusted to 7.0 by means of 1.0 ml of 0.1N HCl. The mixture was boiled for about 10 seconds, whereupon a homogeneous clear solution was obtained. 0~3 ml of -;
~ a 0.lM toluidine blue 0 solution which distributed homogenously - ~ 25 in the first solution was then added to the hot solution.
After cooling, the DMA substrate was ready for activity determinations. Should flakes appear on storage for a pro-'`! ; longed time, the substrate solution is to be boiled up again for a short time prior to use.
:.
L;~ ~
....
Preparation of the streptodornase B antigen DNase which had been obtained from a 16-hour culture of an A streptococci strain forming DNase B and purified according to known methods was standardized to 30 units per ml (measured in a Brookfield microrotation viscosimeter) (Calculated on DNase I from pancreas). Immediately priox to the test, the antigen solution was diluted at a ratio of l : 30.
E X A M P L E 3:
. _ Preparation of the d_lution liquid 400 ml of an aqueous solution of bovine albumin con-taining 250 mg of bovine albumin per l00 ml were mixed with l00 ml of a 0.25 M imidazole-HCl buffer of pH 8.0, containing -1.47 g of calcium chloride dihydrate and 0.6 g of magnesium sulfate per liter. ~ :
E X A M P L E 4: -: Determination of the streptodornase B antibodies in sera Determination was made as follows:
In micro titer plates, 25 /ul of the dilution liquid as : : 20 prepared in Example 3 were filled into Cup 2 of the first ~ ;series, two times 25 /ul of the same dilution liquid were : ~filled into Cup 3, and 25 /ul each of the dilution liquid were :111ed into the following cups. Subsequently, 25 /u1 each o : a~ 50 dilution of a serum taken from healthy persons were 2~5 illed into cups l, 2 and 3. Starting from cup 2, 25 /ul each of the serum~dilution obtained were conveyed by pipet to cup 4 : and ~urther to cup 6 and so on to cups 8, l0 and 12. Starting :; from oup 3, 25 /u1 each~of the-corresponding serum dilution were conveyed over to cup 5 and also further to cups 7, g and ll.
~ ~ .
~ 8 - ~
~3i6~V~ ~ HOE 73/B 012 Subsequently, 25 /ul each of the serum dilution were eliminated from cups 3, 11 and 12. Thus, startin~ Erom cup 1, a dilution series of 1 : 50, 100, 150, ~00, 300, 400, 600, 800, 1200, 1600. 2500 and 3200 was obtained. The same dilution series were made for the sera of patients to be determined in series 2 and 3 of the micro titer plate. Then, 25 /ul of the standard antigen dilution as obtained in Example 2 were added to each of the 25 /ul serum dilution and allowed to stand for 4 minutes on a shaking device. Finally, 50 /ul of the substrate solution as prepared in Example 1 were added to each of the dilutions.
The mixture was then incubated at 37C. The formation of a blue-colored precipitate indicated the dilution of the anti-bodies present in the serum, which was no longer able to bind ; the DNase added. As soon as a control serum accompanying the test mixture and having a fixed titer of 1 : 150 caused the precipitate to form up to the dilution of 1 : 150, the values were read off. In this exemplified specimen, a precipitate formed throughout the first series. ~ence, the titer was less than 1 : 50. ~n the second series, a precipitate formed in cup 4; hence, the titer was 1 : 150. In the third series, a precipitate formed starting from cup 8, corresponding to a titer of 1 : 600.
E X A M P L E 5;
Determination of desoxy-ribonuclease In the first series of a micro titer plate, 50 micro-liters of a culture solution of streptococci, which had been filtered under sterile conditions, were filled into cup 1.
The fo~lowing cups were filled with 2~ microli~ers of the dilution liquid as prepared accordi~g to Example 3.
_ g ._ .:
~ . ., . ~ . . : -. HOE 73/B 012 ;4~
By conveying 25 microliters each from the ~irst to the second cup, further from the second to the third cup and so on, a dilution series of the ~ollowing ratio was obtained: 1 : 2, 1 : 4, 1 : 8, and so on.
A DNase o~ known activity was diluted in the second series of the micro titer plate in the same manner. To 25 micro-liters each of the enzyme dilutions, 50 microliters of the substrate solution as prepared in Example 1 were added, where-upon the mixture was incubated at 40C for 3 hours. The formation of a blue-colored precipitate was taken as an indi-cator for the enzyme activity. The streptococci culture filtrate formed a precipitate up to cup 8, corresponding to a dilution of 1 : 128.
The accompanying standard DNase in this test system always caused precipitation up to cup 10, corresponding to : a dilution of 1 : 512.
The result of this test shows that the culture filtrate to be tested has a DNase activity of 25~ of the standard~
.
.
:~ :
:~::: ~ :: ::
, ' . ,.. :
- 10 -: '' ... j. : .. " ., ~, ... ~,. .. ~ . ... .. ,.,: .. . ,.. : ,.. .... .
In many cases, it may be convenient in carrying out the test to dilute the solution wi~h a dilution liquid containing a buffer system, which is to be selected in accordance wi~h the pH optimum of the DNase to be determined. In addition to the buffer substances proposed by Good et al. (1966) and other buffer systems useful for biochemical work, imidazole-hydro chloric acid has proved to be suitable for the determination of DNases at a pH of from 6.0 to 8Ø The dilution liquid advantageously contains one of the stabilizers as frequently used for operations in dilute enzyme solutions. Proteins, for example albumins, gelatins and derivatives thereof, are as well ; useful for this purpose as are polyhydroxy compounds, for example dextrans and su~ar~
~ he substrate~of the invention is especially sultable in micro msthods~for~the determination of streptodornase anti-bodies in the clinical diagnosis, in particular for the micro ;~ -titer plates used in serology.
.
There is, however, no reason why the substrate should not be used in a macro test using increased volumes in the test tubes. The substrate may also be used as disclosed above to determine the DNase.
In a comparative test for the antistreptodornase B titers in healthy people and patients who have gone through an in-fection caused by streptococci, the test system cited in the Examples is used to find a marked increase of antistrepto-dornase B in the patients.
Being normally below a ratio of 1 : 200 in healthy per-sons, serum titers of the streptodornase antibodies exceeding this ratio may, inter alia, allow the diagnosis of rheumatic fever, infection of the upper air passages or Glomerulo-nephritis.
The following Examples illustrate the invention~
`~ E X A M P L E 1:
Preparation of the DNA substrate O.3 Gram of trishydroxymethylamino-me-thane, 0.5 y of NaCl, 15 mg of purified DNA and 0.05 ml of a 0.01M calcium chloride solution were added to 100 ml of distilled water.
The pH-value of the solution was adjusted to 7.0 by means of 1.0 ml of 0.1N HCl. The mixture was boiled for about 10 seconds, whereupon a homogeneous clear solution was obtained. 0~3 ml of -;
~ a 0.lM toluidine blue 0 solution which distributed homogenously - ~ 25 in the first solution was then added to the hot solution.
After cooling, the DMA substrate was ready for activity determinations. Should flakes appear on storage for a pro-'`! ; longed time, the substrate solution is to be boiled up again for a short time prior to use.
:.
L;~ ~
....
Preparation of the streptodornase B antigen DNase which had been obtained from a 16-hour culture of an A streptococci strain forming DNase B and purified according to known methods was standardized to 30 units per ml (measured in a Brookfield microrotation viscosimeter) (Calculated on DNase I from pancreas). Immediately priox to the test, the antigen solution was diluted at a ratio of l : 30.
E X A M P L E 3:
. _ Preparation of the d_lution liquid 400 ml of an aqueous solution of bovine albumin con-taining 250 mg of bovine albumin per l00 ml were mixed with l00 ml of a 0.25 M imidazole-HCl buffer of pH 8.0, containing -1.47 g of calcium chloride dihydrate and 0.6 g of magnesium sulfate per liter. ~ :
E X A M P L E 4: -: Determination of the streptodornase B antibodies in sera Determination was made as follows:
In micro titer plates, 25 /ul of the dilution liquid as : : 20 prepared in Example 3 were filled into Cup 2 of the first ~ ;series, two times 25 /ul of the same dilution liquid were : ~filled into Cup 3, and 25 /ul each of the dilution liquid were :111ed into the following cups. Subsequently, 25 /u1 each o : a~ 50 dilution of a serum taken from healthy persons were 2~5 illed into cups l, 2 and 3. Starting from cup 2, 25 /ul each of the serum~dilution obtained were conveyed by pipet to cup 4 : and ~urther to cup 6 and so on to cups 8, l0 and 12. Starting :; from oup 3, 25 /u1 each~of the-corresponding serum dilution were conveyed over to cup 5 and also further to cups 7, g and ll.
~ ~ .
~ 8 - ~
~3i6~V~ ~ HOE 73/B 012 Subsequently, 25 /ul each of the serum dilution were eliminated from cups 3, 11 and 12. Thus, startin~ Erom cup 1, a dilution series of 1 : 50, 100, 150, ~00, 300, 400, 600, 800, 1200, 1600. 2500 and 3200 was obtained. The same dilution series were made for the sera of patients to be determined in series 2 and 3 of the micro titer plate. Then, 25 /ul of the standard antigen dilution as obtained in Example 2 were added to each of the 25 /ul serum dilution and allowed to stand for 4 minutes on a shaking device. Finally, 50 /ul of the substrate solution as prepared in Example 1 were added to each of the dilutions.
The mixture was then incubated at 37C. The formation of a blue-colored precipitate indicated the dilution of the anti-bodies present in the serum, which was no longer able to bind ; the DNase added. As soon as a control serum accompanying the test mixture and having a fixed titer of 1 : 150 caused the precipitate to form up to the dilution of 1 : 150, the values were read off. In this exemplified specimen, a precipitate formed throughout the first series. ~ence, the titer was less than 1 : 50. ~n the second series, a precipitate formed in cup 4; hence, the titer was 1 : 150. In the third series, a precipitate formed starting from cup 8, corresponding to a titer of 1 : 600.
E X A M P L E 5;
Determination of desoxy-ribonuclease In the first series of a micro titer plate, 50 micro-liters of a culture solution of streptococci, which had been filtered under sterile conditions, were filled into cup 1.
The fo~lowing cups were filled with 2~ microli~ers of the dilution liquid as prepared accordi~g to Example 3.
_ g ._ .:
~ . ., . ~ . . : -. HOE 73/B 012 ;4~
By conveying 25 microliters each from the ~irst to the second cup, further from the second to the third cup and so on, a dilution series of the ~ollowing ratio was obtained: 1 : 2, 1 : 4, 1 : 8, and so on.
A DNase o~ known activity was diluted in the second series of the micro titer plate in the same manner. To 25 micro-liters each of the enzyme dilutions, 50 microliters of the substrate solution as prepared in Example 1 were added, where-upon the mixture was incubated at 40C for 3 hours. The formation of a blue-colored precipitate was taken as an indi-cator for the enzyme activity. The streptococci culture filtrate formed a precipitate up to cup 8, corresponding to a dilution of 1 : 128.
The accompanying standard DNase in this test system always caused precipitation up to cup 10, corresponding to : a dilution of 1 : 512.
The result of this test shows that the culture filtrate to be tested has a DNase activity of 25~ of the standard~
.
.
:~ :
:~::: ~ :: ::
, ' . ,.. :
- 10 -: '' ... j. : .. " ., ~, ... ~,. .. ~ . ... .. ,.,: .. . ,.. : ,.. .... .
Claims (5)
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. A process for the preparation of a solubilized complex compound of high-molecular-weight desoxy-ribonucleic acid (DNA) and 2-amino-7-dimethyl-amino-3-methyl-diphenazthionium chloride (toluidine blue 0), which comprises boiling and homogenizing an aqueous dispersion of high-molecular weight DNA and toluidine blue 0 in a weight ratio of about 2:1, until a clear homogenous blue-colored solution is obtained.
2. A process as claimed in claim 1 wherein alkali metal ions are added to the dispersion in a concentration from 0.1 to 0.5 percent.
3. A process as claimed in claim 1 wherein the concen-tration of DNA is from 0.01 to 0.03 percent and that of toluidine blue 0 is from 0.005 to 0.015 percent in said solution.
4. A solubilized complex compound of DNA and toluidine blue 0, whenever obtained according to a process as claimed in claim 1, claim 2 or claim 3 or by an obvious chemical equivalent thereof.
5. Method for the determination of the enzymatic activity of desoxy-ribonuclease which comprises contacting a sample of desoxyribonuclease with a solution as claimed in claim 1 until a blue colored precipitate is formed and then determining the enzymatic activity from this result.
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE2344444A DE2344444C3 (en) | 1972-09-08 | 1973-09-04 | Washing and high pressure jet cleaning device |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1064024A true CA1064024A (en) | 1979-10-09 |
Family
ID=5891542
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA208,328A Expired CA1064024A (en) | 1973-09-04 | 1974-09-03 | Substrate for the determination of desoxy-ribonuclease |
Country Status (1)
| Country | Link |
|---|---|
| CA (1) | CA1064024A (en) |
-
1974
- 1974-09-03 CA CA208,328A patent/CA1064024A/en not_active Expired
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