Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
HOE 73/B 012
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.
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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
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HOE 73/B 012
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-
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HOE 73/B 012
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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,
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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.
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HOE 73/B 012
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
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~ 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.
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HOE 73/B 012
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E X A M P L E 2-
....
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.
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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.
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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~
.
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