Note: Descriptions are shown in the official language in which they were submitted.
11~8S~9
REAGENT AND METHOD FOR THE DETERMINATION
OF THE HUMAN-MUSCLE TYPE ALDOLASE
This invention relates to reagents and a method for the
determination of the human-muscle type aldolase.
The aldolase is a general term of the enzymes which
catalize the aldol condensation and cleavage between dihydro-
xyacetone phosphate and a series of aldehydes.
The aldolase according to this invention is meant for a
fructose diphosphate aldolase (E.C. 4. 1. 2. 13. fructose-l,
6-phosphate D-glycelaldehyde-3-phosphate lyase) which
reversibly decomposes fructose-1,6-diphosphate into dihydro-
xyacetone phosphate and D-glycelaldehyde-3-phosphate. This
enzyme occupies the main course of glycolysis system, and
particularly, distributes mainly in muscle, thereby contri-
buting importantly to the energy metabolism.
Mammal aldolase comprises three types of isoenzymes,
that is, the muscle type (A type), the liver type (B type)
and the brain type (C type). It is known that theconstruction
ratio of these isoenzymes varies in accordance with the
metamorphosis of the living body, such as differentiation of
the fetal liver in rat, canceration of organism in human,
rat, and the like.
There have been conventionally known two methods for
determining the human-muscle type aldolase, one of which is
the electrophoresi~ method, and the other is a method which
comprises measuring the decomposition activities of two
sub~trata, that is, fructose-1,6-diphosphate (FDP) and
fructose-l-phosphate (FIP~ for the aldolase, [FDP-ALD activity
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and FIP-ALD activity], thereby determining the activity ratio
(FDP/FIP).
These methods, however, involve various problems.
In the electrophoresis method, the problem is that the
migrated points of the human aldolase isoenzymes are so
closely positioned one another that the discrimination is
difficult and the precise quantitative determination can not
be achieved.
In the measuring method of the activity ratio FDP/FIP,
it is pointed out that the operation is particularly compli-
cated for the measurement of the FIP-ALD activity, accompanied
by no quantitative determination.
Recently, a method by means of radio-immunoassay has been
reported. Comparing with the aforementioned methods, this
method is more excellent in the sensibility or sensitivity
and also in the quantitative matter. However, this method
also has some drawbacks that the reagent can not be stored
because of the quick decrement of radioisotope to be used
for labeling; special machines and apparatuses and a particu-
lar room are required for measuring the radioisotope; handling
of the radioisotope in the measurement and the disposal of
waste matters thereof are difficult, because the use of the
radioisotope is accompanied with a health hazard.
We have developed a reagent to be used for the enzyme-
immunoassay of the human-muscle type aldolase, and a method
for the determination of the human-muscle type aldolase using
~aid reagent. According to this method, various drawbacks in
the methods of prior art can be dissolved or diminished.
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1158~ ~g
The method according to this invetnion is one of the
enzyme-immunoassay, so-called "sandwich method". In this
method, measurement is effected through the following steps:
(a) contacting a specimen with an insolubilized muscle
type,aldolase antibody, followed by separation of
the solid phase;
(b) contacting the solid phase with a muscle type
aldolase antibody labeled with enzyme, followed by
separation of the solid phase;
(c) contacting the solid phase with the substrate of
said enzyme; and
(d) measuring absorbance of the decomposition product
of said substrate.
The following will illustrate more particularly this
method.
In the step (a), an antibody insolubilized by combi-
nation with a carrier is incubated together with a specimen,
such as serum, etc., so that the antigen (human muscle type
aldolase) in the specimen may re~ct with said insolubilized
antibody. After the reaction is over, the reaction solution
is removed and the solid phase is washed with buffer solution,
distilled water or the like.
In the ~tep (b),, the solid phase obtained in the step
(a) is incubated together with an enzyme-labeled antibody,
resulting in that said enzyme-labeled antibody aombines
with the antigen previously,combined with the insolubilized
antibody. After the reaction is over, the reaction solution
is removed, and the solid phase is washed with a buffer
solution, distilled water or the like.
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In the step (c), the solid phase obtained in the step
(b) is incubated together with a substrate of enzyme to
effect the enzyme reaction. After the predetermined time,
the reaction is terminated by~adding a stop solution of the
enzyme reaction thereto.
In the step (d), the absorbance of the decomposition
product of the substrate in the reaction solution of the step
(c) is measured. The measurement of the absorbance is
effected with an absorption photometer using a suitable wave
length for quantitative determination of the decomposition
product of the substrate.
The absorbance of the known amount of the standard
specimen for control is previously measured with said
measuring system so as to set up a working curve of the
amount of antigen versus the absorbance. The amount of
antigen in the specimen can be determined from said working ~-
curve by measuring the absorbance of an unknown amount of
specimen with the same measuring system.
Figs. 1-4 in the attached drawings, show working curves
of the human-muscle type aldolase obtained by the enzyme-
immunoassay in the following Example 5.
Fig. 5 shows a working curve of the human-muscle type
aldolase obtained by the enzyme-immunoassay in the following
Example 6.
Fig. 6 shows the results of determination of the human-
muscle type aldolase in the human serums obtained by the
enzyme-lmmunoassay in the following Example 7.
The following description illustrates the reagent for
determination to the used with the present method.
, ~
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A) The muscle type aldolase antibody
Antiserum is obtained by immunizing the muscle type
aldolase of a mammal such as human, rabbi~, dog, moneky,
bovine, etc. to the other animal.
The immune animal is preferably selected from the
different species of birds, because the muscle type aldolases
do not show significant immunological distinction between the
mammals.
Illustrative of the prefered birds include, for example,
fowl, turkey, duck and the like.
It is preferable to add the inactivated serum of the
animal which is the source of the muscle type aldolase, to
the resulting anti-serum, thereby removing by absorption the
other impure antibody. :~
The purification of these antiserum may be effected by
means of affinity chromatography using support combined with
the mammal-muscle type aldolase, the mammal being selected
from human, rabbit, monkey, bovine, etc., for example.
Support materials to be used for~this purpose are agarose,
bridged dextran, etc. The combined support can be prepared
by activating a support with bromocyane or the like, adding
the activated support to the solution of the muscle type
aldolase, and stirring the mixed solution at a low temperature.
The purified muscle type aldolase antibody is provided by
adding the antiserum to a column filled with said combined
Qupport, and effecting the affinity chromatography. Elution
of the mu~cle type aldolase antibody from the column may be
carried out with a weak alkaline solution.
-` ~1S85~
In this affinity chromatography, the sensitivity varies
in the measuring method of the present invention, depending
on the combination of a kind of the muscle type aldolase
combined with the support and a kind of the muscle type
aldolase used for the preparation of the anti~serum.
In the case of the human muscle type aldolase antiserum,
a preferable working curve is obtained as shown in Fig. 1 by
using the human-muscle type aldolase antibody which is prepared
by purifying with the support combined with the human muscle
type aldolase. In the cases of the rabbit muscle type
aldolase antiserum, more preferable working curves are ob-
tained by purifying with the use of the support combined
with the human muscle type aldolase and the support combined
with the bovine muscle type aldolase, than the support
combined with the rabbit muscle type aldolase as shown in
Figs. 2, 3 and 4. - --
B) The enzyme-labeled muscle type aldolase antibodY
As the enzymes to be used for the labeling, there may
be mentioned the enzymes which are generally used for the
enzyme-immunoassay, for example,~alkaliphosphatase, peroxidase,
~-D-galactosidase, glucoamylase, glucose oxydase, and the like.
The labeling may be achieved by any conventional methods,
such, Por example, as Glutalaldehyde method, Nakane method,
Maleimide method, Mixed acid unhydrides method, Carbodiimid
method, etc. In Clutalaldehyde method, the labeling is
achieved by adding enzyme to the antibody, and adding glutal-
aldehyde so that the concentration may reach to 0.2 - 0.8%,
and finally effecting the reaction at a room temperature.
The reaction ratio of the enzyme to the antibody is preferably
1 : 1 by concentration, although it may be 1 : 2 to 2 : 1.
' :' , ' " : ' :
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llS85'~g
The enzyme-labeled antibody is generally used as a
reagent by diluting it with a buffer solution and the like.
It is also desirable to add an inactivated rabbit serum in
an amount of above 20% to said diluted solution. As shown in
Fig. 4, the addition of the inactivated rabbit serum provides
more preferable working curve than those provided by a buffer
solution alone or by the buffer solution containing the bovine
serum albumin.
C) The insolubilized muscle tyPe aldolase antibody
As a carrier, there are used an insoluble solid which
is combinative with an antibody. Illustrative of such solid
include, for example, polystyrene, cellulose, agarose, glass,
bridged dextran, silicone rubber, metal, ~tc. There may be
mentioned a form such as tube, microtiter plate, powder,
sphere, disc, plate, foil, etc.
In the case of the polystyrene microtiter plate, the
antibody may combine with the wall surface of the plate,
when the antibody is properly diluted with a buffer solution
or the like, the diluted solution is then added to the plate,
and the whole is finally allowed to stand.
D) The others
As a substrate, there are used substrates of enzymes
which have been used for the enzyme-labeled antibody. When
the enzyme used is alkaliphosphatàse, there are used as
substrate~ p-nitrophenyl phosphate, ~-glycerol phosphate,
phenyl phosphate, ~-naphthyl phosphate, phenolphthalein
phosphate, or the like.
As the stop solution for the enzyme reactions, there can
be used known solutions to the respective enzymes. In the
~i585~1t9
case of the alkaliphosphatase, the suitable stop solution
is lN sodium hydroxide solution.
The above descriptions have been made to the sandwich
method, but the human muscle type aldolase according to this
invention can be also determined by the immunoenzymometrîc
assay. This method comprises reacting the enzyme-labeled
antibody with a specimen ~antigen), followed by separation
of the enzyme-labeled antibody-antigen reactant (B) from
unreacted enzyme-labeled antibody (F), and determining the~
enzyme activity of either said (B) or (F) with the substrate.
As the method for the separation of (B) and (F), there may be
mentioned the gel filtration method, the absorption method
by means of insolubilized antigen, etc. Also in determination
of the human-muscle type aldolase by means of the immunoenzy-
mometric assay, the enzyme-labeled human-muscle type aldolase
antibody as described above is used as the reagent.
Follwoing experiments and examples will further illustrate
this invention.
Experiment 1
Human muscle type aldolase anti-serum
The human-muscle type aldolase (1 mg) was dissolved in
0.5 ml of physiological salt water, and to the solution was
admixed the equal volume of Freund complete adjuvant. The
product was administered by injection to the muscle of a fowl
(White Leghorn). The human-muscle type aldolase was admini-
~tered respectively in the same procedure as described above,
two week-~, and also three weeks thereafter. One week after
the last administration, the blood was collected from the
fowl to obtain the human-muscle type aldolase anti-serum.
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1158S'~9
The normal-human serum (NHS) inactivated at 56C for
2 hours was added to this anti-serum, so that it may contain
the content of 10%. The mixture was incubated at 37C for one
hour, and allowed to stand overnight at 4C. The product
was centrifuged at 3000 rpm for 20 minutes. The supernatant
was collected to obtain the human-muscel type aldolase anti-
serum containing absorbed NHS.
Experiment ?
Rabbit-muscle type aldolase anti-serum
The rabbit-muscle type aldolase (2 mg) was dissolved in
1 ml of physiological salt water. Thereafter, the same
procedure as described in Experiment l was-repeated to obtain
the rabbit-muscle type aldolase anti-serum.
To this anti-serum, was added normal-rabbit serum (NRS)
inactivated at 56C for 3 hours, so as to contain the content
of 10%. Thereafter, the same procedure as described in
Experiment 1 was repeated to obtain the rabbit-muscle type
aldolase anti-serum containing absorbed NRS.
Experiment 3
B Human-muscle type aldolase antibody
Sepharose 4B- ~(Pharmacia Fine Chemicals AB; Agarose)
(lOg), and the human-muscle type aldolase (10 mg) were allowed
to stand at 4C overnight in 15 ml of O.lM sodium carbonate
buffer solution (pH 9.0).
~'a ~I~a de~n4 r ~
The resulting Sepharose 4B~gel combined with the human-
muscle type aldolase was packed into a column, and washed
with 0.05 M tris-hydrochloric acid buffer solution (pH 8.o)
containing 0.5 N sodium chloride. To this column were added
3 ml of the NHS-absorbed human-muscle type aldolase anti-serum
*3
obtained in the Experiment 1, and the anti-serum was flowed
out with 0.05 M tris-hydrochloric acid buffer solution
(pH 8.o) containing 0.5 N sodium chloride. Thereafter, 0.1 N
aqueous sodium carbonate solution was added to the column to
elute the human-muscle type aldolase antibody. The fraction
of antibody eluate was subjected to dialysis with 0.05 M
tris-hydrochloric acid buffer solution (pH 8.o) to obtain
3 mg of the human-muscle type aldolase antibody.
Experiment 4
Rabbit-muscle type aldolase antibody
The procedure described in Experiment 3 was repeated
except that 3 ml of the NRS-absorbed rabbit-muscle type
aldolase ar,ti-serum described in Experiment 2 were added to
the Sepharose 4B gel combined with the rabbit-muscle type
aldolase. The rabbit-muscle type aldolase anti-cerum (3 mg)
was obtained.
Experiment 5
Rabbit-muscle type aldolase antibody
The same procedure as described in Experiment 3 was
repeated except that 3 ml of the NRS~absorbed rabbit-muscle
type aldolase anti-serum described in Experiment 2 were added
to the Sepharose 4B gel combined with the human-muscle type
aldolase. The rabbit-muscle type aldolase antibody (3 mg)
was obtained.
Experiment 6
Rabbit-muscle type aldolase antibody
The same procedure as described in Experiment 3 was
repeated excep~ that 3 ml of the NRS-absorbed rabbit-muscle
type aldolase anti-serum described in Experiment 2 were added
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to the Sepharose 4B gel combined with the bovine-muscle type
aldolase. There were thus obtained the rabbit-muscle type
aldolase antibody (3 mg).
Experiment 7
Insolubilized human-muscle type aldolase antibody
Into each of the holes provided on a polystylene
microtiter plate, 200Jul of 0.05 M tris-hydrochloric acid
buffer solution (pH 8.o) containing 25Jug/ml of the human-
muscle type aldolase antibody described in Experiment 3 was
added, and then allowed to stand overnight at 4C. The solu-
tion was removed from the plate. The plate was washed with
distilled water to obtain the microtiter plate combined with
the human-muscle type aldolase antibody.
Experiment 8
Insolubilized rabbit-muscle type aldolase antibody
Into each oP the holes provided on a polystylene
microtiter plate, 2OOJU1 of 0.05 M tris-hydrochloric acid
buffer solution (pH 8.o) containing 25~ug/ml of the rabbit-
muscle type aldolase antibody described in Experiment 4 was
added, and then allowed to stand overnight at 4C. The
solution was removed from the plate. The plate was washed
with di~tilled water to obtain the microtiter plate combined
with the rabbit-muscle type aldolase antibody.
Experiment 9
Insolubilized rabbit-muscle type aldolase antibody
Into each oP the holes provided in a polystylene
microtiter, 200Jul of 0.05 M tris-hydrochloric acid buffer
solution (pH 8.o) containing 25 ~g/ml oP the rabbit-muscle
type aldolase antibody described in Experiment 5 was added,
i~585 ~9
and then allowed to stand overnight at 4C. The solution
was removed from the plate. The plate was washed with
distilled water to obtain the microtiter plate combined with
the rabbit-muscle type aldolase antibody.
Experiment 10
Insolubilized rabbit-muscle type aldolase antibody
Using the rabbit-muscle type aldolase antibody which
was obtained in Experiment 6, there was obtained the micro-
titer plate combined with the rabbit-muscle type aldolase
antibody, in a similar manner as described in the preceding
Experiment 9.
Example 1
Alkaliphosphatase-labeled human-muscle
type aldolase antibody
Into 0.3 ml of water containing 1 mg of alkaliphosphatase
(relative activity 1000 unit/mg), were added 0.2 ml of 0.05 M
phosphoric acid buffer solution (pH 7.0) containing 1 mg of
the human-muscle type aldolase antibody described in Experi-
ment 3 and 50Jul of 2.5% glutalaldehyde solution. The mixture
was allowed to stand for 30 minutes at the room temperature,
followed by subjection to di~alysis with 0.05 M tris-hydro-
chloric acid buffer solution (pH 8.0) overnight. The
alkaliphosphatase-labeled human-muscle type aldolase anti-
body wa~ obtained.
Example 2
Alkaliphosphatase-labeled rabbit-
muscle type aldolase antibody
The same procedure as described in Examp`le 1 was re-
peated, except that 1 mg of the rabbit-muscle type aldolase
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~S8S~
antibody described in Experiment 4 was used with 1 mg of
alkaliphosphatase. The alkaliphosphatase-labeled rabbit-
muscle type aldolase antibody was obtained.
ExamPle 3
Alkaliphosphatase-labeled rabbit-muscle
type aldolase antibody
The same procedure as described in Example 1 was re-
peated, except that the rabbit-muscle type aldolase antibody
described in Experiment 5 was used with 1 mg of alkaliphos- -~
phatase. The alkaliphosphatase-labeled rabbit-muscle type
aldolase antibody was obtained.
Example 4
Alkaliphosphatase-labeled rabbit-muscle
type aldolase antibody
The same procedure as described in Example 1 was re-
peated, except that the rabbit-muscle type aldolase antibody
described in Experiment 6 was used with 1 mg of alkaliphos-
phatase. There was thus obtained the alkaliphosphatase-
labeled rabbit-muscle type aldolase antibody.
ExamPle 5
Enzyme-immunoassay
A standard antigen solution of 500 ng/ml - 7.6 mg/ml was
prepared by diluting the human-muscle type aldolase serially
two-fold with the normal rabbit serum inactivated at 56C for
2 hours. Said standard solution (0.1 ml) was added to the
microtiter plate combined with the muscle type aldolase
antibody, then stood for 60 minutes at 37C. The reaction
~olution was removed. The-plate was washed 4 times with
distilled water. On the other hand, the alkaliphosphatase-
'. : . .
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labeled musele type aldolase antibody was diluted to 150-fold
with 0.05 M tris-hydrochloric acid buffer solution (pH 8.0)
containing 30% of the normal rabbit serum inactivated at
56C for 2 hours. Said diluted solution (0.1 ml) was added
to said plate, and then allowed to stand for 60 minutes at
37C. The reaction solution was removed. The plate was
washed 4 times with distilled water. A solution of
5 mg/ml of p-nitrophenyl phosphate in o.i M sodium carbonate
buffer solution (pH 9.0) containing 0.001 M magnesium
chloride was prepared separately. This solution (0.1 ml) was
added to said plate and allowed to react at 37C for 60
minutes, followed by addition of 0.1 ml of 1 N sodium hydro-
xide to terminate the reaction. The reaction solution was
diluted to 10-fold with distilled water. Absorbance on 405
m~u was measured with a spectrophotometer to set up the working
curve between the absorbance and the concentration of the
human-muscle type aldolase.
Fig. 1 shows the working curve in the case of the above
mentioned assay sy~tem, wherein the microtiter plate combined
with the human-muscle type aldolase antibody in Experiment 7
was used as a microtiter plate combined with muscle type
aldolase antibody, and the alkaliphosphatase-labeled human-
muscle type aldolase antigen in Example 1 was used as an
alkaliphosphatase-labeled muscle type aldolase antigen, res-
pectively.
Fig. 2 shows the working curve in the case where the
mlcrotlter plate comblned with the rabbit-muscle type
aldolase antibody in Experiment 8 was used as~a microtiter
combined with muscle type aldolase antibody, and the alkali-
14
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1~58~g
-
phosphatase-labeled rabbit-muscle type aldolase antibody
in Example 2 was used as an alkaliphosphatase-labeled muscle
type aldolase antibody, respectively.
Fig. 3 shows the working curve in the case where the
microtiter plate combined with the rabbit-muscle type
aldolase antibody in Experiment 9 was used as a microtiter
plate combined with muscle type aldolase antibody, and the
alkaliphosphatase-labeled rabbit-muscle type aldolase
antibody in Example 3 was used as an alkaliphosphatase-
labeled muscle type aldolase antibody, respectively.
Fig. 4 shows the working curve in the case where the
microtiter plate combined with the rabbit-muscle type
aldolase antibody in Experiment lO was used as a microtiter
plate combined with muscle type aldolase antibody, and the
alkaliphosphatase-labeled rabbit-muscle type aldolase
antibody in Example 4 was used as an alkaliphosphatase-
labeled muscle type aldolase antibody, respectively.
In Figs. 1-4, the horizontal axes indicate the con-
centration (ng/ml) of the human-muscle type aldolase, and
the vertical axes indicate the absorbance on 405 m~u
( OD405 mJU )
As shown in Figs. 1-4, the most preferable working curve
is obtained in the insolubilized antibody and the enzyme-
labeled antibody wherein the human-mùscle type aldolase
antibody (Fig. l) is used, and followed by the order of the
insolubilized antibody and the enzyme-labeled antibody wherein
the rabbit-muscle type aldolase antibody (Figs. 4, 3 and 2)
is used. It is noted that, when comparing thè cases wherein
the rabbit-muscle type aldolase antibody is used, the more
ilS8~
preferable working curves are respectively obtained in the
case (Fig. 4) wherein the support combined with the bovine-
muscle type aldolase is used, as well as well as the case
(Fig. 3) wherein the support combined with the human-muscle
type aldolase is used, for the purification of the antibody
by means of affinity chromatography, than the curve obtained
in another case (Fig. 2) wherein the support combined with
the rabbit-muscle type aldolase is used.
Example 6
Enzyme-immunoassay
Effects of the type of diluent on the enzyme-labeled
antibodies were studied by means of the immunoassay system
according to Example 5.
There were used the alkaliphosphatase-labeled human-
muscle type aldolase antibody described in Example 1 as an
enzyme-labeled antibody, and the microtiter plate combined
with the human-musGle type aldolase described in Experiment
7 as an insolubilized antigen.
Following diluents were used in this Example:
(A) 0.05 M tris-hydrochloric acid buffer solution
(pH 8.o) containing 30% concentration of the
normal rabbit serum inactivated at 56C for 2
hours.
(B) 0.05 M tris-hydrochloric acid buffer solution
(pH 8.o) containing 1% concentration of the bovine
serum albumin inactivated at 56C for 30 minutes.
(C) 0.05 M tris-hydrochloric acid buffer solution
(pH 8.o) as control.
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The enzyme-labeled antibodies diluted with each of such
diluents to 150-fold respectively were used. The working
curve was set up by the same procedure as described in Example
5 using the standard antigen-$olution. Each working curve
obtained is shown in Fig. 4. The horizontal axis in Fig. 4
indicates the concentration of the human-muscle type aldolase
(ng/ml) and the vertical axis indicates the absorbance on
405 mJu (D40s mu) The symbols A, B and C indicate the
respective working curves obtained in each case where the
diluents (A), (B) and (C) are used respectively. As shown
in the drawing, the most preferable working curve is working
curve A which is obtained in the case where the diluent
containing the inactivated rabbit-serum is used.
Example 7
Enzyme-immunoassay
Determination was achieved for the concentrations of the
human-muscle type aldolase in the serums of various cancer
patients, of benign ill patients and of healthy humans by
means of the immunoassay system as described in Example 5.
The enzyme-labeled antibody useq in this Example was
the alkaliphosphatase-labeled human-muscle type aldolase
antibody described in Example 1, and the insolubilized
antibody used was the microtiter plate combined with the
human-muscle type aldolase described in Experiment 7. The
serum of specimen was directly used without diluting.
Results are shown in Fig. 6. As shown in the drawings,
the human-muscle type aldolase tends to be contained in the
~erum of the cancer patients in a higher concèntration than
those in the other benign ill patients and those in the
healthy human.
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