Note: Descriptions are shown in the official language in which they were submitted.
f' ;
1 106Zt~0~ ~
The invention relates to a method, a ~ompo-
sition and a test ki.t for deter1rlining the ac-~ivi-ty of
creatinekinase-MB in human body fluids.
The determination of the activity of creatinekinase
S (ATP: creatine-phosphotransferase, E.C. 2.7.3.2; abbreviation:
CK) in serum is considered the most sensitive laboratory method
for diagnosing diseases of skeletal muscles and the myocardium,
~"~
especially myocardial infarction. However, differentiation ~,~
between trauma of skeletal muscles and the myocardium is dif~
cult, especially in making a differential diagnosis of myocardial
infarction. Determination of total CK activity results in
unreliable differentiation.
Prior attempts to improve the reliability of evidence
provided by determination of CK activity in differential diag- ~ ~
nosis have used measurements of activity of other enzymes in the ~ "
serum and correlation of the resulting measurement with each
other, for example, quotient CK/glutamic oxalacetic transaminase.
However quotients of this type cannot beused to differentiate
between cardiac infarction and pulmonary infarction or between
cardiac infarction and secondary shock resulting from other
causes. ~ ,
CK occurs in the body in the form of three isoenzymes,
namely CK-MM, for example, in muscles; CK-BB, for example, in
i
the brain; and hydrid CK-MB, consisting of an M and a B subunit,
for example, in the myocardiumO CK activity in blood serum
.. . ._
10ti260~
is normally due to the CK-MM isoenzyme, because CK-BB does not
pass through the fluid/blood barrier and CK-MB is restricted to
certain organs, for example, the myocardium. However, when the
myocardium is damaged, as in cardiac infarction, CK-Ms is
released into the blood serum and can be detected there.
Quantitative determination of this isoenzyme along
with CK-MM in the serum is considered the most sensitive
laboratory method and provides the greatest evidence in differ-
ential diagnosis of cardiac infarction. It is true that CK-MB
10 is present in other organs, for example, the pancreas, the dia- ~
phragm, the aorta, the lungs and the uterus, as well as in the -
myocardium but the activity thereof in these organs is about
100 times less than in the myocardium, so that any CK-MB ~;
activity liberated from these other organs is below the
limits of detection.
Determination of CK-MB activity previously was based
essentially on one of three methods:
1. Electrophoresis on various carriers. The
results obtained by this method are sometimes contradictory.
Frequently the number of bands appearing is greater than the
number of isoenzymes present, that is, artifacts cause unre-
liability.
2. Column chromatography on various materials. This
method is time-consuming, has an actual operating time of several
hours and ls therefore not suitable for routine investigations.
Results obtained by different investigators are sometimes
contradictory.
3. Immunological determination by antibodies causing
precipitation. This method, described in German Patent Appli-
cations P 21 28 670, (U.S. 3,932,221), and P 22 58 822,
(U.S. S.N.~419,283, filed November 27, 1973) gives good results, for
- 2 -
1C~6Z609
example, in the quantitative determination of aldolase and
alkaline phosphatase isoenzymes. However, the sensitivity
of the method is inadequate for determination of relatively
low activities of CK and especially of CK-MB.
In the processes described in German Patent Applications
P 21 28 670 and P 22 58 822, the isoenzymes of CK are pre-
cipitated by antibodies which effect precipitation and act
specifically on isoenzymes. In each case the residual
activity in the supernatant liquor from the immune precipita-
tion is determined. Apart from the effort involved in this
method, which as a rule necessitates the preparation of
several specific antisera, it is necessary to carry out at
least two different tests, that is, determination of total
CK activity and determination of residual CK activity after
precipitation. Thus, the CK-MB activity can be determined
only by measuring a difference. The result is therefore
subject, in accordance with the rules of the theory of errors,
to the uncertainty of both measurements. In the method of
the invention, the addition of errors is avoided by making
a direct measurement.
Another disadvantage of the precipitation process is
that the immune precipitation, which is a secondary reaction,
takes from about 60 minutes to several hours, so that the
process is not suitable as a rapid test.
..
3LO f';
(~w:ing to -the relatively long time
required to carry out all the above processes, they are
not suitable for rapid diagnosis of cardiac infarction.
Thus, there is a continuing need for a
simple, rapid and reproducible process and composition for
determining the activity of CK-MB in a sample of a body
fluid.
Aceording to one aspect of this invention
a procedure is provided which operates by using specifie
antibodies substantially completely to enzymatlc activity
of the M subunit in CK-MM and CK-MB without substantially
inactivating enzymatic activity of the B subunit in any
CK-MB which may be presènt.
In one aspect, this invention provides
a method for determining the enzyma*ic activity of
creatinekinase-MB in a biologieal liquid sample, which
comprises the steps of:
(a) incubating the creatinekinase-containing
sample with antibodies which completely inhibit the
enzymatic activity of the M subunit of creatinekinase (CK)
isoenzymes MB and MM in solution without precipitation but
whieh do not inhibit the enzymatic activity of the B subunit
of any creatinekinase-MB which may be present in the
sample to immunologieally inhibit the M subunit thereof,
wherein eomplete inhibit.ion of said enzymatic activity of
said M subunit leaves less than 5 U./l. of said enzymatic
activity intact and non-inhibition of said enzymatie activity
of said B subunit is less than 10 U./l.; and then
:~ `; .
-
1~i2609
l,) determi~ g th~ ellzym,~ti~ ~IC~iVity
of the crea-tinekinase B subun:it in the resultant sample.
By one variant, the enzymatic activity jJ
is determined photome-trically in step (b).
By another variant, the biological liquid
sample is blood serum and~le blood serum and antibodies
are incubated in the presence of a CK substrate.
By yet another variant, the method includes
a preliminary step, prior to incubation of said sample with
said antibodies, of determining total CK activity in said
sample.
By still another variant, the complete
inhibition of said enzymatic activity of said M subunit
leaves less than 3 U./l. of said enzymatic activi-ty lntact
and wherein said non-inhibition of said enzymatic activity
of said B subunit isinhibition of less than 5 U.~l.
By a further variant~ the antibodies
are able to inhibit completely up to 2,500 U./l. of
M subunit of creatinekinases MM and MB in said sample.
By yet a further variant, the biological
liquid sampIe is blood serum and the blood serum and antibodies
are incubated in the presence of a CK substrate; wherein
~said complete inhibition of said enzymatic activity of said
M subunit leaves less than 3 U./l. of said enzymatic activity
intact and non-inhibition of said enzymatic activity of
said B subunit is inhibition of less than 5 U./l.; wherein
said antibodies are anti-human skeletal muscle CK-MM from
goats; wherein said antibodies are able to inhibit completely
up to 2,500 U./l. of M subunit of creatinekinases MM and MB
in said sample and including a preliminary step, prior to
.,
~ 5-
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l~Z60~
incubation of said sample with said antibodies, of
determining total CK ac-~ivity in said sample.
By s-till another variant, -the biological
liquid sample, e.g. blood serum is derived from a patient
suspected of a myocardial infarction.
By yet another variant, the antibodies
are substan-tially free of CK-BB isoenzyme activity and
have a molecular weight of 130,000-21,000 and a sedimentation
constant between 6 S and 8 S; and said antibodies are
able to effect complete inhibition of said enzymatic activity
of said M subunit in CK-MM and CK-MB without inhibiting said
enzymatic activity of said B subunit, even in -the presence
of CK substrates.
By a variation thereof, the molecular
weight is about 160,000 and the sedimentation constant is
about 7 S.
In an~ther aspect, this invention provides r
a composition for determining the activity of creatinekinase-
MB in a liquid biological sample comprising antibodies,
being substantially free of CK-BB isoenzyme activity and
being able to inhibit completely the enzymatic activity of
the M subunit of creatinekinases MM and MB in solution without
precipitation and without inactivating the enzymatic activity
of the B subunit of any creatine]cinase-MB in the sample,
wherein complete inhibition of said M subunit leaves less ~ ;
than 5 U./l. of said enzymatic activity and non-inhibition
of said enzymatic activity of said B subunit is less than
10 U./l., and an enzyme-coenzyme and substrate reagent for
determining CK activity.
, ~ -5a~
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i2609
13y olle variant- -tllereor, the <~ntiboclies
are able to effect complete inhibition of said enzyma-tic
activity of said M subunit in CK-MM and CK-MB without
inhibiting said enzymatic activity of said B subunit,
even in the presence of CK substrates.
By yet another variant, the an-tibodies are
anti-skeletal muscle CK-MM obtained by immwlization of
goats. t
By still another variant, the antibodies
are substantially free of CK-BB isoenzyme activity and have
a molecular weight of 130,000 - 210,000 and a sedimentation
constant between 6 S and 8 S; and said antibodies are able
to effect complete inhibition of said enzymatic activity
of said M subunit in CK-MM and CK-MB
without inhibiting said enzymatic activity of said B subunit,
even in the presence of CK substrates.
By a still further variant, the molecular
weight is about 160,000 and the sedimentation constant
is about 7 S.
By one further aspect of this invention, a i-
test kit is provided for the determination of the activity
of creatinekinase-MB in a biological sample, which comprises-
(a) antibodies being substantially free of CK-BB isoenzyme
activity and being able to inhibit completely the enzymatic
activity of the M subunit of creatinekinases MM and MB in
solution without precipitation and without inactivating the
enzymatic activity of the B subunit of any creatinekinase-
MB in the sample, wherein complete inhibition of said M
subunit leaves less than 5 U.~l. of said enzymatic activity
intact and non-inhibition of said enzymatic activity of
said B subunit is less than 10 U./l., and (b) an enzyme-
.- ~ - - , . . ................ , . . - ~ .
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1~6Z609
coenzyllle an~ su~s~r~ate reagenl. ~`or ~CterJninillg Cl<
activity.
By yet another aspect of this invention,
a test kit is provided for the determination of the activity
of creatinekinase-MB in a biological sample, which comprises:
(a) antibodies being substantially free of CK-BB isoenzyme
activity and being able to inhibit completely the enzymatic
activity of the M subunit of creatinekinases MM and MB in
solution without precipitation and without inactivating
the enzymatic activity of the B subunit of any creatinekinase-
MB in the sample, wherein complete inhibition of said M
subunit leaves less than S U./l. of said enzymatic activity
inta~t and non-inhibition of said enzymatic activity of
said B subunit lS less than 10 U./l.; (b) an enzyme-
coenzyme and substrate mixture comprising disodium creatine
phosphate hexahydrate, reduced glutathione or N-acetyleysteine,
disodium adenosine diphosphate hexahydrate, disodium nicotin-
amideadeninedinucleotide phosphate, disodium adenosine t
~ monophosphate, hexokinase, glucose-6-phosphatehydrogenase,
: 20 glucose and magnesium acetate; and (c) a buffer solution
of triethanolamine or imidazole acetate. ~-
By yet another aspect of this invention,
~- ~ antibodies are provided which are capable of completely
inhibiting the enzymatic activity of the M subunit of
: creatinekinases MM and MB withou* inactivating the enzymatic
~:~ activity of the B subunit of any creatinekinase-MB in
the sample, wherein complete inhibition of said M subunit
leaves less than 5 U./l. of~said enzymatic activity intact
; a~nd non-inhibition of said enzymatic activity of said B
subunit is less than 10 U./l., wherein said antibodies are
~ -5c-
.... ~ . ,, . . , ~
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. - , , ~ , . . .
~ 106;2~0~
s~lbs-tal~t;ally fre~ of CK-BB i;oenzyme activity and
have a molecular weight of 130,000 - 210,000 and a
sedimentation constant between 6 S and 8 S.
By yet another aspect, a process is
provided for the preparation of such antibodies, by the
steps of activating a CK-MM antigen by an activator for
-SH groups selected from N-acetylcysteine, mercaptoethanol,
dithioerythritol, glutathione, cysteine, dithiothreitol,
S-(2-aminoethyl)-isothiouronium bromide hydrobromide or
thioglycolic acid or a divalent metal ion ac-tivator
selected from magnesium,manganese, calcium or eobalt, or
a mixture of activators; inoculating an animal with the
thus-aetivated CK-MM antigen aetivity; withdrawing blood
from the animal; and isolating said an-tibodies from the
withdrawn blood.
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... . . .. .. . . . . .... . .
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` 106Z6~9
A sui-table body fluid for tlle method
of an aspect of the invention is human serum. It is also
possible to use biological liquids, e.g., whole blood,
plasma, lymph, urine, sputum and sweat of humans, or other
animals, as well as enzyme preparations, for the determination.
CK-BB interferes with the method of an aspect of the
invention and therefore must not be present in the
biological fluids being tes-ted.
Antibodies according to yet another
aspect of this invention requiredIfor the method of
an aspect of~the invention are obtained from animals
inoculated with CK-MM antigens from skeletal muscle
tissue. Human CK-MM is preferably used as antigen. CK-MM
from animals can be used if the antisera are able to inhibit
completely enzymatic activity of the M subunit in human
CK-MM and CK-MB, if neeessary, in the presence of CK
substrates, without inactivating enzymatic activity of the r
B subunit in any CK-MB which is present. Animals donors
of CK-MM antigens are, for example, various species of
monkeys, preferably Rhesus monkeys and chimpanzees;
domestic animals, e.g., pigs, horses, cattle, rabbits
and guinea-pigs; and other animals, e.g., rats and mice and
birds, e.g., geese, ducks and hens.
The CK-MM antigen used to produce the
antibodies should be free from CK-MB and CK-BB activity.
A sensitive criterion of this purity requirement is
immunological analysis advantageously carried out by
diffusion of electrophoresis techniques. In addition,
analytical disc electrophoresis and polyacrylamide-gel
electrofocussing are useful. Purity with respect to CK-MB
~A~
..... . . . . . .. . . . . . . .... ... . . . . ..
.
- .. . . .
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106Z60~
ancl CK-BI~ takes prececlence o~er absolute puri-ty with
respect to other proteins, wh; ch, for
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r~
~6Z609
example, can be determined by the two latter methods.
Microheterogeneity of types of CK-isoenzymes manifested,
for example, in slight differences in amino acid composition
of theindividual isoenzymes, is as a rule of no importance
as a criterion of purity.
Animals used for the immunization are those which,
after inoculation with activated CK-MM, form antibodies which
are able completely to inhibit enzymatic activity of the M
subunit in the creatinekinases MM and MB without inactl- i
vating the enzymatic activity of the B subunit in CK-MB.
Goats are preferred, particularly for production of antibodies
able to effect complete inhibition of the M subunit in CK-MM
and CK-MB, even in the presence of CK substrates. However,
other animals, especially vertebrates, can also be used as ~-r,~,
antibody donors, for example, species of monkeys, horses,
cattle and cattle-like animals, sheep, dogs, pigs, rabbits,
birds, e.g., hens, turkeys, geese and ducks, and also rats, ~;~
1. ,.. ~.
mlce and guinea-pigs.
It is possible to activate the antigen by treating
CK-MM with activating reagents which stabilize and activate
SH-groups and/or with divalent metal ions. Conventional
dialysis methods are preferably utilized. ~t is thus possible,
for example, to dialyze the antigens employed against a buffered
solution containing the activating substances during a period
of 1-200 hours, preferably during 12 hours, at tempera-
tures of between l and 30 C., preferably at 4 C.
...~
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106260g ,__
Suitably, the dialysis solution is based on physiological sodium
chloride solution. In principle, any buffer suitable for
setting a p~ of 6.5 - 7.5, preferably 6.8 - 7.0, can be used.
In addition to sodium phosphate buffers, triethanolamine (TRAM), ~-i`;;
tris(hydroxyethyl)-amine (TRIS), and imidazole buffer p~ 6.8,
can be used. The solution containing the antigen is to con-
tain 10-500 millimoles of the compound which stabilizes
and activates SH-groups and/or 50-500 millimoles of the
divalent metal ions per milligram of C~-MM.
Immunization of the test animals is carried out with
activated human or animal CK-MM. Activation of CK-MM can be
~ffected by known reagents which stabilize and activate SH
~_ .
groups and/or by divalent metal ions, preferably by a combina-
tion of reagents and metal ions. Examples of preferred reagents l f
r;~ i
which stabilize and activate SH groups are N-acetylcysteine, .
mercaptoethanol and dithioerythritol, as well as glutathione,
cysteine, dithiothreitol, S-(2-aminoethyl)-isothiouronium
bromide hydrobromide (AET) and/or thioglycollic acid.
Divalent metal ions originate from corresponding
water-soluble salts, for example, chlorides or acetates, ~ ;
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1~6Z609
preferably of magnesium as well as manganese, calcium and/or
cobalt. Activators of this type are known in principle in
other fields and those skilled in the art are conversant
with them.
Subsequent immunization and separation to obtain anti-
sera or`antibodies are carried out in a known manner. The
processing and storage of the antisera or antibodies is
also carried out by methods known in immunology.
Antibodies used for the method of aspects of this in-
vention are preferably of the IgG immune globulin (bivalent
antibodies) category. Their molecular weight is between
130,000 and 210,0~0, preferably 160,000. The approximate
sedimentation constant is between 6 S and 8 S, preferably
7 S, as determined by analytical ultracentrifugation. Their
carbohydrate content is 3% of total weight, determined by
the methods described in "Handbook of Experimental Immunology"
ed. by D.M. Weir, 2nd edition 1973, pages 10.45 - 10.58,
Blackwell Scientific Publications. Monovalent IgG fragments
"
(=Fab) and IgM antibodies can also be used according to the
method of aspects of this invention.
The antibodies used should completely inhibit enzyme
activity of M subunit of creatinekinase. In this context,
!'complete inhibition" means inhibition with which, on an
average, at most 5 U./l. and preferably less than 3 U.jl.
of the enzyme activity of the M subunit in CK-MM and CK-MB
remain intact in a sample, as determined by conventional
enzyme activity tests.
-8-
. . .
1~t;260~
The antibodies should have no ef~ect on enzymatic
activity of the B subunit in CK-MB. In this contextl this
means that at most 10 U./l., and preferably less than 5 U./l.,
of the enzyme activity of the B subunit in CK-MB are inhibited
in a sample, as determined by conventional enzyme activity
tests.
In a preferred embodiment of the process, in which the
sample of body fluid being analyzed and the antibodies are
incubated in the presence of CK substrates, the antibodies
should be able to develop their inhibitory action agsinst
the en~ymatic activity of the M subunit in CK-MM and CK-
~to the maximum, even in the presence of CK substrates, without
influencing enzymatic activity of the B subunit in CK-MB.
- This property is present, in addition to properties charac-
terized above, in, for example, antibodies which are obtained
from goats by immunization with fully activated CK-MM. This
requirement is not absolutely essential in carrying out the
method according to an aspect of this invention in the normal
manner by first incubating with antibodies and then adding the
CK substrate and measuring the residual activity of CK in the
resultant sample.
CK substrates which can be employed are the substrates
and effectors customarily used. Creatine or creatine phosphate,
respectively, adenosine triphosphate or adenosine diphosphate,
respectively, and magnesium ions are preferred.
Determination of the activity or the residual activity
of CK and of its isoenzymes can be carried out by any processes
which are rapid and precise, for example, photometry. This is
carried out after auxiliary reactions.
~`
106Z609
It is also possi ~e to use colorimetric methods,
as described, for example, in "Methoden der enzymatischen
Analyse", edited by H.U. Bergmeyer, 3rd edition (1974),
Volume 1, page 145 et seq. Kinetic methods in which enzyme
activity is determined by measurement in VV at, for example,
334, 340 or 366 nm are preferred for this purpose. Especially
preferred is a standard method in which CK is determined
using creatine phosphate and adenosine diphosphate, Z. Klin.
Chem. Rlin. siochem.~ Volume 8, page 658 et seq. (1970) and
Volume 10, page 182 (1972). Test packs for determining CK
activity by this method are available commercially.
CK can also be determined fluorometrically.
Creatine is liberated from creatine phosphate by CK and this
creatine can be measured fluorometrically in a process developed
by R.B. Conn, Clin. Chem., Volume 6, page 537 et seq. (1960),
by reaction with ninhydrin in a strongly alkaline solution. ,.
See also, Sax-et al, Clin. Chem., Volume 11, page 951 et seq.
( 1 9 6 5 ) ~
In a typical embodiment of the method of an aspect of the
invention, C~-MM antibodies are added to a sample of body fluid
being analyzed, preferably a sample of human serum, in an amount
sufficient to inhibit completely up to 2,500 U./l., preferably
1,000 U./l., of the M subunit in CK-MM and CK-MB,
With body fluids having higher total CK activities, preliminary ~T
dilution to 1,000 U./l. is appropriately carried out
before actual determination and taken into account in the calcu-
lations. ~he mixture thus prepared is incubated for
to 30, preferably 5, minutes at temperatures between +10
and +40 C., preferably at about room temperature, most
-- 10
.
' , ~ ' .
106Z~(~9
preferably, 25 or 30 C. Residual enzyme activity of the
reaction mixture is then determined by a known process,
preferably the UV method described above.
The serum/antibody mixture is then added to a known
enzyme-coenzyme/substrate mixture containing all the enzymes,
coenzymes and substrates necessary for carrying out the method
and an adequate amount of a buffer solution, pH 7,is
then added. Customary commercially available formulations
contain, for example, hexokinase, glucose-6-phosphate-dehydro-
genase, adenosine diphosphate and nicotinamide-adenine-
dinucleotide-phosphate as the enzyme-coenzyme mixture and
creatine phosphate and glucose as substrates.
'
~;;.'~'
.
The serum/antibody mixture can be added to a mixture
of the coenzyme and the enzyme, or vice versa, and then a buffer/
substrate mixture can be added. Buffers which are suitable
for the reaction are neutral buffers, for example, triethanol-
amine, imidazole acetate, morpholinepropanesulfonic acid and
morpholineethanesulfonic acid buffers. Triethanolamine and
imidazole acetate buffers are preferred.
The mixture is prepared and inc~bated for 1 to
10, preferably 5, minutes at 15 - 40 , preferably at 25 or 30
C. and the change in extinction is determined at about room
temperature. The activity of the subunit B in CK-MB is cal- P
culate~.
- 11 - !
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1~;)6Z60~
In a pre~erred embodiment, a sample of the bioloyical
fluid being analyzed is incu~ated together with antibodies
and CK substrates in the presence of a buffer and the
substances required for the identification reaction without
5 previous incubation of the sample with the antibodies. ~h
In this embodiment, the antibodies used must
completely inhibit enzymatic activity of the M subunit in
CK-MM and CK-MB even in the presence of CK substances.
Antibodies having this property are, for example, obtained
lO from goats using fully activated CK-MM. The antibodies ~ A'
enable the method of an aspect of the invention to be carried out
in a simple and rapid manner.
For example, antibodies previously lyophilized with
the known coenzyme/enzyme/substrate mixture used for the
15 identification reaction are dissolved in a selected amount .~
of buffer solution. Body fluid, for example, serum, being r
analyzed is added and determination of the activity of the '`~
~.
B content of CK-MB is made. In a variation of this process,
the antibodies are incorporated into the lyophilizate with a
20 mixture consistiny only of coenzymes and enzymes. The
substrates are then added to the buffer solution.
In a further preferred embodiment, simultaneous
determination of total CK activity and of CK-MB activity càn
~'
be carried out 1n a single batch by the preferred process just
25 described. Thus,
(1) total CK activity of the sample is first deter-
mined by a known photometric process;
(2) a lyophilizate consisting of CK-MM antibodies ~
is dissolved in water and added to the same batch;: -
.
- 12 -
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~' .
10~i2~0~ 1
(3) The mixture is then incubated at C. for ~
r~,., ~,
1 to 10, preferably 5, minutes and residual activity
of the sample is determined photometrically.
In this embodiment, the CR-MM antibodies must
5 completely inhibit enzymatic activity of the M subunit in ,~
~P~
CK-MM and CK-MB, even in the presence of CK substrates. P;~
In this preferred embodiment, antibody capacity
of the antisera can be adiusted so as completely to inhibit
up to 2,500 U./l., preferably 1,000 U./l., of the
10 M subunit in CK-r~M and C~-MB. If total CK activities of ~!~
the sample being analyzed are too high for this inhibitory
capacity of the antibodies, preliminary dilutions must be
carried out, for example, to activities of M subunit in
CK-MM and CK-MB of 1,000 U./l. i~.
In a most preferred embodiment, the method of an aspect of
this invention is the foregoing, wherein said biological
fluid is blood serum and the blood serum and antibodies
are incubated in the presence of a CK substrate; including a
preliminary step, prior to incubation of said sample with
said antibodies, of determining total CK ac~ivity in said
sample; complete inhibition o~ said enzymatic activity of
said M subunit leaves less than 3 U./l. of said enzymatic activity
intact and non-inhibition of said enzymatic activity of
said B subunit is inhibition of less than 5 U./l.; said
antibodies are anti-human skeletal muscle CK-MM from goats;
and said antibodies are able - completelyto inhibit up to
2,500 U./l. of M subunit of creatinekinases MM and MB in
said sample.
- 13 -
~06Z609
~. ,~;,
In a most preferred embodiment, the preferred
method above is used to determine CK-B isoenzyme activity
in an improved method of diagnosing myocardial infarction.
In a most preferred embodiment, the composition
5 of an aspect of thi.s invention is one wherein -the antibodies ~ .
are able to effect complete inhibi-tion of the enzymatic activity of r~--
the M subunit in CK-MM and C~-MB without inhibiting
the enzymatic activity of the B subunit, even in the pre-
sence of a CK substrate; and the antibodies are anti-skeletal
muscle CK-MM obtained from goats.
In a preferred embodiment, the test kit of another aspect of this r
invention is one in which (b) the CK substrate comprises
disodium creatine phosphate hexahydrate, reduced glutathione
or N-acetylcysteine, disodium adenosine diphosphate
hexahydrate, disodium nicotinamideadenine dinucleotide
phosphate, disodium adenosine monophosphate, hexokinase, ~~
glucose-6-phosphate dehydrogenase, glucose and magnesium
acetate; and said kit further contains (c) a buffer solu-
tion of triethanolamine or imidazole acetate.
The me-thod of aspects of the inven-tion has several advantages
over known art, including highly precise results and the
rapidity and simplicity with which the process is carried out.
The precision of the metho~ of aspec-ts of the Lnvention is
thought to.be due to the use of antibodies which act specific- f
25. ally on the M subunit of CK-MM and CK-MB and therefore enable
the CK-MB activity in body fluids, e.g., human serum, to be
determined directly.
- 14 -
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.
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1~6260~
Inhibiting antibodies against CK isoenzymes have also
been described in Clin. Chem. Acta, Volume 58, pages 223-232 r
(1975). In addition to a 100~ inhibition of CK-MM, these
antibodies also effect 80~ inhibition of CK-MB. In addition
5 to inhibition of the M subunit of CK-MB, a substantial propor-
tion of the B subunit is also inhibited by the antibodies used, I
since the ratio of activity of each of the M and B subunits in - j
CK-MB to total activity of this isoenzyme is approximately
50 : 100. Even if the residual activity of ~ 20~, deter-
10 mined using these antibodies were reproducible, the values
obtained would nevertheless be too low to be detec*ed reliably r
in an accurante measurement of CK-MB, owing to the already low
total CK activity in serum. Accordingly, inhibiting anti-
bodies described above have not been used to determine CK
15 isoenzyme concentration by the inhibition principle. Using
the method of aspects of this invention, 50% of CK-MB activity, t
i.e., approximately the entire activity of the B subunit, i
reamins available for measurement. This represents a consi-
derable advance in the art.
~ particular advantage of the method of an aspect of the invention
is the speed with which the process can be carried out. This
is especially true of the preferred embodiment, in which inhi-
bition of M content of CK-MM and CK-MB and determination of
-15- '
`~ ~06~6(~
residua~ activ;t~ dre carried out simllltarleously. In this
em~odi~nent, a~l exact test result for making a dia~nosis
can be available within 5 to 30 minutes, usually between
5 and 15 minutes at 2 to 40~ preferably 25 to 37C.
The simplicity with which the process can
be carried out is also a noteworthy advantage. The *est
method can be carried out in larger institutes or hospitals,
using customary mechanized equipment for determining enzyme
activities, or in smaller institutes or in a doctor's
laboratory using a photometer.
Test packs according to an aspect of this
invention containing all of the reagents necessary for
carrying out the method of an aspect of this invention, that r
is, a conventional mixture of coenzyme, enzyme and substrate,
CK-MM antibodies of another aspect of this invention and
buffer solution, are suitable for individual determinations.
A test pack of this type makes it possible to determine CK- r
MB with minimal effort.
It was unexpected that the problem of
determinlng CK-MB by a method operating as simply and
rapidly as the method of an aspect of this invention, could
be solved. The production of specific antisera which, while
completely inhibiting enzymatic activity of the M subunit
in CK-MM and CK-MV, do not affect enzymatic activity of the
B subunit of CK-MB could not be foreseen. The availability
of antibodies which have these properties makes the reaction _
according to aspects of the invention possible.
It is also surprising that the antibodies
empIoyed according to an aspect of this invèntion retain full
inhibitory power even in the presence of substrates. This
~ T' -~6-
. . .
..
., . ,. . ,, ,~ . : . : :.
- ~ :
~-` 106;~609
is not to be expected. Antibodies which do not ef~ect
100% inactivation of CK-MM in the presence of CK
substrates have been described in the
;~ ~ ' , . . . .
.
~ -16a- :
A~
.... " ,. ,., . . .. . ,, ._
,, , , ~. ,.--. . . . . . . . . . . . . . . . .. . . . . . .
. . .. . . ... . . . . .. . . . .
't. . "` '~ . .' ' ` ' ' ' ' `` "' , ,; ,'' ' ' .'. '' ' '
`" . ' . : . ' ' ' '
6Z~O~
~itnrtture, Ann. N.Y. Acad. Sci., Volume 103, pages 858-889
(1963). Antibodies of this type would be completely useless
in the preferred embodiments of the process of the inven-
tion, in accordance with which inhibition by antibodies and
S addition of CK subs~rates takes place simultaneously.
Uninhibited proportions of the M activities would falsely
increase the measured value for CK-MB and in some cases woul
simulate the CK-MB activity which was not present at all. In
this way, false laboratory data for the diagnosis would result.
Determination of CK-MB activity with a rapidity and
precision not achievable hitherto with immunological methods
is made possible by the surprising property of the antibodies
employed according to the invention, that is, complete inhibi-
t;on of enzymatic activity of the M subunit of CK-MM and CK-MB
lS without affecting enzymatic activity ~f the B subunit of CK-MB
and, at the same time, of completely developing their inhibitory ~,
power with respec~ to the M subunit in CK-MM and CK-MB in the
presence of substrates. This permits practical determination
of CK-MB activity by a rapid test.
It becomes possible, from laboratory findings of an
increase in CK-MB activity in a patent~ to differentiate between
a disease or traumatism of skeletal muscles ~nd of the myocardium.
Owing to release of CK-MB into the blood stream and other body
fluids following trauma to the myocardium, it is apparent that
CK-MB isoenzyme activity is an extremely reliable indication of
whether myocardial damage has occured and that a differential
diagnosis of myocardial damage can be made on the basis of ele-
vation of CK-MB levels in the body fluids. This gives impor-
i3 -/ 7-
... .. ~ ., ., , . .. , ~ ~ . ,
, ~- . . .
. ,, . . . .. .. , , .. ` , .. . . . . . . .
. . . .. , . -~ .. - . - , .
. - . . . . . . . .
` ~062609
tant additio~al data for differenti~ting cardiac infarction ~.
~ from pulmonary infarctîon and/or from secondary shock and other
d;seases of, or damage to, the heart.
- Specific and exact determination of activity of CK-MB
gives data on the extent to which the myocardium is involved
in, or damaged by, other extracardiac disease processes, for
example, poisoning or accidents; thereapeutic intervention,
for example, resuscitation; or diagnostic operations, for ex-
ample, cardiac catheterization or coronary angiographs.
Without further elaboration, it is believed that one
skilled in the art can, using the preceding description, ut-
ilize the present invention to its fullest extent. The fol-
lowing preferred specific embodiments are, therefore, to be
construed as merely illustrative.
In the examples which follow "M" (and "mM") are the
c~n~er-trations in moles and millimoles, respectively, per
liter. :
'
~/ 5~ ~ '
., .. ~ . . . , . - ,, i . ... . . .. ...
; : , . ~ : . ' '' ~- :: ' . '- ' '' ,- , ` ' ' '
., . , : :. . , .. : : ....... . - . . .
~06Z609
Preearation of startin~ mater1als
EXAMPLE A
Preparation of CR-MM
(a) 1.2 kg. of deep frozen human skeletal muscle
S are thawed at room temperature and broken up by machine.
The tissue is suspended in 2.5 1. of cQld 0.05 M tris/ Cl
buffer of pH 8.0 (tris-(hydroxymethyl)-aminomethane/HCl buffer)
which contains 0.01 M. of KCl, 1 mM of EDTA (ethylenediamine-
tetraacetic acid) and l mM of dithioerythritol and the sus-
pension is homogenized with a mixer. The homogenized materialis stirred for 45 minutes while being cooled with ice and is
then centrifuged at 12,000 g for 60 minutes. The clear
supernatant liquor (2.680 1.) is subjected to ammonium sulfate
fractionation at pH 8.0 between limits of 40 - 75~ saturation.
The 0.75 s-precipitate is taken up in 0.04 M tris/HCl buffer
at pH 8.0 and dialyzed against the same buffer. In order to
adsorb myoglobin and acid ballast proteins, 500 g. of a moist
basic ion exchanger resin derived from crosslinked dextran and
equilibrated against the same buffer are added. After 30 `
minutes, the exchange resin is filtered off and washed twice
with 400 ml. of 0.04 M tris/HCl buffer containing 0.02 M of ;~
NaCl at pH 8Ø The filtrate and wash water are combined and
brought to 0.75 s with ammonium sulfate. The precipitate is
centrifuged off, dissolved in lO0 ml. of 0.04 M tris/HCl buffer
at pH 8.0 and dialyzed against the same buffer until no further
ammonium sulfate can be detected. The clear dialyzate is equi-
librated on a column packed with a basic ion exchange resin
derived from a crosslinked dextran (6 x 60 cm.) using the same
buffer. The column is washed with the initial buffer until
- 19 -
:.
-
~06Z~Og
the eluate is protein-free. The enzyme is then eluted from
the column with 0 . 04 M tris/HCl buffer containing 0 . 02 M of
NaCl, 1 mM of EDTA and 1 mM of dithioerythritol at pH 8Ø
Fractions having an enzyme content of at least 20 U./ml. are
combined and 80%-satura~ed with ammonium sulfate and the
precipitated enzyme is centrifuged down. For final purifi-
cation, the enzyme is again subjected to chromatography in
the same system, using a smaller volume in the column. The
enzyme is precipitated from the combined active fractions with
0.8 s-ammonium sulfate and dissolved in 50 ml. of 0.04 M
tris/HC1 buffer containing 0.02 M of NaC1, 1 mM of EDTA and
lmM of dithioerythritol at pH 8.0, to give a concentrated
solution. The latter is filtered under sterile conditions and
again brought to 0.8 s with ammonium sulfate. In this way,
an enzyme suspension, which is stable at 4 C., of CK-MM with
a specific activity of 26-30 U./mg., measured with creati~e
as substrate at 25 C., is obtained.
Volume: 86 ml.; activity: 316 U./ml.; protein:
11.8 mg./ml.; Yield: about 30%, based on organ extract.
~ tb) Analogously to Example (Aa), CK-MM is isolated
from muscle tissue from the following animals: Rhesus monkeys,
pigs and cattle.
EXAMPLE B
Preparation of anti-CK-MM.
(a) CK-MM from human muscle is dialyzed agalnst a
physiological NaCl solution buffered with 0.07 M of triethanol-
amine, containing 10 mM of mercaptoethanol and 10 mM of MgC12
at pH 7Ø The enzyme solution is freed from aggregates by
- 20 -
.
106260~
ultracentrifugation and the protein content is adjusted to
2 mg./ml. using the dialysis buffer. 1 ml. of this solution
is emulsified with 1 ml. of complete Freund's adjuvant, a
water/mineral oil suspension additionally containing 2 mg.
of destroyed M-tuberculosis bacillae. This emulsion is in-
jected intramuscularly into a goat. Three injections of the
sametype at intervals of 3 weeks and 3 further booster injec-
tions, each at intervals of 16 weeks,are made. Blood is taken
from the animal 21 days after the last injection. The pH
of the serum, obtained by known methods, is adjusted to 8.4
using a mixture containing 3~ sheep serum albumin and 0.1%
of sodium azide in 0.1 M borate bufer and the serum is filtered
under sterile conditions. The resulting solution, which con-
tains anti-human muscle CK-MM, is charged in 0.5 ml. portions
to brown glass bottles and freeze-dried. The antibodies have
a molecular weight of about 160,000 to 180,000.
.
(b) CK-MM from human muscle obtained according to
Example (Aa) is dialyzed against physiological NaCl solution,
buffered with 0.1 M of imidazole at pH 6.8 and containing 7.5 mM
of N-acetyl-cysteine and 25 mM of magnesium acetate. The enzyme
solution is then freed of aggregates by ultracentrifugation
and the protein content is adjusted to 0.2 mg./ml. with the
dialysis buffer. 1 ml. of this solution is emulsifed with 1 ml.
of complete Freund's adjuvant. This emulsion is injected
intradermally into a wether. This injection is followed by
2 intramuscular injections after 3 and 6 weeks and 3 further
booster injections, each at intervals of 14 weeks. Blood is
taken 19 days after the last injection. The work up is analogous
to Example tBa). Anti-human muscle CK-MM is obtained in
- 21 -
: . . . . . . .. .
106Z609
freeze-dried form. The mulecular weight of the antibodies
is about 160,000 to 180,000.
(c) CK-MM from Rhesus monkey muscle is thoroughly
dialyzed against physiological NaCl solution buffered with
0.15 M of imidazole, and containing 25 mM of dithioerythritol
and 15 mM of manganese-II chloride at pH 6.8. After aggre-
gates have been removed by ultracentrifugation, the protein
content is adjusted to 5 mg./ml. with the dialysis buf~er.
1 ml. of this solution is emulsified with 1 ml. of complete
Freund's adjuvant. Injections, withdrawal of blood and the
work up are carried out as in Example (Ba). Anti-Rhesus
monkey muscle CK-MM is obtained in freeze-dried form. The
sedimenta~ion constant of the antibodies is about 7S.
(d) CK-MM from pig muscle is activated as in
Example (Bb) and the antigen emulsion with complete Freund's
adjuvant is injected into rabbits. Another subcutaneous
antigen injection is made after 3 weeks. The injection is
repeated after 3 weeks more and blood is taken 19 days after
this injection. Isolation is carried out as in Example (Ba). ;
Anti-pig muscle CK-MM is obtained in freeze-dried form. The
molecular weight of the antibodies is about 160,000 to 180,000.
(e) Anti-cattle muscle CK-MM (molecular weight about
160,000 to 180,000) is obtained from cattle muscle in an
analogous manner.
- 22 -
,
lO~Z609
EXAMPLE 1
Inhibition of CK-MML CK-MB and CK-BB by
___________________ __________________
anti-human-CK-MM
Pure CX-MM, CK-~B or CK-BB is added to human serum
inactivated in respect of its own CK activity and CK activities
of individual samples are determined. 0.1 ml. of anti-CK-MM
solution, obtained according to Example Ba, is then added to
0.1 ml. of a sample and the two are mixed and incubated for
5 minutes at 25 C. Residual CK activity is then determined
in a known way. Results are given in the table:
Residual activites of CK isoenzymes after incu-
bation with inhibiting anti-human-CK-MM (average
values i 1 s obtained from 5 determinations in
each case) (s = standard deviation)
T A B L E
Isoenzyme Activity of the Residual activity
isoenzymes added after incubation
(U./1.)with anti-CK-MM
~U./l.)
CK-MM 98 - 1.9 0.3 + 2.1
1043 - 220.5 - 2.5
CK-MB 103 - 2.0 53 ~ 1.7
410 + 7.8 206 + 6.2
CK-BB 197 - 3.8 199 - 4.1 -
Within errors of measurement, inhibition of activity
is 100% for CK-MM, 0~ for CK-BB and 50% for CK-MB, correspond-
25 ing to the content of 50% of M subunits. Results are constant ~ ;
over a wide range of activities of isoenzymes added.
- 23 -
; . . . . . .
iO6260~
E~AMPLE 2
Test I for guantitative determination of
activity of CK-MB in body fluids
____~__ ______________.._ _______
(a) Composition of the test pack:
The test pack is sufficient for 10 activity
determinations. The pack contains 1 bottle of buffer suffic-
ient for 10 determinations, 10 bottles of coenzyme/enzyme/
; substrate mixture and 1 bottle of anti-CK-MM, obtained accord-
ing to Example (Ba).
The bottle of coenzyme/enzyme/substrate mixture
contains:
disodium creatine phosphate hexahydrate 27.24 mg.
reduced glutathione 6.4 mg.
or N-acetyl cysteine 3.4 mg.
15 disodium adenosine diphosphate hexahydrate 1.25 mg.
disodium nicotinamide adenine dinucleotide
phosphate 1.7 mg.
disodium adneosine monophosphate 8.47 mg.
hexokinase 5 U.
20 glucose-6-phosphate dehydrogenase 3 U.
glucose 8.32 mg.
magnesium acetate 4.52 mg.
The bottle of buffer solution contains:
triethanolamine acetate (in water) 105 mM
The lyophilized antibodies are dissolved in 2 ml. of
distilled water. The resuiting antibody solution is adjusted
so that it totally inhibits up to 1,000 U./l. of creatinekinase-
MM. Sera having extremely high total creatinekinase activities
must therefore be diluted previously to about 1,000 U./l.
~he antibody solution is stable for at least 7 days at +4 C.
- 24 -
-
106260~
(b) Method for determining the activity of CK-MB:
(bl) Method:
Pipette 0.1 ml. of serum and 0.1 ml. of antibody
solution into a reaction vessel. Mix well and incubate
for 5 minutes at 25 C. 0.1 ml. of this reaction mixture
and 2.0 ml. of buffer solution are then transferred to a
bottle containing the mixture of coenzyme, enzyme and substrate.
Mix and incubate for 5 minutes at 25 C., then pour
into a cuvette and measure the extinction at 25 C. and then
determine the change in extinction per minute. Wavelength:
334, 340, or 366 nm; layer thickness: 1 cm.
(b2) Calculation:
The CK activity determined for the sample must be
multiplied (a) by the dilution factor 2 and (b) by the CK-MB
hybrid factor 2, since only the B subunits of CK-MB are
measured in the test.
An average value is obtained from the difference
in extinction per minute (~E/minute) and this is used in the
appropriate formula for calculation~
Measurement at 334 nm: activity of CK-MB per unit volume =
~/minute x 4 x 3,500 U./l.
Measurement at 340 nm: activity of CK-MB per unit volume =
QE/minute x 4 x 3,376 U./l.
Measurement at 366 nm: activity of CK-MB per unit volume =
25 ~E/minute x 4 x 6,364 U./l.
- 25 -
106Z609
EXAMPLE 3
Test II for ~uantltatlve determlnatlon
of actiVity of CK-MB in body fluids
__________ _____________ __ _______
(a) composition of the test pack:
The test pack is sufficient for 30 activity determin-
ations. The pack contains 1 bottle of buffer solution for 30
determinations and 30 bottles of a lyophilized mixture con-
sisting of coenzyme, enzyme, substrate and anti-CK-MM of
Example (Ba).
The amount of triethanolamine acetate in the bottle
of buffer solution corresponds to the amount of Example 2a.
The composition of the mixture of coenzyme, enzyme, substrate
and anti-CK-MM of Example (Ba) in the individual bottles p
corresponds to that in Example 2a with respect to the three
lS first-mentioned components and each bottle additionally con-
ta~ns anti-CK-MM which totally inhibits up to 1,000 U./l.
of CK-NM. ,
(b) Determination of the activity of CX-MB.
(bl) Method.
2.0 ml. of buffer solution and 0.1 ml. of serum are
20 pipetted into the contents of a bottle of coenzyme/enzyme/ ~-
substrate/anti-CK-MM mixture. Mix and incubate for 5 minutes
at ~5 C. Then pour into a cuvette and measure the extinc-
tions at 25 C. over a period of 5 minutes. Wavelength:
~~ ~ 334, 340, 366 nm.; layer thickness: 1 cm.
~ (b2) Calculation:
; An average value is obtained from the differences
; in extinction per minute ( E/minute) and this is used in
the appropriate formula for calculation:
.
- 26 -
-
: . ~ : , . :
. . . - . .
106Z60~
Measurement at 334 nm~ activit~ of CK-MB per unit volume =
QE/minute x 7,000 U./l.
Measurement at 340 nm: activity of CK MB per unit volume =
~E/minute x 6,752 U./l.
S Measurement at 366 n~: activity of CK-MB per unit volume =
~E/minute x 12,728 U./l.
~.
; EXAMPLE 4
.
Simultaneous determination of the total CK
________________________
activity and of the CK-MB activity
_______ _________________________
(a) Composition of the test pack:
The composition of the test pack corresponds to
; that of Example 2a.
(b) Determination of total CK activity and of
CK-MB activity of CK-MB.
(bl) Method:
2.0 ml. of buffer solution and 0.1 ml. of serum
or diluted serum are pipetted into a bottle of coenzyme/
enzyme/substrate mixture. Mix and incubate for 5 minutes
at 25 C., then pour into a cuvette and determine the change
in extinction (~El) at 25 C. over a period of 2 minutes.
0.1 ml. of antibody solution is then added. Mixing is carried
out at once and after 3 minutes the change in extinction
(~E2) at 25 C. is again determined. Wavelength: 334, 340,
366 nm: layer thickness: 1 cm.
(b2) Calculation:
The total CK activity is calculated as follows:
Measurement at 334 nm: total activity of CK per unit volume =
~El/minute x 3,500 U./l.
- 27 -
~O~Z60~
Measurement at 340 nm: total activity of CK per unit volume =
QEl/minute x 3,376 U./l.
Measurement at 366 nm: total activity of CK per unit volume =
~El/minute x 6,364 U./l.
The activity of CK-MB is obtained using the following
formulae for calculation:
Measurement at 334 nm: activity of CK-MB per unit volume =
~E2/minute x 7,350 U./1.
Measurement at 340 nm: activity of CK-MB per unit volume =
~E2/minute x 7,090 U./l.
Measurement at 366 nm: activity of CK-MB per unit volume =
~E2/minute x 13,364 U./l.
EXAMPLE 5
Determinatlon of CK-MB activlties ln ~atients with
and without cardiac lnfarction usin~ the test ~ack
of Exam~le 3
_______ ____
(a) CK activities in different groups of patients:
Number Average values
of cases Total CK CK-Ms
: (U./l.) (U./l.)
. ~ I
Patients with increased
CK activities without 48 480 Cl.7
cardiac infarction
.____
: 25 Patients with cardiac 5 510 44
infarctions . _ I .'
The tables shows that an indication of a cardiac
infarction can be obtained rapidly and clearly with the method
of determination of the invention.
- 28 -
lO~;Z609
(b) Change .in CK MB activities in a patient with
cardiac infarction:
Hours after the onset of infarction CK-MB U./l.
3.5 < 5
4.5 ~ 5
; 5.5 6
: 7.5 . 22
8.5 - 23
: 9.5 29
10.5 50
11.5 46
13.5 56
15.5 ~5
17.5 64
21.5 62
25.5 50
29.5 42
33.5 25
43.5 ` 1~
53.5 < 5
61.5 < 5
The figures show the rise and subsequent fall ~ '
in CX-MB activity in a patient suffering from cardiac infarc-
tion determined by the process of the invention.
- 29 -
106Z6a9
The preceding examples can be repeated with similar
success by substituting the generically or specifically
described react~nts and/or operating conditions of this
invention for those used in the preceding examples.
-30-
'~ ` ' `' ~' " ; `: :
