Note: Descriptions are shown in the official language in which they were submitted.
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STABII,IZEI) ISOENZYME CONTROL PRODUCTS
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Field of the Invention
This invention relates to stabilized control products
useful in the clinical environment and in particular
relates to stabilized isoenzyme control reagents including
CK and LDH isoenzymes.
Background of the Invention
This invention is related to a co-pending Canadian appli-
cakion S~N. 455,085 entitled "Stabilized Multi-Parameter
Control Product" filed 25 May 1984 by the inventor hereof.
Isoenzymes, or isozymes, as they are alternatively
referred to, are enzymes in multiple forms which are
capable of performing the same general function but at
different rates. They are sufficiently different in
chemical composition so that they are generally separable
electrophoretically. One such isoenz~me, lactate
dehydrogenase (LDH) is found in five electrophoretically
distinct fractions. Each of these electrophoretic species
of LDH is a tetramer ~onsisting of two polypeptide chain
units, H and M, present in different proportions: H4,
2~ MH3, M2H2, HM3, and M~ These five isoenzymes differ in
catalytic activity (affinity for the substrate, pyruvate
as measured by the Michaelis constant), amino acid
composition, heat lability, and immunological res~nses.
The two peptides ~ and M are coded by different genes.
Thus the type of enzyme present is under genetic control
and regulated by the conditions of the environment imposed
upon the cell.
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Similarly, creatinine kinase (CK) is another isoenzyme
which contains subunits of either M's or Bls and thus may
be present as MM, BB, or MB. The MB form is clinically
significant as an indicator of myocardial information,
however, this form is unstable and tends to disassociate
to reform the MM or BB types. It is an object to
stabilize a control reagent having the ~ form.
The various proportions or combinations of isoenzymes
present in the tissue may be rela.ed to the specific
requirements of the cell in question and is thus affected
by such factors as the extent of differentiation and
development of the cell, as well as the level and type of
metabolism occurring within the cell. Accordingly, the
distribution between the various forms of isoenzymes
provides diagnostically signiicant data. For instance,
LDH exhibits significant control over cellular glycolosis.
Specifically, MH3 and H~ isoenzyme types predominant in
tissues with purely aerobic or respiratory metabolism.
Accordingly, they may be used as diagnostic tools in
determining the condition of muscles such as the heart
particularly with CK isoenzymes, the brain, liver and
other organs in the case of alanine aminotransferase tALT)
or aspartase aminotransferase (AST). Yet another
isoenzyme of significance is alkaline phosphatase gamma-
glutamyl transpeptidase.
With such attention being placed on the determination of
isoenzyme levels, particularly important in the case of
cardiac critical care patients, it is axiomatic that
adequate controls must be available in order to ensure the
proper operation of manual and automated methods designed
to determine these levels. ~eretofore, such controls as
were available, have been typically unstable due to the
highly unstable nature of the enz~mes themselues.
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It is an object of the present invention to provide
control reagents having the necessary levels of isoenzymes
present therein in a stabilized forma~.
It is another object of the present invention to provide
methods whereby isoenzyme control reagents may be
stabilized.
Brief Summary of the InvPntion
1~
In accordance with certain aspects of the
present invention, isoenzyme control reagents are provided
for creatinine kinase, lactate dehydrogenase, alanine
aminotransferase and aspartase aminotransferase which are
substantially stabilized by the addition of plexiform
stabilizing means. The preferred plexiform stabilizin~
means is selected from the group consisting of maltose,
mannitol, cellobiose and lactose with the latter most
being the most preferred. The plexiform stabilizing means
is advantageously provided in a final concentration range
of abo~t 2% - % with the ideal concentration occurring at
abou~ 6%~ The ideal isoenzyme control reagent will have
substantially all water removed, s~ch as by lyophiliza-
tion, to assist in long term storage and stability.
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As a result, the control reagent products of the present
invention may b~ made ~o act and behave n a more similar
fashion as those of a patient and indeed, the instant
reagents are capable of being run on any of the three
separation ~ystems presently in use column chroma~
tography, and the immuno-based separation type systems.
Thus, in one embodiment of the present invention there is
provid~d a CK isoenzyme control reagent stabilized against
; 35 disassociation or rearrangement of subunits comprising:
(a) CK isoenzyme containing subunits of M, B and MB forms;
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and (b) plexiform stabilizing means selected from the group
consisting of reducing monosaccharide sugars and reducing
disaccharide sugars, the ple~iform stabilizing means present
in the range of about 2% to 8% final volume percent, whereby
activity of said CK enzymes in solution is maintained for
at least 10 days.
In another embodiment of the present invention a CK isoenzyme
control reagent stabilized against disassociation or
rearrangement of subunits is provided, the reagent comprising:
an aqueous solution of CK isoenzyme and of protein and nonprotein
analytes and plexiform stabilizing means selected from the
group consisting of maltose, mannitol, cellobiose and lactose,
the plexi~orm stabilizing means present in a concentration
optimized to preserve maximum enzyme activity.
In yet another embodiment of the present invention there is
.: provided a LDH isoenz~me control reagent s~abilized against
disassociation or rearrangement of subunits comprising:
(a) isolated LDH isoenzymes from tissue; (b) a non-phosphate
containing buffer present in a concentration in the range of
about 2% to 8% final volume percent, whereby activity of the
LDH enzymes in solution is maintained for at least 10 days;
(c) human sera;and (d) plexiform stabilizing means selected
from the group consisting of reducing monosaccharide sugars
and reducing disaccharide sugars, the plexiform stabilizing
means present in the range of about 2% to 8% final volume
percent.
In still a further embodiment of the present invention
an LDH isoenzyme control reagent stabilized against disassoc-
iation or rearrangement of subunits is provided r the reagent
comprising: an aqueous solution of LDH isoenzyme and of
protein and nonprotein analytes and plexiform stabilizing
means selected from the group consisting of maltose, mannitol,
cellobiose and lactose, the plexiform stabilizing means
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present in a concentration optimized to preserve maximum
enzyme activity.
In yet a further embodiment of the present invention an ALT
isoenzyme control reagent stabilized against disassociation
or rearrangement of subunits is provided comprising: (a)
isolated ALT isoenzyme from tissue; (b) a non-phosphate
containing buffer; (c) humansexa; and (d) plexiform stabilizing
means selected from the group of reducing monosaccharide sugars
and reducing disaccharide sugars, the plexiform stabilizing
means present in the range of about 2% to 8% final volume
percent.
In still a further embodiment an ALT isoenzyme control reagent
stabilized against disassociation or rearrangement of subunits
is provided comprising an aqueous solution of ALT isoenzyme
and of protein and nonprotein analytes and plexiform stabilizing
means selected from the group consisting of maltose, mannitol,
cellobiose and lactose, the plexiform stabilizing means present
in a concentration optimized to preserve maximum enzyme
activity.
In yet another embodiment of the present invention an AST
isoenzyme control reagent stabilized against disassociation
or rearrangement of subunits is provided comprising: (a)
isolated AST isoenzyme from tissue; (b) a non-phosphate
containing bu~fer; (c~ human sera; and (d) plexiform
stabilizing means selected from the group consisting of reducing
monosaccharide sugars and reducing disaccharide sugars, the
plexiform stabilizing means present in the range of about 2%
to ~% final volume percent.
In yet another embodiment of the present invention an AST
isoenzyme control reagent stabilized against disassocation or
rearrangement of subunits is provided comprising an aqueous
solution of AST isoenzyme and of protein and nonprotein analytes
cii~
and plexiform stabilizing means selected from the group consis~ing
of maltose, mannitoll cellobiose and lactose, the plexiform
stabilizing means present in a concentration optimized to
- preserve maximum enzyme activity.
s
In another embodiment of the present invention an isoenzyme
control reagent stabilized against disassociation or rearrangemnt
of subunits is provided comprising a solution containing the
isoenzyme isolated from human tissue and present at a
~iagnostically significan~ concentration and plexiform stabilizing
means present in the range of about 2% to 8% final volume
percent and selected from the group consisting of maltose,
mannitol, cellobiose and lactose.
In another embodiment of the present invention a method of
stabilizing an isoenzyme control reagent against disassociation
or rearrangement of subunits is provided, the method comprising
adding to an aqueous solution containing the isoenzymes to be
stab.ilized against disassociation or rearrangement of subunits
a plexiform sta~ilizing means selected from the group
consisting o~ reducing monosaccharide sugars and reducing
disaccharide sugars r the plexiform stabilizing means present
in the range of about 2% to 8~ weight per volume.
Detailed Descri ti ~est Mode
~s has been previously intimated, there has heretofore
been great di~iculty in stabilizing such an isoenzyme
control reagent and in parti~ular, stabiliziny the various
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isoenzyme subunits such as the CK isoenzyme unit r~B
without deleteriously affectina other subunit components
or the tests therefor. With the addition of the plexiform
stabilizing means of the present invention, these
isoenzymes and their subunit constituents have now been
stabilized and may be maintained in solution for
significantly greater periods of time than previously
possible. In dry form, obtained when substantialiy all
water has been removed such as by lyophilization, the
stability period is increased to an even greater extent.
The addition of the plexiform stabilizing means of the
present invention results in depression of the actual
freezing point. Depression of the freezing point is
generally associated with slower freezing rates, however,
faster freezing of the product of the present invention
has been observed. It would appear, however, that the
plexiform stabilizing means when added to the material of
the present product results in the loss of the eutectic
point plateau thus actually increasing the freezing rate.
Associated with this phenomenon is the observation that
the cakes formed during freezing are uniformly
crystalline, as opposed to the often occurring powder
forms. This would imply that the plexiform stabilizing
~5 means is holding the constituents in a stable, three
dimensional "crystallineN structure thereby assisting
in the removal of water, the stabilization of he
constituents themselves, as well as speeding the
reconstitution of the lyophilized material. These and
other complex interactions are more fully described in the
co~pending application referred to earlier and for a
fuller explanation, reference is made thereto.
In order to make the isoenzyme control reagents of the
present invention, a specific human tissue was selected in
accordance with the type of enzyme containing product to
be produced. For instance, for the CK MB subunit, heart
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tissue is selected, for the CK ~M enzyme subunit muscle
tissue is employed while for the CK BB subunit ~rain
tissue is used. Similarly, these and other tissues
including non-human tissues may be used as the source
material for the isoenzymes or their subunits in
accordance with well-known knowledge.
The tissue is then treated by grinding etc. to a form
suitable for isoenzyme isolation by either chromatography,
electrophoresis or other immunologically based system in
accordance with techniques well-known. The thusly
isolated enzyme components may be added in diagnostically
significant proportions to a preferably pr~clarified,
human sera base. The human sera is advantageously
clarified to remove lipids by either filtering, freezing,
reconstituting and filtering or by the addition of silica
compounds such as aerosil all of which are methods well-
known.
It is then advantageous to add to this material, a
stabilizing cofactor as may be necessary to assist in the
maintenance of binding site activity. For instance, for
the CK isoenzymes, sulfhydryl containing compounds such as
glutathione or dithiotreotol or n-actyl-cysteine are
useful. Further, the ideal product also includes
chelating means for removing metallic ions, if present,
which may interfere with enzyme activity. If the enzyme's
activity is not affected by the presence of metallic ions,
this agent need not be included. Similarly, if no
- 30 metallic ions are present this agent may again be
eliminated. An example of such a chelating agent is
ethylenediamine tetraacetic acid (ED~A).
Lastly, the isoenzyme product of the present invention
will further comprise a weak, nonphosphate buffer. It is
~` preferred that a nonphosphate buffer is employed in order
to avoid phosphorus containing compounds which may
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otherwise interfere with some of the reactions,
particularly those involving the adenine tripho~phate
(ATP) cycles. The buffer should be ~weak" i.e., within
the range of about 10-200 millimolar concentration. m e
pH of the buffer should ideally be selected or adjusted
in order to preferably optimize activity and maximize
stability. For instance, with the CK and LDH isoenzymes,
a pH of approximately 7 is advantageous while the ideal pH
for the alkaline phosphatase isoenzymes is in the range of
about 7.6 to 7.8.
Finally, plexiform stabilizing means is added in order to
provide the stability and other aforementioned advantages
described herein. Such a
plexiform stabilizing means is selected from the group
consisting of monosaccharide and disaccharide reducing
sugars and preferably will be selected from the group
consisting of mannitol, maltose, cellobiose and lactose.
As described in the referenced application, the n~ost
preferred, in terms of stability advantages and economic
considerations, is lactose. The inventor hereof has found
it desirable to present the plexiform stabilizing means in
a final concentration range of about 2%-8% with the most
- preferred embodiment comprising approximately 6%.
The plexiform stabilizing means not only retards the
disassociation of isoenzymes whose subunits tend to
disassociate rather e~sily, but it also stabilizes the
isoenzyme's electrophoretic patterns. The concentration
30 of the plexiform stabilizing mean is selected to retain
the enzyme activity even if the prodwt is freeze dried
and may be preferably optimized for maximum enzyme
activity. If too little plexiform stabilizing means is
incorporated into the final product, then insufficient
3~ protection is derived, however, it is undesirable to add
too much plexiform stabilizing means which in high
concentrations not only may precipitate out of solution
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but may also block the active site of the protein. Thus,
high concentrations of the plexiform stabilizing means
actually tend to reduce enzyme activity and are conse-
quently avoided.
An example of increased stability is presented in
accompanying Table l. The data therein represents the
activity of a CR isoenzyme control stored in aqueous form
at 37C. It compares the data observed with an
unstabilized isoenzyme control against the s~abilized
isoenzyme control from day 0 to day 30. Still greater
increases in stability may be expected at lower
temperatures i.e., 5 storage as well as in lyophilized
storage format.
It may be readily appreciated by those skilled in the art
that various modifications and substitutions may be made
to the constituents listed above without departing from
the spirit or scope of the present invention.
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TABLE 1
CK ISOENZYME CONTROL
37C WET STABILITY STUDY
. . . ~ . _ .
DAY VNSTABILIZED STABILIZED
CK CK
10 0 984 945
9~4 931
3 944 945
944 956
981 918
972 330
9 938 9~1
942 672i
12 961 942
9~5 948
25 16 695 679
702 670
403~ 4~6
408' 4~2
1 short sample
2 dilution results
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