CA1230300A - Stabilized isoenzyme control product - Google Patents
Stabilized isoenzyme control productInfo
- Publication number
- CA1230300A CA1230300A CA000453361A CA453361A CA1230300A CA 1230300 A CA1230300 A CA 1230300A CA 000453361 A CA000453361 A CA 000453361A CA 453361 A CA453361 A CA 453361A CA 1230300 A CA1230300 A CA 1230300A
- Authority
- CA
- Canada
- Prior art keywords
- reagent
- stabilizing means
- lactose
- isoenzyme
- plexiform
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired
Links
- 108010044467 Isoenzymes Proteins 0.000 title claims abstract description 77
- 230000000087 stabilizing effect Effects 0.000 claims abstract description 73
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 70
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 claims abstract description 30
- 239000008101 lactose Substances 0.000 claims abstract description 30
- 235000000346 sugar Nutrition 0.000 claims abstract description 22
- 238000000034 method Methods 0.000 claims abstract description 11
- 239000002738 chelating agent Substances 0.000 claims abstract description 5
- 150000008163 sugars Chemical class 0.000 claims abstract description 3
- 102000004190 Enzymes Human genes 0.000 claims description 27
- 108090000790 Enzymes Proteins 0.000 claims description 27
- 230000000694 effects Effects 0.000 claims description 26
- 230000008707 rearrangement Effects 0.000 claims description 21
- -1 monosaccharide sugars Chemical class 0.000 claims description 19
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 claims description 17
- GUBGYTABKSRVRQ-CUHNMECISA-N D-Cellobiose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-CUHNMECISA-N 0.000 claims description 17
- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 claims description 16
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 claims description 16
- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 claims description 16
- 229930195725 Mannitol Natural products 0.000 claims description 16
- 239000000594 mannitol Substances 0.000 claims description 16
- 235000010355 mannitol Nutrition 0.000 claims description 16
- GUBGYTABKSRVRQ-QUYVBRFLSA-N beta-maltose Chemical compound OC[C@H]1O[C@H](O[C@H]2[C@H](O)[C@@H](O)[C@H](O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@@H]1O GUBGYTABKSRVRQ-QUYVBRFLSA-N 0.000 claims description 15
- 239000007864 aqueous solution Substances 0.000 claims description 10
- 108090000623 proteins and genes Proteins 0.000 claims description 10
- 102000004169 proteins and genes Human genes 0.000 claims description 9
- 239000000243 solution Substances 0.000 claims description 8
- 150000001455 metallic ions Chemical class 0.000 claims description 6
- 229910019142 PO4 Inorganic materials 0.000 claims description 4
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims description 4
- 239000010452 phosphate Substances 0.000 claims description 4
- 230000000845 anti-microbial effect Effects 0.000 claims 1
- 230000002401 inhibitory effect Effects 0.000 claims 1
- 230000000813 microbial effect Effects 0.000 claims 1
- 150000002016 disaccharides Chemical class 0.000 abstract description 2
- 150000002772 monosaccharides Chemical class 0.000 abstract description 2
- 102000003855 L-lactate dehydrogenase Human genes 0.000 description 12
- 108700023483 L-lactate dehydrogenases Proteins 0.000 description 12
- 210000001519 tissue Anatomy 0.000 description 12
- 239000000047 product Substances 0.000 description 9
- 238000007710 freezing Methods 0.000 description 7
- 230000008014 freezing Effects 0.000 description 7
- 235000018102 proteins Nutrition 0.000 description 5
- 239000000470 constituent Substances 0.000 description 4
- DDRJAANPRJIHGJ-UHFFFAOYSA-N creatinine Chemical compound CN1CC(=O)NC1=N DDRJAANPRJIHGJ-UHFFFAOYSA-N 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- 102100036475 Alanine aminotransferase 1 Human genes 0.000 description 2
- 108010082126 Alanine transaminase Proteins 0.000 description 2
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 2
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 2
- 108700016171 Aspartate ammonia-lyases Proteins 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical class O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- 108090000340 Transaminases Proteins 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 229940109239 creatinine Drugs 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 238000004108 freeze drying Methods 0.000 description 2
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 description 2
- 230000004060 metabolic process Effects 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 210000003205 muscle Anatomy 0.000 description 2
- 108090000765 processed proteins & peptides Proteins 0.000 description 2
- 102000004196 processed proteins & peptides Human genes 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 102000014898 transaminase activity proteins Human genes 0.000 description 2
- 229930024421 Adenine Natural products 0.000 description 1
- GFFGJBXGBJISGV-UHFFFAOYSA-N Adenine Chemical compound NC1=NC=NC2=C1N=CN2 GFFGJBXGBJISGV-UHFFFAOYSA-N 0.000 description 1
- 229910002012 Aerosil® Inorganic materials 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical group OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 101710107035 Gamma-glutamyltranspeptidase Proteins 0.000 description 1
- 108010024636 Glutathione Proteins 0.000 description 1
- 101710173228 Glutathione hydrolase proenzyme Proteins 0.000 description 1
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 1
- 108091000080 Phosphotransferase Proteins 0.000 description 1
- 102000001253 Protein Kinase Human genes 0.000 description 1
- LCTONWCANYUPML-UHFFFAOYSA-M Pyruvate Chemical compound CC(=O)C([O-])=O LCTONWCANYUPML-UHFFFAOYSA-M 0.000 description 1
- 229960000643 adenine Drugs 0.000 description 1
- 235000001014 amino acid Nutrition 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- 230000000747 cardiac effect Effects 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 230000005496 eutectics Effects 0.000 description 1
- 239000012467 final product Substances 0.000 description 1
- 102000006640 gamma-Glutamyltransferase Human genes 0.000 description 1
- 230000008571 general function Effects 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 229960003180 glutathione Drugs 0.000 description 1
- 238000000227 grinding Methods 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000002107 myocardial effect Effects 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 229910052698 phosphorus Inorganic materials 0.000 description 1
- 239000011574 phosphorus Substances 0.000 description 1
- 102000020233 phosphotransferase Human genes 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 108060006633 protein kinase Proteins 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 230000000241 respiratory effect Effects 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 230000006641 stabilisation Effects 0.000 description 1
- 238000011105 stabilization Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 125000003396 thiol group Chemical group [H]S* 0.000 description 1
Landscapes
- Enzymes And Modification Thereof (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
- Fats And Perfumes (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
Abstract Isoenzyme control reagents and methods for making same stabilized by means of plexiform stabilizing means. The preferred plexiform stabilizing means is selected from the group consisting of monosaccharide and disaccharide reducing sugars. The most preferred isoenzyme control reagent comprises the isoenzyme of interest obtained from selected tissue, preclarified human sera, stabilizing cofactor, a chelating agent means, a weak nonphospate buffer and lactose as the most preferred plexiform stabilizing means.
Description
3~J~'~
STABII,IZEI) ISOENZYME CONTROL PRODUCTS
-- , _ 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,
STABII,IZEI) ISOENZYME CONTROL PRODUCTS
-- , _ 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.
~ Z3~lJko 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.
ORD~
~303¢~
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.
~5 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;
.~
~.~3(3~
- 3a -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 .. ..
~Z~30~
- 3b -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 ~3(:~3$J~
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 OR.~-51 ~2~3~
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 ~3(~3~
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
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.
~ Z3~lJko 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.
ORD~
~303¢~
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.
~5 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;
.~
~.~3(3~
- 3a -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 .. ..
~Z~30~
- 3b -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 ~3(:~3$J~
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 OR.~-51 ~2~3~
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 ~3(~3~
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 ORD~
""" ~
~23(~3~?~
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.
~3(~
CK ISOENZYME CONTROL
. . . ~ . _ .
DAY VNSTABILIZED STABILIZED
CK CK
9~4 931 9 938 9~1 942 672i 9~5 948 403~ 4~6 408' 4~2 1 short sample 2 dilution results
""" ~
~23(~3~?~
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.
~3(~
CK ISOENZYME CONTROL
. . . ~ . _ .
DAY VNSTABILIZED STABILIZED
CK CK
9~4 931 9 938 9~1 942 672i 9~5 948 403~ 4~6 408' 4~2 1 short sample 2 dilution results
Claims (47)
1. A CK isoenzyme control reagent stabilized against disassociation or rearrangement of subunits comprising:
a) CK isoenzyme containing subunits of M, B and MB
forms; and b) plexiform stabilizing means selected from the group consisting of reducing monosaccharide sugars and reducing disaacharide sugars, said plexiform 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.
a) CK isoenzyme containing subunits of M, B and MB
forms; and b) plexiform stabilizing means selected from the group consisting of reducing monosaccharide sugars and reducing disaacharide sugars, said plexiform 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.
2. The reagent of Claim 1 wherein the plexiform stabilizing means is selected from the group consisting of maltose, mannitol, cellobiose, and lactose.
3. The reagent as provided in Claim 1 wherein the plexiform stabilizing means is lactose.
4. The reagent as provided in Claim 3 wherein the lactose is present in a final concentration of about 6%.
5. A CK isoenzyme control reagent stabilized against disassociation or rearrangement of subunits 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, said plexiform stabilizing means present in a concentration optimized to preserve maximum enzyme activity.
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, said plexiform stabilizing means present in a concentration optimized to preserve maximum enzyme activity.
6. The reagent as provided in Claim 1 in lyophilized form.
7. The reagent as provided in Claim 3 in lyophilized form.
8. The reagent as provided in Claim 4 in lyophilized form..
9. The reagent as provided in Claim 5 in lyophilized form.
10. A LDH isoenzyme control reagent stabilized 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 said 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, said plexiform stabilizing means present in the range of about 2% to 8% final volume percent.
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 said 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, said plexiform stabilizing means present in the range of about 2% to 8% final volume percent.
11. The reagent of Claim 10 wherein the plexiform stabilizing means is selected from the group consisting of maitose, mannitol, cellobiose, and lactose.
12. The reagent as provided in Claim 10 wherein the plexiform stabilizing means is lactose, said tissue is human tissue, and further comprising stabilizing co-factor means for maximizing enzyme activity and chelating agent means added as necessary to substantially bond with metallic ions, if present, for preserving enzyme binding site activity.
ORD-5l
ORD-5l
13. The reagent as provided in Claim 12 wherein the lactose is present in the final concentration of about 6%.
14. An LDH isoenzyme control reagent stabilized against disassociation or rearrangement of subunits 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, said plexiform stabilizing means present in a concentration optimized to preserve maximum enzyme activity.
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, said plexiform stabilizing means present in a concentration optimized to preserve maximum enzyme activity.
15. The reagent as provided in Claim 10 in lyophilized form.
16. The reagent as provided in Claim 12 in lyophilized form.
17. The reagent as provided in Claim 13 in lyophilized form.
18. The reagent as provided in Claim 14 in lyophilized form.
19. An ALT isoenzyme control reagent stabilized against disassociation or rearrangement of subunits comprising:
a) isolated ALT isoenzyme from tissue;
b) a non-phospate containing buffer;
c) human sera; and d) plexiform stabilizing means selected from the group of reducing monosaccharide sugars and reducing disaccharide sugars, said plexiform stabilizing means present in the range of about 2% to 8% final volume percent.
a) isolated ALT isoenzyme from tissue;
b) a non-phospate containing buffer;
c) human sera; and d) plexiform stabilizing means selected from the group of reducing monosaccharide sugars and reducing disaccharide sugars, said plexiform stabilizing means present in the range of about 2% to 8% final volume percent.
20. The reagent of Claim 19 wherein the plexiform stabilizing means is selected from the group consisting of maltose, mannitol, cellobiose, and lactose.
21. The reagent as provided in Claim 19 wherein the plexiform stabilizing means is lactose, said tissue is human tissue, and further comprizing stabilizing co-factor means for maximizing enzyme activity and chelating agent means added as necessary to substantially bond with metallic ions, if present, for preserving enzyme binding site activity.
22. The reagent as provided in Claim 21 wherein the lactose is present in the final concentration of about 6%.
23. An ALT isoenzyme control reagent stabilized against dis-association or rearrangement of subunits 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, said plexiform stabilizing means present in a concentration optimized to preserve maximum enzyme activity.
24. The reagent as provided in Claim l9 in lyophilized form,
25. The reagent as provided in Claim 21 in lyophilized form.
26 The reagent as provided in Claim 22 in lyophilized form.
27. The reagent as provided in Claim 23 in lyophilized form.
28. An AST isoenzyme control reagent stabilized against disassociation or rearrangement of subunits comprising:
a) isolated AST isoenzyme from tissue;
b) a non-phospate containing buffer;
c) human sera; and d) plexiform stabilizing means selected from the group consisting of reducing monosaccharide sugars and reducing disaccharide sugars, said plexiform stabilizing means present in the range of about 2% to 8% final volume percent.
a) isolated AST isoenzyme from tissue;
b) a non-phospate containing buffer;
c) human sera; and d) plexiform stabilizing means selected from the group consisting of reducing monosaccharide sugars and reducing disaccharide sugars, said plexiform stabilizing means present in the range of about 2% to 8% final volume percent.
29. The reagent of Claim 28 wherein the plexiform stabilizing means is selected from the group consisting of maltose, mannitol, cellobiose, and lactose.
30. The reagent as provided in Claim 28 wherein the plexiform stabilizing means is lactose, said tissue is human tissue, and further comprising stabilizing co-factor means for maximizing enzyme activity and chelating agent means added as necessary to substantially bond with metallic ions, if present, for preserving enzyme binding site activity.
31. The reagent as provided in Claim 30 wherein the lactose is present in the final concentration of about 6%.
32. An AST isoenzyme control reagent stabilized against disassociation or rearrangement of subunits comprising an aqueous solution of AST isoenzyme and of protein and nonprotein analytes and plexiform stabilizing means selected from the group consisting of maltose, mannitol, cellobiose and lactose, said plexiform stabilizing means present in a concentration optimized to preserve maximum enzyme activity.
33. The reagent as provided in Claim 28 in lyophilized form.
34. The reagent as provided in Claim 30 in lyophilized form.
35. The reagent as provided in Claim 31 in lyophilized form .
36. The reagent as provided in Claim 32 in lyophilized form.
37. An isoenzyme control reagent stabilized against dis-association or rearrangement of subunits comprising a solution containing the isoenzyme isolated from human tissue and present at a diagnostically significant 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.
38. The reagent as provided in Claim 37 further comprising antimicrobial means for substantially inhibiting microbial growth.
39. A method of stabilizing an isoenzyme control reagent against disassociation or rearrangement of subunits comprising adding to an aqueous solution containing the isoenzymes to be stabilized against disassociation or rearrangement of subunits a plexiform stabilizing means selected from the group consisting of reducing monosaccharide sugars and reducing disaccharide sugars, said plexiform stabilizing means present in the range of about 2% to 8%
weight per volume.
weight per volume.
40. The method as provided in Claim 39 wherein the plexiform stabilizing means is selected from the group consisting of maltose, mannitol, cellobiose and lactose.
41. The method as provided in Claim 39 wherein the plexiform stabilizing means is lactose.
42. The method as provided in Claim 41 wherein the lactose is present in the final concentration of about 6%.
43. The reagent as provided in Claim 37 wherein the plexiform stabilizing means is lactose.
44. The reagent as provided in Claim 37 in lyophilized form.
45. The reagent as provided in Claim 38 in lyophilized form.
46. The reagent as provided in Claim 39 further comprising the step of lyophilizing the reagent so formed.
47. The reagent as provided in Claim 41 further comprising the step of lyophilizing the reagent so formed.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US50121383A | 1983-06-06 | 1983-06-06 | |
| US501,213 | 1983-06-06 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1230300A true CA1230300A (en) | 1987-12-15 |
Family
ID=23992572
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000453361A Expired CA1230300A (en) | 1983-06-06 | 1984-05-02 | Stabilized isoenzyme control product |
Country Status (6)
| Country | Link |
|---|---|
| JP (1) | JP2596911B2 (en) |
| AU (1) | AU583474B2 (en) |
| CA (1) | CA1230300A (en) |
| DK (1) | DK275184A (en) |
| IL (1) | IL72030A0 (en) |
| NO (1) | NO842258L (en) |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA1226795A (en) * | 1983-06-06 | 1987-09-15 | Michael K. Hoskins | Stabilized coagulation control products |
| CA1226794A (en) * | 1983-06-06 | 1987-09-15 | Michael K. Hoskins | Stabilized multiparameter control product |
| US4663295A (en) * | 1983-06-29 | 1987-05-05 | Ciba Corning Diagnostics Corp. | Estrogen-progesterone control reagents and methods for making same |
| JP2575648B2 (en) * | 1986-04-24 | 1997-01-29 | 国際試薬株式会社 | Method for stabilizing creatine kinase |
| IT1394539B1 (en) * | 2009-05-19 | 2012-07-05 | Sentinel Ch S P A | USE OF A POLYMERASE STABILIZER FOR THE LIOFILIZATION AND PREPARATION OF READY-TO-USE KITS |
Family Cites Families (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS4834912A (en) * | 1971-09-09 | 1973-05-23 | ||
| JPS589688A (en) * | 1981-07-06 | 1983-01-20 | Toyobo Co Ltd | Stable enzymic composition |
| JPS5865218A (en) * | 1981-10-13 | 1983-04-18 | Kowa Co | Preparation of tissual plasminogen activator |
| JPS58107178A (en) * | 1981-12-22 | 1983-06-25 | Mitsui Toatsu Chem Inc | Stable freeze-dried product of beta-galactosidase or its composite |
| JPS58134991A (en) * | 1981-12-28 | 1983-08-11 | Takeda Chem Ind Ltd | Stabilization of serratiopeptidase |
| CA1226795A (en) * | 1983-06-06 | 1987-09-15 | Michael K. Hoskins | Stabilized coagulation control products |
| CA1226794A (en) * | 1983-06-06 | 1987-09-15 | Michael K. Hoskins | Stabilized multiparameter control product |
| US4663295A (en) * | 1983-06-29 | 1987-05-05 | Ciba Corning Diagnostics Corp. | Estrogen-progesterone control reagents and methods for making same |
-
1984
- 1984-05-02 CA CA000453361A patent/CA1230300A/en not_active Expired
- 1984-06-04 DK DK275184A patent/DK275184A/en not_active Application Discontinuation
- 1984-06-05 IL IL72030A patent/IL72030A0/en unknown
- 1984-06-05 AU AU29085/84A patent/AU583474B2/en not_active Ceased
- 1984-06-05 NO NO842258A patent/NO842258L/en unknown
- 1984-06-06 JP JP59114690A patent/JP2596911B2/en not_active Expired - Lifetime
Also Published As
| Publication number | Publication date |
|---|---|
| JP2596911B2 (en) | 1997-04-02 |
| IL72030A0 (en) | 1984-10-31 |
| AU583474B2 (en) | 1989-05-04 |
| DK275184A (en) | 1984-12-07 |
| DK275184D0 (en) | 1984-06-04 |
| JPS606192A (en) | 1985-01-12 |
| AU2908584A (en) | 1984-12-13 |
| NO842258L (en) | 1984-12-07 |
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