EP0187749A1 - Novel reagent and method employing same - Google Patents
Novel reagent and method employing sameInfo
- Publication number
- EP0187749A1 EP0187749A1 EP19840903034 EP84903034A EP0187749A1 EP 0187749 A1 EP0187749 A1 EP 0187749A1 EP 19840903034 EP19840903034 EP 19840903034 EP 84903034 A EP84903034 A EP 84903034A EP 0187749 A1 EP0187749 A1 EP 0187749A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- bilirubin
- reagent
- sodium
- dodecyl sulfate
- fluid
- 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.)
- Withdrawn
Links
- 239000003153 chemical reaction reagent Substances 0.000 title claims abstract description 61
- 238000000034 method Methods 0.000 title claims abstract description 30
- BPYKTIZUTYGOLE-IFADSCNNSA-N Bilirubin Chemical compound N1C(=O)C(C)=C(C=C)\C1=C\C1=C(C)C(CCC(O)=O)=C(CC2=C(C(C)=C(\C=C/3C(=C(C=C)C(=O)N\3)C)N2)CCC(O)=O)N1 BPYKTIZUTYGOLE-IFADSCNNSA-N 0.000 claims abstract description 114
- NRHMKIHPTBHXPF-TUJRSCDTSA-M sodium cholate Chemical compound [Na+].C([C@H]1C[C@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC([O-])=O)C)[C@@]2(C)[C@@H](O)C1 NRHMKIHPTBHXPF-TUJRSCDTSA-M 0.000 claims abstract description 38
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 claims abstract description 34
- 108010015428 Bilirubin oxidase Proteins 0.000 claims abstract description 29
- 239000000203 mixture Substances 0.000 claims abstract description 25
- 239000004094 surface-active agent Substances 0.000 claims abstract description 20
- 239000000872 buffer Substances 0.000 claims abstract description 19
- 241000223251 Myrothecium Species 0.000 claims abstract description 15
- 238000008050 Total Bilirubin Reagent Methods 0.000 claims abstract description 6
- 230000001590 oxidative effect Effects 0.000 claims abstract description 4
- 239000012530 fluid Substances 0.000 claims description 27
- 238000005259 measurement Methods 0.000 claims description 23
- 239000011541 reaction mixture Substances 0.000 claims description 12
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims description 10
- 239000007983 Tris buffer Substances 0.000 claims description 10
- 238000002835 absorbance Methods 0.000 claims description 10
- 239000003755 preservative agent Substances 0.000 claims description 10
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims description 10
- 230000003139 buffering effect Effects 0.000 claims description 9
- 230000002335 preservative effect Effects 0.000 claims description 9
- 239000007864 aqueous solution Substances 0.000 claims description 8
- 230000002452 interceptive effect Effects 0.000 claims description 8
- 239000000243 solution Substances 0.000 claims description 7
- 239000004067 bulking agent Substances 0.000 claims description 6
- 238000011088 calibration curve Methods 0.000 claims description 6
- QXNVGIXVLWOKEQ-UHFFFAOYSA-N Disodium Chemical class [Na][Na] QXNVGIXVLWOKEQ-UHFFFAOYSA-N 0.000 claims description 4
- SNRUBQQJIBEYMU-UHFFFAOYSA-N Dodecane Natural products CCCCCCCCCCCC SNRUBQQJIBEYMU-UHFFFAOYSA-N 0.000 claims description 4
- 229910052708 sodium Inorganic materials 0.000 claims description 4
- 239000011734 sodium Substances 0.000 claims description 4
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 3
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 claims description 3
- 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 3
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 claims description 3
- 229930195725 Mannitol Natural products 0.000 claims description 3
- 229940099352 cholate Drugs 0.000 claims description 3
- BHQCQFFYRZLCQQ-OELDTZBJSA-N cholic acid Chemical compound C([C@H]1C[C@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(O)=O)C)[C@@]2(C)[C@@H](O)C1 BHQCQFFYRZLCQQ-OELDTZBJSA-N 0.000 claims description 3
- 125000003438 dodecyl group Chemical group [H]C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])* 0.000 claims description 3
- 239000000594 mannitol Substances 0.000 claims description 3
- 235000010355 mannitol Nutrition 0.000 claims description 3
- 239000000600 sorbitol Substances 0.000 claims description 3
- 244000005700 microbiome Species 0.000 claims 2
- 229940083575 sodium dodecyl sulfate Drugs 0.000 abstract 2
- 235000019333 sodium laurylsulphate Nutrition 0.000 abstract 2
- 239000000523 sample Substances 0.000 description 8
- 108090000790 Enzymes Proteins 0.000 description 6
- 102000004190 Enzymes Human genes 0.000 description 6
- 238000003556 assay Methods 0.000 description 4
- 230000002255 enzymatic effect Effects 0.000 description 4
- 241000894007 species Species 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- GWZYPXHJIZCRAJ-UHFFFAOYSA-N Biliverdin Natural products CC1=C(C=C)C(=C/C2=NC(=Cc3[nH]c(C=C/4NC(=O)C(=C4C)C=C)c(C)c3CCC(=O)O)C(=C2C)CCC(=O)O)NC1=O GWZYPXHJIZCRAJ-UHFFFAOYSA-N 0.000 description 2
- RCNSAJSGRJSBKK-NSQVQWHSSA-N Biliverdin IX Chemical compound N1C(=O)C(C)=C(C=C)\C1=C\C1=C(C)C(CCC(O)=O)=C(\C=C/2C(=C(C)C(=C/C=3C(=C(C=C)C(=O)N=3)C)/N\2)CCC(O)=O)N1 RCNSAJSGRJSBKK-NSQVQWHSSA-N 0.000 description 2
- QBUVFDKTZJNUPP-UHFFFAOYSA-N biliverdin-IXalpha Natural products N1C(=O)C(C)=C(C=C)C1=CC1=C(C)C(CCC(O)=O)=C(C=C2C(=C(C)C(C=C3C(=C(C=C)C(=O)N3)C)=N2)CCC(O)=O)N1 QBUVFDKTZJNUPP-UHFFFAOYSA-N 0.000 description 2
- 239000013610 patient sample Substances 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 102000009027 Albumins Human genes 0.000 description 1
- 108010088751 Albumins Proteins 0.000 description 1
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 1
- FBPFZTCFMRRESA-JGWLITMVSA-N D-glucitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-JGWLITMVSA-N 0.000 description 1
- 229920001030 Polyethylene Glycol 4000 Polymers 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 238000010420 art technique Methods 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 229910052802 copper Inorganic materials 0.000 description 1
- 239000010949 copper Substances 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 229940068918 polyethylene glycol 400 Drugs 0.000 description 1
- 239000000376 reactant Substances 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 238000007430 reference method Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
Definitions
- This invention relates to an enzymatic bilirubin assay and to a reagent and kit for use therein.
- bilirubin oxidase from Myrothecium species does not measure all fractions of bilirubin in a sample, even when sodium dodecyl sulfate or sodium cholate is employed to disassociate the albumin-bound bilirubin. More, particularly, the present inventors have discovered that bilirubin oxidase from Myrothecium species does not measure ⁇ bilirubin, even with the use of. sodium dodecyl sulfate or sodium cholate.
- bilirubin oxidase from Myrothecium species is capable of measuring all bilirubin fractions present in a sample to be assayed, including ⁇ bilirubin.
- enzymatic methodologies and reagents and kits for use therein which employ bilirubin oxidase from Myrocthecium species and which are capable of measuring substantially all bilirubin present in a sample to be assayed, including ⁇ bilirubin.
- the present invention encompasses an improved reagent.
- the reagent is of the type comprising bilirubin oxidase from Myrothecium species, a buffer, and a surfactant, and having a pH of about 8 to about 8.4.
- the reagent of the present invention is characterized in that the surfactant comprises a mixture of sodium cholate and sodium dodecyl cholate in respective amounts such that the reagent is capable of oxidizing ⁇ , ⁇ , ⁇ , and ⁇ bilirubin.
- the present invention also encompasses a method for the determination of total bilirubin in a fluid.
- the method of the present invention is of the type comprising (a) reacting each of a series of aqueous bilirubin solution having varying known bilirubin concentration with a reagent to form reaction mixtures in order to construct a calibration curve representing a relationship between absorbance of the reaction mixtures and the concentrations of the respective bilirubin solutions; (b) reacting a fluid having an unknown bilirubin concentration with the reagent and measuring the observance of the resultant reaction mixture; and (c) determining the bilirubin concentration in the fluid by comparing the measured value obtained in step (b) with the calibration curve.
- the method of the present invention is characterized in the above reagent employed therein.
- the instant invention also encompasses a method for determining ⁇ bilirubin.
- This method comprises (a) contacting a first sample of a fluid with a reagent capable of only assaying o, b, and ⁇ bilirubin and obtaining a first measurement indicative of the concentration of ⁇ , ⁇ , and ⁇ bilirubin; (b) contacting a second sample of the fluid with the above reagent and obtaining a second measurement indicative- of the concentration of ⁇ , ⁇ , ⁇ , and ⁇ bilirubin present in the fluid; and (c) subtracting the first measurement from the second measurement.
- the present invention also encompasses another method for determining ⁇ bilirubin.
- This latter method comprises (a) contacting a sample of a fluid with a reagent comprising bilirubin oxidase from Myrothecium species; a buffer; and a surfactant selected from a group consisting of sodium cholate, sodium dodecyl sulfate, and mixtures thereof, the surfactant being present in an amount only capable of assaying for ⁇ , ⁇ , and ⁇ bilirubin; (b) obtaining a first measurement indicative of the concentration of ⁇ , ⁇ , and ⁇ bilirubin present in the fluid; (c) adding to the reaction mixture formed in step (a) a surfactant selected from a group consisting of sodium cholate, sodium dodecyl sulfate, and mixtures thereof, the surfactant present in this step (c) being in an amount capable of assaying for ⁇ , ⁇ , ⁇ , and ⁇ bilirub
- kits comprising, in association, an aqueous solution and a composition.
- the aqueous solution comprises a buffer having a buffering capacity in the pH range of about 8.2 ⁇ 0.2; about 4 ⁇ 2 mM sodium cholate; about 15 ⁇ 5 mM sodium dodecyl sulfate; and an effective amount of the non-interfering preservative.
- the composition comprises bilirubin oxidase from Myrothecium species; an effective amount of a bulking agent; and a buffer having a buffering capacity in the pH range of 9.0 ⁇ 0.2.
- the pH of the reagent of the instant invention is preferably 8.2 ⁇ 0.5, more preferably 8.2 ⁇ 0.1, and optimally 8.2 ⁇ 0.05.
- the mixture of sodium cholate and sodium dodecyl sulfate is formulated such that it comprises about 4 ⁇ 2 mM sodium cholate and about 15 ⁇ 5 mM sodium dodecyl sulfate. More preferably, the mixture comprises about 4 ⁇ 1 mM sodium cholate and about 15 ⁇ 2 mM sodium dodecyl sulfate. Optimally, the mixture comprises about 4 ⁇ 0.5 mM sodium cholate and about 15 ⁇ 1 mM sodium dodecyl sulfate.
- the reagent of the instant invention preferably also comprises an effective amount of a non-intefering preservative.
- non-interfering is meant a preservative which neither adversely affects the bilirubin oxidase nor other reactants present in the assay.
- Virtually any non- interfering preservative can be employed in the reagent of the present invention.
- Such non-interfering preservatives include, but are not limited to, EDTA, disodium salt.
- EDTA disodium salt
- M EDTA, disodium salt is employed in the present invention.
- any buffer having a buffering capacity in the desired pH range can be employed in the reagent of the present invention.
- buffers include, but are not limited to, Tris buffer.
- M Tris buffer is employed in the reagent of the present invention.
- the amount of bilirubin oxidase from Myrocthecium species employed in the present invention is not critical and is determined primarily by the speed at which one desires the reaction to proceed.
- at least about 1, more preferably at least about 5, and yet more preferably at least about 10, IU/ml bilirubin oxidase is employed in the reagent of the present invention.
- at least about 25 IU/ml bilirubin oxidase from Myrothecium species is employed in the reagent of the present invention.
- the reagent of the instant invention can be prepared via any technique known to those skilled in the art.
- One convenient technique employs a kit.
- This kit comprises, in association, an aqueous solution and a composition.
- the aqueous solution comprises a buffer having a buffering capacity in the pH range of about 8.2 ⁇ 0.2; about 4 ⁇ 2 mM sodium cholate; about 15 ⁇ 5 mM sodium dodecyl sulfate; and an effective amount of the non-interfering preservative.
- the composition comprises bilirubin oxidase from Myrothecium species; an effective amount of a bulking agent; and a buffer having a buffering capacity in the pH range of 9.0 ⁇ 0.2.
- any bulking agent can be employed in the kit of the present invention.
- Such bulking agents include, but are not limited to, mannitol, sorbitol, any polyethylene glycol 400 (PEG 4000).
- Example 1 The following enzymatic procedures were employed to assay total bilirubin in serum.
- Buffer - an aqueous solution comprising 7.08 gm Tris base, 6.68 gm Tris HCl, 1.72 gm Sodium Cholate, 4.33 gm Sodium Dodecyl Sulfate, 0.372 gm EDTA, disodium salt, deionized water qs. 1,000 ml.
- Enzyme Reagent - take 100 mg of a composition comprising 80 gm mannitol, 10 gm sorbitol, 5.475 gm Tris base, 0.763 gm Tris HCl, and 5 gm bilirubin oxidase (6 IU/mg) and dissolve in 1 ml water.
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- Life Sciences & Earth Sciences (AREA)
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- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- Physics & Mathematics (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
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- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
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- Enzymes And Modification Thereof (AREA)
Abstract
Réactif, réactif en kit et procédé d'utilisation pour détecter la bilirubine totale et la delta-bilirubine. Le réactif a un pH d'environ 8 à 8,4 et comprend: a) de l'oxydase de bilirubine de l'espèce Myrothecium; b) un tampon, et c) un agent tensio-actif. L'agent tensio-actif comprend un mélange de cholate de sodium et de dodécylsulfate de sodium, les quantités respectives dans le mélange de cholate de sodium et de dodécylsulfate de sodium étant telles que le réactif est susceptible d'oxyder de la bilirubine alpha, beta, gamma et delta.Reagent, kit reagent and method of use for detecting total bilirubin and delta-bilirubin. The reagent has a pH of about 8 to 8.4 and includes: a) bilirubin oxidase of the species Myrothecium; b) a buffer, and c) a surfactant. The surfactant comprises a mixture of sodium cholate and sodium dodecylsulfate, the respective amounts in the mixture of sodium cholate and sodium dodecylsulfate being such that the reagent is capable of oxidizing bilirubin alpha, beta , gamma and delta.
Description
NOVEL REAGENT AND METHOD EMPLOYING SAME
Background of the Invention
1. Field of the Invention
This invention relates to an enzymatic bilirubin assay and to a reagent and kit for use therein.
2. Description of the Prior Art
Kosaka et al. (1) reported a novel method for the determination of total bilirubin (which consists of four fractions, namely, α , β, γ, and δ bilirubin) in serum using a new enzyme, bilirubin oxidase, which had been isolated and purified from Myrothecium species. Bilirubin oxidase is a copper-containing enzyme which catalyzes the oxidation of bilirubin to biliverdin and water. The biliverdin formed is successfully oxidized to color substances. As the enzyme does not work on albumin-bilirubin, Kosaka et al, found it necessary to disassociate the bound bilirubin with sodium dodecyl sulfate or sodium cholate.
The present inventors have discovered that contrary to the position of Kosaka et al., bilirubin oxidase from Myrothecium species does not measure all fractions of bilirubin in a sample, even when sodium dodecyl sulfate or sodium cholate is employed to disassociate the albumin-bound bilirubin. More, particularly, the present inventors have discovered that bilirubin oxidase from Myrothecium species does not measure δ bilirubin, even with the use of. sodium dodecyl sulfate or sodium cholate.
Accordingly, it would be very desirable to have an enzymatic bilirubin assay and a reagent and kit for use therein wherein bilirubin oxidase from Myrothecium species is capable of measuring all bilirubin fractions present in a sample to be assayed, including δ bilirubin.
Summary of the Invention In accordance with the present invention, there are provided enzymatic methodologies and reagents and kits for use therein which employ bilirubin oxidase from Myrocthecium species and which are capable of measuring substantially all bilirubin present in a sample to be assayed, including δ bilirubin.
More particularly, the present invention encompasses an improved reagent. The reagent is of the type comprising bilirubin oxidase from Myrothecium species, a buffer, and a surfactant, and having a pH of about 8 to about 8.4. The reagent of the present invention is characterized in that the surfactant comprises a mixture of sodium cholate and sodium dodecyl cholate in respective amounts such that the reagent is capable of oxidizing α, β, γ, and δ bilirubin.
The present invention also encompasses a method for the determination of total bilirubin in a fluid. The method of the present invention is of the type comprising (a) reacting each of a series of aqueous bilirubin solution having varying known bilirubin concentration with a reagent to form reaction mixtures in order to construct a calibration curve representing a relationship between absorbance of the reaction mixtures and the concentrations of the respective bilirubin solutions; (b) reacting a fluid having an unknown bilirubin concentration with the reagent and measuring the observance of the resultant reaction mixture; and (c)
determining the bilirubin concentration in the fluid by comparing the measured value obtained in step (b) with the calibration curve. The method of the present invention is characterized in the above reagent employed therein.
In addition, the instant invention also encompasses a method for determining δ bilirubin. This method comprises (a) contacting a first sample of a fluid with a reagent capable of only assaying o, b, and γ bilirubin and obtaining a first measurement indicative of the concentration of α, β, and γ bilirubin; (b) contacting a second sample of the fluid with the above reagent and obtaining a second measurement indicative- of the concentration of α, β, γ, and δ bilirubin present in the fluid; and (c) subtracting the first measurement from the second measurement.
The present invention also encompasses another method for determining δ bilirubin. This latter method comprises (a) contacting a sample of a fluid with a reagent comprising bilirubin oxidase from Myrothecium species; a buffer; and a surfactant selected from a group consisting of sodium cholate, sodium dodecyl sulfate, and mixtures thereof, the surfactant being present in an amount only capable of assaying for α, β, and γ bilirubin; (b) obtaining a first measurement indicative of the concentration of α, β, and γ bilirubin present in the fluid; (c) adding to the reaction mixture formed in step (a) a surfactant selected from a group consisting of sodium cholate, sodium dodecyl sulfate, and mixtures thereof, the surfactant present in this step (c) being in an amount capable of assaying for α, β, γ, and δ bilirubin; (d) obtaining a second measurement indicative of the concentration of α, β, γ, and δ bilirubin present in the fluid; and (e) substrating the first measurement from the second measurement.
Furthermore, the present invention also encompasses a kit. The kit comprises, in association, an aqueous solution and a composition. The aqueous solution comprises a buffer having a buffering capacity in the pH range of about 8.2 ± 0.2; about 4 ± 2 mM sodium cholate; about 15 ± 5 mM sodium dodecyl sulfate; and an effective amount of the non-interfering preservative. The composition comprises bilirubin oxidase from Myrothecium species; an effective amount of a bulking agent; and a buffer having a buffering capacity in the pH range of 9.0 ± 0.2.
Still other features and attendant advantages of the present invention will become apparent to those skilled in the art from a reading of the following detailed description of the preferred embodiments.
Description of the Preferred Embodiments The pH of the reagent of the instant invention is preferably 8.2 ± 0.5, more preferably 8.2 ± 0.1, and optimally 8.2 ± 0.05.
Preferably, the mixture of sodium cholate and sodium dodecyl sulfate is formulated such that it comprises about 4 ± 2 mM sodium cholate and about 15 ± 5 mM sodium dodecyl sulfate. More preferably, the mixture comprises about 4 ± 1 mM sodium cholate and about 15 ± 2 mM sodium dodecyl sulfate. Optimally, the mixture comprises about 4 ± 0.5 mM sodium cholate and about 15 ± 1 mM sodium dodecyl sulfate.
The reagent of the instant invention preferably also comprises an effective amount of a non-intefering preservative. By non-interfering is meant a preservative which neither adversely affects the bilirubin oxidase nor
other reactants present in the assay. Virtually any non- interfering preservative can be employed in the reagent of the present invention. Such non-interfering preservatives include, but are not limited to, EDTA, disodium salt. Preferably, from about 0.05 to about 0.2, more preferably about 0.1 ± 0.5, and more preferably about 0.1 ± 0.02, M EDTA, disodium salt is employed in the present invention.
Virtually any buffer having a buffering capacity in the desired pH range can be employed in the reagent of the present invention. Such buffers include, but are not limited to, Tris buffer. Preferably, about 0.1 ± 0.01, and more preferably about 0.1 ± 0.005, M Tris buffer is employed in the reagent of the present invention.
The amount of bilirubin oxidase from Myrocthecium species employed in the present invention is not critical and is determined primarily by the speed at which one desires the reaction to proceed. Preferably, at least about 1, more preferably at least about 5, and yet more preferably at least about 10, IU/ml bilirubin oxidase is employed in the reagent of the present invention. Optimally at least about 25 IU/ml bilirubin oxidase from Myrothecium species is employed in the reagent of the present invention.
The reagent of the instant invention can be prepared via any technique known to those skilled in the art. One convenient technique employs a kit. This kit comprises, in association, an aqueous solution and a composition. The aqueous solution comprises a buffer having a buffering capacity in the pH range of about 8.2 ± 0.2; about 4 ± 2 mM sodium cholate; about 15 ± 5 mM sodium dodecyl sulfate; and an effective amount of the
non-interfering preservative. The composition comprises bilirubin oxidase from Myrothecium species; an effective amount of a bulking agent; and a buffer having a buffering capacity in the pH range of 9.0 ± 0.2.
Virtually any bulking agent can be employed in the kit of the present invention. Such bulking agents include, but are not limited to, mannitol, sorbitol, any polyethylene glycol 400 (PEG 4000).
Exemplary kit formulations are set forth in
Table I.
The following examples are provided for the purpose of further illustration only and are not intended to be limitations on the disclosed invention.
Example 1 The following enzymatic procedures were employed to assay total bilirubin in serum.
Materials
Buffer - an aqueous solution comprising 7.08 gm Tris base, 6.68 gm Tris HCl, 1.72 gm Sodium Cholate, 4.33 gm Sodium Dodecyl Sulfate, 0.372 gm EDTA, disodium salt, deionized water qs. 1,000 ml.
Enzyme Reagent - take 100 mg of a composition comprising 80 gm mannitol, 10 gm sorbitol, 5.475 gm Tris base, 0.763 gm Tris HCl, and 5 gm bilirubin oxidase (6 IU/mg) and dissolve in 1 ml water.
System Parameters Wavelength 465 nm
Incubation Temperature 37 °C. Mode Absorbance
Absorbance Range 0 to 1.5A Optical Pathlength 1.0 cm Reaction Time 5 minutes Sample Volume 0.050 ml Buffer Volume 1.00 ml Enzyme Reagent Volume 0.020 ml
1. To 1 ml buffer solution placed in a spectrophotometer at 37°C.,
2. Add 50 μl sample or standard.
3. Record the absorbance at 465 nm (Ai).
4. Add 20 μl of the enzyme solution and
5. Incubate for 5 minutes.
6. Read absorbance at 465 nm (Ae).
Calculations:
From Ai (0.280) and Ae (0.055) of the standard (Beckman Instruments, Inc.'s Ultimate" C-8 brand calibrator (7.7 mg/dl assigned valve)), computed mg/dl bilirubin per unit absorbance at 465 nm (factor = 34.2). Multiply (Ai-Ae) of unknown by the factor.
Patient samples (n=42) were assayed and the results are set forth in Table II.
Y = MX + B
M = 0.981
B = -0.13 = 7.3
= 7.1
SY.X = 0-21 r2 = 0.999
Example 2
The same 42 patient samples were also assayed by a Jendrassik-Grof reference method and the results are also set forth in Table II.
As indicated by the data in Table II, the reagent of the instant invention correlates well with a recognized prior art technique.
Based upon this disclosure, many other modifications and ramifications will naturally suggest themselves to those skilled in the art. These are intended to be within the scope of this invention.
Bibliography 1. Kosaka et al., Clin. Biochem., 16: abstract A108 (October 1983).
Claims
1. A reagent of the type having a pH of about 8 to about 8.4 and comprising:
(a) bilirubin oxidase from Myrothecium species;
(b) a buffer; and
(c) a surfactant; characterized in that:
(a) said surfactant comprises a mixture of sodium cholate and sodium dodecyl sulfate, said sodium cholate and sodium dodecyl sulfate being present in said mixture in respective amounts such that said reagent is capable of oxidizing α, β, γ, and δ bilirubin.
2. The reagent of claim 1 having a pH of about 8.2 ± 0.5 and comprising:
(a) about 4 ± 2 mM sodium cholate; and
(b) about 15 ± 5 mM sodium dodecyl sulfate.
3. The reagent of claim 1 having a pH of about 8.2 ± 0.1 and comprising:
(a) about 4 ± 1 mM sodium cholate; and
(b) about 15 ± 2 mM sodium dodecyl sulfate.
4. The reagent of claim 1 having a pH of about 8.2 ± 0.05 and comprising:
(a) about 4 ± 0.5 mM sodium cholate; and
(b) about 15 ± 1 mM sodium dodecyl sulfate.
5. The reagent of claim 1 further comprising an effective amount of a non-interfering preservative.
6. The reagent of claim 5 comprising:
(a) at least about 1 IU/ml bilirubin oxidase;
(b) about 4 ± 2 mM sodium cholate;
(c) about 15 ± 5 mM sodium dodecyl sulfate; and
(d) from about 0.05 to about 0.2 mM EDTA.
7. The reagent of claim 5 having a pH of about 8.2 ± 0.1 and comprising:
(a) at least about 5 IU/ml bilirubin oxidase;
(b) about 4 ± 1 mM sodium cholate;
(c) about 15 ± 2 mM sodium dodecyl sulfate;
(d) about 0.1 ± 0.5 mM EDTA; and
(e) about 0.1 ± 0.01 M Tris buffer.
8. The reagent of claim 5 having a pH of about 8.2 ± 0.05 and comprising:
(a) at least about 10 IU/ml bilirubin oxidase;
(b) about 4 ± 0.5 mM sodium cholate;
(c) about 15 ± 1 mM sodium dodecyl sulfate;
(d) about 0.1 ± 0.02 mM EDTA; and
(e) about 0.1 ± 0.005 M Tris buffer.
9. A method for the determination of total bilirubin in a fluid, said method being of the type comprising:
(a) reacting each of a series of aqueous bilirubin solutions having varying known bilirubin concentrations with a reagent to form reaction mixtures in order to construct a calibration curve representing a relationship between absorbance of said reaction mixtures and the concentrations of the respective bilirubin solutions;
(b) reacting a fluid having an unknown bilirubin concentration with said reagent and measuring the absorbance of the resulting reaction mixture; and
(c) determining the bilirubin concentration in said fluid by comparing the measured value obtained in step (b) with said calibration curve.
characterized in that the reagent employed therein is the reagent of claim 1.
10. The method of claim 9 wherein said reagent having a pH from about 8.2 ± 0.5 and comprises:
(a) about 4 ± 2 mM sodium cholate; and
(b) about 15 ± 5 mM sodium dodecyl sulfate.
11. The method of claim 9 wherein said reagent has a pH of about 8.2 ± 0.1 and comprises:
(a) about 4 ± 1 mM sodium cholate; and
(b) about 15 ± 2 mM sodium dodecyl sulfate.
12. The method of claim 9 wherein said reagent has a pH of about 8.2 ± 0.05 and comprises:
(a) about 4 ± 0.5 mM sodium cholate; and
(b) about 15 ± 1 mM sodium dodecyl sulfate.
13. The method of claim 9 wherein said reagent further comprises an effective amount of a non-intefering preservative.
14. The method of claim 13 wherein said reagent comprises:
(a) at least about 1 IU/ml bilirubin oxidase;
(b) about 4 ± 2 mM sodium cholate;
(c) about 15 ± 5 mM sodium dodecyl sulfate; and
(d) from about 0.05 to about 0.2 mM EDTA.
15. The method of claim 13 wherein said reagent has a pH of about 8.2 ± 0.1 and comprises:
(a) at least about 5 IU/ml bilirubin oxidase;
(b) about 4 ± 1 mM sodium cholate;
(c) about 15 ± 2 mM sodium dodecyl sulfate;
(d) about 0.1 ± 0.5 mM EDTA; and
(e) about 0.1 ± 0.01 M Tris buffer.
16. The method of claim 13 wherein said reagent has a pH of about 8.2 ± 0.05 and comprises:
(a) at least about 10 IU/ml bilirubin oxidase;
(b) about 4 ± 0.5 mM sodium cholate;
(c) about 15 ± 1 mM sodium dodecyl sulfate;
(d) about 0.1 ± 0.02 mM EDTA; and
(e) about 0.1 ± 0.005 M Tris buffer.
17. A method for determining δ bilirubin comprising:
(a) contacting a first sample of a fluid with a reagent capable of only assaying α, β, and γ bilirubin and obtaining a first measurement indicative of the concentration of α, β, and γ bilirubin;
(b) contacting a second sample of said fluid with the reagent of claim 1 and obtaining a second measurement indicative of the concentration of α, β, γ, and δ bilirubin present in said fluid; and
(c) subtracting said first measurement from said second measurement.
18. A method for determining δ bilirubin comprising:
(a) contacting a sample of a fluid with a reagent comprising bilirubin oxidase produced by a microorganism of the genus Myrothecium; a buffer; and a surfactant selected from a group consisting of sodium cholate, sodium dodecyl sulfate, and mixtures thereof, said surfactant being present in an amount only capable of assaying for α, β, and γ bilirubin;
(b) obtaining a first measurement indicative of the concentration of α, β, and γ bilirubin present in said fluid;
(c) adding to the reaction mixture formed in step (a) a surfactant selected from a group consisting of sodium cholate, sodium dodecyl sulfate, and mixtures thereof, said surfactant present in this step (c) being in an amount capable of assaying for α, β, γ, and δ bilirubin;
(d) obtaining a second measurement indicative of the concentration of α, β, γ, and δ bilirubin present in said fluid; and
(e) subtracting said first measurement from said second measurement.
19. A kit comprising in association:
(a), an aqueous solution comprising:
(i) a buffer having a buffering capacity in the pH range of about 8.2 ± 0.2; (ii) 4 ± 2 mM sodium cholate; (iii) 15 ± 5 mM sodium dodecyl cholate; and (iv) an effective amount of a non-interfering preservative; and (b) a composition comprising:
(i) bilirubin oxidase from the Myrothecium species; (ii) an effective amount of a bulking agent; and (iii) a buffer having a buffering capacity in the pH range of 9.0 ± 0.2.
20. The kit of claim 19 wherein:
(a) said aqueous solution comprises:
(i) about 0.1 ± 0.01 M Tris buffer; (ii) about 4 ± 1 mM sodium cholate; (iii) about 15 ± 2 mM sodium dodecyl suifate; and
(iv) from about 0.05 to about 0.2 mM EDTA, disodium salt; and (b) said composition comprises:
(i) at least about 10 IU/gm bilirubin oxidase; (ii) about 0.8 ± 0.1 gm/gm mannitol and about 0.1 ± 0.01 gm/gm sorbitol; and (iii) about 0.055 ± 0.0055 gm/gm Tris base and about 0.0076 ± 0.0008 gm/gm Tris HCl.
NOVEL REAGENT AND METHOD EMPLOYING SAME
Abstract of the Disclosure A reagent of the type having a pH of about 8 to about 8.4 and comprising:
(a) bilirubin oxidase from Myrothecium species;
(b) a buffer; and
(c) a surfactant; characterized in that:
(a) the surfactant comprises a mixture of sodium cholate and sodium dodecyl sulfate, the sodium cholate and sodium dodecyl sulfate being present in the mixture in respective amounts such that the reagent is capable of oxidizing α, β, γ, and δ bilirubin.
A method for the determination of total bilirubin in a fluid, the method being of the type comprising:
(a) reacting each of a series of aqueous bilirubin solutions having varying known bilirubin concentrations with a reagent to form reaction mixtures in order to construct a calibration curve representing a relationship between absorbance of the reaction mixtures and the concentrations of the respective bilirubin solutions;
(b) reacting a fluid having an unknown bilirubin concentration with the reagent and measuring the absorbance of the resulting reaction mixture; and
(c) determining the bilirubin concentration in the fluid by comparing the measured value obtained in step (b) with the calibration curve,
characterized in that the above reagent is employed therein.
A method for determining δ bilirubin comprising:
(a) contacting a first sample of a fluid with a reagen capable of only assaying α, β, and γ bilirubin;
(b) contacting a second sample of said fluid with the reagent of claim 1 and obtaining a second measurement indicative of the concentration of α, β, γ, and δ biliruin present in said fluid; and
(c) subtracting the first measurement from the second measurement.
A mehod for determining δ bilirubin comprising:
(a) contacting a sample of a fluid with a reagent comprising bilirubin oxidase produced by a microorganism of the genus Myrothecium; a buffer; and a surfactant selected from a group consisting of sodium cholate, sodium dodecyl sulfate, and mixtures thereof, the surfactant being present in an amount only capable of assaying for α, β, and γ bilirubin;
(b) obtaining a first measurement indicative of the concentration of α, β, and q bilirubin present in the fluid;
(c) adding to the reaction mixture formed in step (a) a surfactant selected from a group consisting of sodium cholate, sodium dodecyl sulfate, and mixtures thereof, the surfactant present in this step (c) being in an amount capable of asaying for α, β, q, and w bilirubin;
(d) obtaining a second measurement indicative of the concentration of α, β, q, and w bilirubin present in said fluid; and
(e) subtracting the first measurement from the second measurement.
A kit comprising in association:
(a) an aqueous solution comprising:
(i) a buffer having a buffering capacity in the pH range of about 8.2 ± 0.2; (ii) 4 ± mM sodium cholate; (iii) 15 ± 5 mM sodium dodecyl cholate; and (iv) an effective amount of a non-interfering preservative; and
(b) a composition comprising:
(i) bilirubin oxidase from Myrothecium species; (ii) an effective amount of a bulking agent; and (iii) a buffer having a buffering capacity in the pH range of 9.0 ± 0.2.
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| PCT/US1984/001215 WO1986000933A1 (en) | 1984-07-28 | 1984-07-28 | Novel reagent and method employing same |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| EP0187749A1 true EP0187749A1 (en) | 1986-07-23 |
Family
ID=22182218
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| EP19840903034 Withdrawn EP0187749A1 (en) | 1984-07-28 | 1984-07-28 | Novel reagent and method employing same |
Country Status (3)
| Country | Link |
|---|---|
| EP (1) | EP0187749A1 (en) |
| JP (1) | JPS61502443A (en) |
| WO (1) | WO1986000933A1 (en) |
Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4751190A (en) * | 1985-07-22 | 1988-06-14 | Abbott Laboratories | Fluorescence polarization immunoassay and reagents for use therein |
| DE3608453A1 (en) * | 1986-03-14 | 1987-09-17 | Boehringer Mannheim Gmbh | METHOD FOR ENZYMATICALLY DETERMINING BILIRUBIN IN SERUM |
| FR2653891B1 (en) * | 1989-10-31 | 1994-01-14 | Fathi Moussa | METHOD FOR DETERMINING BILIRUBINS AND / OR BILIVERDINES PRESENT IN A BIOLOGICAL MEDIUM BY ELECTROCHEMICAL OXIDATION. |
| US5783407A (en) * | 1996-04-05 | 1998-07-21 | Beckman Instruments, Inc. | Method of measuring bilirubin |
| JP3734115B2 (en) * | 1997-02-28 | 2006-01-11 | 日東紡績株式会社 | Method for measuring bilirubin fraction |
| EP2108703B1 (en) * | 2007-04-27 | 2012-08-08 | ARKRAY, Inc. | Method for bilirubin determination and analytical instrument used for bilirubin determination |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE3249743C2 (en) * | 1982-02-18 | 1990-10-04 | Amano Pharmaceutical Co., Ltd., Nagoya, Aichi, Jp | |
| JPS59125899A (en) * | 1982-12-29 | 1984-07-20 | Nippon Shoji Kk | Reagent for determination of direct-acting bilirubin by the enzymatic method and its determination procedure |
-
1984
- 1984-07-28 EP EP19840903034 patent/EP0187749A1/en not_active Withdrawn
- 1984-07-28 JP JP59503001A patent/JPS61502443A/en active Pending
- 1984-07-28 WO PCT/US1984/001215 patent/WO1986000933A1/en not_active Ceased
Non-Patent Citations (1)
| Title |
|---|
| See references of WO8600933A1 * |
Also Published As
| Publication number | Publication date |
|---|---|
| WO1986000933A1 (en) | 1986-02-13 |
| JPS61502443A (en) | 1986-10-30 |
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