NOVEL REAGENT AND METHOD EMPLOYING SAME

Abstract: A reagent, a reagent kit and method for use in the determination of total bilirubin and delta -bilirubin. The reagent has a pH of about 8 to 8.4 and comprises: a) bilirubin oxidase from Myrothecium species; b) a buffer, and c) a surfactant; whereby the surfactant comprises a mixture of sodium cholate and sodium dodecyl sulphate, the sodium cholate and sodium dodecyl sulphate being present in the mixture in respective amounts such that the reagent is capable of oxidising alpha , beta , gamma and delta -bilirubin.

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, <_ , ø, 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. OMPI 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 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 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 M 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 OMPI IPO 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 M sodium cholate; about 15 ± 5 mM sodium dodecyl sulfate; and an effective amount of the - !TRE OMPI 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. TABLE I Constituent Amount Aqueous solution More (1000 ml) : Optimal Preferred Preferred Tris base, gm 7.08 ± 10% ± 15% Tris HC1, gm 6.68 ± 10% ± 15% Sodium Cholate, gm 1.72 ± 10% ± 15% Sodium Dodecyl Sulfate, gm 4.33 ± 10% ± 15% EDTA, disodium, gm 0.372 ± 10% ± 15% Deionized Water qs. 1,000 ml Composition (101.238g) Mannitol , gm 80 ± 10% ± 15% Sorbitol, gm 10 _= 10% 4- 15% Tris base, gm 5.475 ± 10% 15% Tris HC1, gm 0.763 ± 10% 15% Bilirubin Oxidase (~6 IU/mg) , gm 5 ± 10% ± 15% OMPI 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/ g) and dissolve in 1 ml water. System Parameters Wavelength 465 n 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. TABLE II Jend:rassik-Grof Present Invention X Y 1 0.4 0.4 2 0.5 0.5 3 0.3 0.3 4 0.4 0.5 5 0.6 0.6 6 0.4 0.3 7 0.2 0.2 8 0.3 0.2 9 0.3 0.2 10 0.3 0.2 11 0.7 0.7 12 0.2 0.2 13 0.2 0.2 14 0.3 0.2 15 0.3 0.2 16 0.4 0.3 17 0.3 0.4 18 0.4 0.3 19 0.6 0.3 20 0.5 ' 0.4 OMPI 21 4.7 4.7 22 15.2 14.7 23 11.3 10.7 24 * 10.2 9.7 25 11.5 11.1 26 9.1 8.6 27 18.9 18.3 28 33.0 32.6 29 19.1 18.5 30 10.1 9.4 31 12.5 12.3 32 6.6- 6.4 33 11.0 10.4 34 11.7 10.9 35 14.1 13.7 36 12.1 11.8 37 6.7 6.3 38 10.6 9.9 39 30.0 29.7 40 12.0 11.6 41 13.5 12.7 42 16.7 16.8 Y = MX + B M = 0.981 B = -0.13 x " = 7.3 7 = 7.1 S Y.X = 0-21 r 2 = 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). " * . * * J - Λ V'-' " V, : - r "

Claims: The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows: 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 M 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; - OMP PΪI (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 M 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 M 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 M EDTA; and (e) about 0.1 -fc 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 o , β , and γ bilirubin and obtaining a first measurement indicative of the concentration of _ , 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 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 o , β , γ, and δ bilirubin; (d) obtaining a second measurement indicative of the concentration of α, β, γ, and 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 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, / ' _ OMPI 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 o, 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 <_ , β, and γ bilirubin; (b) obtaining a first measurement indicative of the concentration of < _ , 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 , β, q, and w bilirubin; (d) obtaining a second measurement indicative of the concentration of , 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. ' -i ' C- i EmT _OM * pi ■ "' ' "^ ~"

Inventors: ANDERSON SUSANNE MARY, OSAKI SHIGEMASA, ANDERSON, SUSANNE, MARY, OSAKI, SHIGEMASA
Applicants: BECKMAN INSTRUMENTS INC, BECKMAN INSTRUMENTS, INC.
Application year: 1984
Main classification: C12Q1
IPC codes: C12N9, C12Q1, C12R1