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US4211844A
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Bilirubin-specific fungal enzyme preparation
Assignee: Clinical Diagnostic Systems Inc
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Filing: 1978-05-19
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Granted: 1980-07-08
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Expires: 1998-05-19
Abstract
Abstract A fungal enzyme preparation has specific bilirubin degrading activity and, in a preferred embodiment, generates hydrogen peroxide. Extraction methods for the enzyme preparation are disclosed. Assay compositions, elements and methods using the aforementioned enzyme preparation are also disclosed.
Inventors
- 👤 Tai-Wing Wu
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-07 18:01
CPC Classifications
C; C12; C12N; C12N9/00; C12N9/0004; C12Q; C12Q1/00; C12Q1/34; G; G01; G01N; G01N33/00; G01N33/48; G01N33/50; G01N33/72; G01N33/728; Y; Y10; Y10S; Y10S435/00; Y10S435/814; Y10S435/816; Y10S435/8215; Y10S435/911
Claims Analysis
Total Claims: 32
Claim 1
Independent Claim
44 words
A bilirubin-specific enzyme preparation comprising a fungal enzyme which, in the presence of a bilirubin-containing aqueous liquid having a pH of about 7.4 and a temperature of about 37° C., degrades at least about 0.02 micromoles of bilirubin per minute per milligram of protein.
Claim 2
Dependent Claim
44 words
A bilirubin-specific enzyme preparation comprising a fungal enzyme which, in the presence of a bilirubin-containing aqueous liquid having a pH of about 7.4 and a temperature of about 37° C., degrades at least about 0.02 micromoles of bilirubin per minute per milligram of protein.
Claim 3
Dependent Claim
60 words
A bilirubin-specific enzyme preparation as described in claim 1 which, in the presence of a bilirubin-containing aqueous liquid maintained at a pH of about 7.4 and a temperature of 37° C., yields a detectable reaction product that exhibits an absorption peak at about 510 nm and that, upon excitation with 450 nm radiation, exhibits fluorescence emission at about 525 nm.
Claim 4
Dependent Claim
12 words
A bilirubin-specific enzyme preparation as described in claim 1 derived from mushroom.
Claim 5
Dependent Claim
48 words
A bilirubin-specific enzyme preparation comprising a fungal enzyme which, in the presence of a bilirubin-containing aqueous liquid having a pH of about 7.4 and a temperature of about 37° C., degrades at least about 0.02 micromoles of bilirubin per minute per milligram of protein and generates hydrogen peroxide.
Claim 6
Dependent Claim
48 words
A bilirubin-specific enzyme preparation comprising a fungal enzyme which, in the presence of a bilirubin-containing aqueous liquid having a pH of about 7.4 and a temperature of about 37° C., degrades at least about 0.02 micromoles of bilirubin per minute per milligram of protein and generates hydrogen peroxide.
Claim 7
Dependent Claim
60 words
A bilirubin-specific enzyme preparation as described in claim 4 which, in the presence of a bilirubin-containing aqueous liquid maintained at a pH of about 7.4 and a temperature of 37° C., yields a detectable reaction product that exhibits an absorption peak at about 510 nm and that, upon excitation with 450 nm radiation, exhibits fluorescence emission at about 525 nm.
Claim 8
Dependent Claim
12 words
A bilirubin-specific enzyme preparation as described in claim 4 derived from mushroom.
Claim 9
Dependent Claim
68 words
An assay composition for the determination of bilirubin in an aqueous liquid, said composition comprising a hydrogen-peroxide detection composition and a bilirubin-specific enzyme preparation comprising a mushroom enzyme which, in the presence of a bilirubin-containing aqueous liquid having a pH of about 7.4 and a temperature of about 37° C., degrades at least about 0.02 micromoles of bilirubin per minute per milligram of protein and generates hydrogen peroxide.
Claim 10
Dependent Claim
68 words
An assay composition for the determination of bilirubin in an aqueous liquid, said composition comprising a hydrogen-peroxide detection composition and a bilirubin-specific enzyme preparation comprising a mushroom enzyme which, in the presence of a bilirubin-containing aqueous liquid having a pH of about 7.4 and a temperature of about 37° C., degrades at least about 0.02 micromoles of bilirubin per minute per milligram of protein and generates hydrogen peroxide.
Claim 11
Dependent Claim
15 words
An assay composition as described in claim 7 wherein said assay composition contains a buffer.
Claim 12
Dependent Claim
24 words
An assay composition as described in claim 7 wherein said hydrogen peroxide detection composition comprises a material having peroxidative activity and a chromogenic indicator.
Claim 13
Dependent Claim
20 words
An assay composition as described in claim 7 wherein said hydrogen peroxide detection composition comprises peroxidase and a chromogenic indicator.
Claim 14
Dependent Claim
69 words
A dry test element for the assay of an aqueous liquid, said element having a reagent zone containing an assay composition for bilirubin, said assay composition for bilirubin comprising a mushroom enzyme preparation which, in the presence of a bilirubin-containing aqueous liquid having a pH of about 7.4 and a temperature of about 37° C., degrades at least about 0.02 micromoles of bilirubin per minute per milligram of protein.
Claim 15
Dependent Claim
69 words
A dry test element for the assay of an aqueous liquid, said element having a reagent zone containing an assay composition for bilirubin, said assay composition for bilirubin comprising a mushroom enzyme preparation which, in the presence of a bilirubin-containing aqueous liquid having a pH of about 7.4 and a temperature of about 37° C., degrades at least about 0.02 micromoles of bilirubin per minute per milligram of protein.
Claim 16
Independent Claim
83 words
A method for the determination of bilirubin in an aqueous liquid sample comprising (a) contacting said liquid sample with a bilirubin-specific enzyme preparation to interact bilirubin with said enzyme preparation and produce a detectable change, said enzyme preparation containing a mushroom enzyme which, in the presence of a bilirubin-containing aqueous liquid having a pH of about 7.4 and a temperature of about 37° C., degrades at least about 0.02 micromoles of bilirubin per minute per milligram of protein; and (b) detecting said change.
Claim 17
Independent Claim
83 words
A method for the determination of bilirubin in an aqueous liquid sample comprising (a) contacting said liquid sample with a bilirubin-specific enzyme preparation to interact bilirubin with said enzyme preparation and produce a detectable change, said enzyme preparation containing a mushroom enzyme which, in the presence of a bilirubin-containing aqueous liquid having a pH of about 7.4 and a temperature of about 37° C., degrades at least about 0.02 micromoles of bilirubin per minute per milligram of protein; and (b) detecting said change.
Claim 18
Independent Claim
96 words
A method for the determination of bilirubin in an aqueous liquid sample comprising (a) contacting said liquid sample with an assay composition containing a hydrogen-peroxide detection composition and a bilirubin-specific enzyme preparation to interact bilirubin with said enzyme preparation and produce a detectable change, said enzyme preparation containing a mushroom enzyme which, in the presence of a bilirubin-containing aqueous liquid having a pH of about 7.4 and a temperature of about 37° C., degrades at least about 0.02 micromoles of bilirubin per minute per milligram of protein and generates hydrogen peroxide; and (b) detecting said change.
Claim 19
Independent Claim
96 words
A method for the determination of bilirubin in an aqueous liquid sample comprising (a) contacting said liquid sample with an assay composition containing a hydrogen-peroxide detection composition and a bilirubin-specific enzyme preparation to interact bilirubin with said enzyme preparation and produce a detectable change, said enzyme preparation containing a mushroom enzyme which, in the presence of a bilirubin-containing aqueous liquid having a pH of about 7.4 and a temperature of about 37° C., degrades at least about 0.02 micromoles of bilirubin per minute per milligram of protein and generates hydrogen peroxide; and (b) detecting said change.
Claim 20
Dependent Claim
37 words
The method as described in claim 13 wherein said contacting step is carried out at a pH within the range of from about 7.3 to about 8.0 and a temperature of from about 20° to 50° C.
Claim 21
Dependent Claim
127 words
An assay method for the determination of an analyte other than bilirubin in an aqueous liquid sample containing bilirubin, bilirubin being an interferent for said assay method, said method comprising (a) treating said samples with an interactive composition for said analyte to produce a detectable change, (b) prior to or during step (a), contacting said sample with a bilirubin-specific enzyme preparation containing a mushroom enzyme which, in the presence of a bilirubin-containing aqueous liquid having a pH of about 7.4 and a temperature of about 37° C., degrades at least about 0.02 micromoles of bilirubin per minute per milligram of protein, thereby degrading bilirubin and reducing its potential for interference with the detectable change produced in step (a), and (c) detecting said change produced in step (a).
Claim 22
Dependent Claim
127 words
An assay method for the determination of an analyte other than bilirubin in an aqueous liquid sample containing bilirubin, bilirubin being an interferent for said assay method, said method comprising (a) treating said samples with an interactive composition for said analyte to produce a detectable change, (b) prior to or during step (a), contacting said sample with a bilirubin-specific enzyme preparation containing a mushroom enzyme which, in the presence of a bilirubin-containing aqueous liquid having a pH of about 7.4 and a temperature of about 37° C., degrades at least about 0.02 micromoles of bilirubin per minute per milligram of protein, thereby degrading bilirubin and reducing its potential for interference with the detectable change produced in step (a), and (c) detecting said change produced in step (a).
Claim 23
Independent Claim
139 words
A method for the extraction of a bilirubin-specific, fungal enzyme preparation comprising (a) blending mushroom with an aqueous buffer solution having a pH of about 6.8 to 7.8 to form a homogeneous aqueous mixture, (b) separating insoluble solids from said aqueous mixture to obtain an aqueous liquid supernatant, (c) treating said supernatant with a water-soluble salt to produce a precipitate, (d) suspending said precipitate in an aqueous buffer solution having a pH of about 6.8 to 7.8 to form a suspension, (e) heating said suspension above about 22° C. but below the boiling point thereof, (f) cooling said suspension and separating solids from said suspension to recover an aqueous liquid supernatant representing a bilirubin-specific fungal enzyme preparation, each of said steps a, b, c, d, and f carried out at a temperature of from about 0° to 4° C.
Claim 24
Independent Claim
139 words
A method for the extraction of a bilirubin-specific, fungal enzyme preparation comprising (a) blending mushroom with an aqueous buffer solution having a pH of about 6.8 to 7.8 to form a homogeneous aqueous mixture, (b) separating insoluble solids from said aqueous mixture to obtain an aqueous liquid supernatant, (c) treating said supernatant with a water-soluble salt to produce a precipitate, (d) suspending said precipitate in an aqueous buffer solution having a pH of about 6.8 to 7.8 to form a suspension, (e) heating said suspension above about 22° C. but below the boiling point thereof, (f) cooling said suspension and separating solids from said suspension to recover an aqueous liquid supernatant representing a bilirubin-specific fungal enzyme preparation, each of said steps a, b, c, d, and f carried out at a temperature of from about 0° to 4° C.
Claim 25
Independent Claim
168 words
A method for the extraction of a bilirubin-specific, fungal enzyme preparation comprising the following steps carried out at a temperature of from about 0° to 4° C.: (a) blending mushroom with an aqueous buffer solution having a pH of about 6.8 to 7.8 to form a homogeneous aqueous mixture, (b) separating insoluble solids from said aqueous mixture to obtain an aqueous liquid supernatant, (c) treating said supernatant with a water-soluble salt to produce a precipitate, (d) suspending said precipitate in an aqueous buffer solution having a pH of about 6.8 to 7.8 to form a suspension, (e) treating said suspension with a water-miscible organic liquid chilled to a temperature effective to produce a precipitate from said resulting aqueous-organic liquid mixture, (f) repeating step (d) and (e) at least one time using as said precipitate in said repeat of step (d) the precipitate produced in step (e), and (g) recovering the precipitate remaining following the last repeat of step (e) to obtain a bilirubin-specific, hydrogen peroxide-generating fungal enzyme preparation.
Claim 26
Independent Claim
168 words
A method for the extraction of a bilirubin-specific, fungal enzyme preparation comprising the following steps carried out at a temperature of from about 0° to 4° C.: (a) blending mushroom with an aqueous buffer solution having a pH of about 6.8 to 7.8 to form a homogeneous aqueous mixture, (b) separating insoluble solids from said aqueous mixture to obtain an aqueous liquid supernatant, (c) treating said supernatant with a water-soluble salt to produce a precipitate, (d) suspending said precipitate in an aqueous buffer solution having a pH of about 6.8 to 7.8 to form a suspension, (e) treating said suspension with a water-miscible organic liquid chilled to a temperature effective to produce a precipitate from said resulting aqueous-organic liquid mixture, (f) repeating step (d) and (e) at least one time using as said precipitate in said repeat of step (d) the precipitate produced in step (e), and (g) recovering the precipitate remaining following the last repeat of step (e) to obtain a bilirubin-specific, hydrogen peroxide-generating fungal enzyme preparation.
Claim 27
Dependent Claim
68 words
An assay composition for the determination of bilirubin in an aqueous liquid, said composition comprising a hydrogen-peroxide detection composition and a bilirubin-specific enzyme preparation comprising a fungal enzyme which, in the presence of a bilirubin-containing aqueous liquid having a pH of about 7.4 and a temperature of about 37° C., degrades at least about 0.02 micromoles of bilirubin per minute per milligram of protein and generates hydrogen peroxide.
Claim 28
Dependent Claim
68 words
An assay composition for the determination of bilirubin in an aqueous liquid, said composition comprising a hydrogen-peroxide detection composition and a bilirubin-specific enzyme preparation comprising a fungal enzyme which, in the presence of a bilirubin-containing aqueous liquid having a pH of about 7.4 and a temperature of about 37° C., degrades at least about 0.02 micromoles of bilirubin per minute per milligram of protein and generates hydrogen peroxide.
Claim 29
Dependent Claim
69 words
A dry test element for the assay of an aqueous liquid, said element having a reagent zone containing an assay composition for bilirubin, said assay composition for bilirubin comprising a fungal enzyme preparation which, in the presence of a bilirubin-containing aqueous liquid having a pH of about 7.4 and a temperature of about 37° C., degrades at least about 0.02 micromoles of bilirubin per minute per milligram of protein.
Claim 30
Dependent Claim
69 words
A dry test element for the assay of an aqueous liquid, said element having a reagent zone containing an assay composition for bilirubin, said assay composition for bilirubin comprising a fungal enzyme preparation which, in the presence of a bilirubin-containing aqueous liquid having a pH of about 7.4 and a temperature of about 37° C., degrades at least about 0.02 micromoles of bilirubin per minute per milligram of protein.
Claim 31
Independent Claim
83 words
A method for the determination of bilirubin in an aqueous liquid sample comprising (a) contacting said liquid sample with a bilirubin-specific enzyme preparation to interact bilirubin with said enzyme preparation and produce a detectable change, said enzyme preparation containing a fungal enzyme which, in the presence of a bilirubin-containing aqueous liquid having a pH of about 7.4 and a temperature of about 37° C., degrades at least about 0.02 micromoles of bilirubin per minute per milligram of protein; and (b) detecting said change.
Claim 32
Independent Claim
83 words
A method for the determination of bilirubin in an aqueous liquid sample comprising (a) contacting said liquid sample with a bilirubin-specific enzyme preparation to interact bilirubin with said enzyme preparation and produce a detectable change, said enzyme preparation containing a fungal enzyme which, in the presence of a bilirubin-containing aqueous liquid having a pH of about 7.4 and a temperature of about 37° C., degrades at least about 0.02 micromoles of bilirubin per minute per milligram of protein; and (b) detecting said change.
References Cited (Backward Citations)
No citation records indexed.
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1983-07-08 | 1984-01-07 | 1984-07-08 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1987-07-08 | 1988-01-07 | 1988-07-08 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1991-07-08 | 1992-01-07 | 1992-07-08 | $7700.00 | Window Closed / Lapsed |