US4963469A
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US
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Enzymatically inactive, immunologically-active β-galactosidase muetin, process for making and uses thereof
Assignee: Roche Diagnostics GmbH
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Filing: 1988-07-12
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Granted: 1990-10-16
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Expires: 2008-07-12
Abstract
Abstract The present invention provides an enzymatically-inactive, immunologically-active β-galactosidase mutein, wherein, in the region between the amino acids 430 and 550, at least one amino acid of the natural sequence is changed to another amino acid and the enzymatic activity does not amount to more than 1%, referred to the native enzyme. The present invention also provides a process for the production of this mutein. Furthermore, the present invention is concerned with the use of this mutein in the immunological determination of serum proteins by the enzyme immunoassay principle.
Inventors
- 👤 Ralf Mattes
- 👤 Helmut Lenz
- 👤 Werner Stock
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-04 07:26
CPC Classifications
C; C12; C12Y; C12Y302/00; C12Y302/01; C12Y302/01023; C12N; C12N9/00; C12N9/14; C12N9/24; C12N9/2402; C12N9/2468; C12N9/2471; G; G01; G01N; G01N33/00; G01N33/48; G01N33/50; G01N33/53; G01N33/531; G01N33/532; G01N33/535; G01N33/573; G01N33/58; G01N33/581; Y; Y10; Y10S; Y10S435/00; Y10S435/962
Claims Analysis
Total Claims: 26
Claim 1
Independent Claim
42 words
A mutein of β-galactosidase which is enzymatically inactive and immunologically active, characterized by an amino acid mutation at amino acid 503 which is normally Tyr of said β-galactosidase, said mutein having no more than 1% of the enzymatic activity of native β-galactosidase.
Claim 2
Dependent Claim
42 words
A mutein of β-galactosidase which is enzymatically inactive and immunologically active, characterized by an amino acid mutation at amino acid 503 which is normally Tyr of said β-galactosidase, said mutein having no more than 1% of the enzymatic activity of native β-galactosidase.
Claim 3
Dependent Claim
18 words
The mutein of claim 1, further comprising an additional mutation at amino acid 461 which is normally Glu.
Claim 4
Dependent Claim
24 words
The mutein of claim 1, wherein said mutation is a change of Tyr at amino acid 503 by one of Phe, Glu or Ser.
Claim 5
Dependent Claim
47 words
The mutein of claim 2, wherein said mutein has a mutation at amino acid 461 in which Glu is changed to one of Ser, Ala, Asp or Tyr, and a mutation at amino acid 503 in which Tyr is changed to one of Phe, Glu, or Ser.
Claim 6
Dependent Claim
61 words
Process for producing an enzymatically inactive, immunologically active β-galactosidase mutein which contains a mutation at amino acid 503 which is normally Tyr, comprising treating a plasmid coding for native β-galactosidase causing site directed mutagenesis in said plasmid resulting in a mutation in the portion of said plasmid coding for β-galactosidase amino acid 503, and expressing said plasmid to produce said mutein.
Claim 7
Dependent Claim
61 words
Process for producing an enzymatically inactive, immunologically active β-galactosidase mutein which contains a mutation at amino acid 503 which is normally Tyr, comprising treating a plasmid coding for native β-galactosidase causing site directed mutagenesis in said plasmid resulting in a mutation in the portion of said plasmid coding for β-galactosidase amino acid 503, and expressing said plasmid to produce said mutein.
Claim 8
Dependent Claim
27 words
Process of claim 5, further comprising causing site directed mutagenesis of said plasmid at the portion thereof coding for β-galactosidase amino acid 461, which is normally Glu.
Claim 9
Dependent Claim
31 words
Process of claim 6, wherein said site directed mutagenesis comprises mutagenesis of the portion coding for Glu at amino acid 461 to code for one of Ser, Ala, Asp or Tyr.
Claim 10
Dependent Claim
30 words
Process of claim 5, wherein said site directed mutagenesis comprises mutagenesis of the portion coding for Tyr at amino acid 503 to code for one of Phe, Glu, or Ser.
Claim 11
Dependent Claim
50 words
Process of claim 5, wherein said site directed mutagenesis comprises mutagenesis of the portion coding for Glu at amino acid 461 to code for one of Ser, Ala, Asp or Tyr and the portion coding for Tyr at amino acid 503 to code for one of Phe, Glu, or Ser.
Claim 12
Dependent Claim
125 words
An immunoassay method for determining an analyte in a liquid sample suspected of containing a potentially interfering binding substance, comprising: contacting said liquid sample with (i) a labeled conjugate of (a) enzymatically acitve B-galactosidase and (b) an antibody or bindable fragment thereof which specifically binds to the analyte to be determined, and (ii) an amount of enzymatically inactive, immunologically active B-galactosidase mutein characterized by an amino acid mutation at amino acid 503 which is normally Tyr, said mutein having no more than 1% of the enzymatic activity of native B-galactosidase and effective to specifically bind any said potentially interfering binding substance capable of binding immunologically active B-galactosidase; and measuring bound or free labeled conjugate in order to determine the amount of analyte in said sample.
Claim 13
Dependent Claim
125 words
An immunoassay method for determining an analyte in a liquid sample suspected of containing a potentially interfering binding substance, comprising: contacting said liquid sample with (i) a labeled conjugate of (a) enzymatically acitve B-galactosidase and (b) an antibody or bindable fragment thereof which specifically binds to the analyte to be determined, and (ii) an amount of enzymatically inactive, immunologically active B-galactosidase mutein characterized by an amino acid mutation at amino acid 503 which is normally Tyr, said mutein having no more than 1% of the enzymatic activity of native B-galactosidase and effective to specifically bind any said potentially interfering binding substance capable of binding immunologically active B-galactosidase; and measuring bound or free labeled conjugate in order to determine the amount of analyte in said sample.
Claim 14
Dependent Claim
47 words
Method of claim 10 wherein said contacting step further comprises adding a sufficient amount of a Fab fragment, which does not bind to said analyte, effective to specifically bind any said potentially interfering binding substance capable of binding said antibody or bindable fragment of said labeled conjugate.
Claim 15
Dependent Claim
29 words
Method of claim 10, wherein said mutein is added in the form of a chemically bound conjugate of said mutein and a Fab fragment of a non-analyte specific immunoglobulin.
Claim 16
Dependent Claim
20 words
Method of claim 10, wherein said mutation further comprises an additional mutation at amino acid 461 which is normally Glu.
Claim 17
Dependent Claim
23 words
Method of claim 10, wherein said mutation is a change of Tyr at amino acid 503 by one of Phe, Glu or Ser.
Claim 18
Dependent Claim
24 words
Method of claim 13, wherein said mutation is a change of Glu at amino acid 461 to one of Ser, Ala, Asp or Tyr.
Claim 19
Dependent Claim
40 words
Method of claim 10, wherein said mutein comprises a change of Tyr at amino acid 503 by one of Phe, Glu or Ser and a change of Glu at amino acid 461 to one of Ser, Ala, Asp or Tyr.
Claim 20
Dependent Claim
115 words
Reagent useful in eliminating a potentially interfering binding substance in a B-galactosidase labeled immunoassay for determining an analyte in a liquid sample, comprising: (i) an enzymatically inactive, immunologically active B-galactosidase mutein characterized by an amino acid mutation at amino acid 503 which is normally Tyr, said mutein having no more than 1% of the enzymatic activity of native B-galactosidase and wherein said mutein specifically binds to any said potentially interfering binding substance capable of binding to immunologically active B-galactosidase, and (ii) a Fab fragment which does not bind to said analyte but which specifically binds to any said potentially interfering binding substance capable of binding to the antibody or bindable fragment thereof said labeled conjugate.
Claim 21
Dependent Claim
115 words
Reagent useful in eliminating a potentially interfering binding substance in a B-galactosidase labeled immunoassay for determining an analyte in a liquid sample, comprising: (i) an enzymatically inactive, immunologically active B-galactosidase mutein characterized by an amino acid mutation at amino acid 503 which is normally Tyr, said mutein having no more than 1% of the enzymatic activity of native B-galactosidase and wherein said mutein specifically binds to any said potentially interfering binding substance capable of binding to immunologically active B-galactosidase, and (ii) a Fab fragment which does not bind to said analyte but which specifically binds to any said potentially interfering binding substance capable of binding to the antibody or bindable fragment thereof said labeled conjugate.
Claim 22
Dependent Claim
15 words
Reagent of claim 17, wherein said mutein and said Fab fragment are chemically bound together.
Claim 23
Dependent Claim
20 words
Reagent of claim 17, wherein said mutein further comprises an additional mutation at amino acid 461 which is normally Glu.
Claim 24
Dependent Claim
23 words
Reagent of claim 17, wherein said mutation is a change of Tyr at amino acid 503 by one of Phe, Glu or Ser.
Claim 25
Dependent Claim
24 words
Reagent of claim 19, wherein said mutation is a change of Glu at amino acid 461 to one of Ser, Ala, Asp, or Tyr.
Claim 26
Dependent Claim
40 words
Reagent of claim 19, wherein said mutein comprises a change of Tyr at amino acid 503 by one of Phe, Glu or Ser and a change of Glu at amino acid 461 to one of Ser, Ala, Asp, or Tyr.
References Cited (Backward Citations)
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| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
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| 3.5 Years (Year 4) | 1993-10-15 | 1994-04-16 | 1994-10-16 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1997-10-15 | 1998-04-16 | 1998-10-16 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2001-10-15 | 2002-04-16 | 2002-10-16 | $7700.00 | Window Closed / Lapsed |