US4410660A
Expired / Lapsed
US
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Binding assay for the detection of mycobacteria
Assignee: Montefiore Medical Center
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Filing: 1980-05-05
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Granted: 1983-10-18
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Expires: 2000-05-05
Abstract
Abstract A method for detecting the presence of Mycobacteria in a fluid or tissue which comprises mixing the fluid or tissue containing a secretory product of Mycobacteria with a complex of a tracer-containing molecule and a binding macromolecule having reversible binding affinity for the tracer-containing molecule and detecting the tracer-containing molecule, wherein the tracer-containing molecule is a charcoal-adsorbable protein from Mycobacterium tuberculosis which has a molecular weight of 20,000-30,000 and which is immunochemically stable from 4°0 C. to 250° C. and has a pH range from 3.0 to 9.0. The method is particularly applicable to the detection of infectious tuberculosis in humans and determining the antibiotic sensitivity of infecting Mycobacteria.
Inventors
- 👤 Eugene W. Straus
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-27 23:28
CPC Classifications
G; G01; G01N; G01N33/00; G01N33/48; G01N33/50; G01N33/53; G01N33/569; G01N33/56911; G01N33/5695; C; C12; C12N; C12N1/00; C12N1/20; C12N1/205; C12P; C12P1/00; C12P1/06; C12Q; C12Q1/00; C12Q1/02; C12Q1/04; C12Q1/18; G01N33/543; G01N33/54313; G01N33/54326; G01N33/54353; C12R; C12R2001/00; C12R2001/01; C12R2001/32; G01N2333/00; G01N2333/195; G01N2333/35; Y; Y10; Y10S; Y10S435/00; Y10S435/8215; Y10S435/822; Y10S435/863; Y10S436/00; Y10S436/807; Y10S436/808; Y10S436/811; Y10S530/00; Y10S530/81; Y10S530/812; Y10S530/815; Y10S530/816; Y10S530/82; Y10S530/825
Claims Analysis
Total Claims: 39
Claim 1
Independent Claim
75 words
A heat stable charcoal-adsorbable labeled protein obtained from the growth media of M. tuberculosis, said protein having a molecular weight of between 20,000 and 30,000, immunochemical stability over a temperature range of 4° C.-250° C., a pH range of 3.0-9.0 and exhibiting comigration towards the anode in the presence of bovine serum albumin and borate buffer at a pH of 8.6 with an applied voltage of 80 volts on starch gel electrophoresis for 12 hours.
Claim 2
Dependent Claim
75 words
A heat stable charcoal-adsorbable labeled protein obtained from the growth media of M. tuberculosis, said protein having a molecular weight of between 20,000 and 30,000, immunochemical stability over a temperature range of 4° C.-250° C., a pH range of 3.0-9.0 and exhibiting comigration towards the anode in the presence of bovine serum albumin and borate buffer at a pH of 8.6 with an applied voltage of 80 volts on starch gel electrophoresis for 12 hours.
Claim 3
Dependent Claim
20 words
A heat stable charcoal-absorbable protein as defined in claim 1 wherein the molecular weight is determined by Sephadex G75 chromatography.
Claim 4
Dependent Claim
22 words
The protein of claim 1, which contains a tracer selected from the group consisting of a radiolabel, a chromophore and an enzyme.
Claim 5
Dependent Claim
28 words
The protein of claim 3, wherein said chromophore is selected from the group consisting of a fluorescent label, a visible light absorbing label and an ultraviolet light-absorbing label.
Claim 6
Dependent Claim
11 words
The protein of claim 3, wherein said tracer is a radiolabel.
Claim 7
Dependent Claim
11 words
The protein of claim 1, having a molecular weight of 24,000.
Claim 8
Dependent Claim
14 words
The protein of claims 1 or 3, which is attached to an insoluble support.
Claim 9
Dependent Claim
15 words
The protein of claim 7, wherein said protein is covalently attached to said insoluble support.
Claim 10
Dependent Claim
12 words
The protein of claim 7, wherein said support is a latex particle.
Claim 11
Dependent Claim
12 words
The protein of claim 9, wherein said latex is a polystyrene latex.
Claim 12
Dependent Claim
13 words
The protein of claim 9, wherein said latex is a styrene butadiene latex.
Claim 13
Dependent Claim
27 words
The protein of claim 7, wherein said support is a material selected from the group consisting of dispersed coccal bacteria, cell membrane fragments, silica, silica-alumina and alumina.
Claim 14
Dependent Claim
17 words
The protein of claims 1 or 3, which is in reversible complexation with a binding macromolecule therefor.
Claim 15
Dependent Claim
24 words
The protein of claim 13, wherein said binding macromolecule is an antibody raised against tubercular protein secreted by a bacterium of the genus Mycrobacterium.
Claim 16
Dependent Claim
11 words
The protein of claim 14, wherein said bacterium is Mycobacterium tuberculosis.
Claim 17
Dependent Claim
15 words
The protein of claim 13, wherein said binding macromolecule is attached to an insoluble support.
Claim 18
Dependent Claim
11 words
The protein of claim 16, wherein said attachment is covalent attachment.
Claim 19
Dependent Claim
28 words
The protein of claim 16, wherein said support is a material selected from the group consisting of a cross-linked resin, a latex particle, glass, silica, silica-alumina, and alumina.
Claim 20
Dependent Claim
19 words
The protein of claim 18, wherein said latex is selected from the group consisting of sytrene-butadiene and polystyrene latexes.
Claim 21
Dependent Claim
12 words
The protein of claim 16, wherein said support is a polyolefin disc.
Claim 22
Dependent Claim
15 words
The protein of claim 16, wherein said support is the inner wall of a tube.
Claim 23
Dependent Claim
12 words
The protein of claim 21, wherein said tube is a test tube.
Claim 24
Dependent Claim
18 words
The protein of claim 16, wherein said binding macromolecule is entrapped in the interstices of a polyacrylamide gel.
Claim 25
Dependent Claim
13 words
The protein of claim 16, wherein said solid support is a magnetic particle.
Claim 26
Dependent Claim
68 words
A heat stable tracer containing protein which is prepared by: (a) labeling a mixture of tubercular proteins isolated from the growth media of Mycobacterium tuberculosis with a tracer and chromatographically separating a labeled tubercular protein having a molecular weight of between 20,000 and 30,000, stability over a pH range of from 3.0-9.0 stability over a temperature range of 4° C.-250° C. and well-defined mobility on starch gel electrophoresis.
Claim 27
Dependent Claim
68 words
A heat stable tracer containing protein which is prepared by: (a) labeling a mixture of tubercular proteins isolated from the growth media of Mycobacterium tuberculosis with a tracer and chromatographically separating a labeled tubercular protein having a molecular weight of between 20,000 and 30,000, stability over a pH range of from 3.0-9.0 stability over a temperature range of 4° C.-250° C. and well-defined mobility on starch gel electrophoresis.
Claim 28
Dependent Claim
17 words
A heat stable tracer containing protein as defined in claim 25 wherein the tracer is a radiolabel.
Claim 29
Dependent Claim
17 words
A heat stable tracer containing protein as defined in claim 26 wherein the tracer is 125 I.
Claim 30
Dependent Claim
22 words
A heat stable tracer containing protein as defined in claim 27 wherein the mixture of tubercular proteins is tuberculin purified protein derivatives.
Claim 31
Dependent Claim
78 words
A process for preparing a tracer-containing protein purified from Mycobacterium tuberculosis which comprises: labeling a mixture of tubercular proteins isolated from the growth media of Mycobacterium tuberculosis; chromatographing from said mixture of labeled tubercular proteins, a labeled, charcoal-adsorbable protein having a molecular weight of between 20,000 and 30,000, which is stable over a pH range of from 3.0 to 9.0 and over a temperature range of 4° C.-250° C. and has well-defined mobility characteristics on starch gel electrophoresis.
Claim 32
Dependent Claim
78 words
A process for preparing a tracer-containing protein purified from Mycobacterium tuberculosis which comprises: labeling a mixture of tubercular proteins isolated from the growth media of Mycobacterium tuberculosis; chromatographing from said mixture of labeled tubercular proteins, a labeled, charcoal-adsorbable protein having a molecular weight of between 20,000 and 30,000, which is stable over a pH range of from 3.0 to 9.0 and over a temperature range of 4° C.-250° C. and has well-defined mobility characteristics on starch gel electrophoresis.
Claim 33
Dependent Claim
11 words
The process of claim 29, wherein said tracer is a radiolabel.
Claim 34
Dependent Claim
11 words
The process of claim 30, wherein said radiolabel is 125 I.
Claim 35
Dependent Claim
13 words
The process of claim 29, wherein said mixture of tubercular proteins is PPD.
Claim 36
Dependent Claim
24 words
The process of claim 29, wherein said mixture of tubercular proteins is isolated from the filtrates of growth medium of actively growing Mycobacterium tuberculosis.
Claim 37
Dependent Claim
32 words
The process of claim 15, wherein said chromatographic step comprises: fractionating said mixture on a material containing beads of dextran cross-linked with epichlorohydrin, and having a fractionation range for peptides of 3,000-80,000.
Claim 38
Dependent Claim
125 words
The process of claim 29, wherein said chromatographic step comprises: (A) firstly, fractionating said mixture on a material containing allyl dextran, cross-linked with N,N'-methylene-bis-acrylamide with a fractionation range for proteins of 5×10 3 -2.5×10 5 ; (B) secondly, refractionating the fractions of said first fractionation which are most immunoreactive towards antibody raised against tubercular protein from Mycobacterium, on a material containing neutral agarose beads, said beads being substantially free of charged groups and having a protein fractionation range of 10 4 -4×10 6 ; and (C) thirdly, refractionating the fractions of said second fractionation which are most immunoreactive towards antibody raised against tubercular proteins isolated from Mycobacterium, on a material containing dextran cross-linked with epichlorohydrin, having a moleclar weight fractionation range for proteins of 3,000-80,000.
Claim 39
Dependent Claim
26 words
The process of claim 29, wherein said chromatographic step comprises: adsorbing said mixture on a material comprising microfine precipitated silica; and desorbing said tracer-containing protein therefrom.
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) | 1986-10-17 | 1987-04-18 | 1987-10-18 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1990-10-17 | 1991-04-18 | 1991-10-18 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1994-10-17 | 1995-04-18 | 1995-10-18 | $7700.00 | Window Closed / Lapsed |