US4863852A
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US
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Method of detecting, isolating and purifying clostridium difficile toxin A and its receptors
Assignee: Virginia Tech Intellectual Properties Inc
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Filing: 1986-02-11
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Granted: 1989-09-05
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Expires: 2006-02-11
Abstract
Abstract A method is provided for detecting the presence of C. difficile toxin A. Stool or other appropriate specimen is contacted with a reagent containing an available non-reducing galactose-alpha-1-3-galactosyl structure, which is a specific receptor for toxin A. The reagent may be intact cells, cell membranes, membrane fractions containing the toxin A receptor structure, glycoconjugates, as well as the purified toxin A receptor per se. Binding of toxin A is determined by conventional assay techniques. The method may also be used to isolate and purify toxin A. Conversely, immobilized toxin A is used to detect, isolate, or purify biological materials of interest containing the receptor structure.
Inventors
- 👤 Tracy D. Wilkins
- 👤 Howard C. Krivan
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-28 10:34
CPC Classifications
G; G01; G01N; G01N33/00; G01N33/48; G01N33/50; G01N33/53; G01N33/569; G01N33/56911; Y; Y10; Y10S; Y10S435/00; Y10S435/803; Y10S435/8215; Y10S435/822; Y10S435/842; Y10S436/00; Y10S436/829; Y10S530/00; Y10S530/82; Y10S530/825
Claims Analysis
Total Claims: 33
Claim 1
Independent Claim
42 words
A method of detecting Clostridium difficile toxin A, which method does not detect toxin B, comprising: (a) contacting a specimen with a reagent containing an available non-reducing galactose-alpha-1-3-galactose-beta-1-4-Nacetyl glucosamine structure; (b) assaying for binding of C. difficile toxin A to the reagent.
Claim 2
Independent Claim
42 words
A method of detecting Clostridium difficile toxin A, which method does not detect toxin B, comprising: (a) contacting a specimen with a reagent containing an available non-reducing galactose-alpha-1-3-galactose-beta-1-4-Nacetyl glucosamine structure; (b) assaying for binding of C. difficile toxin A to the reagent.
Claim 3
Dependent Claim
26 words
A method according to claim 1 wherein the reagent is selected from the group consisting of erythrocytes, erythrocyte ghosts, brush border membranes, thyroglobulin, laminin and collagen.
Claim 4
Dependent Claim
18 words
A method according to claim 2 wherein the erythrocytes and erythrocyte ghosts are rabbit erythrocytes and erythrocyte ghosts.
Claim 5
Dependent Claim
17 words
A method according to claim 2 wherein the brush border membrane is a hamster brush border membrane.
Claim 6
Dependent Claim
16 words
A method according to claim 1 wherein the reagent comprises purified C. difficile toxin A receptor.
Claim 7
Dependent Claim
35 words
A method according to claim 1 wherein the assaying means of step (b) is selected from the group consisting of enzyme-linked immunosorbent assay, hemagglutination, slide agglutination, slide coagglutination, indirect immunofluorescence, latex agglutination, and liposome agglutination.
Claim 8
Dependent Claim
50 words
A method according to claim 2, wherein the reagent is selected from the group consisting of erythrocytes and erythrocyte ghosts and step (b) comprises adding antisera containing toxin A antibody from another species, whereby agglutination of said erythrocyte or erythrocyte ghosts indicates the presence of toxin A in the specimen.
Claim 9
Dependent Claim
16 words
A method according to claim 1 wherein contact in step (a) is below about 20° C.
Claim 10
Dependent Claim
16 words
A method according to claim 8 wherein contact is between about 0° C. and 15° C.
Claim 11
Dependent Claim
11 words
A method according to claim 1 wherein the sample is feces.
Claim 12
Independent Claim
48 words
A method for detecting C. difficile toxin A, which method does not detect toxin B, comprising: (a) contacting a specimen with a material selected from the group consisting of rabbit erythrocytes, rabbit erythrocyte ghosts, and brush border membranes; and (b) assaying for binding of C. difficile toxin A.
Claim 13
Independent Claim
48 words
A method for detecting C. difficile toxin A, which method does not detect toxin B, comprising: (a) contacting a specimen with a material selected from the group consisting of rabbit erythrocytes, rabbit erythrocyte ghosts, and brush border membranes; and (b) assaying for binding of C. difficile toxin A.
Claim 14
Dependent Claim
16 words
A method according to claim 11 wherein the reagent is rabbit erythrocyte or rabbit erythrocyte ghosts.
Claim 15
Dependent Claim
18 words
A method according to claim 11 wherein the reagent is rabbit is a erthrocyte or rabbit erythrocyte ghosts.
Claim 16
Independent Claim
60 words
A method for isolating or purifying a reagent containing an available non-reducing galactose-alpha-1-3-galactose-beta-1-4-N-acetyl glucosamine structure comprising: (a) contacting materials which may contain the reagent with immobilized C. difficile toxin A at a temperature favoring reversible binding of the reagent to the toxin A; (b) increasing the temperature to release the reagent from the immobilized toxin A; (c) eluting the reagent.
Claim 17
Independent Claim
60 words
A method for isolating or purifying a reagent containing an available non-reducing galactose-alpha-1-3-galactose-beta-1-4-N-acetyl glucosamine structure comprising: (a) contacting materials which may contain the reagent with immobilized C. difficile toxin A at a temperature favoring reversible binding of the reagent to the toxin A; (b) increasing the temperature to release the reagent from the immobilized toxin A; (c) eluting the reagent.
Claim 18
Dependent Claim
17 words
A method according to claim 14 wherein the temperature in step (a) is below about 20° C.
Claim 19
Dependent Claim
15 words
A method according to claim 15 wherein the temperature in step (a) is 0-15° C.
Claim 20
Dependent Claim
19 words
A method according to claim 16 wherein the temperature is increased in step (b) to above about 30° C.
Claim 21
Dependent Claim
23 words
A method according to claim 17 wherein the temperature in step (b) is increased to between about 30° C. and about 37° C.
Claim 22
Dependent Claim
21 words
A method according to claim 14 wherein the toxin A is immobilized on a substrate to form an affinity chromatography column.
Claim 23
Independent Claim
58 words
A method for isolating or purifying C. difficile toxin A comprising: (a) contacting a source of toxin A with immobilized reagent containing an available non-reducing galactose-alpha-1-3-galactose-beta-1-4-N-acetyl glucosamine structure at a temperature favoring reversible binding of toxin A to the immobilized reagent; (b) increasing the temperature to release toxin A bound to the immobilized reagent; (c) eluting toxin A
Claim 24
Independent Claim
58 words
A method for isolating or purifying C. difficile toxin A comprising: (a) contacting a source of toxin A with immobilized reagent containing an available non-reducing galactose-alpha-1-3-galactose-beta-1-4-N-acetyl glucosamine structure at a temperature favoring reversible binding of toxin A to the immobilized reagent; (b) increasing the temperature to release toxin A bound to the immobilized reagent; (c) eluting toxin A
Claim 25
Dependent Claim
17 words
A method according to claim 20 wherein the temperature in step (a) is below about 20° C.
Claim 26
Dependent Claim
16 words
A method according to claim 21 wherein the temperature in step (a) is 0° C.-15° C.
Claim 27
Dependent Claim
19 words
A method according to claim 22 wherein the temperature is increased in step (b) to above about 30° C.
Claim 28
Dependent Claim
23 words
A method according to claim 23 wherein the temperature is increased in step (b) to between about 30° C. and about 37° C.
Claim 29
Dependent Claim
19 words
A method according to claim 20 wherein the immobilized reagent is disposed in the form of an affinity column.
Claim 30
Dependent Claim
11 words
A method according to claim 25 wherein the reagent is thryroglobulin.
Claim 31
Dependent Claim
20 words
A method according to claim 21 wherein the reagent is immobilized on a substrate to form an affinity chromatography column.
Claim 32
Dependent Claim
11 words
A method according to claim 27 wherein the reagent is thyroglobulin.
Claim 33
Dependent Claim
11 words
A method according to claim 27 wherein the reagent is laminin.
References Cited (Backward Citations)
US4533630A
Cited
EP0153519A1
Cited
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