Patent US5552294A successfully refreshed from Google Patents.
US5552294A
Expired / Lapsed
US
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Rapid detection of virulence-associated factors
Assignee: Boston Children's Hospital
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Filing: 1994-07-25
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Granted: 1996-09-03
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Expires: 2014-07-25
Abstract
Abstract A method for detecting at least one virulence-associated factor (VAF), e.g., a bacterial toxin, in a sample is described. The sample suspected of containing the VAF-producing bacteria is contacted with a VAF releasing solution under conditions which release VAF from the bacteria. The released VAF subsequently is immunochemically detected. The preferred method is a membrane-based enzyme linked immunosorbent assay for immunochemically detecting the well-characterized Shiga family toxins including SLT I and SLT II. Also described is the VAF releasing solution and a kit containing the reagents for conducting the described methods.
Inventors
- 👤 Grace M. Thorne
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-09 02:31
CPC Classifications
G; G01; G01N; G01N33/00; G01N33/48; G01N33/50; G01N33/53; G01N33/569; G01N33/56911; G01N33/56916; Y; Y10; Y10S; Y10S435/00; Y10S435/962; Y10S436/00; Y10S436/825; Y10T; Y10T436/00; Y10T436/25; Y10T436/25375
Claims Analysis
Total Claims: 24
Claim 1
Independent Claim
117 words
A method for immunochemically detecting a virulence-associated factor in feces, comprising: contacting a feces sample suspected of containing a virulence-associated factor producing bacteria with a factor releasing solution under conditions which release at least one virulence-associated factor from the bacteria; and immunochemically detecting the presence or quantity of the released virulence-associated factor, wherein said virulence-associated factor is selected from the group consisting of a bacterial toxin, a surface antigen, an adhesive factor, and a heat-release protein; said factor releasing solution containing: a) a surface-active agent; b) urea; and c) an antibiotic selected from the group consisting of polymyxins and mitomycin C; and said solution releasing the virulence-associated factor without impeding the immunochemical detectability of the virulence-associated factor.
Claim 2
Independent Claim
117 words
A method for immunochemically detecting a virulence-associated factor in feces, comprising: contacting a feces sample suspected of containing a virulence-associated factor producing bacteria with a factor releasing solution under conditions which release at least one virulence-associated factor from the bacteria; and immunochemically detecting the presence or quantity of the released virulence-associated factor, wherein said virulence-associated factor is selected from the group consisting of a bacterial toxin, a surface antigen, an adhesive factor, and a heat-release protein; said factor releasing solution containing: a) a surface-active agent; b) urea; and c) an antibiotic selected from the group consisting of polymyxins and mitomycin C; and said solution releasing the virulence-associated factor without impeding the immunochemical detectability of the virulence-associated factor.
Claim 3
Dependent Claim
15 words
A method as claimed in claim 1 wherein the virulence-associated factor is a bacterial toxin.
Claim 4
Dependent Claim
69 words
A method as claimed in claim 1 wherein the at least one virulence-associated factor in the contacting step is selected from the group consisting of Shiga-like toxin I, Shiga-like toxin II, heat-labile enterotoxin, heat stable enterotoxins a+b, heat stable-like enterotoxins, adhesins, lipopolysaccharide-0157 antigen, hemolysin, cholera toxin, flagella, zot toxin, Shiga toxin, toxin A, toxin B, surface antigen for invasion, cytotoxins, proteases, siderophores, invasins, outer membrane protein, pili, and lipopolysaccharide.
Claim 5
Dependent Claim
19 words
A method as claimed in claim 1 wherein the bacterial toxin is Shiga-like toxin I or Shiga-like toxin II.
Claim 6
Dependent Claim
22 words
A method as claimed in claim 1 wherein the bacterial toxin includes a mixture of Shiga-like toxin I and Shiga-like toxin II.
Claim 7
Dependent Claim
14 words
A method as claimed in claim 1 wherein the antibiotic is polymyxin B sulphate.
Claim 8
Dependent Claim
22 words
A method as claimed in claim 1 wherein the virulence-associated factor releasing solution in the contacting step further comprises a protease inhibitor.
Claim 9
Dependent Claim
40 words
A method as claimed in claim 1 wherein the surface active agent in the virulence-associated factor releasing solution is selected from the group consisting of N,N', N'-polyoxyethylene(10)-N-tallow-1,3-diaminopropane, sulfobetaine derivatives of cholic acid, alkyl glucosides, polyoxyethylenesorbitan monolaurate and octylphenolethylene oxide condensate.
Claim 10
Dependent Claim
14 words
A method as claimed in claim 8 wherein the surface active agent is N,N',N'-polyoxyethylene(10)-N-tallow-1,3-diaminopropane.
Claim 11
Dependent Claim
20 words
A method as claimed in claim 1 wherein said surface-active agent comprises N,N',N'-polyoxyethylene(10)-N-tallow-1,3-diaminopropane, and said antibiotic comprises polymyxin B sulphate.
Claim 12
Dependent Claim
19 words
A method as claimed in claim 1 wherein the virulence-associated factor is immunochemically detected by an enzyme-linked immunosorbent assay.
Claim 13
Dependent Claim
26 words
A method as claimed in claim 1 wherein the contacting and immunochemically detecting steps are completed in a time span of less than about four hours.
Claim 14
Dependent Claim
26 words
A method as claimed in claim 12 wherein the contacting and immunochemically detecting steps are completed in a time span of less than about two hours.
Claim 15
Dependent Claim
26 words
A method as claimed in claim 13 wherein the contacting and immunochemically detecting steps are completed in a time span of less than about thirty minutes.
Claim 16
Dependent Claim
23 words
A method as claimed in claim 1 wherein the presence of at least one virulence-associated factor is detected in the immunochemically detecting step.
Claim 17
Dependent Claim
23 words
A method as claimed in claim 1 wherein the quantity of at least one virulence-associated factor is detected in the immunochemically detecting step.
Claim 18
Independent Claim
67 words
A method for detecting a Shiga-like toxin directly from a stool sample, comprising: contacting a stool sample suspected of containing bacteria producing at least one Shiga-like toxin with a toxin releasing solution under conditions such that at least one Shiga-like toxin is released; and immunochemically detecting any of the at least one Shiga-like toxin released directly from bacteria in the stool sample by the toxin releasing solution.
Claim 19
Independent Claim
67 words
A method for detecting a Shiga-like toxin directly from a stool sample, comprising: contacting a stool sample suspected of containing bacteria producing at least one Shiga-like toxin with a toxin releasing solution under conditions such that at least one Shiga-like toxin is released; and immunochemically detecting any of the at least one Shiga-like toxin released directly from bacteria in the stool sample by the toxin releasing solution.
Claim 20
Dependent Claim
30 words
A method as claimed in claim 17 wherein the at least one Shiga-like toxin in the contacting step includes a mixture of both Shiga-like toxin I and Shiga-like toxin II.
Claim 21
Dependent Claim
18 words
A method as claimed in claim 17 wherein the immunochemically detecting step is an enzyme-linked immunosorbent assay step.
Claim 22
Dependent Claim
15 words
A method as claimed in claim 19 wherein the enzyme-linked immunosorbent assay step is membrane-based.
Claim 23
Independent Claim
125 words
A method for immunochemically detecting Shiga-like toxin I or Shiga-like toxin II in feces, comprising: contacting a feces sample suspected of containing Shiga-like toxin I or Shiga-like toxin II producing bacteria with a toxin releasing solution under conditions which release Shiga-like toxin I or Shiga-like toxin II from the bacteria; and immunochemically detecting the presence or quantity of the released Shiga-like toxin I or Shiga-like toxin II, said toxin releasing solution containing: a) a surface active agent selected from the group consisting of N,N',N'-polyoxyethylene(10)-N-tallow-1,3-diaminopropane, polyoxyethylenesorbitan monolaurate, and octylphenolethylene oxide condensate; b) urea; c) a polymyxin; and d) phenylmethylsulphonyl fluoride and said solution releasing the Shiga-like toxin I or Shiga-like toxin II without impeding the immunochemical detectability of the Shiga-like toxin I or Shiga-like toxin II.
Claim 24
Independent Claim
125 words
A method for immunochemically detecting Shiga-like toxin I or Shiga-like toxin II in feces, comprising: contacting a feces sample suspected of containing Shiga-like toxin I or Shiga-like toxin II producing bacteria with a toxin releasing solution under conditions which release Shiga-like toxin I or Shiga-like toxin II from the bacteria; and immunochemically detecting the presence or quantity of the released Shiga-like toxin I or Shiga-like toxin II, said toxin releasing solution containing: a) a surface active agent selected from the group consisting of N,N',N'-polyoxyethylene(10)-N-tallow-1,3-diaminopropane, polyoxyethylenesorbitan monolaurate, and octylphenolethylene oxide condensate; b) urea; c) a polymyxin; and d) phenylmethylsulphonyl fluoride and said solution releasing the Shiga-like toxin I or Shiga-like toxin II without impeding the immunochemical detectability of the Shiga-like toxin I or Shiga-like toxin II.
References Cited (Backward Citations)
US4677080A
Cited
US5164298A
Cited
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