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1. (WO1993017129) METHODE ET NECESSAIRE A DIAGNOSTIC POUR LA DETERMINATION DE BACTERIES
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WHAT IS CLAIMED IS :
1. A method for the determination of the presence of a specific bacteria in a sample, comprising:
(a) contacting a sample to be tested with
(i) bacteriophage capable of attaching to said specific
bacteria, said bacteriophage having affixed thereto one of
two members of a specific binding pair, and
" (ii) water-insoluble, water-suspendible solid phase carrier
particles having affixed thereto the second member of said
specific binding pair,
to form a test mixture having water-insoluble complexes of at
least (1) said specific bacteria, (2) said bacteriophage, and (3)
said solid phase particles, if said specific bacteria are present in
said fluid sample,
(b) separating said complexes from solid phase particles and bacteriophage
in said test mixture not complexed to said specific bacteria, and
(c) testing to detect said complexes, thereby to determine if said specific _ bacteria are present.

2. A method for the determination of the presence of a specific bacteria in a sample, comprising:
. (a) contacting a sample to be tested with
(i) bacteriophage capable of attaching to said specific
bacteria, said bacteriophage having affixed thereto one of
two members of a specific binding pair, and
(ii) water-insoluble, water-suspendible solid phase carrier
panicles having affixed thereto the second member of said
specific binding pair,
a labeling agent being affixed to at least one of said
bacteriophage or said carrier panicle,
to form a test mixture having water-insoluble complexes of at
least (1) said specific bacteria, (2) said bacteriophage, and (3)
said solid phase particles, if said specific bacteria are present in
said fluid sample.
(b) separating said complexes from solid phase particles and bacteriophage
in said test mixture not complexed to said specific bacteria, and

SUBSTITUTE SHEET (c) testing to detect said complexes, thereby to determine if said specific bacteria are present.

3. The method of claim 2 in which said specific binding pair are biotin and avidin.

4. The method of claim 2 in which said specific binding pair are an antigen and an antibody thereto.

5. The method of claim 2 in which said labeling agent is affixed to said carrier particle.

6. The method of claim 2 in which said particle has a size approximating the size of said specific bacteria.

7. The method of claim 2 in which said particle has a. size of from about 0.2 to about 10 times the size of said specific bacteria.

8. The method of claim 2 in which said particle size is approximately 1 micron.

9. The method of claim 2 in which in step (b) said complexes are separated by filtration retaining said complexes on a filtration medium and passing particles not complexed to said specific bacteria through said medium.

10. A method for the determination of the presence of a specific bacteria in a fluid, comprising:
(a) contacting said fluid with
(i) a measured amount of biotinylated bacteriophage capable
of attaching to said specific bacteria, and
(ii) a measured amount of avidinylated water-insoluble,
water-suspendible. solid phase, labeled carrier particles in
suspension in an aqueous medium,
to form water-insoluble complexes of at least (1) said specific
bacteria, (2) said bacteriophage, and (3) said carrier particles, if
said specific bacteria are present in said fluid, said aqueous
suspension having a concentration of such particles sufficient to

SUBSTITUTE SHEET permit formation of said complexes if said specific bacteria are
present in said fluid, and insufficient for agglutination of said
particles in said suspension to an extent preventing their
separation from said complex in step (b)
(b) separating said complex from said fluid and any canier particles
not complexed with said specific bacteria, to obtain a segregate
of said complex if said specific bacteria are present, and
(c) testing for said segregate to detect said label, thereby to
determine if said specific bacteria are present.

11. The method of claim 10 in which said avidinylated labeled carrier particles are in suspension in an aqueous medium from which suspension said measured amount is taken, said aqueous suspension having a concentration of such particles (i) sufficient to permit formation of said complex in step (a) if said specific bacteria is present in said sample and (ii) insufficient for agglutination of said particles in said suspension to an extent preventing their separation from said complex after said step (a). .

12. The method of claim 10 in which said avidinylated labeled carrier particles are in suspension in an aqueous medium from which said measured amount is taken, said aqueous suspension having a concentration of such particles ranging from about 1 x 10^ to about 1 x 10^ particles per mL.

13. The method of claim 10 in which said avidinylated labeled carrier particle has a size in its largest dimension approximating the size of said specific bacteria.

14. The method of claim 10 in which said particles are polystyrene or other • polymer/copolymer spheres having a particle size of about one micrometer.

15. The method of claim 10 in which said label is a member selected from the group consisting of a radioactive isotope, an enzyme, a luminescent, a chromogenic and a fluorogenic material, and said step (c) is by means selected from the group consisting of radiometric, enzymatic, luminescence, colorimetric and fluorometric means.

SUBSTITUTE SHEET

1 16. The method of claim 15 in which said label is an enzymatic colorimetric

2 indicator.

l 17. The method of claim 16 in which said label is horseradish peroxidase.

1 18. The method of claim 17 in which the method of testing said segregate to

2 detect horseradish peroxidase includes contacting said segregate with a peroxidase

3 chromogenic substrate.

1 19. The method of claim 15 in which said label is a fluorescent

2 chromophore.

1 20. The method of claim 19 in which said label is fluorescein.

1 21. The method of claim 15 in which said label is the radioactive isotope

2 125

1 . 22. The method of claim 10 in which the biotinylated bacteriophage are

2 suspended in an aqueous medium from which said measured amount is taken, said

3 bacteriophage suspension having a concentration of bacteriophage sufficient for a ratio

4 of bacteriophage to specific bacteria, if present, effective to produce sufficient of said

5 complexes for detection of said label in step (c).

1 23. The method of claim 22 in which said ratio is sufficient for detection of

2 bacteria present in said sample at a concentration in the range from as little as 10 CFU.

1 24. The method of claim 10 in which said bacteriophage is species specific.

1 25. A method of claim 10 in which the bacteriophage is genus specific.

1 26. The method of claim 10 in which the bacteriophage is specific for

2 bacteria selected from the group consisting of Salmonella, Listeria, Campylobacter.

3 Bacillus, hemorrhagic Escherichia, and Shigella bacteria or any microorganism that is

4 susceptible to bacteriophage binding.

1 27. The method of claim 10 in which said complex is separated by filtration

2 retaining said complex on a filtration medium.

SUBSTITUTE SHEET

28. The method of claim 27 in which said complex is larger than 3 - 5 μm.

29. The method of claim 28 in which said filtration medium has a pore size of about 3 - 5 μm.

30. A method for the determination of the presence in a fluid of specific bacteria, comprising:
(a) contacting a sample of said fluid with
(i) a measured amount of an aqueous suspension of
biotinylated bacteriophage capable of attaching to specific
bacteria, and
(ii) a measured amount of an aqueous suspension of a water- insoluble, water-suspendible avidinylated solid phase
carrier particle labeled with an enzymatic colorimetric
indicator, said carrier particle having a size approximating
the size of said specific bacteria, thereby, if said specific
bacteria are present in said fluid sample -in a
predetermined minimum concentration, to form a test
mixture containing complexes of at least (1) said specific
bacteria, (2) said bacteriophage, and (3) said solid phase
carrier particles, said complex having a size of at least
about 3 - 5 times, said aqueous suspension of avidinylated
particles having a concentration of such particles sufficient
to permit formation of said complexes if said specific
bacteria are present in said sample, and insufficient for
agglutination of said particles in said suspension to an
extent preventing their separation from said complex in
step (c)
(b) depositing said test mixture onto a filtration medium having a
pore size of about 3 - 5 times the size of said bacteria,
(c) washing said filtration medium after step (b) with an aqueous
medium, whereby paniculate matter smaller than 3 - 5 times the
size of said bacteria is flushed through said filtration medium and
said complexes are retained on said filtration medium if said
complexes were formed in step (a), and

SUBSTITUTE SHEET (d) depositing a color developing reagent including substrate for said
enzymatic colorimetric indicator onto said filtration medium.
whereby an indicator color is formed on said medium if said
complexes are present, thereby indicating the presence of specific
bacteria in said sample.

31. A method of determining the presence of specific bacteria present in a fluid, which comprises:
(a) selecting a bacteriophage which attaches to a specific bacteria,
(b) affixing a first binding agent to said bacteriophage to obtain a
combining bacteriophage,
(c) selecting a water-insoluble, water-suspendible solid phase particle
of approximate size to said specific bacteria,
(d) affixing a second binding agent to said solid phase particle to
obtain a combining particle, said second binding agent being
operative to bind with said first binding agent,
(e) affixing a labeling agent to said solid phase particle or to said
second binding agent to obtain a labeled combining particle,
(f) contacting said combining bacteriophage with a fluid to be
tested for presence of said specific bacteria, whereby if said
specific bacteria are present said combining bacteriophage attach
to said specific bacteria,
(f) contacting said labeled combining particle with said fluid to be
tested, either with or after said combining bacteriophage in step
(e), whereby said labeled combining particles bind with said
combining bacteriophage, resulting, if said specific bacteria is
present, in complexes of at least (1) said specific bacteria, (2)
said bacteriophage, and (3) said solid phase particles, said
complexes being of a larger size than said labeled combining
particle or said labeled combining particle combined with
combining bacteriophage unattached to said specific bacteria,
(g) separating said complexes from said fluid and from labeled
combining particles and labeled combining particles combined
with combining bacteriophage unattached to a said specific
bacteria, and
(h) testing for said complexes, if present, to detect said label, thereby
to determine if said specific bacteria are present.

SUBSTITUTE SHEET

32. The method of claim 31 in which said label is a member selected from the group consisting of a radioactive isotope, an enzyme, a luminescent, a chromogenic and a fluorogenic material, and said step (c) is by means selected from the group consisting of radiometric, enzymatic, luminescence, a colorimetric and fluorometric means.

33. The method of claim 31 in which the bacteriophage is specific for bacteria selected from the group consisting of Salmonella, Listeria, Campylobacter, Bacillus, hemorrhagic Escherichia, and Shigella bacteria.

34. The method of claim 31 in which said first binding agent is biotin and said second binding agent is avidin.

35. A diagnostic kit for the determination of the presence of specific bacteria in a fluid, comprising- (a) a first reagent including a bacteriophage capable of attaching to
specific bacteria, a first member of a specific binding pair being
coupled to said bacteriophage,
(b) a second reagent including a second member of said specific
binding pair coupled to a water-insoluble, water- suspendible
solid phase carrier particle having a size approximating the size
of said specific bacteria, a labeling agent being affixed at least to
said bacteriophage in said first reagent or to said carrier particle
in said second reagent.

36. The diagnostic kit of claim 35, further comprising a filter medium which retains thereon complexes of at least (i) said specific bacteria, (ii) said bacteriophage, and (iii) said solid phase, said complexes being formed and retained on said filter medium, if said specific bacteria are present in said fluid, by contacting said fluid with said first and second reagents and depositing the resulting mixture onto said filter medium.

' 37. The diagnostic kit of claim 35 further comprising a third reagent which tests for the presence of said label.

SUBSTITUTE SHEET

38. The diagnostic kit of claim 36 further comprising a third reagent which tests for the presence of said label, for deposit onto filter medium after said mixture is poured onto the said filter medium.

39. The diagnostic kit of claim 35 in which said first member of a specific binding pair is biotin and said second member of such specific binding pair is avidin, and in which said particles are in suspension in an aqueous medium, such aqueous suspension having a concentration of such particles (i) sufficient to permit formation of said complexes if the specific bacteria are present in said fluid, and (ii) insufficient for agglutination of said particles in said suspension to an extent preventing their separation from said complexes.

40. The diagnostic kit of claim 35 . in which said particles are suspended in an aqueous medium, such aqueous suspension having a concentration of said particles ranging from about 1 x 10^ to about 1 x 10^ particles per mL.

41. The diagnostic kit of claim 35 in which said bacteriophage are suspended in an aqueous medium, said bacteriophage suspension having a concentration of bacteriophage sufficient for a ratio of bacteriophage to specific bacteria, if present in a test sample, effective to produce sufficient of said complex for detection of said label.

42. The diagnostic kit of claim 41 in which said ratio is sufficient for detection of bacteria present in said sample at a concentration in the range from as little as 10 CFU.

43. The diagnostic kit of claim 35 in which said first member of a specific binding pair is biotin and said second member of such specific binding pair is avidin.

44. The diagnostic kit of claim 35 in which said labeling agent is a member selected from the group consisting of a radioactive isotope, an enzyme, a luminescent, a chromogenic and a fluorogenic material.

45. The diagnostic kit of claim 35 in which said labeling agent is affixed to said particle.

SUBSTITUTE SHEET

46. The diagnostic kit of claim 37 in which said labeling agent is an enzymatic colorimetric indicator and said third reagent includes a substrate for said enzymatic colorimetric indicator.

47. The diagnostic kit of claim 46 in which said indicator is horseradish peroxidase and said substrate is a peroxidase chromogenic substrate.

48. The diagnostic kit of claim 35 in which said bacteria-type is selected from the group consisting of Salmonella, Listeria, Campylobacter, Bacillus, hemorrhagic Escherichia, and Shigella bacteria.

49. The diagnostic kit of claim 36 in which said filter medium has a pore size of about 3 - 5 microns .

50. A diagnostic kit for the determination of the presence of specific bacteria in a fluid sample, said bacteria being selected from the group consisting of Salmonella, Listeria, Campylobacter, Bacillus, hemorrhagic Escherichia, and Shigella bacteria, comprising:
(a) a first reagent including a biotinylated bacteriophage capable of
attaching to said selected bacteria, and
(b) a second reagent including water-insoluble, water-suspendible
avidinylated solid phase particles in aqueous suspension labeled
with an enzymatic colorimetric indicator and having a size
approximating the size of said specific bacteria, said aqueous
suspension having a concentration of such particles sufficient to
permit formation of said complex of (1) the specific bacteria, (2)
the bacteriophage, and (3) the solid phase particles if said specific
bacteria are present in said sample, and insufficient for
agglutination of said particles in said suspension to an extent
preventing their separation from said complex.

51. The diagnostic kit of claim 50 further comprising a filter medium having a pore diameter of about 3 - 5 microns.

52. The diagnostic kit of claim 50 further comprising a third reagent which includes a substrate for said enzvmatic colorimetric indicator.

SUBSTITUTE SHEET

53. The method of claim 1 in which said first member of a binding pair is biotin and said second member is avidin.

54. The method of claim 2 in which said first member of a binding pair is biotin and said second member is avidin.

SUBSTITUTE SHEET