Method of efficient extraction of protein from cells

• US 20090123910A1
• Filed: 11/14/2007
• Published: 05/14/2009
• Est. Priority Date: 11/14/2007
• Status: Active Grant

• Current Assignee: Arbor Vita Corporation
• Original Assignee: Becton, Dickinson & Co
• Inventors: Ferguson, Carrie S., Beadenkopf, Robert J., Bruton, Jeff H., Rosales, Julie L., Crews, Virginia M., Malick, Adrien P.
• Granted Patent: US 9,207,240 B2
• Time in Patent Office: Days
• US Class Current: 435/5
• CPC Class Codes: C07K 1/145 by extraction or solubilisation
• CPC Class Codes: C12N 15/52 Genes encoding for enzymes or proenzymes
• CPC Class Codes: C12N 2710/20022 New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
• CPC Class Codes: G01N 1/4044 by chemical techniques; Digestion; Chemical decomposition
• CPC Class Codes: G01N 2333/025 Papovaviridae, e.g. papillomavirus, polyomavirus, SV40, BK virus, JC virus
• CPC Class Codes: G01N 2333/16 HIV-1, HIV-2
• CPC Class Codes: G01N 2469/10 Detection of antigens from microorganism in sample from host
• CPC Class Codes: G01N 33/54393 Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
• CPC Class Codes: G01N 33/56983 Viruses

Abstract

Methods for producing a protein extract from cells, such as cells or cellular samples containing viral proteins, are provided. In general terms, the methods may involve: increasing the pH of the cells to a pH of at least about pH 10.0 to produce an intermediate composition, and then, in the presence of a non-ionic detergent such as a polyoxyethylene alkyl ether, neutralizing the pH of the intermediate composition to produce the protein extract. Such methods can be used in conjunction with methods for detecting one or more target proteins in a sample, such as viral proteins. Systems, kits and compositions for practicing the subject methods are also provided.

Claims

1. A method for producing a protein extract from fixed or unfixed cells, comprising:

a) contacting said cells with an extraction reagent to produce an intermediate composition having a pH of at least about pH 10.0; and

b) contacting said intermediate composition with a neutralizing reagent to neutralize said pH of said intermediate composition and produce said protein extract,

wherein one or both of said extraction reagent and said neutralizing reagent comprises a polyoxyethylene alkyl ether.

2. The method of claim 1, wherein said method comprises:

a) contacting said cells with an extraction reagent to produce an intermediate composition having a pH of at least about pH 10.0, wherein said extraction reagent comprises a polyoxyethylene alkyl ether; and

b) contacting said intermediate composition with a neutralizing reagent to neutralize said pH of said intermediate composition and produce said protein extract at a pH of about 6-9.

3. The method of claim 1, wherein said method comprises:

a) contacting said cells with an extraction reagent to produce an intermediate composition having a pH of at least about pH 10.0; and

b) contacting said intermediate composition with a neutralizing reagent to neutralize said pH of said intermediate composition and produce said protein extract at a pH of about 6-9, wherein said neutralization reagent comprises a polyoxyethylene alkyl ether.

4. The method of claim 1, wherein said polyoxyethylene alkyl ether is a Brij™

surfactant.

5. The method of claim 4, wherein said Brij™

surfactant is Brij™

35.

6. The method of claim 5, wherein the concentration of said Brij™

35 in the extraction reagent is within about 2-6% (v/v).

7. The method of claim 6, wherein said extraction reagent further comprises about 0.1N NaOH, 50 mM TriSodium Citrate, and has a pH of 12.5 to 12.9.

8. The method of claim 7, wherein said extraction reagent further comprises Triton™

detergent or Tween™

detergent, or mixture thereof.

9. The method of claim 7, wherein said extraction reagent further comprises Triton™

X-100 detergent or Tween™

-20 detergent, or mixture thereof.

10. The method of claim 9, wherein the concentration of said Triton™

X-100 or TWEEN-20™

detergent or both is within about 2-6% (v/v).

11. The method of claim 1, wherein the neutralization reagent is a Tris-based buffer.

12. The method of claim 1, wherein the samples are incubated between 10 and 30 minutes after the addition of the extraction reagent.

13. The method of claim 1, wherein the samples are incubated between 10 and 30 minutes after the addition of the neutralization reagent.

14. The method of claim 4, wherein said Brij™

surfactant is one or more of Brij™

35 polyoxyethylene (23) lauryl ether, (CH₃(CH₂)₁₀CH₂(OCH₂CH₂)_nOH, n˜

23);

Brij™

30 polyoxyethylene (4) lauryl ether (CH₃(CH₂)₁₀CH₂(OCH₂CH₂)_nOH, n˜

4);

Brij™

52, polyoxyethylene (2) cetyl ether (C₁₆H₃₃(OCH₂CH₂)nOH, n˜

2);

Brij™

56 polyoxyethylene (10) cetyl ether (C₁₆H₃₃(OCH₂CH₂)nOH, n˜

10);

Brij™

58, polyoxyethylene (20) cetyl ether (C₁₆H₃₃(OCH₂CH₂)_nOH, n˜

20);

Brij™

72 polyoxyethylene (2) stearyl ether, (C₁₈H₃₇(OCH₂CH₂)nOH, n˜

2);

Brij™

76, polyoxyethylene (10) stearyl ether (C₁₈H₃₇(OCH₂CH₂)nOH, n˜

10);

Brij™

78 polyoxyethylene (20) stearyl ether (C₁₈H₃₇(OCH₂CH₂)nOH, n˜

20);

Brij™

92, polyoxyethylene (2) oleyl ether (C₁₈H₃₅(OCH₂CH₂)nOH, n˜

2);

Brij™

93, polyoxyethylene (2) oleyl ether;

(C₁₈H₃₅(OCH2CH2)nOH, n˜

2);

Brij™

97, polyoxyethylene (10) oleyl ether (C₁₈H₃₅(OCH₂CH₂)nOH, n˜

10);

Brij™

98, polyoxyethylene (20) oleyl ether (C₁₈H₃₅(OCH₂CH₂)_nOH, n˜

20);

Brij™

700, Polyoxyethylene (100) stearyl ether, (C₁₈H₃₇(OCH₂CH₂)₂₁OH, n˜

100);

or Brij™

721, Polyoxyethylene (21) Stearyl Ether (C₁₈H₃₇(OCH₂CH₂)_nOH, n˜

21).

19. The method of claim 1, wherein said pH is in the range of about pH 11.0 to about pH 13.

20. The method of claim 1, wherein said extraction reagent further comprises a denaturant.

21. The method of claim 20, wherein said denaturant is sodium dodecyl sulphate (SDS), urea or sarkosyl.

22. A method for detecting a target protein which is previously known or otherwise characterized, comprising:

a) producing a protein extract from fixed or unfixed cells according to the method of claim 1; and

b) testing for the presence of said protein in said protein extract.

23. The method of claim 22, wherein said testing employs a capture agent for said target protein.

24. The method of claim 22, wherein said testing includes an immunological assay.

25. The method of claim 24, wherein said assay is an enzyme linked immunoabsorbant assay (ELISA).

26. The method of claim 24, wherein said assay is an immunochromatographic assay or lateral flow (LF) assay.

27. The method of claim 26, wherein said assay employs moncoclonal antibody-conjugated gold particles.

28. The method of claim 24, wherein said assay employs fluorescently-labeled monoclonal antibodies.

29. The method of claim 24, wherein said assay employs chemiluminescent particles, colored/dyed latex particles, SERS Raman particles, silica-coated gold cores with various reporter dyes, or silica-coated silver cores with reporter dyes.

30. The method of claim 24, wherein said assay employs consecutive use of one or more monoclonal antibodies.

31. The method of claim 22, wherein said testing employs a cytometric bead array (CBA) or multiplexed bead assay.

32. The method of claim 22, wherein said target protein is a human papillomavirus (HPV) protein and said testing employs a PDZ domain polypeptide that binds to the HPV E6 protein.

33. The method of claim 22, wherein said unfixed cells are exfoliated cervical cells.

34. The method of claim 22, wherein said fixed cells are exfoliated cervical cells.

35. The method of claim 22, wherein said fixed or unfixed cells are received from a remote location prior to said contacting step a).

36. The method of claim 22, further comprising:

d) communicating results of said testing to a remote location.

37. A system for producing a protein extract, according to the method of claim 1, said system comprising:

a) a cellular sample comprising fixed or unfixed cells;

b) an extraction reagent that has a pH of at least about pH 10.0, and

c) a neutralizing reagent,

wherein one or both of said extraction reagent and said neutralizing reagent comprises a polyoxyethylene alkyl ether.

38. The system of claim 37, wherein the polyoxyethylene alkyl ether is a Brij™

detergent.

39. The system of claim 38, wherein said Brij™

detergent is Brij™

35.

40. The system of claim 37, further comprising reagents for detecting a target protein in said protein extract.

41. A kit for producing a protein extract from fixed or unfixed cells, said kit comprising:

a) an extraction reagent that has a pH of at least about pH 10.0,

b) a neutralizing reagent; and

c) instructions for performing the method of claim 1 using said extraction reagent and said neutralizing reagent;

wherein one or both of said extraction reagent and said neutralizing reagent comprises a polyoxyethylene alkyl ether.

42. The kit of claim 41, wherein the polyoxyethylene alkyl ether is a Brij™

detergent.

43. The kit of claim 42, wherein said Brij™

detergent is Brij™

35.

44. The kit of claim 41, further comprising reagents for detecting a target protein in said protein extract.

45. The kit of claim 44, wherein said reagents include a capture agent for said target protein.

46. The kit of claim 45, wherein said target protein is an HPV E6 protein, and the capture agent is a monoclonal antibody against the HPV E6 protein or a PDZ domain polypeptide that binds to the HPV E6 protein.

15. The method of claim, further comprising:

obtaining a cellular sample comprising said fixed or unfixed cells prior to step a), wherein the cellular sample contains or is suspected of containing a target protein.

16. The method of claim 15, wherein said cells are fixed cervical cells, and the target protein is a human Papillomavirus (HPV) E6 protein.

17. The method of claim 16, wherein said fixed cells are present in SUREPATH™

, CYTOLYT™

, THINPREP™

or PRESERVCYT™

transport medium.

18. The method of claim 15, wherein said cellular sample is obtained from an individual.

47. A method for extracting a target viral protein from a cell sample, comprising:

a) contacting a cell sample containing cells in which a target viral protein is present or suspected of being present with an extraction reagent to produce an intermediate composition having a pH of at least about pH 10.0; and

b) contacting said intermediate composition with a neutralizing reagent to neutralize said pH of said intermediate composition and produce an extract of the target viral protein

wherein one or both of said extraction reagent and said neutralizing reagent comprises a polyoxyethylene alkyl ether.

48. The method of claim 47, wherein said polyoxyethylene alkyl ether is a Brij™

surfactant.

49. The method of claim 48, wherein the Brij™

surfactant is Brij™

35 which is comprised by the extraction reagent at a concentration of about 2-6% (v/v).

50. The method of claim 49, wherein said extraction reagent further comprises about 0.1N NaOH, 50 mM TriSodium Citrate, and a pH of 12.5 to 12.9.

51. The method of claim 50, wherein said extraction reagent further comprises Triton™

detergent or Tween™

detergent, or a mixture thereof.

52. The method of claim 51, wherein said extraction reagent further comprises Triton™

X-100 detergent or Tween™

-20 detergent, or a mixture thereof.

53. The method of claim 52, wherein the concentration of said Triton™

X-100 or TWEEN-20™

detergent or both is within about 2-6% (v/v).

54. The method of claim 47, wherein the neutralization reagent is a Tris-based buffer.

55. The method of claim 47, wherein the samples are incubated between 10 and 30 minutes after the addition of the extraction reagent.

56. The method of claim 47, wherein the samples are incubated between 10 and 30 minutes after the addition of the neutralization reagent.

57. The method of claim 48, wherein said Brij™

surfactant is one or more of;

Brij™

35 polyoxyethylene (23) lauryl ether, (CH₃(CH₂)₁₀CH₂(OCH₂CH₂)_nOH, n˜

23);

Brij™

30 polyoxyethylene (4) lauryl ether (CH₃(CH₂)₁₀CH₂(OCH₂CH₂)_nOH, n˜

4);

Brij™

52, polyoxyethylene (2) cetyl ether (C₁₆H₃₃(OCH₂CH₂)nOH, n˜

2);

Brij™

56 polyoxyethylene (10) cetyl ether (C₁₆H₃₃(OCH₂CH₂)nOH, n˜

10);

Brij™

58, polyoxyethylene (20) cetyl ether (C₁₆H₃₃(OCH₂CH₂)_nOH, n˜

20);

Brij™

72 polyoxyethylene (2) stearyl ether, (C₁₈H₃₇(OCH₂CH₂)nOH, n˜

2);

Brij™

76, polyoxyethylene (10) stearyl ether (C₁₈H₃₇(OCH₂CH₂)nOH, n˜

10);

Brij™

78 polyoxyethylene (20) stearyl ether (C₁₈H₃₇(OCH₂CH₂)nOH, n˜

20);

Brij™

92, polyoxyethylene (2) oleyl ether (C₁₈H₃₅(OCH₂CH₂)nOH, n˜

2);

Brij™

93, polyoxyethylene (2) oleyl ether;

(C₁₈H₃₅(OCH2CH2)nOH, n˜

2);

Brij™

97, polyoxyethylene (10) oleyl ether (C₁₈H₃₅(OCH₂CH₂)nOH, n˜

10);

Brij™

98, polyoxyethylene (20) oleyl ether (C₁₈H₃₅(OCH₂CH₂)_nOH, n˜

20);

Brij™

700, Polyoxyethylene (100) stearyl ether, (C₁₈H₃₇(OCH₂CH₂)₂₁OH, n˜

100);

or Brij™

721, Polyoxyethylene (21) Stearyl Ether (C₁₈H₃₇(OCH₂CH₂)_nOH, n˜

21).

58. The method of claim 47, wherein the cells in the cell sample are fixed with a chemical fixative.

59. The method of claim 58, wherein the chemical fixative is selected from the group consisting of alcohols, aldehydes, ketones, osmium tetroxide, acetic acid, picric acid, heavy metal ion salts, and propylene glycol.

60. The method of claim 59, wherein the alcohol is methanol or ethanol;

the aldehyde is gluteraldehyde or formaldehyde; and

the ketone is acetone.

61. The method of claim 58, wherein said fixed cells are present in SUREPATH™

, CYTOLYT™

, THINPREP™

or PRESERVCYT™

transport medium.

62. The method of claim 47, further comprising:

receiving the cell sample prior to step a).

63. The method of claim 47, wherein the target viral protein is encoded by a pathogenic virus.

64. The method of claim 63, wherein the pathogenic virus is selected from the group consisting of HIV, Ebola virus, Marburg virus, hepatitis virus, respiratory syncytial virus (RSV), herpes simplex virus (HSV), and human papilloma virus (HPV).

65. The method of claim 47, wherein the target viral protein is E6 or E7 protein of HPV.

66. The method of claim 65, wherein the HPV is HPV strain 4, 11, 20, 24, 28, 36, 48, 50, 16, 18, 31, 35, 30, 39, 45, 51, 52, 56, 59, 58, 33, 66, 68, 69, 26, 53, 73, or 82.

67. The method of claim 65, wherein the HPV is an oncogenic HPV strain selected from the group consisting of HPV 26, HPV 53, HPV 66, HPV 73, HPV 82, HPV 16, HPV 18, HPV 31, HPV 35, HPV 30, HPV 39, HPV 45, HPV 51, HPV 52, HPV 56, HPV 59, HPV 58, HPV 33, HPV 66, HPV 68, HPV 69, and BPV 82.

68. The method of claim 47, further comprising:

detecting the presence of the target viral protein in the extract.

69. The method of claim 68, wherein the detecting employs a capture agent for the target viral protein.

70. The method of claim 69, wherein the viral protein is E6 protein of HPV;

and the capture agent is an antibody against the E6 protein.

71. The method of claim 69, wherein the viral protein is E6 protein of HPV;

and the capture agent comprises a polypeptide containing a PDZ domain.

72. The method of claim 69, wherein the viral protein is E6 protein of HPV and the capture agent comprises both an antibody against the E6 protein and a polypeptide containing a PDZ domain.

73. The method of claim 72, wherein the PDZ domain is the second domain of MAGI-1, or the PDZ domain of DLG or TIP1.

74. The method of claim 47, where the cell sample further contains mucous, blood or sputum.

75. The method of claim 47, where the cell sample contains exfoliated cervical cells.

76. The method of claim 68, wherein the detecting includes an immunological assay.

77. The method of claim 76, wherein said assay is an enzyme linked immunoabsorbant assay (ELISA).

78. The method of claim 76, wherein said assay is an immunochromatographic assay or lateral flow (LF) assay.

79. The method of claim 76, wherein said assay employs moncoclonal antibody-conjugated gold particles.

80. The method of claim 76, wherein said assay employs fluorescently-labeled monoclonal antibodies.

81. The method of claim 76, wherein said assay employs chemiluminescent particles, colored/dyed latex particles, SERS Raman particles, or silica-coated gold or silver cores with reporter dyes.

82. The method of claim 76, wherein said assay employs consecutive use of one or more monoclonal antibodies.

83. The method of claim 68, wherein the detecting employs a cytometric bead array (CBA) or multiplexed bead assay.