METHOD AND KIT FOR THE DETECTION OF HEPATITIS-SPECIFIC ANTIBODIES

• US 20150111767A1
• Filed: 03/07/2013
• Published: 04/23/2015
• Est. Priority Date: 03/08/2012
• Status: Active Grant

• Current Assignee: Dobroslav Melamed, Dror Atzmon
• Original Assignee: Smart Biotech Ltd.
• Inventors: Jehuda-Cohen, Tamar
• Granted Patent: US 9,651,557 B2
• Time in Patent Office: Days
• US Class Current: 506/9
• CPC Class Codes: G01N 33/576 for hepatitis
• CPC Class Codes: Y02A 50/30 Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

The present invention relates to an improved method for detecting antibodies to a hepatitis virus in a tissue sample from individuals, which can reliably detect antibodies in recently infected individuals and which provides much lower false positive results in individuals that have cleared their hepatitis infections. More particularly, the present invention relates to an improved method and kit which utilizes an activator of (i) hepatitis virus-primed lymphocytes, (ii) memory cells specific for said hepatitis virus, (iii) hepatitis virus-specific antibody production, or (iv) a combination thereof in a tissue sample to stimulate the production of antibodies from newly primed B cells, if present.

Claims

1. A method for the detection of hepatitis virus-specific antibodies in a sample from a subject comprising the steps of:

a) incubating a sample from said subject in a culture in the presence of a medium comprising a) one or more activators of (i) hepatitis virus-primed lymphocytes, (ii) memory cells specific for said hepatitis virus, (iii) hepatitis virus-specific antibody production, or (iv) a combination thereof, and b) an antibody against a B-lymphocyte membrane domain;

b) exposing the resultant culture of step a) to a hepatitis virus antigen, thereby allowing an antigen-antibody immune complex to form; and

c) detecting the antigen-antibody immune complex of step b);

thereby detecting the presence of hepatitis virus-specific antibodies.

2. The method according to claim 1, wherein said method further comprises the step of d) exposing a separate aliquot from said sample from said subject to said hepatitis virus antigen.

3. The method according to claim 2, wherein if said antigen-antibody immune complex is detected in step c) but not in step d), said subject has a very early hepatitis infection.

4. The method according to claim 2, wherein if the level of said antigen-antibody immune complex is higher in step c) than in step d), said subject has a recent hepatitis infection.

5. The method according to claim 2, wherein if the level of said antigen-antibody immune complex is higher in step d) than in step c), said subject has a cleared hepatitis infection.

6. The method according to claim 2, wherein if the levels of said antigen-antibody immune complexes are in the same range in step d) and in step c), said subject has a chronic and/or long term hepatitis infection.

7. The method according to any preceding claim, wherein said tissue sample is a whole blood sample.

8. The method according to claim 7, further comprising the step of collecting a whole blood sample from said subject, prior to step a) into a container.

9. The method according to claim 8, wherein said container comprises a polystyrene polymer, a polypropylene polymer, or a combination thereof.

10. The method according to any one of claims 8-9, wherein said container has a first and second positioning mechanism, wherein said first positioning mechanism renders the container fully closed, thereby stably maintaining liquid and vacuum pressure inside said container and wherein said second positioning mechanism provides ventilation to the contents of said container while preserving a sterile environment inside said container.

11. The method according to any preceding claim, wherein said hepatitis virus is a hepatitis C virus (HCV).

12. The method according to any one of claims 1-10, wherein said hepatitis virus is a hepatitis B virus (HBV).

13. The method according to any one of claims 1-10, wherein said hepatitis virus is a hepatitis A virus (HAV), hepatitis D virus (HDV), or hepatitis E virus (HEV).

14. The method according to any preceding claim, wherein said medium comprises a non-carbon dioxide-dependent buffering agent.

15. The method according to claim 14, wherein said buffering agent is HEPES ((4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) or a phosphate-based buffering agent.

16. The method according to any preceding claim, wherein the culture of step a) results in a supernatant, and the supernatant is exposed to a viral antigen in step b), thereby allowing an antigen-antibody immune complex to form.

17. The method according to any preceding claim, wherein said antibody is against a non-secreted protein.

18. The method according to claim 17, wherein said antibody is anti-CD19.

19. The method according to any preceding claim, wherein said activator is pokeweed mitogen.

20. The method according to any preceding claim, wherein said activator is a viral-derived peptide, lectin, bacterial endotoxin, a virus, lipid A, a cytokine, a chain of nucleotides, or a lymphokine.

21. The method according to any preceding claim, wherein said hepatitis virus-primed lymphocytes or memory cells are B-lymphocytes.

22. The method according to any preceding claim, wherein said hepatitis virus-primed lymphocytes or memory cells are T-lymphocytes.

23. The method according to any preceding claim, wherein said hepatitis virus-primed lymphocytes or memory cells are both B-lymphocytes and T-lymphocytes.

24. The method according to any preceding claim, wherein said antigen-antibody immune complex is detected on a solid phase support.

25. The method according to claim 24, wherein said solid phase support is a nitrocellulose strip, polymer beads, synthetic beads, or a combination thereof.

32. The method according to any preceding claim, wherein said tissue sample is a whole blood sample.

33. The method according to claim 32, further comprising the step of collecting a whole blood sample from said subject, prior to step a) into a container.

34. The method according to claim 33, wherein said container comprises a polystyrene polymer, a polypropylene polymer, or a combination thereof.

35. The method according to any one of claims 33-34, wherein said container has a first and second positioning mechanism, wherein said first positioning mechanism renders the container fully closed, thereby stably maintaining liquid and vacuum pressure inside said container and wherein said second positioning mechanism provides ventilation to the contents of said container while preserving a sterile environment inside said container.

36. The method according to any preceding claim, wherein said hepatitis virus is a hepatitis C virus (HCV).

39. The method according to any preceding claim, wherein said medium comprises a non-carbon dioxide-dependent buffering agent.

40. The method according to claim 39, wherein said buffering agent is HEPES ((4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) or a phosphate-based buffering agent.

41. The method according to any preceding claim, wherein the culture of step a) results in a supernatant, and the supernatant is exposed to a viral antigen in step b), thereby allowing an antigen-antibody immune complex to form.

42. The method according to any preceding claim, wherein said antibody is against a non-secreted protein.

43. The method according to claim 42, wherein said antibody is anti-CD19.

44. The method according to any preceding claim, wherein said activator is pokeweed mitogen.

45. The method according to any preceding claim, wherein said activator is a viral-derived peptide, lectin, bacterial endotoxin, a virus, lipid A, a cytokine, a chain of nucleotides, or a lymphokine.

46. The method according to any preceding claim, wherein said hepatitis virus-primed lymphocytes or memory cells are B-lymphocytes.

47. The method according to any preceding claim, wherein said hepatitis virus-primed lymphocytes or memory cells are T-lymphocytes.

48. The method according to any preceding claim, wherein said hepatitis virus-primed lymphocytes or memory cells are both B-lymphocytes and T-lymphocytes.

49. The method according to any preceding claim, wherein said antigen-antibody immune complex is detected on a solid phase support.

50. The method according to claim 49, wherein said solid phase support is a nitrocellulose strip, polymer beads, synthetic beads, or a combination thereof.

53. The method according to any preceding claim, wherein said tissue sample is a whole blood sample.

54. The method according to claim 53, further comprising the step of collecting a whole blood sample from said subject, prior to step a) into a container.

55. The method according to claim 54, wherein said container comprises a polystyrene polymer, a polypropylene polymer, or a combination thereof.

56. The method according to any one of claims 54-55, wherein said container has a first and second positioning mechanism, wherein said first positioning mechanism renders the container fully closed, thereby stably maintaining liquid and vacuum pressure inside said container and wherein said second positioning mechanism provides ventilation to the contents of said container while preserving a sterile environment inside said container.

57. The method according to any preceding claim, wherein said hepatitis virus is a hepatitis C virus (HCV).

60. The method according to any preceding claim, wherein said medium comprises a non-carbon dioxide-dependent buffering agent.

61. The method according to claim 60, wherein said buffering agent is HEPES ((4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) or a phosphate-based buffering agent.

62. The method according to any preceding claim, wherein the culture of step a) results in a supernatant, and the supernatant is exposed to a viral antigen in step b), thereby allowing an antigen-antibody immune complex to form.

63. The method according to any preceding claim, wherein said antibody is against a non-secreted protein.

64. The method according to claim 63, wherein said antibody is anti-CD19.

65. The method according to any preceding claim, wherein said activator is pokeweed mitogen.

66. The method according to any preceding claim, wherein said activator is a viral-derived peptide, lectin, bacterial endotoxin, a virus, lipid A, a cytokine, a chain of nucleotides, or a lymphokine.

67. The method according to any preceding claim, wherein said hepatitis virus-primed lymphocytes or memory cells are B-lymphocytes.

68. The method according to any preceding claim, wherein said hepatitis virus-primed lymphocytes or memory cells are T-lymphocytes.

69. The method according to any preceding claim, wherein said hepatitis virus-primed lymphocytes or memory cells are both B-lymphocytes and T-lymphocytes.

70. The method according to any preceding claim, wherein said antigen-antibody immune complex is detected on a solid phase support.

71. The method according to claim 70, wherein said solid phase support is a nitrocellulose strip, polymer beads, synthetic beads, or a combination thereof.

77. The method according to any preceding claim, wherein said hepatitis virus is a hepatitis C virus (HCV).

80. The method according to any preceding claim, wherein said medium comprises a non-carbon dioxide-dependent buffering agent.

81. The method according to claim 80, wherein said buffering agent is HEPES ((4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) or a phosphate-based buffering agent.

83. The method according to any preceding claim, wherein said antibody is against a non-secreted protein.

84. The method according to claim 83, wherein said antibody is anti-CD19.

85. The method according to any preceding claim, wherein said activator is pokeweed mitogen.

86. The method according to any preceding claim, wherein said activator is a viral-derived peptide, lectin, bacterial endotoxin, a virus, lipid A, a cytokine, a chain of nucleotides, or a lymphokine.

87. The method according to any preceding claim, wherein said hepatitis virus-primed lymphocytes or memory cells are B-lymphocytes.

88. The method according to any preceding claim, wherein said hepatitis virus-primed lymphocytes or memory cells are T-lymphocytes.

89. 23. The method according to any preceding claim, wherein said hepatitis virus-primed lymphocytes or memory cells are both B-lymphocytes and T-lymphocytes.

90. The method according to any preceding claim, wherein said antigen-antibody immune complex is detected on a solid phase support.

91. The method according to claim 90, wherein said solid phase support is a nitrocellulose strip, polymer beads, synthetic beads, or a combination thereof.

97. The method according to any preceding claim, wherein said hepatitis virus is a hepatitis C virus (HCV).

100. The method according to any preceding claim, wherein said medium comprises a non-carbon dioxide-dependent buffering agent.

101. The method according to claim 100, wherein said buffering agent is HEPES ((4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) or a phosphate-based buffering agent.

102. The method according to any preceding claim, wherein the culture of step a) results in a supernatant, and the supernatant is exposed to a viral antigen in step b), thereby allowing an antigen-antibody immune complex to form.

103. The method according to any preceding claim, wherein the culture of step a) further comprises an antibody against a B-lymphocyte membrane domain.

104. The method according to claim 103, wherein said antibody is against a non-secreted protein.

105. The method according to any one of claims 103-104, wherein said antibody is anti-CD19.

106. The method according to any preceding claim, wherein the culture of step a) further comprises an anti-IgD antibody.

107. The method according to any preceding claim, wherein the culture of step a) further comprises an anti-IgG, anti-IgA, or anti-IgE antibody.

108. The method according to any preceding claim, wherein said activator is pokeweed mitogen.

109. The method according to any preceding claim, wherein said activator is a viral-derived peptide, lectin, bacterial endotoxin, a virus, lipid A, a cytokine, a chain of nucleotides, or a lymphokine.

110. The method according to any preceding claim, wherein said hepatitis virus-primed lymphocytes or memory cells are B-lymphocytes.

111. The method according to any preceding claim, wherein said hepatitis virus-primed lymphocytes or memory cells are T-lymphocytes.

112. The method according to any preceding claim, wherein said hepatitis virus-primed lymphocytes or memory cells are both B-lymphocytes and T-lymphocytes.

113. The method according to any preceding claim, wherein said antigen-antibody immune complex is detected on a solid phase support.

114. The method according to claim 113, wherein said solid phase support is a nitrocellulose strip, polymer beads, synthetic beads, or a combination thereof.

117. The kit according to any preceding claim, wherein said tissue sample is a whole blood sample.

118. The kit according to any preceding claim, wherein said container is made of a plastic, glass, silicon, synthetic membrane, or metal.

119. The kit according to any preceding claim, wherein said container is a test tube, a bottle, a well or a flask.

120. The kit according to any preceding claim, wherein said container is vacuum sealed.

121. The kit according to any preceding claim, wherein said container is a vacutube.

122. The kit according to any preceding claim, wherein said container comprises a polystyrene polymer, a polypropylene polymer, or a combination thereof.

123. The kit according to any preceding claim, wherein said container has a first and second positioning mechanism, wherein said first positioning mechanism renders the container fully closed, thereby stably maintaining liquid and vacuum pressure inside said container and wherein said second positioning mechanism provides ventilation to the contents of said container while preserving a sterile environment inside said container.

124. The kit according to any preceding claim, wherein said hepatitis virus is a hepatitis C virus (HCV).

127. The kit according to any preceding claim, wherein said medium comprises a non-carbon dioxide-dependent buffering agent.

128. The kit according to claim 127, wherein said buffering agent is HEPES ((4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) or a phosphate-based buffering agent.

129. The kit according to any preceding claim, wherein said antibody is against a non-secreted protein.

131. The kit according to any preceding claim, wherein said activator is pokeweed mitogen.

132. The kit according to any preceding claim, wherein said activator is a viral-derived peptide, lectin, bacterial endotoxin, a virus, lipid A, a cytokine, a chain of nucleotides, or a lymphokine.

133. The kit according to any preceding claim, wherein said hepatitis virus-primed lymphocytes or memory cells are B-lymphocytes.

134. The kit according to any preceding claim, wherein said hepatitis virus-primed lymphocytes or memory cells are T-lymphocytes.

135. The kit according to any preceding claim, wherein said hepatitis virus-primed lymphocytes or memory cells are both B-lymphocytes and T-lymphocytes.

136. The kit according to any preceding claim, wherein said kit additionally comprises an assay for the detection of hepatitis viral-specific antibodies.

137. The kit according to claim 136, wherein said assay is an enzyme linked immunosorbent assay, a western blot, or an immunofluorescence assay.

138. The kit according to any one of claims 136-137, wherein said assay comprises a solid phase support.

139. The kit according to claim 138, wherein said solid phase support is a nitrocellulose strip, polymer beads, synthetic beads, or a combination thereof.

140. The kit according to any preceding claim, further comprising a means for drawing blood from said subject.

141. The kit according to any preceding claim, further comprising instructions for use.

26. A method for the detection of a hepatitis infection in a subject comprising the steps of:

a. incubating a sample from said subject in a culture in the presence of a medium comprising a) one or more activators of (i) hepatitis virus-primed lymphocytes, (ii) memory cells specific for said hepatitis virus, (iii) hepatitis virus-specific antibody production, or (iv) a combination thereof, and b) an antibody against a B-lymphocyte membrane domain;

b. exposing the resultant culture of step a) to a hepatitis virus antigen, thereby allowing an antigen-antibody immune complex to form; and

c. detecting the antigen-antibody immune complex of step b);

thereby detecting a hepatitis infection in said subject.

27. The method according to claim 26, wherein said method further comprises the step of d) exposing a separate aliquot from said sample from said subject to said hepatitis virus antigen.

28. The method according to claim 27, wherein if said antigen-antibody immune complex is detected in step c) but not in step d), said subject has a very early hepatitis infection.

29. The method according to claim 27, wherein if the level of said antigen-antibody immune complex is higher in step c) than in step d), said subject has a recent hepatitis infection.

30. The method according to claim 27, wherein if the level of said antigen-antibody immune complex is higher in step d) than in step c), said subject has a cleared hepatitis infection.

31. The method according to claim 27, wherein if the levels of said antigen-antibody immune complexes are in the same range in step d) and in step c), said subject has a chronic and/or long term hepatitis infection.

37. The method according to any one of claims 26-35, wherein said hepatitis virus is a hepatitis B virus (HBV).

38. The method according to any one of claims 26-35, wherein said hepatitis virus is a hepatitis A virus (HAV), hepatitis D virus (HDV), or hepatitis E virus (HEV).

51. A method for the detection of a very early hepatitis infection in a subject comprising the steps of:

a. incubating a sample from said subject in a culture in the presence of a medium comprising a) one or more activators of (i) hepatitis virus-primed lymphocytes, (ii) memory cells specific for said hepatitis virus, (iii) hepatitis virus-specific antibody production, or (iv) a combination thereof, and b) an antibody against a B-lymphocyte membrane domain;

b. exposing the resultant culture of step a) to a hepatitis virus antigen, thereby allowing an antigen-antibody immune complex to form; and

c. detecting the antigen-antibody immune complex of step b);

thereby detecting a very early hepatitis infection in said subject.

52. The method according to claim 51, wherein said method further comprises the step of d) exposing a separate aliquot from said sample from said subject to said hepatitis virus antigen, and wherein if an antigen-antibody immune complex is detected in step c) but not in step d), said subject has a very early hepatitis infection.

58. The method according to any one of claims 51-56, wherein said hepatitis virus is a hepatitis B virus (HBV).

59. The method according to any one of claims 51-56, wherein said hepatitis virus is a hepatitis A virus (HAV), hepatitis D virus (HDV), or hepatitis E virus (HEV).

72. A method for detecting a hepatitis infection in a seronegative subject comprising the steps of:

a. incubating a sample from said subject in a culture in the presence of a medium comprising a) one or more activators of (i) hepatitis virus-primed lymphocytes, (ii) memory cells specific for said hepatitis virus, (iii) hepatitis virus-specific antibody production, or (iv) a combination thereof, and b) an antibody against a B-lymphocyte membrane domain;

b. exposing the resultant culture of step a) to a hepatitis virus antigen, thereby allowing an antigen-antibody immune complex to form; and

c. detecting the antigen-antibody immune complex of step b);

wherein if the antigen-antibody immune complex is detected, said seronegative subject has a hepatitis infection, thereby detecting a hepatitis infection in said seronegative subject.

73. The method according to claim 72, wherein said tissue sample is a whole blood sample.

74. The method according to claim 73, further comprising the step of collecting a whole blood sample from said subject, prior to step a) into a container.

75. The method according to claim 74, wherein said container comprises a polystyrene polymer, a polypropylene polymer, or a combination thereof.

76. The method according to any one of claims 73-74, wherein said container has a first and second positioning mechanism, wherein said first positioning mechanism renders the container fully closed, thereby stably maintaining liquid and vacuum pressure inside said container and wherein said second positioning mechanism provides ventilation to the contents of said container while preserving a sterile environment inside said container.

78. The method according to any one of claims 72-76, wherein said hepatitis virus is a hepatitis B virus (HBV).

79. The method according to any one of claims 72-76, wherein said hepatitis virus is a hepatitis A virus (HAV), hepatitis D virus (HDV), or hepatitis E virus (HEV).

82. The method according to claim 72, wherein the culture of step a) results in a supernatant, and the supernatant is exposed to a viral antigen in step b), thereby allowing an antigen-antibody immune complex to form.

92. A method of differentiating between a cleared and chronic hepatitis infection in a subject comprising the steps of:

a. obtaining a tissue sample from said subject;

b. determining the anti-hepatitis virus antibody level in a first aliquot of said sample, wherein a detectable anti-hepatitis virus antibody level indicates that a subject is seropositive;

c. stimulating a second aliquot of said sample to produce anti-hepatitis virus antibodies in vitro by incubating said second aliquot in a culture in the presence of a medium comprising a) one or more activators of (i) hepatitis virus-primed lymphocytes, (ii) memory cells specific for said hepatitis virus, (iii) hepatitis virus-specific antibody production, or (iv) a combination thereof, and b) an antibody against a B-lymphocyte membrane domain;

d. determining the anti-hepatitis virus antibody level in said second aliquot of said sample;

e. dividing a value representing the stimulated anti-hepatitis virus antibody level obtained in step (d) by a value representing the anti-hepatitis virus antibody level obtained in step (b) for each subject;

f. determining if the quotient obtained in step (d) is above a pre-determined threshold value for each subject,

wherein a value below said threshold value indicates that the subject has a cleared hepatitis virus infection and a value close to said threshold value indicates that the subject has a chronic hepatitis infection, thereby differentiating between a cleared and chronic hepatitis infection in said subject.

93. The method according to claim 92, wherein said tissue sample is a whole blood sample.

94. The method according to claim 93, further comprising the step of collecting a whole blood sample from said subject, prior to step a) into a container.

95. The method according to claim 94, wherein said container comprises a polystyrene polymer, a polypropylene polymer, or a combination thereof.

96. The method according to any one of claims 94-95, wherein said container has a first and second positioning mechanism, wherein said first positioning mechanism renders the container fully closed, thereby stably maintaining liquid and vacuum pressure inside said container and wherein said second positioning mechanism provides ventilation to the contents of said container while preserving a sterile environment inside said container.

98. The method according to any one of claims 92-96, wherein said hepatitis virus is a hepatitis B virus (HBV).

99. The method according to any one of claims 92-96, wherein said hepatitis virus is a hepatitis A virus (HAV), hepatitis D virus (HDV), or hepatitis E virus (HEV).

115. A kit for the detection of hepatitis virus-specific antibodies in a subject comprising:

a container for retaining a tissue sample, wherein said container comprises a medium, said medium comprising a) one or more activators of (i) lymphocytes activated by said hepatitis virus, (ii) memory cells specific for said hepatitis virus, (iii) antibodies against said hepatitis virus, or (iv) a combination thereof, and b) an antibody against a B-lymphocyte membrane domain.

116. The kit according to claim 115, wherein said kit further comprises a second container lacking said medium for retaining a second tissue sample.

125. The kit according to any one of claims 115-123, wherein said hepatitis virus is a hepatitis B virus (HBV).

126. The kit according to any one of claims 115-123, wherein said hepatitis virus is a hepatitis A virus (HAV), hepatitis D virus (HDV), or hepatitis E virus (HEV).

130. The kit according to claim 126, wherein said antibody is anti-CD19.