Method and apparatus for immunoassays

• US 4,727,019 A
• Filed: 05/10/1985
• Issued: 02/23/1988
• Est. Priority Date: 05/11/1984
• Status: Expired due to Term

• Current Assignee: Hybritech, Inc. (Danaher Corporation)
• Original Assignee: Hybritech, Inc. (Danaher Corporation)
• Inventors: Valkirs, Gunars E., Owen, Newton C., Levinson, Philip A.
• Primary Examiner(s): NOT, DEFINED
• Application Number:
  US06/733,292
• Time in Patent Office: 1,019 Days
• Field of Search: 435/5, 435/7, 435/287, 435/805, 435/21, 436/513, 436/527, 436/531, 436/548, 436/807, 436/808, 436/824, 422/56-58
• US Class Current: 435/5
• CPC Class Codes: G01N 33/5302 Apparatus specially adapted for immunological test procedures
• CPC Class Codes: G01N 33/54366 Apparatus specially adapted for solid-phase testing
• CPC Class Codes: Y10S 435/805 Test papers
• CPC Class Codes: Y10S 435/974 Aids related test
• CPC Class Codes: Y10S 436/807 Apparatus included in process claim, e.g. physical support structures
• CPC Class Codes: Y10S 436/818 Human chorionic gonadotropin
• CPC Class Codes: Y10S 436/82 Hepatitis associated antigens and antibodies
• CPC Class Codes: Y10S 436/824 Immunological separation techniques

Abstract

Disclosed herein is an apparatus and process for conducting ligand receptor assays. The apparatus comprises a first member which is a membrane or a filter to which is bound a receptor for the ligand or which is capable of extracting cells carrying the ligand from a fluid sample. The apparatus further comprises a second member which is composed of absorbent material which acts when in contact with the first member to induce flow through the first member when a fluid sample is added to it. The apparatus is used to conduct assays by applying a sample to the upper surface of the first member to bind ligand in the sample by means of receptor fixed to the first member or, in certain cases, by extracting cellular material which has ligand associated with it. Addition of the sample is typically followed by addition of labeled antibody against the antigen being assayed followed by a washing step to remove unbound labeled receptor. The presence of labeled antibody on the first member after washing is indicative of the presence of the antigen in the sample being assayed. In a preferred embodiment of the invention, the ligand is an antigen and the receptor is an antibody.

Claims

1. An apparatus for use in a ligand-receptor assay process for determination of the presence or concentration of a target ligand in a liquid sample comprising:

(a) a porous first member which has upper and lower surfaces, the sample being applied to the upper surface, and wherein at least one first receptor capable of binding the target ligand is directly or indirectly bound within an area smaller than the area of the member to which the sample is applied; and

(b) a second member which is a body of absorbent material having a surface over which the lower surface of the first member is placed and having capillaries therethrough which are in communication with the pores on the lower surface of the first member so as to draw liquid added to the upper surface of the first member which has permeated the first member into the capillaries of the second member, the communication between the first and second members having been established prior to, and maintained during, addition of liquids to the apparatus during the assay process.

2. An apparatus according to claim 1 wherein the target ligand is selected from the group consisting of antigens, antibodies, enzymes, and nucleic acid oligomers.

3. An apparatus according to claim 2 wherein the first receptor is selected from the group consisting of antibodies, antigens, enzyme receptors, and nucleic acid oligomers.

4. An apparatus according to claim 1 wherein the porous first member is a membrane or filter.

5. An apparatus according to claim 4 wherein the membrane or filter is of a material selected from glass or nylon.

6. An apparatus according to claim 4 wherein the first and second members are separated by at least one porous member.

7. An apparatus according to claim 4 wherein the apparatus further comprises a container for the first and second members having an opening sufficient to permit the application of assay reagents to the first member.

8. An apparatus according to claim 7 wherein the opening further comprises a section having sides which slope inwardly to define a funnel for direction of the assay reagents applied to the first member.

9. An apparatus according to claim 7 wherein the bottom end of the container is closed and the container is of a volume sufficient to contain all of the assay reagents applied to the first member during the assay, the container being vented to allow escape of displaced air.

10. An apparatus according to claim 9 wherein the apparatus is disposable.

11. An apparatus according to claim 9 wherein a signal is generated to indicate the presence or concentration of the target ligand following the application of the assay reagents to the first member, the signal being either visual or determined by instrumental means.

12. An apparatus according to claim 11 wherein the signal is visual.

13. An apparatus according to claim 11 wherein the signal is generated within about five minutes following the application of the assay reagents to the first member.

14. An apparatus according to claim 13 wherein the signal is visual.

15. An apparatus according to claim 1 wherein the assay is a nucleic acid probe assay.

16. An apparatus according to claim 15 wherein the first receptor is a nucleic acid oligomer.

17. An apparatus according to claim 16 wherein the nucleic acid oligomer is a DNA oligomer.

18. An apparatus according to claim 1 wherein the assay is an immunoassay.

19. An apparatus according to claim 18 wherein the first receptor is an antibody.

20. An apparatus according to claim 19 wherein the antibody is a polyclonal antibody preparation.

21. An apparatus according to claim 19 wherein the antibody is a monoclonal antibody.

22. An apparatus according to claim 18 wherein the first receptor is an antigen.

23. An apparatus according to claim 20 wherein the target ligand is human choriogonadotropin.

24. An apparatus according to claim 21 wherein the target ligand is human choriogonadotropin.

25. An apparatus according to claim 20 wherein the target ligand is an antigen associated with group A or group B streptococcus.

26. An apparatus according to claim 21 wherein the target ligand is an antigen associated with group A or group B streptococcus.

27. An apparatus according to claim 18 wherein the target ligand is an antigen or antibody associated with a virus.

28. An apparatus according to claim 27 wherein the virus is a hepatitis virus.

29. An apparatus according to claim 27 wherein the virus is associated with AIDS.

30. A ligand-receptor assay process for determination of the presence or concentration of a target ligand in a liquid sample employing the apparatus of claim 1 comprising:

(a) applying the liquid sample suspected of containing the target ligand to the upper surface of the porous first member of the apparatus;

(b) applying to the upper surface a second receptor capable of binding the target ligand, the second receptor being labeled to permit its detection;

(c) separating unbound labeled second receptor from any labeled second receptor bound to the target ligand within the first member; and

(d) determining the presence or concentration of the target ligand by detection of bound labeled second receptor.

31. A ligand-receptor assay process for determination of the presence or concentration of a target ligand in a liquid sample employing the apparatus of claim 1 comprising:

(a) mixing the liquid sample suspected of containing the target ligand with a second receptor capable of binding the target ligand, the second receptor being labeled to permit its detection;

(b) applying the mixture of the liquid sample and the labeled second receptor to the upper surface of the porous first member of the apparatus;

(c) separating unbound labeled second receptor from any labeled second receptor bound to the target ligand within the first member; and

(d) determining the presence or concentration of the target ligand by detection of bound labeled second receptor.

32. A process according to claims 30 or 31 wherein the target ligand is selected from the group consisting of antigens, antibodies, enzymes, and nucleic acid oligomers.

33. A process according to claim 32 wherein the first receptor is selected from the group consisting of antibodies, antigens, enzyme receptors and nucleic acid oligomers.

34. A process according to claims 30 or 31 wherein separation of unbound second labeled receptor from the first member is accomplished by washing.

35. A process according to claim 34 wherein the second labeled receptor is labeled with an enzyme and the washing step is followed by application to the first member of a substrate for the enzyme, the enzyme and substrate being selected to generate a color change which is either visual or determined by instrumental means.

36. A process according to claim 35 wherein the color change is visual.

37. A process according to claims 30 or 31 wherein the assay is a nucleic acid probe assay.

38. A process according to claim 37 wherein the target ligand is a nucleic acid oligomer and the first and second receptors are complementary nucleic acid oligomers.

39. A process according to claim 38 wherein the first and second receptors are DNA oligomers.

40. A process according to claim 39 wherein the target ligand is a DNA oligomer.

41. A process according to claim 39 wherein the target ligand is an RNA oligomer.

42. A process according to claims 30 or 31 wherein the assay is an immunoassay.

43. A process according to claim 42 wherein the target ligand is an antigen and the first and second receptors are antibodies.

44. A process according to claim 43 wherein at least one of the antibodies is a polyclonal antibody preparation.

45. A process according to claim 43 wherein at least one of the antibodies is a monoclonal antibody.

46. A process according to claim 45 wherein the first and second antibodies are monoclonal antibodies which bind, respectively, to non-interfering epitopes of the antigen.

47. A process according to claim 42 wherein the target ligand is an antibody and at least one of the first and second receptors is an antigen.

48. A process according to claim 47 wherein the target ligand is an antibody, the first receptor is an antigen, and the second receptor is an antibody against the target antibody.

49. A process according to claim 48 wherein the antigen is an allergen and the target antibody is an IgE antibody.

50. A process according to claim 49 wherein the antibody against the IgE antibody is an anti-IgE monoclonal antibody.

51. A process according to claim 44 wherein the target ligand is human choriogonadotropin.

52. A process according to claim 45 wherein the target ligand is human choriogonadotropin.

53. A process according to claim 46 wherein the target ligand is human choriogonadotropin.

54. A process according to claim 44 wherein the target ligand is an antigen associated with group A or group B streptococcus.

55. A process according to claim 45 wherein the target ligand is an antigen associated with group A or group B streptococcus.

56. A process according to claim 46 wherein the target ligand is an antigen associated with group A or group B streptococcus.

57. A process according to claim 42 wherein the target ligand is an antigen or antibody associated with a virus.

58. A process according to claim 57 wherein the virus is a hepatitis virus.

59. A process according to claim 57 wherein the virus is associated with AIDS.

60. A process according to claim 42 wherein the liquid sample is urine, serum, plasma, or other liquid media.

132. An apparatus according to claim 11 wherein the assay is an immunoassay.

133. An apparatus according to claim 132 wherein the first receptor is an antibody and the target ligand is human choriogonadotropin.

134. An apparatus according to claim 133 wherein the antibody is a monoclonal antibody.

135. An apparatus according to claim 132 wherein the first receptor is an antibody and the target ligand is an antigen associated with Group A streptococcus.

136. An apparatus according to claim 135 wherein the antibody is a monoclonal antibody.

137. A process according to claim 35 wherein the assay is an immunoassay.

138. A process according to claim 137 wherein at least one of the first and second receptors is a monoclonal antibody.

139. A process according to claim 138 wherein the target ligand is human choriogonadotropin.

140. A process accoring to claim 138 wherein the target ligand is an antigen associated with Group A streptococcus.

61. An apparatus for use in a ligand-receptor assay process for determination of the presence or concentration of a target ligand in a liquid sample comprising:

(a) a porous first member which has upper and lower surfaces, the sample being applied to the upper surface, and wherein at least one anti-receptor capable of binding a first receptor is directly or indirectly bound within an area smaller than the area of the member to which the sample is applied, the first receptor being capable of binding the target ligand; and

(b) a second member which is a body of absorbent material having a surface over which the lower surface of the first member is placed and having capillaries therethrough which are in communication with the pores on the lower surface of the first member so as to draw liquid added to the upper surface of the first member which has permeated the first member into the capillaries of the second member, the communication between the first and second members having been established prior to, and maintained during, addition of liquids to the apparatus during the assay process.

62. An apparatus according to claim 61 wherein the target ligand is selected from the group consisting of antigens, antibodies, enzymes, and nucleic acid oligomers.

63. An apparatus according to claim 62 wherein the first receptor is selected from the group consisting of antibodies, antigens, enzyme receptors, and nucleic acid oligomers.

64. An apparatus according to claim 61 wherein the porous first member is a membrane or filter.

65. An apparatus according to claim 64 wherein the membrane or filter is of a material selected from glass or nylon.

66. An apparatus according to claim 64 wherein the first and second members are separated by at least one porous member.

67. An apparatus according to claim 64 wherein the apparatus further comprises a container for the first and second members having an opening sufficient to permit the application of assay reagents to the first member.

68. An apparatus according to claim 67 wherein the opening further comprises a section having sides which slope inwardly to define a funnel for direction of the assay reagents applied to the first member.

69. An apparatus according to claim 67 wherein the bottom end of the container is closed and the container is of a volume sufficient to contain all of the assay reagents applied to the first member during the assay, the container being vented to allow escape of displaced air.

70. An apparatus according to claim 69 wherein the apparatus is disposable.

71. An apparatus according to claim 69 wherein a signal is generated to indicate the presence or concentration of the target ligand following the application of the assay reagents to the first member, the signal being either visual or determined by instrumental means.

72. An apparatus according to claim 71 wherein the signal is visual.

73. An apparatus according to claim 71 wherein the signal is generated within about five minutes following the application of the assay reagents to the first member.

74. An apparatus according to claim 73 wherein the signal is visual.

75. An apparatus according to claim 61 wherein the assay is a nucleic acid probe assay.

76. An apparatus according to claim 75 wherein the first receptor is a nucleic acid oligomer.

77. An apparatus according to claim 76 wherein the nucleic acid oligomer is a DNA oligomer.

78. An apparatus according to claim 61 wherein the assay is an immunoassay.

79. An apparatus according to claim 78 wherein the first receptor is an antibody.

80. An apparatus according to claim 79 wherein the antibody is a polyclonal antibody preparation.

81. An apparatus according to claim 79 wherein the antibody is a monoclonal antibody.

82. An apparatus according to claim 78 wherein the first receptor is an antigen.

83. An apparatus according to claim 80 wherein the target ligand is human choriogonadotropin.

84. An apparatus according to claim 81 wherein the target ligand is human choriogonadotropin.

85. An apparatus according to claim 80 wherein the target ligand is an antigen associated with group A or group streptococcus.

86. An apparatus according to claim 81 wherein the target ligand is an antigen associated with group A or group streptococcus.

87. An apparatus according to claim 78 wherein the target ligand is an antigen or antibody associated with a virus.

88. An apparatus according to claim 87 wherein the virus is a hepatitis virus.

89. An apparatus according to claim 87 wherein the virus is associated with AIDS.

90. An apparatus according to claim 78 wherein the first receptor is an antibody and the anti-receptor is an antibody against the first receptor.

91. An apparatus according to claim 90 wherein at least one of the antibodies is a polyclonal antibody preparation.

92. An apparatus according to claim 90 wherein at least one of the antibodies is a monoclonal antibody.

93. An apparatus according to claim 92 wherein the first receptor is a monoclonal antibody.

94. An apparatus according to claim 92 wherein the anti-receptor is a monoclonal antibody.

95. An apparatus according to claim 78 wherein the first receptor is conjugated with a hapten and the anti-receptor is an antibody against the hapten.

96. An apparatus according to claim 78 wherein the hapten is fluorescein.

97. An apparatus according to claim 78 wherein the first receptor is conjugated to biotin and the anti-receptor is avidin.

98. A ligand-receptor asasy process for determination of the presence or concentration of a target ligand in a liquid sample employing the apparatus of claim 61 comprising:

(a) applying the first receptor capable of binding the target ligand and the liquid sample suspected of containing the target ligand to the upper surface of the porous first member of the apparatus;

(b) applying to the upper surface a second receptor capable of binding the target ligand, the second receptor being labeled to permit its detection;

(c) separating unbound labeled second receptor from any labeled second receptor bound to the target ligand within the first member; and

(d) determining the presence or concentration of the target ligand by detection of bound labeled second receptor.

99. A ligand-receptor assay process for determination of the presence or concentration of a target ligand in a liquid sample employing the apparatus of claim 61 comprising:

(a) applying the first receptor capable of binding the target ligand to the upper surface of the porous first member of the apparatus;

(b) mixing the liquid sample suspected of containing the target ligand with a second receptor capable of binding the target ligand, the second receptor being labeled to permit its detection;

(c) applying the mixture of the liquid sample and the labeled second receptor to the upper surface of the porous first member of the apparatus;

(d) separating unbound labeled second receptor from any labeled second receptor bound to the target ligand within the first member; and

(e) determining the presence or concentration of the target ligand by detection of bound labeled second receptor.

100. A process according to claims 98 or 99 wherein the target ligand is selected from the group consisting of antigens, antibodies, enzymes, and nucleic acid oligomers.

101. A process according to claim 100 wherein the first receptor is selected from the group consisting of antibodies, antigens, enzyme receptors and nucleic acid oligomers.

102. A process according to claims 98 or 99 wherein separation of unbound labeled receptor from the first member is accomplished by washing.

103. A process according to claim 102 wherein the second labeled receptor is labeled with an enzyme and the washing step is followed by application to the first member of a substrate for the enzyme, the enzyme and substrate being selected to generate a color change which is either visual or determined by instrumental means.

104. A process according to claim 103 wherein the color change is visual.

105. A process according to claims 98 or 99 wherein the assay is a nucleic acid probe assay.

106. A process according to claim 105 wherein the target ligand is a nucleic acid oligomer and the first and second receptors are complementary nucleic acid oligomers.

107. A process according to claim 106 wherein the first and second receptors are DNA oligomers.

108. A process according to claim 107 wherein the target ligand is a DNA oligomer.

109. A process according to claim 108 wherein the target ligand is an RNA oligomer.

110. A process according to claims 98 or 99 wherein the assay is an immunoassay.

111. A process according to claim 110 wherein the target ligand is an antigen and the first and second receptors are antibodies.

112. A process according to claim 111 wherein at least one of the antibodies is a polyclonal antibody preparation.

113. A process according to claim 111 wherein at least one of the antibodies is a monoclonal antibody.

114. An process according to claim 113 wherein the first and second antibodies are monoclonal antibodies which bind, respectively, to non-interfering epitopes of the antigen.

115. A process according to claim 110 wherein the target ligand is an antibody and at least one of the first and second receptors is an antigen.

116. A process according to claim 115 wherein the target ligand is an antibody, the first receptor is an antigen, and the second receptor is an antibody against the target antibody.

117. A process according to claim 116 wherein the antigen is an allergen and the target antibody is an IgE antibody.

118. A process according to claim 117 wherein the antibody against the IgE antibody is an anti-IgE monoclonal antibody.

119. A process according to claim 112 wherein the target ligand is human choriogonadotropin.

120. A process according to claim 113 wherein the target ligand is human choriogonadotropin.

121. A process according to claim 114 wherein the target ligand is human choriogonadotropin.

122. A process according to claim 112 wherein the target ligand is an antigen associated with group A or group B streptococcus.

123. A process according to claim 113 wherein the target ligand is an antigen associated with group A or group B streptococcus.

124. A process according to claim 114 wherein the target ligand is an antigen associated with group A or group B streptococcus.

125. A process according to claim 110 wherein the target ligand is an antigen or antibody associated with a virus.

126. A process according to claim 125 wherein the virus is a hepatitis virus.

127. A process according to claim 125 wherein the virus is associated with AIDS.

128. A process according to claim 110 wherein the liquid sample is urine, serum, plasma, or other liquid media.

129. A process according to claim 110 wherein the anti-receptor is an antibody.

130. A process according to claims 98 or 99 wherein application of the first receptor is followed by application of the liquid sample.

131. A process according to claims 98 or 99 wherein the first receptor and the liquid sample are admixed prior to application to the first member.