US4145406A
Expired / Lapsed
US
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Specific binding - adsorbent assay method and test means
Assignee: Bayer Corp
•
Filing: 1977-04-11
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Granted: 1979-03-20
•
Expires: 1997-04-11
Abstract
Abstract A method and test means for determining a specific binding substance, such as an antigen or hapten, in a liquid sample wherein a solid, nonspecific adsorbent is contacted, preferably in sequence, with the sample and a labeled form of a binding partner for the substance to be determined, such as a labeled antibody. The amount of label which becomes associated with the adsorbent by binding of the labeled binding partner to the adsorbed substance to be determined, i.e., in a sandwich fashion, is a function of the amount of the substance to be determined in the liquid sample. Preferably, the adsorbent is an ion exchange material, and the assay steps are carried out under pH conditions between the isoelectric points (pI values) of the substance to be determined and the labeled binding partner. The method and test means are particularly suited for the detection of hepatitis B surface (HB s ) antigen, especially where the label is a radioactive material and a column format is employed. The resultant radioimmunoadsorbent (RI-Ad™) assay and test means for HB s antigen offers significant manufacturing and user advantages over the prior art methods and test means.
Inventors
- 👤 Lloyd A. Schick
- 👤 Stephen K. Carpenter
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-25 03:46
CPC Classifications
G; G01; G01N; G01N33/00; G01N33/48; G01N33/50; G01N33/53; G01N33/536; G01N33/537; G01N33/538; Y; Y10; Y10S; Y10S436/00; Y10S436/804; Y10S436/82
Claims Analysis
Total Claims: 68
Claim 1
Independent Claim
173 words
A method for determining a specific binding substance in a liquid sample, comprising the steps of: (a) contacting said sample with (1) a specific binding partner of the substance to to determined, which binding partner is incorporated with a label and which labeled binding partner has a pI value different from that of said substance to be determined, and (2) a solid ion exchange adsorbent (i) of an anionic type when the pI value of said substance to be determined is less than that of said binding partner, or (ii) of a cationic type when the pI value of said substance to be determined is greater than that of said labeled binding partner; and (b) determining the amount of the label which either (i) becomes associated with the adsorbent by binding of the labeled binding partner with adsorbed substance to be determined or (ii) remains unassociated therefrom, wherein step (a) is performed under conditions of pH between the pI value of the substance to be determined and that of said labeled binding partner.
Claim 2
Independent Claim
173 words
A method for determining a specific binding substance in a liquid sample, comprising the steps of: (a) contacting said sample with (1) a specific binding partner of the substance to to determined, which binding partner is incorporated with a label and which labeled binding partner has a pI value different from that of said substance to be determined, and (2) a solid ion exchange adsorbent (i) of an anionic type when the pI value of said substance to be determined is less than that of said binding partner, or (ii) of a cationic type when the pI value of said substance to be determined is greater than that of said labeled binding partner; and (b) determining the amount of the label which either (i) becomes associated with the adsorbent by binding of the labeled binding partner with adsorbed substance to be determined or (ii) remains unassociated therefrom, wherein step (a) is performed under conditions of pH between the pI value of the substance to be determined and that of said labeled binding partner.
Claim 3
Dependent Claim
30 words
A method as in claim 1 wherein step (a) is accomplished by (i) contacting the sample with the adsorbent and (ii) thereafter contacting the adsorbent with the labeled binding partner.
Claim 4
Dependent Claim
21 words
A method as in claim 1 wherein said adsorbent is contacted with the sample and the labeled binding partner substantially simultaneously.
Claim 5
Dependent Claim
51 words
A method as in claim 1 wherein step (b) is accomplished by (i) separating from the adsorbent substantially all of the labeled binding partner not associated therewith by binding with adsorbed substance to be determined, and (ii) measuring the amount of the label either associated with the adsorbent or separated therefrom.
Claim 6
Dependent Claim
36 words
A method as in claim 4 wherein said separation step is accomplished by washing the adsorbent whereby the amount of the label either associated with the washed adsorbent or contained in the washings can be measured.
Claim 7
Dependent Claim
13 words
A method as in claim 1 wherein said label is a radioactive material.
Claim 8
Dependent Claim
27 words
A method as in claim 1 wherein said substance to be determined is an antigen or a hapten and said specific binding partner thereof is an antibody.
Claim 9
Dependent Claim
31 words
A method as in claim 1 wherein said substance to be determined is hepatitis B surface antigen and said specific binding partner thereof is an antibody to hepatitis B surface antigen.
Claim 10
Independent Claim
187 words
A method for determining a specific binding substance in a liquid sample, said substance to be determined having a pI value different from that of a specific binding partner thereof incorporated with a label, said method comprising the steps of: (a) contacting said liquid sample with a solid ion exchange material (i) of an anionic type when the pI value of said substance to be determined is less than that of said labeled binding partner, or (ii) of a cationic type when the pI value of said substance to be determined is greater than that of said labeled binding partner; (b) separating the ion exchange material and the unadsorbed portion of the liquid sample; (c) contacting the ion exchange material with said labeled binding partner; and (d) determining the amount of the label which either (i) becomes associated with the ion exchange material by binding of the labeled binding partner with adsorbed substance to be determined or (ii) remains unassociated therefrom; wherein steps (a)-(c) are performed under conditions of pH between the pI value of said substance to be determined and that of said labeled binding partner.
Claim 11
Independent Claim
187 words
A method for determining a specific binding substance in a liquid sample, said substance to be determined having a pI value different from that of a specific binding partner thereof incorporated with a label, said method comprising the steps of: (a) contacting said liquid sample with a solid ion exchange material (i) of an anionic type when the pI value of said substance to be determined is less than that of said labeled binding partner, or (ii) of a cationic type when the pI value of said substance to be determined is greater than that of said labeled binding partner; (b) separating the ion exchange material and the unadsorbed portion of the liquid sample; (c) contacting the ion exchange material with said labeled binding partner; and (d) determining the amount of the label which either (i) becomes associated with the ion exchange material by binding of the labeled binding partner with adsorbed substance to be determined or (ii) remains unassociated therefrom; wherein steps (a)-(c) are performed under conditions of pH between the pI value of said substance to be determined and that of said labeled binding partner.
Claim 12
Dependent Claim
40 words
A method as in claim 9 wherein step (b) is accomplished by washing the ion exchange material with a liquid having a pH between the pI value of the substance to be determined and that of the labeled binding partner.
Claim 13
Dependent Claim
53 words
A method as in claim 9 wherein step (d) is accomplished by (i) separating from the ion exchange material substantially all of the labeled binding partner not associated therewith by binding with adsorbed substance to be determined, and (ii) measuring the amount of the label either associated with the adsorbent or separated therefrom.
Claim 14
Dependent Claim
63 words
A method as in claim 11 wherein said separation step is accomplished by washing the ion exchange material with a liquid having a pH between the pI value of the substance to be determined and that of the labeled binding partner whereby the amount of the label either associated with the washed ion exchange material or contained in the washings can be measured.
Claim 15
Dependent Claim
13 words
A method as in claim 12 wherein said label is a radioactive material.
Claim 16
Dependent Claim
25 words
A method as in claim 13 wherein step (d) is accomplished by measuring the radioactivity of the washed ion exchange material or of the washings.
Claim 17
Dependent Claim
30 words
A method as in claim 9 wherein the difference between the pI value of the substance to be determined and that of the labeled binding partner is greater than 0.5.
Claim 18
Dependent Claim
30 words
A method as in claim 9 wherein the difference between the pI value of the substance to be determined and that of the labeled binding partner is greater than 2.0.
Claim 19
Dependent Claim
27 words
A method as in claim 9 wherein said substance to be determined is an antigen or a hapten and said specific binding partner thereof is an antibody.
Claim 20
Dependent Claim
17 words
A method as in claim 17 wherein said substance to be determined is hepatitis B surface antigen.
Claim 21
Dependent Claim
15 words
A method as in claim 9 wherein said substance to be determined is an antibody.
Claim 22
Dependent Claim
20 words
A method as in claim 19 wherein said substance to be determined is an antibody to hepatitis B surface antigen.
Claim 23
Dependent Claim
113 words
A method as in claim 9 wherein steps (a)-(d) are accomplished by: (a) introducing said sample to a column of said ion-exchange material in particulate form; (b) washing said ion exchange material in the column to remove substantially all of said substance to be determined not adsorbed to the ion exchange material; (c) introducing to said column a solution of said labeled binding partner; and (d) washing said ion exchange material in the column to remove substantially all of said labeled binding partner not associated with the ion exchange material by binding to adsorbed substance to be determined and measuring the label in the column or in the eluate from the last wash.
Claim 24
Independent Claim
138 words
A method for detecting hepatitis B surface antigen in a liquid sample, comprising the steps of: (a) contacting said sample with a solid adsorbent of a type capable of nonspecifically binding with hepatitis B surface antigen; (b) contacting the adsorbent with an antibody to hepatitis B surface antigen, which antibody is incorporated with a label; and (c) determining the amount of the label which becomes associated with the adsorbent by binding of the labeled antibody with adsorbed hepatitis B surface antigen, wherein steps (a) and (b) are performed under conditions favorable to binding between hepatitis B surface antigen and the adsorbent, and either the adsorbent is of a type incapable of significantly binding with the labeled antibody under any conditions or step (b) is performed under conditions unfavorable to significant binding between the labeled antibody and the adsorbent.
Claim 25
Independent Claim
138 words
A method for detecting hepatitis B surface antigen in a liquid sample, comprising the steps of: (a) contacting said sample with a solid adsorbent of a type capable of nonspecifically binding with hepatitis B surface antigen; (b) contacting the adsorbent with an antibody to hepatitis B surface antigen, which antibody is incorporated with a label; and (c) determining the amount of the label which becomes associated with the adsorbent by binding of the labeled antibody with adsorbed hepatitis B surface antigen, wherein steps (a) and (b) are performed under conditions favorable to binding between hepatitis B surface antigen and the adsorbent, and either the adsorbent is of a type incapable of significantly binding with the labeled antibody under any conditions or step (b) is performed under conditions unfavorable to significant binding between the labeled antibody and the adsorbent.
Claim 26
Dependent Claim
13 words
A method as in claim 22 wherein said adsorbent is a hydrophobic material.
Claim 27
Dependent Claim
25 words
A method as in claim 23 wherein said hydrophobic adsorbent is an ω-amino alkyl-substituted agarose having 4 to 10 carbon atoms in the alkyl group.
Claim 28
Dependent Claim
41 words
A method as in claim 22 wherein said adsorbent is an ion exchange material and wherein steps (a) and (b) are performed under conditions of pH between the pI value of hepatitis B surface antigen and that of the labeled antibody.
Claim 29
Dependent Claim
33 words
A method as in claim 25 wherein said labeled antibody has a pI value greater than that of hepatitis B surface antigen and wherein said ion exchange material is of an anionic type.
Claim 30
Dependent Claim
16 words
A method as in claim 26 wherein said anionic type exchange material is a diethylaminoethyl-substituted polymer.
Claim 31
Dependent Claim
12 words
A method as in claim 27 wherein the polymer is a polysaccharide.
Claim 32
Dependent Claim
16 words
A method as in claim 26 wherein said anionic type exchange material is a diethylaminoethyl-substituted cellulose.
Claim 33
Dependent Claim
35 words
A method as in claim 26 wherein the pI value of the labeled antibody is greater than 8.0 and wherein steps (a) and (b) are performed under conditions of pH between about 6.0 and 8.0.
Claim 34
Dependent Claim
33 words
A method as in claim 25 wherein said labeled antibody has a pI value less than that of hepatitis B surface antigen and wherein said ion exchange material is of a cationic type.
Claim 35
Dependent Claim
28 words
A method as in claim 22 which comprises the additional step between steps (a) and (b) of separating said adsorbent and the unadsorbed portion of the liquid sample.
Claim 36
Dependent Claim
29 words
A method as in claim 32 wherein said separation step is accomplished by washing the adsorbent under conditions favorable to binding between hepatitis B surface antigen and the adsorbent.
Claim 37
Dependent Claim
52 words
A method as in claim 22 wherein step (c) is accomplished by (i) separating from the adsorbent substantially all of the labeled antibody not associated with the adsorbent by binding with adsorbed hepatitis B surface antigen, and (ii) measuring the amount of the label either associated with the adsorbent or separated therefrom.
Claim 38
Dependent Claim
36 words
A method as in claim 34 wherein said separation step is accomplished by washing the adsorbent, whereby the amount of the label either associated with the washed adsorbent or contained in the washings can be measured.
Claim 39
Dependent Claim
13 words
A method as in claim 35 wherein said label is a radioactive material.
Claim 40
Independent Claim
127 words
A method for detecting hepatitis B surface antigen in a sample of serum or plasma, comprising the steps of: (a) contacting said sample with a solid anion exchange material under conditions of pH between 5 and 8; (b) washing the anion exchange material with a liquid having a pH of between 5 and 8; (c) contacting the anion exchange material under conditions of pH between 5 and 8 with an antibody to hepatitis B surface antigen, which antibody is incorporated with a label and has a pI value greater than about 8; and (d) determining the amount of the label which either (i) becomes associated with the anion exchange material by binding of the labeled antibody with adsorbed hepatitis B surface antigen or (ii) remains unassociated therefrom.
Claim 41
Independent Claim
127 words
A method for detecting hepatitis B surface antigen in a sample of serum or plasma, comprising the steps of: (a) contacting said sample with a solid anion exchange material under conditions of pH between 5 and 8; (b) washing the anion exchange material with a liquid having a pH of between 5 and 8; (c) contacting the anion exchange material under conditions of pH between 5 and 8 with an antibody to hepatitis B surface antigen, which antibody is incorporated with a label and has a pI value greater than about 8; and (d) determining the amount of the label which either (i) becomes associated with the anion exchange material by binding of the labeled antibody with adsorbed hepatitis B surface antigen or (ii) remains unassociated therefrom.
Claim 42
Dependent Claim
45 words
A method as in claim 37 wherein step (d) is accomplished by washing the anion exchange material with a liquid having a pH between 5 and 8 and measuring the amount of the label either associated with the washed adsorbent or contained in the washings.
Claim 43
Dependent Claim
13 words
A method as in claim 38 wherein said label is a radioactive material.
Claim 44
Dependent Claim
12 words
A method as in claim 39 wherein said label is 125 I.
Claim 45
Independent Claim
225 words
A method for detecting hepatitis B surface antigen in a sample of serum or plasma, comprising the steps of: (a) introducing said sample to a column of an anion exchange material in particulate form equilibrated in a liquid having a pH of between 5 and 8; (b) passing through the column a liquid having a pH of between 5 and 8 to remove substantially all hepatitis B surface antigen not adsorbed to the anion exchange material; (c) introducing to the anion exchange material a predetermined volume of a solution of radiolabeled antibody to hepatitis B surface antigen having a predetermined level of radioactivity; (d) incubating the solution of radiolabeled antibody in contact with the anion exchange material for between 1/2 and 18 hours at a temperature of between 4° and 50° C.; (e) passing through the column a liquid having a pH of between 5 and 8 to remove substantially all radiolabeled antibody not associated with the anion exchange material by binding to adsorbed hepatitis B surface antigen; (f) measuring the radioactivity of the anion exchange material or of the eluate from the next previous step; and (g) comparing the radioactivity measured in the next previous step to that measured following the same procedure using a liquid sample containing no significant amount of hepatitis B surface antigen in place of said serum or plasma sample.
Claim 46
Independent Claim
225 words
A method for detecting hepatitis B surface antigen in a sample of serum or plasma, comprising the steps of: (a) introducing said sample to a column of an anion exchange material in particulate form equilibrated in a liquid having a pH of between 5 and 8; (b) passing through the column a liquid having a pH of between 5 and 8 to remove substantially all hepatitis B surface antigen not adsorbed to the anion exchange material; (c) introducing to the anion exchange material a predetermined volume of a solution of radiolabeled antibody to hepatitis B surface antigen having a predetermined level of radioactivity; (d) incubating the solution of radiolabeled antibody in contact with the anion exchange material for between 1/2 and 18 hours at a temperature of between 4° and 50° C.; (e) passing through the column a liquid having a pH of between 5 and 8 to remove substantially all radiolabeled antibody not associated with the anion exchange material by binding to adsorbed hepatitis B surface antigen; (f) measuring the radioactivity of the anion exchange material or of the eluate from the next previous step; and (g) comparing the radioactivity measured in the next previous step to that measured following the same procedure using a liquid sample containing no significant amount of hepatitis B surface antigen in place of said serum or plasma sample.
Claim 47
Dependent Claim
15 words
A method as in claim 41 wherein said anion exchange material is a diethylaminoethyl-substituted polymer.
Claim 48
Dependent Claim
12 words
A method as in claim 42 wherein the polymer is a polysaccharide.
Claim 49
Dependent Claim
15 words
A method as in claim 41 wherein said anion exchange material is a diethylaminoethyl-substituted cellulose.
Claim 50
Dependent Claim
20 words
A method as in claim 41 wherein said liquids referred to in steps (a), (b), and (e) are pH buffers.
Claim 51
Dependent Claim
18 words
A method as in claim 45 wherein said pH buffers have a pH of between 6.5 and 7.5.
Claim 52
Dependent Claim
13 words
A method as in claim 46 wherein said pH buffers are phosphate buffers.
Claim 53
Dependent Claim
14 words
A method as in claim 41 wherein said antibody is radiolabeled with 125 I.
Claim 54
Independent Claim
75 words
A method for detecting hepatitis B surface antigen in the form of its immune complex with antibody in a liquid sample, comprising the steps of: (a) combining said sample, an immunochemical-bond weakening agent, and a solid adsorbent of a type capable of and under conditions favorable to nonspecific binding with hepatitis B surface antigen; (b) separating said bond weakening agent from the adsorbent; and (c) determining the hepatitis B surface antigen bound to the adsorbent.
Claim 55
Independent Claim
75 words
A method for detecting hepatitis B surface antigen in the form of its immune complex with antibody in a liquid sample, comprising the steps of: (a) combining said sample, an immunochemical-bond weakening agent, and a solid adsorbent of a type capable of and under conditions favorable to nonspecific binding with hepatitis B surface antigen; (b) separating said bond weakening agent from the adsorbent; and (c) determining the hepatitis B surface antigen bound to the adsorbent.
Claim 56
Dependent Claim
14 words
A method as in claim 49 wherein said adsorbent is an anion exchange material.
Claim 57
Dependent Claim
15 words
A method as in claim 50 wherein said bond weakening agent is a chaotropic agent.
Claim 58
Dependent Claim
15 words
A method as in claim 5 wherein said chaotropic agent is urea, guanidine, or thiocyanate.
Claim 59
Dependent Claim
12 words
A method as in claim 51 wherein said chaotropic agent is urea.
Claim 60
Dependent Claim
13 words
A method as in claim 53 wherein said anion exchange material is DEAE-cellulose.
Claim 61
Dependent Claim
19 words
A method as in claim 54 wherein urea is present at a concentration of between 2 and 8 molar.
Claim 62
Dependent Claim
48 words
A method as in claim 49 wherein step (c) is accomplished by contacting the adsorbent with antibody to hepatitis B surface antigen, which antibody is incorporated with a label, and determining the amount of label which either (i) becomes associated with the adsorbent or (ii) remains unadsorbed therefrom.
Claim 63
Dependent Claim
13 words
A method as in claim 56 wherein said label is a radioactive material.
Claim 64
Independent Claim
121 words
A method for detecting an antibody to hepatitis B surface antigen in a liquid sample, comprising the steps of: (a) providing a solid adsorbent having nonspecifically bound thereto hepatitis B surface antigen; (b) contacting the adsorbent with said sample and with an antibody to hepatitis B antigen, which antibody is incorporated with a label; and (c) determining the amount of the label which either (i) becomes associated with the adsorbent by binding of the labeled antibody to adsorbed hepatitis B surface antigen or (ii) remains unadsorbed therefrom, wherein either the adsorbent is of a type incapable of significantly binding with the labeled antibody or step (b) is performed under conditions unfavorable to significant binding between the labeled antibody and the adsorbent.
Claim 65
Independent Claim
121 words
A method for detecting an antibody to hepatitis B surface antigen in a liquid sample, comprising the steps of: (a) providing a solid adsorbent having nonspecifically bound thereto hepatitis B surface antigen; (b) contacting the adsorbent with said sample and with an antibody to hepatitis B antigen, which antibody is incorporated with a label; and (c) determining the amount of the label which either (i) becomes associated with the adsorbent by binding of the labeled antibody to adsorbed hepatitis B surface antigen or (ii) remains unadsorbed therefrom, wherein either the adsorbent is of a type incapable of significantly binding with the labeled antibody or step (b) is performed under conditions unfavorable to significant binding between the labeled antibody and the adsorbent.
Claim 66
Dependent Claim
14 words
A method as in claim 58 wherein said adsorbent is an anion exchange material.
Claim 67
Dependent Claim
57 words
A method as in claim 58 wherein step (c) is accomplished by (i) washing the adsorbent to separate therefrom substantially all of the labeled antibody not associated with the adsorbent by binding with adsorbed hepatitis B surface antigen, and (ii) measuring the amount of the label either associated with the washed adsorbent or contained in the washings.
Claim 68
Dependent Claim
13 words
A method as in claim 60 wherein said label is a radioactive material.
References Cited (Backward Citations)
US4041146A
Cited
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1982-03-19 | 1982-09-18 | 1983-03-20 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1986-03-19 | 1986-09-18 | 1987-03-20 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1990-03-19 | 1990-09-18 | 1991-03-20 | $7700.00 | Window Closed / Lapsed |