Specific binding - adsorbent assay method and test means

• US 4,145,406 A
• Filed: 04/11/1977
• Issued: 03/20/1979
• Est. Priority Date: 04/11/1977
• Status: Expired due to Term

• Current Assignee: Miles Laboratories, Inc. (Bayer AG)
• Original Assignee: Miles Laboratories, Inc. (Bayer AG)
• Inventors: Schick, Lloyd A., Carpenter, Stephen K.
• Primary Examiner(s): Padgett, Benjamin R.
• Assistant Examiner(s): Nucker, Christine M.
• Application Number:
  US05/786,207
• Time in Patent Office: 708 Days
• Field of Search: 424/1, 424/12, 23/230 B
• US Class Current: 436/541
• CPC Class Codes: G01N 33/538 by sorbent column, particles or resin strip , i.e. sorbent materials
• CPC Class Codes: Y10S 436/804 Radioisotope, e.g. radioimmunoassay
• CPC Class Codes: Y10S 436/82 Hepatitis associated antigens and antibodies

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.

Claims

1. 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.

2. 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.

3. A method as in claim 1 wherein said adsorbent is contacted with the sample and the labeled binding partner substantially simultaneously.

4. 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.

5. 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.

6. A method as in claim 1 wherein said label is a radioactive material.

7. 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.

8. 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.

52. A method as in claim 5 wherein said chaotropic agent is urea, guanidine, or thiocyanate.

9. 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.

10. 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.

11. 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.

12. 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.

13. A method as in claim 12 wherein said label is a radioactive material.

14. 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.

15. 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.

16. 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.

17. 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.

18. A method as in claim 17 wherein said substance to be determined is hepatitis B surface antigen.

19. A method as in claim 9 wherein said substance to be determined is an antibody.

20. A method as in claim 19 wherein said substance to be determined is an antibody to hepatitis B surface antigen.

21. 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.

22. 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.

23. A method as in claim 22 wherein said adsorbent is a hydrophobic material.

24. 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.

25. 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.

26. 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.

27. A method as in claim 26 wherein said anionic type exchange material is a diethylaminoethyl-substituted polymer.

28. A method as in claim 27 wherein the polymer is a polysaccharide.

29. A method as in claim 26 wherein said anionic type exchange material is a diethylaminoethyl-substituted cellulose.

30. 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.

31. 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.

32. 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.

33. 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.

34. 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.

35. 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.

36. A method as in claim 35 wherein said label is a radioactive material.

37. 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.

38. 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.

39. A method as in claim 38 wherein said label is a radioactive material.

40. A method as in claim 39 wherein said label is ¹²⁵ I.

41. 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.

42. A method as in claim 41 wherein said anion exchange material is a diethylaminoethyl-substituted polymer.

43. A method as in claim 42 wherein the polymer is a polysaccharide.

44. A method as in claim 41 wherein said anion exchange material is a diethylaminoethyl-substituted cellulose.

45. A method as in claim 41 wherein said liquids referred to in steps (a), (b), and (e) are pH buffers.

46. A method as in claim 45 wherein said pH buffers have a pH of between 6.5 and 7.5.

47. A method as in claim 46 wherein said pH buffers are phosphate buffers.

48. A method as in claim 41 wherein said antibody is radiolabeled with ¹²⁵ I.

49. 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.

50. A method as in claim 49 wherein said adsorbent is an anion exchange material.

51. A method as in claim 50 wherein said bond weakening agent is a chaotropic agent.

53. A method as in claim 51 wherein said chaotropic agent is urea.

54. A method as in claim 53 wherein said anion exchange material is DEAE-cellulose.

55. A method as in claim 54 wherein urea is present at a concentration of between 2 and 8 molar.

56. 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.

57. A method as in claim 56 wherein said label is a radioactive material.

58. 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.

59. A method as in claim 58 wherein said adsorbent is an anion exchange material.

60. 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.

61. A method as in claim 60 wherein said label is a radioactive material.