ACETAMINOPHEN-PROTEIN ADDUCT ASSAY DEVICE AND METHOD

• US 20090263839A1
• Filed: 04/21/2009
• Published: 10/22/2009
• Est. Priority Date: 04/21/2008
• Status: Abandoned Application

• Current Assignee: Arkansas Children's Hospital Research Institute Inc., The Board of Trustees of the University of Arkansas (University of Arkansas System)
• Original Assignee: Arkansas Children's Hospital Research Institute Inc., The Board of Trustees of the University of Arkansas (University of Arkansas System)
• Inventors: James, Laura, Hinson, Jack A., Roberts, Dean
• Application Number:
  US12/427,434
• Publication Number:
  US 20090263839A1
• Time in Patent Office: Days
• US Class Current: 435/7.930
• CPC Class Codes: G01N 33/54386 Analytical elements
• CPC Class Codes: G01N 33/54388 based on lateral flow

Abstract

The present invention describes devices and methods for detecting and measuring the amount of acetaminophen-protein adducts in a sample.

Claims

1. A device for use in conducting a competitive assay, wherein the device detects an amount of APAP-protein adduct in a sample, the device comprising:

a. an amount of anti-APAP antibody coupled to an indicator; and

,

b. an amount of synthetic APAP-protein adduct.

2. The device of claim 1, wherein the synthetic APAP-protein adduct comprises APAP conjugated to a protein, wherein the protein comprises at least one cysteine residue.

3. The device of claim 2, wherein the protein is selected from the group consisting of BSA, ovalbumin, and lactalbumin.

4. The device of claim 1, wherein an antigenic determinant recognized by the anti-APAP antibody is selected from the group consisting of a 3-(cysteine-S-yl) linkage of the APAP-protein adduct, an exposed portion of an APAP molecule in the APAP-protein adduct, and an APAP hapten.

5. The device of claim 1, wherein the device further comprises a substrate, comprising a porous material selected from the group consisting of nitrocellulose, cellulose, paper, glass fiber mesh, silica gel, synthetic resins, and combinations thereof.

6. The device of claim 5, wherein the synthetic APAP-protein adduct is immobilized on the substrate within a test zone.

7. The device of claim 6, wherein inhibition of binding of the synthetic APAP-protein adduct to the anti-APAP antibody coupled to an indicator indicates the amount of APAP-protein adduct in the sample.

8. A device for use in conducting a competitive assay, wherein the device detects an amount of APAP-protein adduct in a sample, the device comprising:

a. an amount of synthetic APAP-protein adduct coupled to an indicator; and

,

b. an amount of anti-APAP antibody.

9. The device of claim 8, wherein the synthetic APAP-protein adduct comprises APAP conjugated to a protein, wherein the protein comprises at least one cysteine residue.

10. The device of claim 9, wherein the protein is selected from the group consisting of BSA, ovalbumin, and lactalbumin.

11. The device of claim 8, wherein an antigenic determinant recognized by the anti-APAP antibody is selected from the group consisting of a 3-(cysteine-S-yl) linkage of the APAP-protein adduct, an exposed portion of the APAP in the APAP-protein adduct, and an APAP hapten.

12. The device of claim 8, wherein the device further comprises a substrate comprising a porous material selected from the group consisting of nitrocellulose, cellulose, paper, glass fiber mesh, silica gel, synthetic resins, and combinations thereof.

13. The device of claim 12, wherein an amount of the anti-APAP antibody is immobilized on the substrate within a test zone.

14. The device of claim 13, wherein an inhibition of the binding of the anti-APAP antibody with the synthetic APAP-protein adduct coupled to an indicator indicates the amount of APAP-protein adduct in the sample.

15. A device for use in conducting a sandwich assay, wherein the device detects an amount of APAP-protein adduct in a sample, the device comprising:

a. an amount of a first anti-APAP antibody coupled to an indicator, wherein an antigenic determinant recognized by the first anti-APAP antibody is selected from the group consisting of a 3-(cysteine-S-yl) linkage of the APAP-protein adduct, an exposed portion of the APAP in the APAP-protein adduct, and an APAP hapten; and

,

b. an amount of a second anti-APAP antibody, wherein a second antigenic determinant recognized by the second anti-APAP antibody is selected from the group consisting of a 3-(cysteine-S-yl) linkage of the APAP-protein adduct, an exposed portion of the APAP in the APAP-protein adduct, and an APAP hapten.

16. The device of claim 15, wherein the device further comprises a substrate comprising a porous material selected from the group consisting of nitrocellulose, cellulose, paper, glass fiber mesh, silica gel, synthetic resins, and combinations thereof.

17. The device of claim 16, wherein the amount of the second anti-APAP antibody is immobilized on the material of the substrate within a test zone.

18. The device of claim 17, wherein an amount of the second anti-APAP antibody that is bound to the APAP-protein adduct, wherein the APAP-protein adduct is bound to the first anti-APAP antibody coupled to an indicator indicates the amount of APAP-protein adducts in the sample.

19. A device for use in conducting a sandwich assay, wherein the device detects an amount of APAP-protein adduct in a sample, the device comprising:

a. an amount of a first anti-APAP antibody coupled to an indicator, wherein the antigenic determinant recognized by the first anti-APAP antibody is selected from the group consisting of a 3-(cysteine-S-yl) linkage of the APAP-protein adduct, an exposed portion of the APAP in the APAP-protein adduct, and an APAP hapten; and

,

b. an amount of a second anti-APAP antibody, wherein the antigenic determinant recognized by the second anti-APAP antibody is an antigenic determinant of a protein selected from the group consisting of a protein forming an APAP-protein adduct and a protein forming an APAP-protein complex.

20. The device of claim 19, wherein the device further comprises a substrate comprising a porous material selected from the group consisting of nitrocellulose, cellulose, paper, glass fiber mesh, silica gel, synthetic resins, and combinations thereof.

21. The device of claim 20, wherein the amount of the second anti-APAP antibody is immobilized on the material of the substrate within a test zone.

22. The device of claim 21, wherein the amount of the second anti-APAP antibody that is bound to the APAP-protein adduct, wherein the APAP-protein adduct is bound to the first anti-APAP antibody coupled to an indicator, indicates the amount of APAP-protein adducts in the sample.

23. A device for detecting an APAP-protein adduct comprising an anti-APAP antibody.

24. A dipstick device for detecting and quantifying an APAP-protein adduct in a sample, the device comprising:

a. an amount of a synthetic APAP-protein adduct coupled to a nanoparticulate gold indicator, wherein the synthetic APAP-protein adduct is diffusively attached at a sample contact end of a substrate; and

,

b. an amount of an anti-APAP antibody, wherein the anti-APAP antibody is adhered to the substrate in a test zone, and wherein an antigenic determinant recognized by the anti-APAP antibody is APAP bound to a protein, wherein the protein comprises at least one cysteine residue.

25. The device of claim 24, wherein the synthetic APAP-protein adduct is selected from the group consisting of APAP bound to BSA, APAP bound to ovalbumin, APAP bound to lactalbumin and combinations thereof.

26. The device of claim 24, wherein the antigenic determinant is selected from the group consisting of a 3-(cysteine-S-yl) linkage of the APAP-protein adduct, an exposed portion of the APAP in the APAP-protein adduct, and an APAP hapten.

27. The device of claim 24, wherein inhibition of binding of the anti-APAP antibody to the synthetic APAP-protein adduct coupled to a nanoparticulate gold indicator indicates the presence of APAP-protein adducts in the sample.

28. A dipstick device for detecting and quantifying an APAP-protein adduct in a sample, the device comprising:

a. an amount of a synthetic APAP-protein adduct , wherein the synthetic APAP-protein adduct is adhered to a substrate in a test zone, and wherein the synthetic APAP-protein adduct is selected from the group consisting of APAP bound to BSA, APAP bound to ovalbumin, APAP bound to lactalbumin and combinations thereof; and

,

b. an amount of an anti-APAP antibody coupled to a nanoparticulate gold indicator, wherein the anti-APAP antibody is diffusively attached at a sample contact end of the substrate, and wherein an antigenic determinant recognized by the anti-APAP antibody is APAP bound to a protein comprising at least one cysteine residue.

29. The device of claim 28, wherein the antigenic determinant is selected from the group consisting of a 3-(cysteine-S-yl) linkage of the APAP-protein adduct, an exposed portion of the APAP in the APAP-protein adduct, and an APAP hapten.

30. The device of claim 28, wherein inhibition of binding of the synthetic APAP-protein adduct to the anti-APAP antibody coupled to a nanoparticulate gold indicator indicates the presence of APAP-protein adducts in the sample.

31. A dipstick device for detecting and quantifying an APAP-protein adduct in a sample, the device comprising:

a. an amount of a first anti-APAP antibody coupled to a nanoparticulate gold indicator, wherein the first anti-APAP antibody is diffusively attached at a sample contact end of a substrate, and wherein an antigenic determinant recognized by the first anti-APAP antibody is selected from the group consisting of a 3-(cysteine-S-yl) linkage of the APAP-protein adduct, an exposed portion of the APAP in the APAP-protein adduct, and an APAP hapten; and

,

b. an amount of a second anti-APAP antibody adhered to the substrate in a test zone, wherein an antigenic determinant of the second anti-APAP antibody is selected from the group consisting of a 3-(cysteine-S-yl) linkage of the APAP-protein adduct, an exposed portion of the APAP in the APAP-protein adduct, and an APAP hapten.

32. The device of claim 31, wherein an amount of the APAP-protein adduct that is bound to both the first anti-APAP antibody and to the second anti-APAP antibody indicates the amount of APAP-protein adducts in the sample.

33. A dipstick device for detecting and quantifying an APAP-protein adduct in a sample, the device comprising:

a. an amount of a first anti-APAP antibody coupled to a nanoparticulate gold indicator, wherein the first anti-APAP antibody is diffusively attached to a sample contact end of a substrate, and wherein an antigenic determinant recognized by the first anti-APAP antibody is selected from the group consisting of a 3-(cysteine-S-yl) linkage of the APAP-protein adduct, an exposed portion of the APAP in the APAP-protein adduct, and an APAP hapten; and

,

b. an amount of a second anti-APAP antibody adhered to the substrate in a test zone, wherein the antigenic determinant recognized by the second anti-APAP antibody is a segment of a protein selected from the group consisting of a protein forming an APAP-protein adduct and a protein forming an APAP-protein complex.

34. The device of claim 33, wherein an amount of the APAP-protein adduct that is bound to both the first anti-APAP antibody and to the second anti-APAP antibody indicates the amount of APAP-protein adducts in the sample.

35. A method of determining an amount of APAP-protein adduct in a sample, comprising:

a. contacting an amount of the sample with a substrate comprising an anti-APAP antibody coupled to an indicator and further comprising a synthetic APAP-protein adduct; and

,

b. determining the amount of APAP-protein adduct in the sample by measuring an indicator change caused by inhibition of binding of the synthetic APAP-protein adduct to the anti-APAP antibody coupled to the indicator.

36. The method of claim 35, wherein the amount of APAP-protein adduct detectable in the sample ranges between about 0.1 nmol/ml of serum and about 100 nmol/ml of serum.

37. The method of claim 36, wherein the amount of APAP-protein adduct detectable in the sample ranges between about 1 nmol/ml of serum and about 40 nmol/ml of serum.

38. The method of claim 35, wherein the sample is selected from the group consisting of blood, urine, saliva, tears, breast milk, lymph, blood plasma, blood serum, bile fluid, cerebrospinal fluid, supernate from cell cultures, tissue extracts, and combinations thereof.

39. The method of claim 35, wherein the sample is conditioned prior to contact with the substrate using methods selected from the group consisting of centrifugation, protein precipitation, fast gel filtration with a molecular weight cutoff of about 5 kDa, and combinations thereof.

40. The method of claim 35, wherein the anti-APAP antibody coupled to an indicator is transported to the synthetic APAP-protein adduct by wicking through the substrate.

41. The method of claim 35, wherein the indicator change is measured using an instrument selected from the group consisting of densitometer, fluorometer, quantitative voltammetry device, and quantitative coulometry device.

42. A method of determining an amount of APAP-protein adduct in a sample, comprising:

a. contacting an amount of the sample with a substrate comprising an amount of a synthetic APAP-protein adduct coupled to an indicator and further comprising an anti-APAP antibody; and

,

b. determining an amount of APAP-protein adduct in a sample by measuring an indicator change caused by inhibition of binding of the anti-APAP antibody to the synthetic APAP-protein adduct coupled to an indicator.

43. The method of claim 42, wherein the amount of APAP-protein adduct detectable in the sample ranges between about 0.1 nmol/ml of serum and about 100 nmol/ml of serum.

44. The method of claim 43, wherein the amount of APAP-protein adduct detectable in the sample ranges between about 1 nmol/ml of serum and about 40 nmol/ml of serum.

45. The method of claim 42, wherein the sample is selected from the group consisting of blood, urine, saliva, tears, breast milk, lymph, blood plasma, blood serum, bile fluid, cerebrospinal fluid, supernate from cell cultures, tissue extracts, and combinations thereof.

46. The method of claim 42, wherein the sample is conditioned prior to contact with the conjugated antibody using methods selected from the group consisting of centrifugation, protein precipitation, fast gel filtration with a molecular weight cutoff of about 5 kDa, and combinations thereof.

47. The method of claim 42, wherein the synthetic APAP-protein adduct is transported to the anti-APAP antibody by wicking through the substrate.

48. The method of claim 42, wherein the indicator change is measured using an instrument selected from the group consisting of densitometer, fluorometer, quantitative voltammetry device, and quantitative coulometry device.

49. A method of determining an amount of an APAP-protein adduct in a sample, comprising:

a. contacting an amount of the sample with a substrate comprising an amount of a first anti-APAP antibody conjugated with an indicator and further comprising a second anti-APAP antibody; and

,

b. determining the amount of APAP-protein adduct in the sample by measuring indicator changes caused by the binding of the APAP-protein adduct to the first anti-APAP antibody and to the second anti-APAP antibody.

50. The method of claim 49, wherein the amount of APAP-protein adduct detectable in the sample ranges between about 0.1 nmol/ml of serum and about 100 nmol/ml of serum.

51. The method of claim 50, wherein the amount of APAP-protein adduct detectable in the sample ranges between about 1 nmol/ml of serum and about 40 nmol/ml of serum.

52. The method of claim 49, wherein the sample is selected from the group consisting of blood, urine, saliva, tears, breast milk, lymph, blood plasma, blood serum, bile fluid, cerebrospinal fluid, supernate from cell cultures, tissue extracts, and combinations thereof.

53. The method of claim 49, wherein the sample is conditioned prior to contact with the conjugated antibody using methods selected from the group consisting of centrifugation, protein precipitation, fast gel filtration with a molecular weight cutoff of about 5 kDa, and combinations thereof.

54. The method of claim 49, wherein the APAP-protein adduct is transported to the first and second anti-APAP antibody by wicking through the substrate.