DETECTION AND MEASUREMENT OF THYROID HORMONE AUTOANTIBODIES

• US 20100120170A1
• Filed: 11/12/2008
• Published: 05/13/2010
• Est. Priority Date: 11/12/2008
• Status: Active Grant

• Current Assignee: Hemopet
• Original Assignee: Hemopet
• Inventors: Dodds, W. Jean, Ongchangco, Ferdie S.
• Granted Patent: US 7,799,532 B2
• Time in Patent Office: Days
• US Class Current: 436/501
• CPC Class Codes: G01N 33/564 for pre-existing immune complex or autoimmune disease , i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
• CPC Class Codes: G01N 33/78 Thyroid gland hormones , e.g. T3, T4, TBH, TBG or their receptors

Abstract

A non-radioisotopic method detects T3AA and T4M thyroid autoantibodies in a sample from a non-human species such as the canine species. Antibodies and autoantibodies are bound, and a precipitated or bound antigen-antibody or antigen-autoantibody complex is formed. The supernatant or surrounding fluid of the bound or precipitated antigen-antibody or antigen-autoantibody complex is then removed. The thyroid activity of the bound complex, precipitate, supernatant or surrounding fluid is measured. The thyroid analyte is at least one of T3, Free T3, T4 or Free T4.

Claims

1-2. -2. (canceled)

4. (canceled)

6. (canceled)

8. (canceled)

10. (canceled)

12-13. -13. (canceled)

27-30. -30. (canceled)

32-35. -35. (canceled)

42. A non-radio isotopic method of determining T3AA canine thyroid autoantibody in a patient sample comprising:

a) measuring Free T3 in a patient sample, wherein the sample is from a canine species;

b) treating the patient sample with a non-radioisotopic precipitating or binding agent;

(c) suspending the non-radioisotopic precipitate or bound complex, and at least one of;

(i) measuring Free T3 in suspended non-radioisotopic precipitate or bound complex using a non-radioisotopic label, and

(ii) subtracting the measurement of i) from a) wherein the result of ii) is directly proportional to the amount of T3AA in the patient sample;

or

(iii) measuring Free T3 in supernatant or surrounding fluid of the non-radioisotopic precipitate or bound complex using a non-radioisotopic label;

(iv) subtracting the measurement of iii) from a) wherein the result of iv) is indirectly proportional to the amount of T3AA in the patient sample; and

effecting detection with a sensitivity in a range between 1 picogram per mL and up to about 1 femtogram per mL.

3. A method as claimed in claim 42 including using a non-radioisotopic first particle or a first chemical or substance to effect precipitation or binding of an precipitate or bind the antigen-antibody or antigen-autoantibody complex.

5. A method as claimed in claim 3 including using either polyethylene glycol or charcoal or binding compound, second chemical, or second particle for effecting separation respectively of a bound antigen or autoantibody from an unbound antigen or antibody.

7. A method as claimed in claim 42 including measuring Free T3 by both of steps i) and ii) and steps iii) and iv).

9. A method as claimed in claim 42 including measuringinihe steps of c) i) or c) iii). Free T3 in an antigen-antibody complex itself in the precipitate or a detachable bound complex, the measuring being by fluorescence, chemical or other tagging, or measuring the mass.

11. A method as claimed in claim 42 including employing direct chemiluminesence, and including a bioluminescent detector and microparticles for immobilizing Free T3 and having a non-radioisotopic label for detection as a solid phase, the detection of the amount of Free T3-being with a sensitivity more than 1 picogram per mL and being a sensitivity of at least up to about 1 femto gram per mL.

21. A method as claimed in claim 42 including:

measuring in the sample the a quantity of non-radioisotopic label in the precipitate or bound complex or in the supernatant or surrounding fluid in a manner substantially independent of the amount of an uncomplexed non-radioisotopic labeled antigen in the precipitate or bound complex by measuring the quantity of the label in the precipitate or bound complex or in the supernatant or surrounding fluid.

22. A method as claimed in claim 21, including:

using a control sample substantially identical to the patient sample to determine the amount of an uncomplexed non-radio isotopic labeled antigen present in the precipitate or bound complex by measuring an amount of an uncomplexed antigen in the control sample after treating the control sample with a precipitating or binding agent,

(a) subtracting the measured amount of the uncomplexed antigen in the control sample from the measured amount of the complexed antigen in the sample, and

(b) using the resulting amount in step (a) to determine the amount in of T3AA in the sample.

23. The method of claim 21, wherein the quantity of the an unlabelled antigen is at least as great as the quantity of a non-radioisotopic labeled antigen.

24. The method of claim 21, wherein the sample is canine serum, the T3AA autoantibody is thyroid autoantibody, and an antigen is thyroid hormone.

25. The method of claim 21, including washing the precipitate or bound complex with a washing agent to dissolve an uncomplexed labeled antigen without dissolving the a non-radioisotopic labeled antigen-autoantibody complex, and washing the precipitate or bound complex with a washing agent to dissolve the uncomplexed labeled antigen without dissolving the non-radioisotopic labeled antigen-autoantibody complex.

26. The method of claim 25, wherein the washing reduces the amount of an uncomplexed labeled antigen to less than 5% of a total amount of a label in the precipitate or bound complex.

36. A method as claimed in claim 42 including having a specificity of the assay at a level at least as specific as an assay performed by known radioimmunoassay technique.

39. A method as claimed in claim 42 including measuring T3AA canine thyroid autoantibody using a Free T3 analyte.

43. A non-radio isotopic method of determining T3AA thyroid autoantibody in a sample:

(a) measuring Free T3 in a patient sample, wherein the sample is from a canine species;

(b) treating the patient sample with a non-radioisotopic precipitating or binding agent;

(c) suspending the non-radioisotopic precipitate or bound complex, and at least one of;

(i) measuring Free T3 in suspended non-radioisotopic precipitate or bound complex using a non-radioisotopic label; and

(ii) subtracting the measurement of i) from a) wherein the result of ii) is directly proportional to the amount of T3AA in the patient sample;

or

(iii) measuring Free T3 in supernatant or surrounding fluid of the precipitate or bound complex using a non-radjoisotopic label;

(iv) subtracting the measurement of iii) from a) wherein the result of iv) is indirectly proportional to the amount of T3AA in the patient sample; and

effecting detection of a), c) i) or c) iii) by employing non-radioisotopic direct chemiluminesence, and including a bioluminescent detector and microparticles for immobilizing Free T3 as a solid phase, thereby to obtain a non-radioisotopic determination of T3AA.

14. A method as claimed in claim 43 the detection range being with a sensitivity between 1 picogram per mL and up to about 1 femtogram per mL.

37. A method as claimed in claim 43 including effecting detection by detecting with a sensitivity range between 1 picogram per mL and up to 1 femtogram per mL, and having a specificity of the assay at a level at least as specific as an assay performed by known radioimmunoassay technique.

40. A method as claimed in claim 43 including measuring T3AA canine thyroid autoantibody using a Free T3 analyte.

45. A method as claimed in claim 43 including measuring Free T3 by both of steps i) and ii) and steps iii) and iv).

47. A method as claimed in claim 43 including:

measuring in the sample the quantity of non-radioisotopic label in the precipitate or bound complex or in the supernatant or surrounding fluid in a manner substantially independent of the amount of an uncomplexed non-radio isotopic labeled antigen in the precipitate or bound complex by measuring the quantity of the label in the precipitate or bound complex or in the supernatant or surrounding fluid.

48. A method as claimed in claim 47, including:

using a control sample substantially identical to the patient sample to determine the amount of uncomplexed non-radioisotopic labeled antigen present in the precipitate or bound complex by measuring an amount of uncomplexed antigen in the control sample after treating the control sample with a precipitating or binding agent,

(a) subtracting the measured amount of the uncomplexed antigen in the control sample from the measured amount of the complexed antigen in the patient sample; and

(b) using the resulting amount in step (a) to determine the amount of T3AA in the patient sample.

44. A non-radio isotopic method of determining T3AA canine thyroid autoantibody in a sample

(a) measuring Free T3 in a patient sample, wherein the sample is from a canine species;

(b) treating the patient sample with a non-radioisotopic precipitating or binding agent;

(c) suspending the non-radioisotopic precipitate or bound complex, and at least one of;

(i) measuring Free T3 in suspended non-radioisotopic precipitate or bound complex using a non-radioisotopic label, and

(ii) subtracting the measurement of i) from a) wherein the result of ii) is directly proportional to the amount of T3 AA in the patient sample;

or

(iii) measuring ffee Free T3 in supernatant or surrounding fluid of the precipitate or bound complex using a non-radioisotopic label;

(iv) subtracting the measurement of iii) from a) wherein the result of iv) is indirectly proportional to the amount of T3AA in the patient sample; and

(d) effecting detection of a), c) i) or c) iii) by employing a non-radioisotopic detection thereby to obtain a non-radioisotopic determination of T3AA.

31. A method as claimed in claim 44 including effecting detection of Free T3 of step a), c) i) or c) iii) by detecting with sensitivity in a range between 1 picogram per mL up to 1 femtogram per mL.

38. A method as claimed in claim 44 including effecting detection by detecting with a sensitivity range between 1 picogram per mL and up to 1 femtogram per mL, and having a specificity of the assay at a level at least as specific as an assay performed by known radioimmunoassay technique.

41. A method as claimed in claim 44 including measuring T3AA canine thyroid autoantibody using a Free T3 analyte.

46. A method as claimed in claim 44 including measuring Free T3 by both of steps i) and ii) and steps iii) and iv).

49. A method as claimed in claim 44 including:

measuring in the sample the quantity of non-radioisotopic label in the precipitate or bound complex or in the supematant or surrounding fluid in a manner substantially independent of the amount of an uncomplexed non-radioisotopic labeled antigen in the precipitate or bound complex by measuring the quantity of the label in the precipitate or bound complex or in the supematant or surrounding fluid.

50. A method as claimed in claim 49, including:

using a control sample substantially identical to the patient sample to determine the amount of uncomplexed non-radioisotopic labeled antigen present in the precipitate or bound complex by measuring an amount of uncomplexed antigen in the control sample after treating the control sample with a precipitating or binding agent,

(a) subtracting the measured amount of the uncomplexed antigen in the control sample from the measured amount of the complexed antigen in the patient sample, and

(b) using the resulting amount in step (a) to determine the amount of T3AA in the patient sample.