TSH IMMUNOASSAYS AND PROCESSES FOR PERFORMING TSH IMMUNOASSAYS IN THE PRESENCE OF ENDOGENOUS CONTAMINANTS IN RESTRICTED WASH FORMATS

• US 20120301905A1
• Filed: 11/29/2011
• Published: 11/29/2012
• Est. Priority Date: 05/27/2011
• Status: Active Grant

• Current Assignee: Abbott Point Of Care Incorporated (Abbott Laboratories Incorporated)
• Original Assignee: Abbott Point Of Care Incorporated (Abbott Laboratories Incorporated)
• Inventors: Tetin, Sergey Y., Wang, Dan, Collier, Gordan Bruce, Skinner, Joseph P., Ruan, Qiaoqiao
• Granted Patent: US 9,201,078 B2
• Time in Patent Office: Days
• US Class Current: 435/7.94
• CPC Class Codes: B01L 3/50273 characterised by the means or forces applied to move the fluids micropumps F04B19/006, of the membrane type F04B43/043
• CPC Class Codes: B01L 3/502738 characterised by integrated valves microvalves F16K99/0001
• CPC Class Codes: C07K 16/26 against hormones ; against hormone releasing or inhibiting factors
• CPC Class Codes: C07K 2317/33 Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
• CPC Class Codes: G01N 2333/59 Follicle-stimulating hormone [FSH]; Chorionic gonadotropins, e.g. HCG; Luteinising hormone [LH]; Thyroid-stimulating hormone [TSH]
• CPC Class Codes: G01N 33/5302 Apparatus specially adapted for immunological test procedures
• CPC Class Codes: G01N 33/543 with an insoluble carrier for immobilising immunochemicals
• CPC Class Codes: G01N 33/74 involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
• CPC Class Codes: G01N 33/76 Human chorionic gonadotropin including luteinising hormone, follicle stimulating hormone, thyroid stimulating hormone or their receptors

Abstract

The invention relates to low wash Thyroid Stimulating Hormone (TSH) immunoassays using an ELISA sandwich assay having limited or no wash step between the antigen capture, detection antibody addition and substrate introduction steps. This invention exhibits low cross reactivity with biologically similar interfering cross reacting species, such as Follicle Stimulating Hormone (FSH), Luteinizing Hormone (LH) and Chorionic Gonadotropin (CG).

Claims

1. A thyroid stimulating hormone (TSH) sandwich immunoassay, comprising at least two epitope-compatible antibodies comprising at least one capture antibody and at least one signal antibody, wherein the dissociation constant (Kd) of both antibodies for TSH is less than or about 1 nM, and wherein the capture antibody Kd for follicle stimulating hormone (FSH), luteinizing hormone (LH) and chorionic gonadotropin (CG) are greater than 1000 nM, greater than 1000 nM and greater than 500 nM, respectively, and wherein the immunoassay uses a single wash step.

2. The immunoassay of claim 1, wherein the capture antibody and the signal antibody each have a Kd for TSH of no greater than 0.5 nM

3. The immunoassay of claim 1, wherein the capture antibody and the signal antibody each have a Kd for TSH of no greater than 0.15 nM.

4. An immunoassay of claim 1, wherein the signal antibody Kd for FSH, LH and CG are greater than 250 nM, greater than 35 nM and greater than 35 nM, respectively.

5. An immunoassay of claim 1, wherein the capture antibody has a first Kd ratio, Kd(FSH):

Kd(TSH), of greater than 6000, and the signal antibody has a second Kd ratio, Kd(FSH);

Kd(TSH), of greater than 1500.

6. An immunoassay of claim 1, wherein the first Kd ratio is greater than 3000 and the second Kd ratio is greater than 1800.

7. An immunoassay of claim 1, wherein the first Kd ratio ranges from 1500 to 50000, and the second Kd ratio ranges from 1500 to 50000.

8. An immunoassay of claim 1, wherein the capture antibody has a Kd(FSH) of greater than 1000, a Kd(LH) of greater than 1000 nM, and a Kd(CG) of greater than 500 nM, and wherein the signal antibody has a Kd(FSH) of greater than 300 nM, a Kd(LH) of greater than 35 nM, and a Kd(CG) of greater than 35 nM.

9. An immunoassay of claim 1, wherein the capture antibody has a Kd(FSH) of from 1000 to 5000 nM, a Kd(LH) of from 1000 to 5000 nM, and a Kd(CG) of from 500 to 5000 nM, and wherein the signal antibody has a Kd(FSH) of from 250 to 5000 nM, a Kd(LH) of from 35 to 5000 nM, and a Kd(CG) of from 35 to 5000 nM.

10. An immunoassay of claim 1, wherein the capture antibody and the signal antibody, respectively, are capable of binding to at least two different epitopes on the TSH-β

subunit of TSH.

11. An immunoassay of claim 1, wherein the capture antibody and the signal antibody are epitope-compatible antibodies.

12. An immunoassay of claim 1, wherein the capture antibody and signal antibody are selected from the following capture/signal antibody pairs 5409/T25C and T25C/5409.

13. An immunoassay of claim 1, wherein the capture antibody and signal antibody are selected from the following capture/signal antibody pairs 5409/10-1179-456 and 10-1179-456/5409.

14. A method for performing a TSH immunoassay, the method comprising the steps of:

(a) inserting a fluid sample into a device comprising an immunosensor in a conduit, said immunosensor having a capture antibody to TSH;

(b) dissolving a signal antibody into said fluid sample;

(c) forming a sandwich complex on said immunosensor, said complex comprising said capture antibody, TSH, and said signal antibody;

(d) washing uncomplexed signal antibody from the sensor with a wash fluid in no more than one washing step; and

(e) detecting a signal associated with said complexed signal antibody,

wherein the capture antibody and the signal antibody each have a dissociation constant (Kd) for TSH of no greater than 1 nM.

15. The method of claim 14, wherein the capture antibody and the signal antibody each have a Kd for TSH of no greater than 0.5 nM

16. The method of claim 14, wherein the capture antibody and the signal antibody each have a Kd for TSH of no greater than 0.15 nM.

17. The method of claim 14, wherein the capture antibody and the signal antibody each have a dissociation constant (Kd) for TSH of no greater than 0.15 nM, wherein the capture antibody Kd for follicle stimulating hormone (FSH), luteinizing hormone (LH) and chorionic gonadotropin (CG) is greater than 1000, greater than 1000 and greater than 500 nM, respectively, and wherein the signal antibody Kd for FSH, LH and CG are greater than 250, greater than 35 and greater than 35 nM, respectively.

18. The method of claim 14, wherein the capture antibody has a first Kd ratio, Kd(FSH):

Kd(TSH), of greater than 6000, and the signal antibody has a second Kd ratio, Kd(FSH);

Kd(TSH), of greater than 1500.

19. The method of claim 14, wherein the sandwich complex is formed by substantially non-sequential contact of said capture and signal antibodies with the fluid sample.

20. The method of claim 14, wherein the sandwich complex is formed without one or more intervening wash steps directed to ameliorating a cross reaction with one or more of FSH, LH and CG.

21. The method of claim 14, wherein the fluid sample is a diluted blood sample, an undiluted blood sample, a diluted plasma sample, or an undiluted plasma sample.

22. The method of claim 14, wherein the capture antibody comprises mouse monoclonal antibody (Biospacific Cat#5409 SPTNE-5) and the signal antibody comprises mouse monoclonal antibody (Fitzgerald Cat#10-T25C).

23. The method of claim 14, wherein the capture antibody is covalently bound to a bead.

24. The method of claim 14, wherein the signal antibody is conjugated to a reporter molecule selected from the group consisting of alkaline phosphatase, horseradish peroxidase and a fluorescent moiety.

25. The method of claim 14, wherein the detecting step is electrochemical.

26. The method of claim 14, wherein the detecting step is optical.

27. The method of claim 14, comprising a single wash step to remove sample and unbound signal antibody from the capture antibody and where the wash fluid also comprises a substrate for a reporter molecule.

28. The method of claim 14, further comprising metering the fluid sample in a metering chamber to form a metered sample having a volume of from 1 to 500 μ

L.

29. The method of claim 14, further comprising bursting a wash fluid pouch, which is in fluid communication with the immunosensor, to release from 5 to 500 μ

L of wash fluid into said conduit.

30. The method of claim 14, further comprising forming a plurality of air segments in said wash fluid.

31. The method of claim 14, wherein the capture antibody and the signal antibody, respectively, are capable of binding to at least two different epitopes on the TSH-β

subunit of TSH.

32. The method of claim 14, wherein the capture antibody and the signal antibody are epitope-compatible antibodies.

33. The method of claim 14, wherein the capture antibody and signal antibody are selected from the following capture/signal antibody pairs 5409/T25C and T25C/5409.

34. The method of claim 14, wherein the capture antibody and signal antibody are selected from the following capture/signal antibody pairs 5409/10-1179-456 and 10-1179-456/5409.

35. A thyroid stimulating hormone (TSH) sandwich immunoassay antibody selection method for non-sequential assays and assays with low wash fluid cycles, comprising selecting at least two epitope-compatible antibodies comprising at least one capture antibody and at least one signal antibody, wherein the capture antibody Kd for follicle stimulating hormone (FSH), luteinizing hormone (LH) and chorionic gonadotropin (CG) is greater than 1000, greater than 1000 and greater than 500 nM, respectively, and wherein the signal antibody Kd for TSH is less than about 1 nM and the Kd for FSH, LH and CG are greater than 250, greater than 35 and greater than 35 nM, respectively, respectively.

36. The method of claim 35, wherein the capture antibody and the signal antibody each have a Kd for TSH of no greater than 0.5 nM

37. The method of claim 35, wherein the capture antibody and the signal antibody each have a Kd for TSH of no greater than 0.15 nM.