WO2000031535A2

WO2000031535A2 - Thyroid peroxidase autoantibody assay compositions, method and kit

Info

Publication number: WO2000031535A2
Application number: PCT/US1999/027563
Authority: WO
Inventors: Martha Louise Garrity; Renato Bundoc Del Rosario
Original assignee: Quest Diagnostics Investments Incorporated
Priority date: 1998-11-25
Filing date: 1999-11-19
Publication date: 2000-06-02
Also published as: AU1740500A; WO2000031535A3

Abstract

Compositions, methods and kits are provided for performing assays for a thyroid peroxidase autoantibody. Solution-stable, indirectly labeled, preferably with an acridinium ester, thyroid peroxidase molecules, portions thereof or synthetic analogs thereof are provided for use with the kits and automated assay platform, using cartridges containing the inventive compositions which provide adequate shelf-life to make practical the production, shipping and storage of the cartridges for users of the automated assay platform.

Description

THYROID PEROXIDASE AUTOANTIBODY ASSAY COMPOSITIONS, METHOD AND KIT

Field of the Invention

The present invention relates to an assay method to detect autoantibodies to thyroid peroxidase, compositions for use with such an assay and a manufacture and a kit containing reagents useful for performing such an assay.

Background of the Invention Antibodies developed by an organism against the organism's own molecular structure, i.e., against itself, are referred to as autoantibodies. Such autoantibodies can cause significant health problems for the organism. In particular, in humans various autoimmune diseases such as Lupus, multiple sclerosis and Graves' disease are caused by autoantibodies. In particular, autoantibodies to an enzyme protein known as thyroid peroxidase ("TPO"), which is found in the thyroid, are known to cause Graves' disease. Therefore, the presence of these TPO autoantibodies is of clinical relevance for the diagnosis of Graves' disease. Assays for TPO autoantibodies are known in the art. For example, see WO93/05072, incorporated herein by reference. Such assays have included the use of at least two modified TPO molecules to sandwich any TPO autoantibody which may be present in a sample being assayed for the presence of such autoantibodies. A first modified TPO molecule has one member of a specific binding pair bound to it. A second modified TPO molecule has a label directly attached to it, such as a chemiluminescent, acridinium ester label. The first TPO molecule with the specific binding pair attached to it binds to some solid phase which has the other member of the specific binding pair attached thereto. The amount of TPO autoantibodies present in a sample is determined by the amount of label detected bound to the TPO autoantibody through the second TPO molecule and simultaneously bound to the first modified TPO molecule. The modified TPO molecules have previously been provided in a freeze-dried state. Additionally, the use of automated test instruments to perform diagnostic assays is known. Further, the use of solid-phase particles, such as, magnetic particles for performing steps, such as separating steps for various biochemical operations, including immunoassays, such as medical diagnostic assays is known in the art. In a particular application, biochemical assays are carried out on an automated assay instrument, which utilizes cartridges, typically disposable cartridges. The cartridge contains vials, which contain various reagents used in carrying out a particular assay. At least one of the vials contains a fluid and magnetic particles or superparamagnetic particles, which, while not themselves magnetic, have dipole moments and can be acted upon by a magnetic field. However, in order to provide a TPO autoantibody assay which is particularly suitable for use on an automated diagnostic assay instrument, it is necessary to provide the reagents, including the acridinium ester-labeled TPO in a liquid medium. Further, these reagents are preferably provided in reagent cartridges for use on the automated diagnostic assay instrument. These reagent cartridges may not be used for some period of time, and accordingly, the reagents must be stable over time. Surprisingly, it was found that acridinium ester-labeled TPO, in contrast to many other acridinium ester-labeled proteins, was very unstable when provided as a liquid reagent. Efforts to stabilize the acridinium ester-labeled TPO by classical methods, discussed in more detail below, failed.

Accordingly, in light of this unexpected problem, it would be desirable to have a TPO autoantibody sandwich antigen assay that utilizes liquid reagents in which all of the liquid reagents are stable for a period of time which allows for their use on an automated diagnostic assay instrument. Further, it would be desirable to provide methods of making and using the compositions, and manufactures, such as cartridges containing the inventive compositions.

Summary of the Invention In accordance with one aspect of the present invention there is provided a composition comprising a thyroid peroxidase molecule associated with a non-destabilizing hapten. In a preferred embodiment the non-destabilizing hapten is non-radioactive. In a more preferred embodiment the non-destabilizing hapten is selected from the following: digoxigenin, a digoxigenin derivative, fluorescein, a fluorescein derivative, rhodamine, a rhodamine derivative, dinitrophenyl or a dinitrophenyl derivative. Preferably NHS esters-of the foregoing are used. In another embodiment there is provided a reagent composition comprising a thyroid peroxidase molecule associated with a non- destabilizing hapten and a fluid medium containing the thyroid peroxidase molecule. Preferably, the reagent composition has a stable shelf-life of at least about one day when stored at about 4 degrees centigrade. In accordance with one aspect of the present invention there is provided a detectable molecular complex which includes: a thyroid peroxidase autoantibody; a thyroid peroxidase molecule; the thyroid peroxidase molecule including a non-destabilizing hapten; an anti-hapten specific binding partner, the anti-hapten specific binding partner labeled with a detectable moiety; and a thyroid peroxidase autoantibody specific binding partner. The thyroid peroxidase autoantibody specific binding partner is adjacent a solid support. The thyroid peroxidase autoantibody is bound at a first site to the thyroid peroxidase autoantibody first specific binding partenr and bound at a second site to the thyroid peroxidase autoantibody specific binding partner adjacent to a solid support. Additionally, the anti-hapten specific binding partner labeled with the detectable moiety is bound to the non-destabilizing hapten moiety of the thyroid peroxidase molecule.

In one embodiment of the present invention, the thyroid peroxidase autoantibody specific binding partner includes a first partner of a specific binding pair and the solid support includes a second partner of the specific binding pair.

In a particular embodiment of the present invention the first partner of the specific binding pair is biotin and the second partner of the specific binding pair is streptavidin.

In a preferred embodiment of the invention, the thyroid peroxidase autoantibody specific binding partner is a thyroid peroxidase molecule. In another preferred embodiment of the invention, the solid support is a particle, more preferably a magnetic bead.

In yet another particular embodiment, the hapten moiety is a digoxigenin derivative and the anti-hapten specific binding partner is a Fab fragment specific to the digoxigenin derivative.

In a further embodiment, the detectable label is a chemiluminescent moiety, preferably an acridinium ester.

In a preferred embodiment, the thyroid peroxidase autoantibody is a human autoantibody. In another aspect of the present invention there is provided a method for quantitation of TPO autoimmune activity and/or disease by assaying for a thyroid peroxidase autoantibody in a sample derived from a subject. The method includes the following steps: reacting the sample which may contain a thyroid peroxidase autoantibody derived from the subject with the following; a thyroid peroxidase molecule, the thyroid peroxidase molecule, including a hapten moiety; an anti-hapten specific binding partner, the anti-hapten specific binding partner labeled with a detectable moiety; and a thyroid peroxidase autoantibody specific binding partner, wherein the thyroid peroxidase autoantibody specific binding partner is adjacent a solid support.

In a further aspect of the present invention there is provided a thyroid peroxidase autoantibody assay kit. The kit includes: a first container containing a thyroid peroxidase molecule, the thyroid peroxidase molecule, including a hapten moiety; a second container containing an anti- hapten specific binding partner, the anti-hapten specific binding partner labeled with a detectable moiety; a third container containing a thyroid peroxidase autoantibody specific binding partner; a fourth container containing a solid support; and, means for retaining the first through the fourth containers. In a particular embodiment the contents of any one or more of the first container through the fourth container maybe combined in one to three containers. In a preferred embodiment, there is provided a fifth container containing a liquid diluent.

In another preferred embodiment the second through fourth containers also contain a liquid. In yet another embodiment of the invention there is provided a human thyroid peroxidase autoantibody assay reagent cartridge for use on an automated, diagnostic assay instrument.

Other features and advantages of the invention will become apparent from the following detailed description, taken in conjunction with the accompanying drawings, which illustrate, by way of example, various features of preferred embodiments of the invention.

Description of the Figures The detailed description of the invention will be made with reference to the accompanying figures, where, as appropriate, like numerals designate corresponding parts of the figures. The figures are meant to be generally illustrative of various examples of the present invention, but are merely examples and are not meant to be limiting of the scope of the invention. FIG. 1 is an illustration of a detectable molecular complex of the present invention. FIGS. 2A-2C illustrate a particular reagent cartridge which can be used with the presently claimed compositions and methods. Detailed Description of the Invention As depicted in Figure 1 , there is a detectable molecular complex 8 which includes a number of elements as follows: a TPO autoantibody 10 which is the analyte being assayed for in a specimen from a subject, typically a human medical patient. TPO autoantibody 10 is bound to a first thyroid peroxidase molecule 1 2. TPO molecule 1 2 includes a non- destabilizing hapten 14. Non-destabilizing hapten 14 may be attached, by for example, chemical conjugation, directly or indirectly to TPO molecule 1 2 as would be known to those of ordinary skill in the art. Active portions of TPO molecule 1 2 which would otherwise be unstable if directly conjugated to an acridinium ester are within the scope of the invention. "Hapten" has the art recognized meaning of a subtance which by itself will not stimulate an immune response, generally because of its relatively small size, but when attached to a larger molecule, such as a protein, can provide a specific antigenic determinant for an antibody to recognize. By non-destabilizing hapten is meant a hapten which will not cause the degradation of TPO molecule 1 2, for a period of time greater than at least about one day at 4 degrees centigrade, particularly when TPO molecule 1 2 is suspended in a fluid medium. Examples of specific haptens which can be used in the present invention include: digoxigenin, a digoxigenin derivative, fluorescein, a fluorescein derivative, rhodamine, a rhodamine derivative, dinitrophenyl or a dinitrophenyl derivative. Specifically, examples of suitable haptens include, but are not limited to: 3-amino-3-deoxydigoxigenin hemisuccinamide, succinimidyl ester; 3-iodoacetylamino-3-deoxydigoxigenin; 5-carboxyfluorescein, succinimidyl ester; 6-carboxyfluorescein, succinimidyl ester; 5-(and-6)-carboxyfluorescein, succinimidyl ester; 6-(fluorescein-5-carboxamido) hexanoic acid, succinimidyl ester ; 6-(fluorescein-5-(and-6)-carboxamido) hexanoic acid, succinimidyl ester; 6-(fluorescein-5-(and-6)-carboxamido) hexanoic acid, succinimidyl ester; Fluorescein-5-EX, succinimidyl ester; 5-(and-6)-carboxy-2', 7'-dichlorofluorescein diacetate, succinimidyl ester; 5-(and-6)-carboxynaphthofluorescein, succinimidyl ester; 5-carboxytetramethylrhodamine, succinimidyl ester; 6-carboxytetramethylrhodamine, succinimidyl ester; 5-carboxy-X-rhodamine, succinimidyl ester; 5-carboxyrhodamine 6G, succinimidyl ester; 6-carboxyrhodamine 6G, succinimidyl ester; 6-carboxy-X-rhodamine, succinimidyl ester; 5-(and-6)-carboxyrhodamine 6G, succinimidyl ester; 5-(and-6)-carboxytetramethylrhodamine, succinimidyl ester; Rhodamine Red™-X, succinimidyl ester; Texas Red^®-X, succinimidyl ester; 4-carboxydihydrotetramethylrosamine, succinimidyl ester; 5-(and-6)-carboxy-X-rhodamine, succinimidyl ester; Rhodamine Green™ carboxylic acid, trifluoroacetamide, succinimidyl ester; 6-(2,4-dinitrophenyl)aminohexanoic acid, succinimidyl ester; Biotin-X 2, 4-dinitrophenyl-X-L-lysine, succinimidyl ester.

In contrast, an acridinium ester directly conjugated to thyroid peroxidase molecule 12 is a destabilizing hapten. That is with the acridinium ester directly conjugated to a thyroid peroxidase molecule which is suspended in a solution even when stored at 4 degrees C the acridinium ester conjugated TPO molecule will begin to degrade in less than a day. If stored at -20 degrees C, the conjugated TPO molecule is stable for about six weeks. Additionally, conventional techniques well know in the art of protein stabilization such as: (a) addition of protein stabilizers and additives including trehalose, glycerol, polyethylene glycol, tween, cyclodextrin, BSA, equine serum, globulins, guanidine, sodium bisulfite, EDTA, etc., (b) buffer selection (PBS, Citrate, TES, CAPS, TAPS, CAPSO, CHES, MOPS, HEPPS, TRIS) and (c) purification by size exclusion HPLC did not significantly improve the shelf life of TPO directly conjugated to an acridinium ester.

In contrast, a TPO molecule conjugated with a non-destabilizing hapten suspended in solution is stable at 4 degrees for at least one year. The results set forth in the table below show the stability of digoxigenin labeles TPO at various times and temperatures. As is known in the art, Arhenius calculations are used to extrapolate the stability of the TPO-digoxigenin for up to a year.

Accelerated Stability of digoxigenin labeled TPO

4° 37 days @ 25° 10 days® 35°

Standard RLU RLU Recovery RLU Recovery

0 lU/mL 1292 1374 106% 1229 95% 2.06 lU/mL 4593 4007 87% 4589 100% 5.34 lU/mL 8986 7876 88% 7488 83% 1.1 lU/mL 14585 13047 89% 13786 95% 20.8 lU/mL 29730 22908 77% 26211 88% 39.8 lU/mL 65016 54408 84% 56955 88% 68.1 lU/ml 87927 71524 81% 84449 96% Mean 88% 92% The destabiliztion of the TPO molecule by the directly conjugated acridinium ester was surprising in light of the fact that direct conjugation of acridinuim esters to ACTH, Cortisol, erythropoietin, calcitonin, parathyroid hormone, human growth hormone, IGF-1 , Free T3, Free T4, thyroid stimulating hormone, thyroglobulin, PSA, DHEA-S, kept in in solution, did not destabilize these molecules. Rather, they had a greater than one year shelf life in solution at 2-8 degrees centigrade.

Adjacent to non-destabilizing hapten 14 is an anti-hapten specific binding partner 1 6. Anti-hapten specific binding partner 1 6 may be an antibody or portion of an antibody such as a Fab fragment, or an aptamer, or any other molecular entity which can specifically recognize and bind to non-destabilizing hapten 14 and does not bind to other entities in an amount which will cause a background signal rendering the compositions and methods of the present invention unusable. Adjacent anti-hapten specific binding partner 16 is a detectable moiety 18. Detectable moiety 18 may be a radioactive isotope, but preferably it is a non-radioactive signal generating moiety, such as a fluorescent, chemiluminescent or detectable enzyme-product-generating moiety and the like all as are known in the art. In a preferred embodiment, detectable moiety 18 is a chemiluminescent acridinium ester. Acridinium esters suitable for use in the present invention include those disclosed in United States Patent Nos. 5,284,951 ; 5,281 ,71 2; 5,283,334; 5,284,952; 5,290,936; 5,321 ,136 and 5,338,847.

TPO autoantibody 10 is also bound to a thyroid peroxidase autoantibody specific binding partner (TPO-AA-SPB) 20. TPO-AA-SPB 20 can be an antibody or a fragment thereof or an aptamer, which is a nucleic acid analog of an antibody, or any other molecular entity which can specifically recognize and bind to TPO autoantibody 10 and does not bind to other entities in an amount which will cause a background signal rendering the compositions and methods of the present invention unusable. In a preferred embodiment, TPO-AA-SPB 20 is a thyroid peroxidase molecule. Active portions of TPO-AA-SPB 20 which would otherwise be unstable if directly conjugated to an acridinium ester are within the scope of the invention.

TPO-AA-SPB 20 includes a first partner of a specific binding pair 22. In a particular embodiment, first partner of a specific binding pair 22 is biotin. Other examples of first partner of a specific binding pair 22 include, but are not limited to biocytin, digoxin, concanavalin A. and other moieties, which can specifically interact with another moiety, such as antibodies, and cell surface and intracellular receptors are also within the scope of the present invention. Detectable molecular complex 8 also includes a solid phase 26.

Solid phase 26 can be the side of a test tube a microtiter well wall and the like. In a preferred embodiment, solid phase 26 is a particle, and in a preferred embodiment solid phase 26 is a magnetic bead. The solid-phase 26 of the present invention can be comprised of, but not limited to: glass, latex, plastic, metal and combinations thereof. If solid phase 26 is a particle, such a particle can have a size of from about 0.01 microns to about 1 cm, more preferably from about 0.05 microns to about 100 microns, and most preferably from about 0.05 microns to about 10 microns. In a preferred embodiment, the solid-phase particle is a magnetic particle, particularly a paramagnetic particle, as are known in the art.

Typically, about 5 ug to about 100 ug of paramagnetic particles are used per assay.

However, the term particle is not meant to be limited to particular shape of solid phase 26. For example, possible shapes include but are not limited to, spherically shaped, irregularly shaped, flat discs, polygonal prisms and solid, and perforated versions of the aforementioned shapes and combinations of all of the above are within the scope of the present invention.

Solid phase 26 includes a second partner of a specific binding pair 24. In a particular embodiment, second partner of a specific binding pair 24 is streptavidin. Other examples of second partner of a specific binding pair 24 can include, for example, avidin, avidin derivatives, or an antibody to the first partner of a specific binding pair, such as an anti-biotin antibody.

An example of an automated test instrument on which assays, such as immunoassays of the present invention, can be run is the Nichols Advantage^®, automated assay platform, available from Nichols Institute Diagnostics, San Juan Capistrano, California. Other automated test instruments, as are known in the art, are also within the scope of the present invention.

An example of an assay for TPO autoantibodies which may be performed on an automated assay platform is as follows:

A patient sample suspected of containing TPO autoantibodies is diluted about 1 :30 by the addition of about 1 5 ul to about 435 ul of a diluent such as about 67% 50mM TES Buffered Saline, pH 7.4 with 33% heat inactivated equine serum and a preseravitve such as 0.01 sodium azide. The following reagents are combined; about 50 ul of biotinylated TPO, about 30 ul of TPO conjugated with digoxegenin or a derivative thereof, about 50 ul of an acridinium ester labeled Fab fragment, about 65 ul of diluted sample. The TPO digoxigenin is stored in about 500mM TES Buffered Saline, pH about 7.4 with about 1 % BSA and a preservative such as about 0.01 % sodium azide. This combination of ingredients is allowed to incubate at about 37 C for about twenty minutes. An additional about 50 ul of diluents are added along with about 20 ul of magnetic particles. Another ten minute incubation follows, followed by a wash step and then a signal detection and counting step. An assay could also be run manually as would be known to one of ordinary skill in the art.

The compositions of the present invention can be made as follows: A solution of TPO protein is concentrated to about 0.2 mL using, for example, a Centricon tube (Millipore, 30K cut off) . The TPO is transferred to a mini centrifuge tube and diluted with about 0.1 M sodium bicarbonate buffer (pH 8.3, < 1 mL). A Digoxigenin NHS ester is thoroughly dissolved in DMF and an aliquot (2-3% of the volume of buffer) of the solution is added to the TPO solution without delay. The TPO reaction mixture is continously vortexed for 2 min and further incubated for another hour. At the end of this period, the reaction tube is centrifuged to remove insoluble material. The supernatant is purified by size exclusion HPLC chromatography (Pharmacia Superose 1 2, 280 nm, PBS eluent). The fractions are pooled, concentrated and the protein concentration determined by UV (280 nm). The Digoxigenin-TPO conjugate is diluted with buffer and stored at 4°C.

A solution of Anti-Digoxigenin Fab (1 -5 mg, Boehringer Manheim) in about 0.1 M sodium bicarbonate (pH 9.6) is prepared in a mini centrifuge tube. Acetonitrile is added to a 2 ml vial containing acridinium methyl ester sulfonyl chloride. After complete dissolution, an aliquot (2-3% of the volume of buffer) of an acridinium ester solution is added to the Anti- Digoxigenin Fab without delay. The reaction mixture is continously vortexed for 2 min and further incubated for a total of 1 5 min. The addition is repeated twice. At the end of this period, the reaction tube is centrifuged to remove insoluble material. The supernatant is purified by size exclusion HPLC chromatography (Pharmacia Superose 1 2, 280 nm detection, PBS eluent). The fractions are pooled, concentrated and the acridinium ester anti-digoxigenin conjugate is diluted with PBS (pH 6) containing 0.4% BSA and stored at 4°C.

FIGS. 2A-2C depict a reagent cartridge 432 of the present invention. Other reagent cartridges for used in automated assay devices are within the scope of the present invention. The cartridge 432 in general is an elongate prismatic body 434 consisting of a base portion 436, and one or more removably attached add-on portions 438, each molded of polymer material. At the forward end 440 of the base portion 436, the base portion 436 defines a vertically extending bore 442 in which an agitation container 444 is rotationally received captively. The agitation container 444 includes an upper radially extending flange portion 446, which is disposed adjacent to an upper flange portion 448 of the body 434. A molded resilient integral hook portion 450 extends from the flange 448 over flange 446 to captively retain agitation container 444 in the bore 442. The body 434 has a lower edge 452, which at the forward end 440 of the module bounds an opening 454. Agitation container 444 includes circumferentially continuous gear portion 456 exposed in this opening. Above the opening 454, the body 434 includes sensor tab 458, which is receivable into a sensor opening 460 at the rear of recess 41 6 to inform a computer system on an automated assay instrument (not shown) where a particular reagent module has been inserted into the slots 430. Above the sensor tab 458, the body 434 includes a pair of spaced apart resilient detent tongues 462. These tongues are receivable on opposite sides of a positioning key 464 at the rear of a recess (not shown) and engage opposite detents (not shown) to removably retain cartridge 432 in a particular slot (not shown)

Internally, agitation container 444 includes vertically extending agitation fins 472, see FIG. 2C, so that rotary oscillation of this container effected by reciprocation of a rack (not shown) stirs the contents of container 444. Container 444 is used to hold a reagent liquid which includes solid-phase particles, such as magnetic beads. The agitation of this container prevents the beads from settling, so that they may be transferred along with the liquid contained therein into a container, such as a cuvette, by a probe of an automated assay instrument (not shown). Container 444 includes a slit septum, 474, as shown in FIG. 2C. Base portion 436 further includes three recesses 476, within which respective reagent containers 478 are removably received. The reagent containers 478 each include a slit septum 478' closing the top opening of these containers, but through which a probe (not shown) may access the reagent liquid in these containers. At the rear of base portion 436 is defined a pair of vertically extending spaced apart guide rails 482. The guide rails 482 cooperate with the remainder of body 434 to define a pair of oppositely opening vertically extending grooves 484, each ending vertically upwardly at an abutment surface 486 adjacent to the upper flange 448 of body 434. The add-on portion 438 has a pair of elongate vertically extending inwardly facing engagement tongues 448, which are vertically receivable in sliding engagement into the grooves 484. Add-on portion 438 includes a lid 490 defining an opening 492 closed by slit septum 494 by which the probe (not shown) may access the liquid reagent in these add-on portions 438.

Add-on portions 438 at their side opposite the base portion 436 define features replicating those of the base portion, so that additional addon portions may be interlocked to form the regent cartridges 432, as is indicated in phantom lines, viewing FIG. 2A. Either the base portion 436 or add-on portions 438 may accept a handle member 496 and configured with engagement tongues like those referenced above with respect to numeral 488. This handle member 496 is engageable with the base portion 436 or with one of the add-on portions 438 in order to facilitate manual grasping and easy insertion and removal of the reagent cartridges 432 at slots 430. At a side surface 498, reagent cartridge 432 can carry a bar code tag 500, which allows for identification by the assay instrument (not shown) of the particular reagents provided to the instrument in each cartridge 432.

Examples of compositions and methods of the present invention are set forth below.

The presently disclosed embodiments are to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims, rather than the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.

Claims

What is claimed is:

1 . A composition comprising a thyroid peroxidase molecule associated with a non-destabilizing hapten.

2. The composition of claim 1 further wherein the non- destabilizing hapten is non-radioactive.

3. The composition of claim 1 wherein the non-destabilizing hapten is selected from the group consisting of digoxigenin, a digoxigenin derivative, fluorescein, a fluorescein derivative, rhodamine, a rhodamine derivative, dinitrophenyl or a dinitrophenyl derivative.

4. A reagent composition comprising a thyroid peroxidase molecule associated with a non-destabilizing hapten and a fluid medium, wherein the thyroid peroxidase molecule associated with the non- destabilizing hapten is contained in the fluid medium, further wherein the reagent composition has a stable shelf-life of at least one day when stored at four degrees centigrade.

5. The composition of claim 4 wherein the non-destabilizing hapten is selected from the group consisting of digoxigenin, a digoxigenin derivative, fluorescein, a fluorescein derivative, rhodamine, a rhodamine derivative, dinitrophenyl or a dinitrophenyl derivative.

6. A detectable molecular complex comprising: a) a thyroid peroxidase autoantibody; b) a thyroid peroxidase molecule, the thyroid peroxidase autoantibody first specific binding partenr including a non-destabilizing hapten; c) an anti-hapten specific binding partner, the anti-hapten specific binding partner labeled with a detectable moiety; and d) a thyroid peroxidase autoantibody specific binding partner, wherein the thyroid peroxidase autoantibody specific binding partner is adjacent a solid support; wherein the thyroid peroxidase autoantibody is bound at a first site to the thyroid peroxidase molecule and bound at a second site to the thyroid peroxidase autoantibody specific binding partner adjacent to a solid support, further wherein the anti-hapten specific binding partner labeled with the detectable moiety is bound to the non-destabilizing hapten moiety of the thyroid peroxidase molecule.

7. The detectable complex of claim 6 wherein the thyroid peroxidase autoantibody specific binding partner includes a first partner of a specific binding pair and the solid support includes a second partner of the specific binding pair.

8. The detectable complex of claim 7 wherein the first partner of the specific binding pair is biotin.

9. The detectable complex of claim 7 wherein the second partner of the specific binding pair is streptavidin.

10. The detectable complex of claim 6 wherein the thyroid peroxidase autoantibody specific binding partner is a second thyroid peroxidase molecule.

1 1 . The detectable complex of claim 6 wherein the solid support is a particle.

1 2. The detectable complex of claim 1 1 wherein the solid particle is a magnetic particle.

1 3. The composition of claim 6 wherein the non-destabilizing hapten is selected from the group consisting of digoxigenin, a digoxigenin derivative, fluorescein, a fluorescein derivative, rhodamine, a rhodamine derivative, dinitrophenyl or a dinitrophenyl derivative.

14. The detectable complex of claim 6 wherein the anti-hapten specific binding partner is an antibody.

1 5. The detectable complex of claim 6 wherein the anti-hapten specific binding partner is a Fab fragment.

16. The detectable complex of claim 6 wherein the detectable label is a chemiluminescent moiety.

17. The detectable complex of claim 1 6 wherein the chemiluminescent moiety is an acridinium ester.

18. A method for assaying for a thyroid peroxidase autoantibody which may be present in a sample derived from a subject, the method comprising the step of: a) reacting the sample containing a thyroid peroxidase autoantibody derived from the subject with the following: i) a thyroid peroxidase molecule, the thyroid peroxidase first specific binding partner including a hapten moiety; ii) an anti-hapten specific binding partner, the anti- hapten specific binding partner labeled with a detectable moiety; and iii) a thyroid peroxidase autoantibody specific binding partner, wherein the thyroid peroxidase autoantibody specific binding partner is adjacent a solid support.

1 9. A thyroid peroxidase autoantibody assay kit comprising: a) a first container containing a thyroid peroxidase molecule, the thyroid peroxidase molecule including a hapten moiety; b) a second container containing an anti-hapten specific binding partner, the anti-hapten specific binding partner labeled with a detectable moiety; c) a third container containing a thyroid peroxidase autoantibody specific binding partner; d) a fourth container containing a solid support; and, e) means for retaining the first through the fourth containers.

20. The kit of claim 19 wherein the contents of any one or more of the first container through the fourth container may be combined in one to three containers.

21 . The kit of claim 20 further comprising a fifth container containing a liquid diluent.

22. The kit of claim 20 wherein the contents of the first through fourth containers are suspended in a liquid.

23. The kit of claim 19 wherein the containers and the means for retaining the containers comprise an assay reagent cartridge for use on an automated diagnostic assay instrument.