Chemistry: analytical and immunological testing – Thyroid hormone tests
Reexamination Certificate
1998-03-09
2004-06-29
Devi, S. (Department: 1645)
Chemistry: analytical and immunological testing
Thyroid hormone tests
C436S501000, C436S504000, C436S542000, C436S531000, C436S543000, C436S545000, C436S817000, C436S804000, C435S007100, C435S007930
Reexamination Certificate
active
06756233
ABSTRACT:
BACKGROUND OF THE INVENTION
For several decades equilibrium dialysis techniques were the only available method for the measurement of free hormones in serum, and until recently were the only methods considered reliable. Equilibrium dialysis methods in this context suffer from several drawbacks including poor precision, tediousness and so on; but above all their results are highly dependent on the purity of the tracers used.
Ellis and Ekins, R. (Acta Endocr. (KbH.) Suppl. 177:106, 1973), disclosed a direct method for free hormone determinations in their paper “Direct Measurement By Radioimmunoassay of the Free Thyroid Hormone Concentration in Serium.” This represented a major improvement over equilibrium dialysis methods because it allowed for the direct measurement by radioimmunoassay (RIA) of free ligand levels in serum dialysates, thus circumventing the problem of tracer purity. This method is now considered by many as the reference methodology for CO free hormone measurements. It is, however, still time consuming and operator-dependent, and it is unavailable to most small laboratories.
Indirect methods for the estimation of free hormone concentrations which were introduced shortly thereafter include the testosterone/steroid hormone binding globulin (SHBG) ratio, the thyroxine (T4)/thyroid binding globulin (TBG) ratio, the free T4 index (based on the product of triiodothyronine (T3) uptake and T4), and the free androgen index.
Ekins, R. (Free Thyroid Hormones; Proceedings of the International Symposium held in Venice, December 1978, Amsterdam: Excerpta Medica, 1979 72-92), introduced the concept of “direct dynamic methods” in which an anti-free ligand antibody is used in direct contact with the biological fluid during dialysis. This constitutes the basis for so-called “immunoextraction” methods.
One such method is taught in U.S. Pat. No. 4,046,870 in which a two-tube immunoassay method measures the rate of transfer of T4 from binding proteins to T4-specific antibody. This method suffered from several analytical and clinical shortcomings which made it virtually just another free T4 index assay.
A second method, introduced by Clinical Assays (Cambridge, Mass. 02139), was a true immunoextraction method. It used a single-tube, two-stage, sequential (back-titration) technique. In this method, a serum sample is incubated with immobilized antibody; then, following a wash step, unoccupied sites on the immobilized antibody are “back-titrated” using labeled ligand. In this approach, the serum is never in contact with the labeled ligand. Although theoretically sound, it suffers from poor sensitivity and precision, and both reactions require exact timing.
Single-step immunoextraction methods for the determination of free ligand concentrations in biological specimens were the obvious next step in the development of free ligand assay systems. These methods rely on chemical rather than physical separation of labeled ligand from nedogenous binders. In order to achieve this objective, several approaches can be adopted, as detailed below.
The prior art discloses that by chemically altering the structure of a given ligand, its binding to endogenous binders is reduced or diminished. This has been amply demonstrated for steroid hormones. (See the discussion of free testosterone below.) In the case of thyroid hormones, Ross, J. E. and Tapley, D. F. (Effect of various analogues on the binding of labeled thyroxine to thyroxine-binding globulin and prealbumin, Endocrinology 79:493, 1966), have shown that the binding of TBG (thyroid binding globulin) to T4 is inhibited if a fairly bulky substitution is made at the 3′ position of the T4 molecule. In addition, Schall, R. F., et al (An enzyme-labeled immunoassay for the measurement of unsaturated thyroid hormone binding capacity in serum and plasma, Clin. Chem. 25:1078 (abstract) 1979), and Kleinhammer, G., et al (Enzyme immunoassay for determination of thyroxine binding index, Clin. Chem. 24:1033, 1978), independently demonstrated that TBG fails to bind to conjugates formed by labeling T4 with horseradish peroxidase. This fact constitutes the basis for the single-step immunoextraction method described in U.S. Pat. No. 4,410,633 to Corning Glass Works, for the measurement of free thyroxine and free 3,5,3′-triiodothyronine wherein horseradish peroxidase is chemically attached to T4 and T3 and later radiolabeled.
In addition, the prior art also discloses that T3 and T4 require the following molecular structure for maximal binding to endogenous binding proteins, viz. TBG, thyroid binding pre-albumin (TBPA), albumin, Snyder, S. M, et al (Binding of thyroid hormones and their analogues to thyroxine-globulin in human serum, J. Biol. Chem. 251:6489, 1976); Sterling, K., et al (Equilibrium dialysis studies of the binding of thyroxine by human serum albumin, J. Clin. Invest. 41:1021, 1962):
1. The L-alanine side chain configuration:
2. The presence of 4′-hydroxyl group (primarily for TBPA and albumin binding); and
3. The presence of two (halogen) substituents in the inner and outer rings (positions 3,5,3′ and 5′).
Several hundred T3 and T4 analogs have been synthesized and studied for their ability to bind to thyroid hormone binding proteins.
U.S. Pat. No. 4,366,143 and its European counterpart, Patent No. 00 26 103, broadly describe the use of such analogs as tracers in a single immunoextraction using simultaneous rather than sequential titration of antibody for the measurement of free hormones. (For convenience, these patents will be collectively referred to hereinafter as the “Amersham” patent.)
An intact alanine side chain is required for optimal binding of T4 and T3 to TBG: the amino group on the analine side chain is the essential constituent. Analogs described in the Amersham patent are T3 and T4 molecules modified at the alanine side chain. Although theoretically these analogs do not bind TBG to any significant extent, they undoubtedly bind albumin and TBPA significantly since the 4′-hydroxyl group on the T3 and the T4 molecules is left intact. It is well established that the binding of albumin and TBPA to the thyronines is quantitative, especially under physiological conditions, Sterling, K. (Molecular structure of thyroxine in relation to its binding by human serum albumin, J.Clin Invest. 43:1721, 1964), and Pages, et al (Binding of thyroxine and thyroxine analogs to human serum prealbumin, Biochem 12:2773, 1973).
The failure of the Amersham patent to recognize the importance of albumin and TBPA binding to the thyronines renders the patent's teachings inadequate for the true measurement of free T3 and free T4 in biological fluids. In fact the commercially available reagents eased on the patent yield misleading and inaccurate free hormone results. This is particularly true in several pathological conditions characterized by significant alterations in the circulating albumin level.
Recent literature has shown that the albumin concentration correlates directly with free T4 concentrations generated by the Amersham assay system. In addition, it is well documented that Amersham's method consistently yields falsely decreased free T4 results in third-trimester pregnancies and in patients suffering from severe non-thyroidal illness, while yielding falsely elevated free T4 levels in cases of familial dysalbuminemic hyperthyroxinemia, a condition in which T4 is abnormally bound to circulating albumin.
During pregnancy, albumin circulates at lower than normal levels, especially during the third trimester. Since Amersham's labeled analog T4 tracer binds albumin and TBPA to a significant extent (greater than 99%), one would expect the Amersham assay system to yield lower than normal free T4 results during the third trimester: more analog tracer is available to bind T4 antibody, resulting in higher binding and lower apparent dose.
Non-esterified free fatty acids are capable of displacing labeled analog from albumin; moreover, they circulate at higher than normal concentrations during pregnancy. This could explain th
Devi S.
Mueth Joseph E.
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