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The binding by serum proteins of circulating thyroid and steroid hormones is a phenomenon whose physiological significance is still not understood, and the validity of the free hormone hypothesis remains in doubt. Indeed, even the most basic physicochemical consequences of these proteins' presence within the microcirculation continue to generate controversy, reflecting disagreement of the rate-limiting effects of hormone-protein interactions on hormone efflux from protein-containing fluid compartments. My colleagues and I have claimed, in particular, that the observations on which Pardridge and coworkers' current ideas crucially depend are entirely explicable using a relatively simple mathematical model of hormone efflux from tissue capillaries differing from the even simpler model relied on by these authors only in that it takes basic hormone-binding kinetics into consideration. The necessity to postulate the local hormone release mechanisms that Pardridge et al. propose in order to account for their observations is thus obviated. Though this conclusion continues to be contested by Pardridge et al. (on the grounds that our own model is invalid), the controversy demonstrates the crucial importance in this area of sound mathematical analysis and the great danger of misinterpreting experimental data by reliance on oversimple theoretical concepts. In reality, the effects of intracapillary protein-binding reactions on target-tissue hormone uptake are of considerably greater complexity than are encompassed in the simple model which is sufficient to explain the observation of Pardridge et al. (1). In particular the assumption made by a number of workers that intracapillary hormone dissociation from binding proteins does not limit the rate of tissue uptake if the latter is substantially less than the intracapillary free hormone generation rate is demonstrably invalid, being incorrectly based on the kinetics of homogeneous (liquid-phase) reactions. By making this assumption, false conclusions may be drawn regarding the kinetics of hormone transport to target tissues, and hence of the effects of the changes in binding protein concentrations that occur in a variety of pathophysiological states. Relying on more detailed analysis, my colleagues and I have suggested that it is plausible, purely on physicochemical grounds, that the characteristic changes in binding protein levels seen in pregnancy serve to redistribute hormone throughout the body, specifically (in the case of the thyroid hormones) directing T4 to the feto-placental unit. Though difficult to verify directly (and perhaps invalid), this proposition has refocused attention on the fetal needs for T4 before the development of the fetal thyroid gland, and on the possible effects on neurological development of an inadequate maternal T4 supply.(ABSTRACT TRUNCATED AT 400 WORDS)

The main developments in the “ligand assay” field in which I have been involved are traced. These include the original development of “first generation” competitive assays relying on radiolabeled analyte markers; the development of the first “second generation”, noncompetitive (ultrasensitive) methods, which rely on the use of labeled (monoclonal) antibodies and high specific activity nonisotopic labels (leading to the transformation of the immunodiagnostic field in the 1980s); and the development of the first “third generation” miniaturized, chip-based, microarray methods, which permit the simultaneous ultrasensitive measurement of many analytes in the same small sample. The latter—applicable both to immunoassay and to DNA/RNA analysis—are likely to revolutionize the diagnostic and pharmaceutical fields in the next decade.

Throughout the 1970s, controversy centered both on immunoassay "sensitivity" per se and on the relative sensitivities of labeled antibody (Ab) and labeled analyte methods. Our theoretical studies revealed that RIA sensitivities could be surpassed only by the use of very high-specificity nonisotopic labels in "noncompetitive" designs, preferably with monoclonal antibodies. The time-resolved fluorescence methodology known as DELFIA--developed in collaboration with LKB/Wallac--represented the first commercial "ultrasensitive" nonisotopic technique based on these theoretical insights, the same concepts being subsequently adopted in comparable methodologies relying on the use of chemiluminescent and enzyme labels. However, high-specific-activity labels also permit the development of "multianalyte" immunoassay systems combining ultrasensitivity with the simultaneous measurement of tens, hundreds, or thousands of analytes in a small biological sample. This possibility relies on simple, albeit hitherto-unexploited, physicochemical concepts. The first is that all immunoassays rely on the measurement of Ab occupancy by analyte. The second is that, provided the Ab concentration used is "vanishingly small," fractional Ab occupancy is independent of both Ab concentration and sample volume. This leads to the notion of "ratiometric" immunoassay, involving measurement of the ratio of signals (e.g., fluorescent signals) emitted by two labeled Abs, the first (a "sensor" Ab) deposited as a microspot on a solid support, the second (a "developing" Ab) directed against either occupied or unoccupied binding sites of the sensor Ab. Our preliminary studies of this approach have relied on a dual-channel scanning-laser confocal microscope, permitting microspots of area 100 microns 2 or less to be analyzed, and implying that an array of 10(6) Ab-containing microspots, each directed against a different analyte, could, in principle, be accommodated on an area of 1 cm2. Although measurement of such analyte numbers is unlikely ever to be required, the ability to analyze biological fluids for a wide spectrum of analystes is likely to transform immunodiagnostics in the next decade.