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Biomedical subjects

R Ekins

Publications and source records attributed to R Ekins.

At least 19 recordsLinked to original sources
PubMed1992

Multianalyte microspot immunoassay. The microanalytical 'compact disk' of the future.

Throughout the 1970s, controversy centered both on immunoassay 'sensitivity' per se and on the relative sensitivities of labelled antibody and labelled analyte methods. Our own theoretical studies in this period revealed that radioimmunoassay (RIA) sensitivities could be surpassed only by the use of very high specific activity non-isotopic labels in 'non-competitive' designs, preferably based on the use of monoclonal antibodies. The time-resolved fluorescence methodology known as Delfia - developed in collaboration with the instrument manufacturer LKB/Wallac - represented the first commercial 'ultra-sensitive' non-isotopic 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, a second advantage of high specific activity labels is that they permit the development of 'multi-analyte' immunoassay systems combining ultra-sensitivity 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 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 (eg fluorescent signals) emitted by two labelled Ab's, the first ('sensor' Ab) deposited as a microspot on a solid support, the second a 'developing' Ab directed against either occupied or unoccupied sensor Ab binding sites.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding, Competitive↗
PubMed1991

Immunoassay standardization.

Assays employed in the biological sciences fall into two categories, which may be respectively termed "comparative" (or "functionally-specific") and "analytical" (or "structurally-specific"). The former are intended to compare the relative effects of substances, or mixtures of substances--not necessarily of identical chemical structure--on a biological system (e.g. whole animal, tissue, cell, etc). Results are represented by units of effect (i.e. they are not units of "amount" of the substance(s) measured), and differ depending on the biological system used. Such assays cannot be "standardised" by the use of a calibrant. In contrast, analytical assays are intended to measure the number of molecules (or mass) of a single substance of unique chemical structure in a test sample, and cannot legitimately be employed to measure mixtures of substances of different structure. Results are represented by units of molecular number or mass, and should be identical for any test sample irrespective of the assay system used. Immunoassays generally fall into this category. Insofar as the antigenic substances present in standards or test samples are dissimilar and/or molecularly heterogeneous, an immunoassay is invalid, and the results it yields have no universal significance. Attempts to standardize "analytically-invalid" immunoassays inevitably fail. Many substances of biological interest (e.g. TSH)--initially defined in terms of their biological function--have subsequently been shown to be molecularly heterogenous. Problems thus arise in the standardization of immunoassays used for their measurement, reflecting the fact that the measurement of a mixture of substances of differing molecular structure (and function) is a meaningless concept. It is thus impossible to "measure TSH"; it is only possible to measure the effect TSH exerts in a particularly assay system. The only long-term solution to this problem is the development of assay systems measuring individual components of such heterogenous mixtures.

Animals↗
PubMed1990

Measurement of free hormones in blood.

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)

Blood Proteins↗
PubMed1990

Multispot, multianalyte, immunoassay.

Consideration of the basic principles of immunoassay design reveals that highly sensitive assays can, in principle, be developed using amounts of "sensor" antibody far smaller than are currently conventional in this field. Furthermore, when using such amounts, the fractional occupancy of antibody binding sites by analyte is independent of both sample volume and antibody concentration. Labelling of both the sensor-antibody and a developing antibody (designed to recognize either occupied or unoccupied sensor-antibody binding sites) permits the development of "ratiometric" immunoassays relying on measurement of the ratio of signals emitted by the two labelled antibodies. Furthermore, the sensor-antibody can be located within a "microspot" a few microns 2 in area. By labelling both sensor and developing antibodies with fluorescent labels, and scanning the microspot using a highly focussed laser beam, microspot immunoassays at least comparable in sensitivity with conventional "macroscopic" immunoassays are made possible. This in turn permits the development of immunoassay "arrays" capable in principle of measuring very large numbers of different substances within small samples (such as a drop of blood). The general principles and theory underlying these concepts are discussed, and preliminary experimental data using currently available instrumentation reported.

Fluorescent Antibody Technique↗
PubMed1989

High specific activity chemiluminescent and fluorescent markers: their potential application to high sensitivity and 'multi-analyte' immunoassays.

The sensitivities of immunoassays relying on conventional radioisotopic labels (i.e. radioimmunoassay (RIA) and immunoradiometric assay (IRMA) permit the measurement of analyte concentrations above ca 10(7) molecules/ml. This limitation primarily derives, in the case of 'competitive' or 'limited reagent' assays, from the 'manipulation errors arising in the system combined with the physicochemical characteristics of the particular antibody used; however, in the case of 'non-competitive' systems, the specific activity of the label may play a more important constraining role. It is theoretically demonstrable that the development of assay techniques yielding detection limits significantly lower than 10(7) molecules/ml depends on: (1) the adoption of 'non-competitive' assays designs; (2) the use of labels of higher specific activity than radioisotopes; (3) highly efficient discrimination between the products of the immunological reactions involved. Chemiluminescent and fluorescent substances are capable of yielding higher specific activities than commonly used radioisotopes when used as direct reagent labels in this context, and both thus provide a basis for the development of 'ultra-sensitive', non-competitive, immunoassay methodologies. Enzymes catalysing chemiluminescent reactions or yielding fluorescent reaction products can likewise be used as labels yielding high effective specific activities and hence enhanced assay sensitivities. A particular advantage of fluorescent labels (albeit one not necessarily confined to them) lies in the possibility they offer of revealing immunological reactions localized in 'microspots' distributed on an inert solid support. This opens the way to the development of an entirely new generation of 'ambient analyte' microspot immunoassays permitting the simultaneous measurement of tens or even hundreds of different analytes in the same small sample, using (for example) laser scanning techniques. Early experience suggests that microspot assays with sensitivities surpassing that of isotopically based methodologies can readily be developed.

Antibody Affinity↗
PubMed1988

The "Pardridge" hypotheses relating to the role of hormone binding proteins in hormone delivery: a critique.

Though the physiological role of specific binding proteins in serum is unknown, and the validity of the "free hormone hypothesis" has been challenged by several authors (including ourselves), we regard Pardridge's widely publicised objections to this hypothesis (and his recent suggestion of the existence of complex biochemical mechanisms causing the release of individual hormones in particular tissues) as deriving from an incorrect analysis of the effects of serum binding proteins on the kinetics of hormone transport, and therefore without foundation. This presentation is intended to substantiate this view.

Chemical Phenomena↗
PubMed1987

Validity of analog free thyroxin immunoassays.

I have briefly illustrated the way in which the main features of analog methods may be readily predicted by consideration of elementary physicochemical laws. (Editorial limitations on the length of this presentation have prevented exploration of other issues such as the reasons for, and effects of, the hitherto unexplained inclusion by manufacturers of large amounts of albumin (12, 18) in kit reagents.) The main implication of our analysis is that, to conform genuinely to the principles of "unbound analog" free hormone immunoassay, an analog must bind to serum proteins to a maximal extent of approximately 10% (but preferably less) in the absence of antibody. The notion that an analog is suitable for use in this context merely if it binds to serum proteins with lower affinities than does the native hormone (6) is demonstrably fallacious. Current analog methods thus neither adhere to the principles of unbound analog free hormone assay nor do they survive classic dilution tests of free hormone assay validity. Demonstrably they do not, in a general sense, measure the free hormone in serum. They may nevertheless yield roughly "correct" values in pregnancy (and thus, arguably, possess clinical value) if the analog fortuitously distributes in an optimal manner between serum proteins, implying approximate balance between the biasing effects caused by the protein changes occurring in pregnancy. However, such methods yield incorrect estimates in all other situations in which the binding characteristics of test samples are disturbed (e.g., by the presence of binding competitors, drugs, abnormal proteins, etc. or when "unbalanced" changes in protein concentration occur), this being the fundamental cause of their diagnostic unreliability. Wilkins, Midgley, and their colleagues have consistently opposed these conclusions, although they have never (using their computer model or by other rigorous means) demonstrated them to be incorrect, nor indeed have they ever formally substantiated the physicochemical propositions on which they themselves claim the methodology rests. Furthermore, they have repeatedly misinterpreted experimental results yielded by the Amerlex kits (such as the effects thereon of serum dilution, NEFA, drugs, etc.), discreetly abandoning (and even totally reversing) their original claims (though not withdrawing them) when these appeared no longer tenable. Perhaps the most serious consequence of these events is the resulting confusion in the literature. This is of major importance in physiological research (28), but it also clearly has major implications in clinical chemistry.(ABSTRACT TRUNCATED AT 400 WORDS)

Fatty Acids, Nonesterified↗
PubMed1985

Roles of serum thyroxine-binding proteins and maternal thyroid hormones in fetal development.

Endocrinologists have speculated for many years about three apparently unrelated topics--the unknown physiological role of specific thyroid (and steroid) hormone-binding proteins present in serum; the extent and significance of placental transport of thyroid hormones from mother to fetus throughout pregnancy; and the specific hormonal role (if any) of thyroxine. A unifying hypothesis is advanced for the existence of subtle endocrine control systems which may profoundly affect early fetal development and ultimate intellectual and behavioural attainments in adults.

Biological Transport↗